ArticlePDF Available

Open Reading Frame-3a gene of the 2019 novel coronavirus inhibits the occurrence and development of colorectal cancer

Authors:

Abstract and Figures

Intestinal microecology is composed of bacteria, fungi and viruses. As a part of intestinal microecology, viruses participate in the occurrence and development of colorectal cancer. The 2019-nCoV was detected in stool samples from patients during COVID-19, suggesting that the 2019-nCoV may be associated with intestinal microecology. However, the relationship of the 2019-nCoV and CRC is unclear. The aim of this study is to explore the role of Open Reading Frame-3a (ORF3a) of the 2019-nCoV in CRC. After the pCDH-CMV-MCS-EF1-Puro vector that provides high expression of ORF3a was transfected into the SW480 CRC cell line, immunofluorescence was used to determine the localization of ORF3a in SW480 cells. The proliferation, migration, invasion, apoptosis, and cell cycle progression of SW480 cells were measured using the Cell Counting Kit-8 (CCK-8), wound healing, Transwell assay, flow cytometry, the TUNEL assay, and propidium iodide single staining. The results showed that ORF3a inhibited the proliferation, invasion, and migration of SW480 cells and induced their apoptosis after 24, 48, 72 h. Meanwhile, ORF3a inhibited the cell cycle and blocked SW480 CRC cells in the G1 phase. In in vivo experiments, high ORF3a expression was associated with decreased tumor volume, tumor weight, relative tumor volume, and tumor activity. ORF3a inhibited the growth and induced apoptosis and necrosis of tumor tissues. Based on this, we demonstrated that ORF3a might play a role in CRC, providing a new direction for the prevention and treatment of CRC.
Content may be subject to copyright.
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
Discover Oncology
Research
Open Reading Frame‑3a gene ofthe2019 novel coronavirus inhibits
theoccurrence anddevelopment ofcolorectal cancer
HanShuwen1· WuYinhang2· MaoJing3· ChenGong4· HouXiaohui5· YangXi1· WuWei6
Received: 20 November 2021 / Accepted: 22 February 2022
© The Author(s) 2022 OPEN
Abstract
Intestinal microecology is composed of bacteria, fungi and viruses. As a part of intestinal microecology, viruses par-
ticipate in the occurrence and development of colorectal cancer. The 2019-nCoV was detected in stool samples from
patients during COVID-19, suggesting that the 2019-nCoV may be associated with intestinal microecology. However, the
relationship of the 2019-nCoV and CRC is unclear. The aim of this study is to explore the role of Open Reading Frame-3a
(ORF3a) of the 2019-nCoV in CRC. After the pCDH-CMV-MCS-EF1-Puro vector that provides high expression of ORF3a
was transfected into the SW480 CRC cell line, immunouorescence was used to determine the localization of ORF3a in
SW480 cells. The proliferation, migration, invasion, apoptosis, and cell cycle progression of SW480 cells were measured
using the Cell Counting Kit-8 (CCK-8), wound healing, Transwell assay, ow cytometry, the TUNEL assay, and propidium
iodide single staining. The results showed that ORF3a inhibited the proliferation, invasion, and migration of SW480 cells
and induced their apoptosis after 24, 48, 72h. Meanwhile, ORF3a inhibited the cell cycle and blocked SW480 CRC cells
in the G1 phase. In invivo experiments, high ORF3a expression was associated with decreased tumor volume, tumor
weight, relative tumor volume, and tumor activity. ORF3a inhibited the growth and induced apoptosis and necrosis of
tumor tissues. Based on this, we demonstrated that ORF3a might play a role in CRC, providing a new direction for the
prevention and treatment of CRC.
Highlights
1. ORF3a was successfully transfected into SW480 cells, and its expression was localized in the cytoplasm of SW480
cells.
Yang Xi and Wu Wei contributed equally to this work
Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12672- 022-
00473-6.
* Yang Xi, yangxi19900601@126.com; * Wu Wei, hchwuwei2018@126.com; ww@hzhospital.com; Han Shuwen,
shuwenhan985@163.com; Wu Yinhang, bawnywuyinhang@163.com; Mao Jing, maojingzd@163.com; Chen Gong,
cg13566746965@163.com; Hou Xiaohui, houxiaohui20130820@163.com | 1Department ofOncology, Huzhou Central Hospital, Aliated
Central Hospital Huzhou University, No. 1558, Sanhuan North Road, Wuxing District, Huzhou313000, Zhejiang, China. 2Graduate School
ofSecond Clinical Medicine Faculty, Zhejiang Chinese Medical University, No. 548 Binwen Road, Binjiang District, Hangzhou310053,
Zhejiang, China. 3Graduate School ofMedical College ofZhejiang University, No. 268 Kaixuan RoadJianggan District, Hangzhou310029,
Zhejiang, China. 4Clinical Medicine ofHuzhou University, Medical College ofHuzhou University, No. 759, Erhuan East Road,
Huzhou313000, Zhejiang, China. 5Graduate School ofNursing, Huzhou University, No. 1 Bachelor Road, Wuxing District, Huzhou313000,
Zhejiang, China. 6Department ofGastroenterology, Huzhou Central Hospital, Aliated Central Hospital Huzhou University, No. 1558,
Sanhuan North Road, Wuxing District, Huzhou313000, Zhejiang, China.
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
2. ORF3a inhibited SW480 cell proliferation, invasion and migration, induced their apoptosis, and blocked them in the
G1 phase.
3. ORF3a inhibited colorectal tumor growth and accelerated cancer cell death invivo.
4. Our study provides new insights into the use of viruses in anti-tumor regimens in colorectal cancer.
Keywords 2019-nCoV· ORF3a· Colorectal cancer· Cell viability· Cell cycle· Apoptosis
Abstract
Intestinal microecology is composed of bacteria, fungi and viruses. As a part of intestinal microecology, viruses par-
ticipate in the occurrence and development of colorectal cancer. The 2019-nCoV was detected in stool samples from
patients during COVID-19, suggesting that the 2019-nCoV may be associated with intestinal microecology. However, the
relationship of the 2019-nCoV and CRC is unclear. The aim of this study is to explore the role of Open Reading Frame-3a
(ORF3a) of the 2019-nCoV in CRC. After the pCDH-CMV-MCS-EF1-Puro vector that provides high expression of ORF3a
was transfected into the SW480 CRC cell line, immunouorescence was used to determine the localization of ORF3a in
SW480 cells. The proliferation, migration, invasion, apoptosis, and cell cycle progression of SW480 cells were measured
using the Cell Counting Kit-8 (CCK-8), wound healing, Transwell assay, ow cytometry, the TUNEL assay, and propidium
iodide single staining. The results showed that ORF3a inhibited the proliferation, invasion, and migration of SW480 cells
and induced their apoptosis after 24, 48, 72h. Meanwhile, ORF3a inhibited the cell cycle and blocked SW480 CRC cells
in the G1 phase. In invivo experiments, high ORF3a expression was associated with decreased tumor volume, tumor
weight, relative tumor volume, and tumor activity. ORF3a inhibited the growth and induced apoptosis and necrosis of
tumor tissues. Based on this, we demonstrated that ORF3a might play a role in CRC, providing a new direction for the
prevention and treatment of CRC.
Highlights
1. ORF3a was successfully transfected into SW480 cells, and its expression was localized in the cytoplasm of SW480
cells.
2. ORF3a inhibited SW480 cell proliferation, invasion and migration, induced their apoptosis, and blocked them in the
G1 phase.
3. ORF3a inhibited colorectal tumor growth and accelerated cancer cell death invivo.
4. Our study provides new insights into the use of viruses in anti-tumor regimens in colorectal cancer.
Keywords 2019-nCoV· ORF3a· Colorectal cancer· Cell viability· Cell cycle· Apoptosis
1 Introduction
Colorectal cancer (CRC) is the third most common malignancy worldwide and the second leading cause of cancer-
related deaths [1]. An increasing amount of data indicate that intestinal flora imbalance may promote the occurrence
of CRC [2]. The gut microenvironment is an extremely complex microecosystem. The diversity of organisms in the
human intestinal tract is enormous. In addition to bacteria and fungi, hundreds of thousands of viruses are present [3].
In a recent study, researchers analyzed the gut metagenomes of 28,060 human samples and 2898 reference genomes
of gut bacteria distributed worldwide. They constructed a database named GDP” containing 142,000 sequences
of intestinal phages [4]. Viruses can be directly involved in the occurrence of CRC by infecting cells or indirectly,
by regulating the composition of the bacterial community [5]. Viral infections have been shown to induce cancer.
The currently known viruses associated with CRC include human papillomavirus [6, 7], some herpes viruses, human
immunodeficiency virus [8], and hepatitis B or hepatitis C virus (HCV) [9]. Studies have shown that cytomegalovirus
herpesvirus infection is considerably associated with the incidence of CRC [7]. Hepatitis B virus infection not only
induces liver cancer, but is also associated with liver metastasis in CRC [10]. However, there are few reports on the
molecular mechanisms or specific effects of viruses on the occurrence and development of CRC.
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
Many coronavirus-related studies have been conducted owing to the novel coronavirus epidemic. The British
Journal of Hematology published a special case in which a patient with Hodgkin’s lymphoma was cured of cancer
4months after being infected with the novel coronavirus [11]. Nonetheless, the exact mechanism by which the
patient’s tumor disappeared is unknown. Meanwhile, studies have shown that COVID-19 infections increase the risk of
death in patients with cancer [12, 13]. Current studies have mostly focused on the effects of the virus on the outcomes
of cancer; however, the understanding of the relationship between coronavirus infection and cancer development,
metastasis, and sensitivity to treatment is still very limited. Fecal samples of COVID-19 patients were tested, and it
was found that 2019-nCoV could still be detected in stool within 35days after respiratory clearance [14]. It remains
to be elucidated whether the 2019-nCoV, as an endogenous retrovirus, can act as an inducer or inhibitor for the
occurrence of CRC, thereby affecting its course. By analyzing the genome sequence of COVID-19, we found that the
novel coronavirus functional gene ORF3a might induce or inhibit CRC by acting on CRC driver genes.
The open reading frame (ORF) is a part of a gene sequence that encodes proteins and is involved in virus replica-
tion and release. The 2019-nCoV genome contains 14 ORFs encoding 27 proteins. The 3 end contains four ORFs that
encode structural proteins (S, E, M, and N) and eight ORFs that encode accessory proteins (ORF3a, 3b, P6, 7a, 7b, 8b,
9b, and ORF14) [15, 16]. ORF3a is a conservative coronavirus protein that contains different functional domains related
to virulence, infectivity, and virus release [17]. ORF3a regulates viral mRNA translation and expression in the host [18,
19]. Although there are no reports on the relationship between ORF3a and tumor diseases, studies have shown that
the ORF3a protein of 2019-nCoV 2 activates the NLRP3 inflammasome by promoting TNF receptor-associated factor
3 (TRAF3)-dependent ASC ubiquitination [20], and the activated NLRP3 inflammasome can improve colitis-related
cancers induced by methoxymethane/dextran sodium sulfate in mice [21]. This suggests that the ORF3a protein may
have an inhibitory effect on colitis-related cancers. Research has also shown that ORF3a of 2019-nCoV can induce
cell apoptosis through exogenous pathways [22, 23].
Based on the above information, we transfected SW480 cells with the ORF3a cDNA using a lentiviral vector. Invivo
and invitro experiments were performed and pathological tissue analysis was conducted to examine the function of
ORF3a in SW480 cell viability and apoptosis and characterize the role of ORF3a in the growth of colorectal tumors.
The results of our present study are expected to provide a new research idea for the anti-tumor therapy of CRC using
viruses.
2 Materials andmethods
2.1 Lentivirus transfection
2.1.1 Construction ofoverexpression vector
We declare that all methods were performed in accordance with the relevant guidelines and regulations. The 2019-
nCoV ORF3a gene was inserted into the expression vector pCDH-CMV-MCS-EF1-PURO vector (ORF3a overexpression
vector). The vector pCDH-CMV-MCS-EF1-PURO was double digested with XbaI and BamHI at 37 for 2h. Then, the
products of enzyme digestion were analyzed using 1% agarose gel electrophoresis, and photographed using a gel
imager (980A, Shanghai Furi Technology, Shanghai, China). Next, the DNA fragment of interest was recovered accord-
ing to the instructions of the agarose gel recovery kit. The DNA solution was then collected, followed by seamless
cloning. The recovered vector and the synthesized DNA fragments were subjected to homologous recombination
and incubated at 50°C for 15min. The recombined product was then used to transform STBl3 competent cells. Trans-
formed bacteria were evenly spread on a plate containing ampicillin and incubated overnight for colony formation.
Three single clones were picked and cultured in 4mL LB medium overnight. Then, a centrifuge was used to obtain
the plasmid solution. The extracted plasmid was digested at 37°C for 2h. Electrophoresis was performed for 30min:
the electrophoresis buffer was 1 × TAE, the agarose concentration was 1%, and the electrophoresis voltage was 130V.
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
Pictures were taken on a gel imager for restriction digestion identification. The plasmids with the correct sequencing
were extracted in large quantities and stored at − 20°C.
2.1.2 Screening forcells stably expressing ORF3a
Briey, 293T cells were plated into a 60mm dish at a density of 1.5 × 106 cells/dish. The next morning, after the cells
adhered to the plate, the lentivirus was packaged. The initial lentivirus solution was stored in a refrigerator at 4°C and
used on the same day. Then, SW480 cells (Jiangsu Kaiji Biotechnology Company, Ltd., Nanjing, China) were infected with
the virus. Cells in the logarithmic phase were washed with PBS, trypsinized to obtain a suspension of single detached
cells, and 3mL of complete culture medium was added for neutralization. The cell suspension was collected and centri-
fuged at 300×g for 3min. The supernatant was discarded, and the precipitated cells were resuspended 1mL complete
medium, counted, and plated in a 6-well plate at a density of 3 × 105 cells/well, and 2mL medium was added to each
well. The next day, after the cells were attached to the plate, the medium in the wells was discarded and replaced with
L-15 complete medium containing 1000μL virus supernatant. After 48h, the infection eciency was observed under a
uorescence microscope (IX73, Olympus, USA). If there were uninfected cells, puromycin was added at a nal concentra-
tion of 0.5µg/mL (pre-experimentally determined) to select for infected cells. The cells were cultured in the presence of
the drug until all the cells showed green uorescence. Some cells were cryopreserved, and some cell precipitates were
collected to verify overexpression.
2.1.3 Confirmation ofORF3a overexpression using qPCR
RNA extraction was carried out with 1mL TRIzol (9109, TaKaRa, Kyoto, Japan). DEPC H2O was used as the control (blank)
for the determination of RNA purity and RNA quantication. Two microliters of RNA solution were aliquoted in an enzyme
label analyzer (Epoch, BioTek, Vermont, USA) to determine the concentration and quality of the sample. Complementary
DNA (cDNA) was synthesized from the extracted RNA using a cDNA synthesis kit (Fermentas; Thermo Fisher Scientic,
Inc.), according to the manufacturer’s protocol. RT-PCR experiments (ABI 7900HT FAST, USA) were performed by using
the Power SYBR Green PCR Master Mix (A25742, Thermo, Waltham, USA). The primers (5-3) used were as follows: ORF3a-
hF: GCA ACG ATA CCG ATA CAA GCC, ORF3a-hR: CCA GCA GCA A CG AGC AAA A; GAPDH-hF: TGA CAA CTT TGG TAT CGT GGA AGG ,
GAPDH-hR: AGG CAG GGA TGA TGT TCT GGA GAG .
2.1.4 Analysis ofcell proliferation using theCCK‑8 assay
The CCK-8 assay was used to detect changes in SW480 cell proliferation. The cells were prepared into a cell suspension of
5 × 104 cells/mL, and 100μL cell suspension was added to each well of a 96-well cell culture plate (Corning Incorporated
3599, State of New York, USA). Then, the cells were cultured in a 5% carbon dioxide incubator (3111, Thermo) at 37
for 24, 48, or 72h. Next, 10μL CCK-8 (KGA317, Jiangsu Kaiji Biotechnology Company, Ltd., Jiangsu, China) was added to
the cells of each well, and the absorbance at λ = 450nm was measured. The growth inhibition rate was calculated. The
experiment was repeated three times, and the dierence in inhibition rates was compared between the three groups.
2.2 Details oftheauthentication ofthecells
SW480 [SW480] (Procell CL-0223) were kindly provided by Procell Life & Technology Co.,Ltd. A proper amount of
SW480[SW-480] cells (NO. PC174, 1 × 106) was taken and DNA was extracted using Chele × 100. 21 CELLID System was
used to amplify 20 STR loci and sex identication loci, and ABI3130 × 1 genetic analyzer was used to produce PCR. The
results were analyzed using GeneMapper IDX software (Applied Biosystems) and compared with ATCC DSMZ, JCRB, Cel-
losaurus Datebases, etc.
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
2.3 Analysis ofORF3a expression using immunofluorescence
The dierential expression of ORF3a protein in CRC tissues was determined using immunouorescence assay. Before
dewaxing, the tissue sections were placed at 25 for 60min or baked in an incubator at 60 for 20min. The tissue
sections were immersed in xylene for 10min and soaked for 10min after replacing xylene. The samples were soaked in
anhydrous ethanol, 95% ethanol, and 70% ethanol for 5min each and washed twice with PBS for 5min each. Antigen
repair and paran-embedded tissue microarray for paraformaldehyde xation. The sodium citrate buer solution (pH
6.0) was heated to approximately 95°C in an electric furnace or water bath. The tissue sections were placed in buer solu-
tion and heated for 10min. Following cooling at room temperature, the sections were removed and washed three times
with PBS (5min each). Normal goat serum (5%) was dropped onto tissue sections and incubated at room temperature
for 30min, after which the excess liquid was discarded. Then, the primary antibody was added and incubated at room
temperature for 1h or at 4 about 12h or at 37 for 1h. The sections were washed three times with PBS (5min each).
Fluorescent-labeled secondary antibodies were added and incubated at room temperature for 1h in the dark. The cells
were washed three times with PBS (5min each). DAPI solution was then added to the section for 10min. The sections
were washed and stained again as above and then washed three times with PBS (2min each). The dierence in protein
levels in CRC was determined. The experiment was repeated three times.
2.4 Analysis ofcell invasion using theTranswell assay
A Transwell assay was used to detect changes in SW480 cell invasion. The SW480 cells were divided into three groups:
ORF3a blank group, ORF3a control group, and ORF3a high expression group. Cells in the logarithmic growth phase were
digested with trypsin and inoculated into a six-well plate. The next day, after the cells adhered to the plate, the serum
was removed from the cells, and the cells were starved in incomplete medium for 24h. Simultaneously, Matrigel matrix
glue (356234, BD, USA) was placed at 4 about 12h to melt, and the melted Matrigel glue was diluted twice with
incomplete culture medium. Diluted Matrigel (30μL) was added into the upper chamber of Transwell and incubated at
37 for 120min. Matrigel was then polymerized into a gel. The cells were digested with 0.25% trypsin and collected,
and their density was adjusted to 1 × 105 cells/mL with incomplete medium. The cell suspension (100µL) was added
into the Transwell chamber (3422, Corning Incorporated), and 500 µL of medium containing 20% FBS was added to the
lower chamber. The plate was placed in a 5% carbon dioxide incubator. After 24h, the matrix glue and cells in the upper
chamber were wiped with a cotton swab. The Transwell was removed, placed upside down, and dried. Then, 500 µL of
0.1% crystal violet solution (C3886, Sigma, Bellefonte, Pennsylvania, USA) was added to a 24-well plate. The chamber was
placed in a plate, and the membrane was immersed in the dye and incubated at 37 for 30min. After PBS washing, an
inverted biological microscope (IX51, Olympus, Japan) was used for observation. Three visual elds were photographed
(magnication 200×), and the cells were counted. The experiment was repeated three times, and the dierence in the
number of cells between the three groups was compared.
2.5 Analysis ofcell migration using wound healing assay
A wound healing assay was used to detect SW480 cell migration. The cells in the logarithmic growth phase were digested
and inoculated into six-well plates. The next day, when the cell adhered to the plate and reached approximately 80%
conuence, the corresponding lentivirus was added according to the group setting. The sterile pipette tip was used to
scratch a line across a six-well plate. The oating cells were washed o with PBS; then, fresh culture medium was added,
and the plates were placed in a 5% carbon dioxide incubator for further culture for 24h. After culture, the cells were
photographed (magnication 100×), and the migration distance of the cells was measured. The experiment was repeated
three times, and the dierence in the migration distance between the three groups of cells was compared.
2.6 Analysis ofcell apoptosis using theannexin V‑FITC/Propidium Iodide assay
FITC Annexin V Propidium Iodide (PI) was used to detect apoptosis in SW480 cells. The cells of the three groups were
resuspended in 500μL binding buer. Then, 5μL Annexin V-FITC and 5μL PI [Annexin V-FITC/PI Apoptosis Detection
Kit (KGA105, Jiangsu Kaiji Biotechnology Company, Ltd., Jiangsu, China] were added and mixed for 5–15min at room
temperature in the dark. Flow cytometry (Becton–Dickinson FACS Calibur, New Jersey, USA) was used to detect apoptosis
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
at 24, 48 and 72h. The percentages of the UL, LL, LR, and UR regions were calculated. The apoptosis rate was calculated
according to the following equation (%) = LR (%) + UR (%).
2.7 Analysis ofcell cycle using PI single staining assay
The changes in cell cycle were detected using PI single staining assay. The three groups of logarithmically growing cells
were trypsin digested and inoculated into well plates. The distribution of cells in the various phases of the cell cycle was
determined using a cell cycle detection kit (KGA511, Jiangsu Kaiji Biotechnology Company, Ltd., Jiangsu, China). When
the cells adhered to the plate after 24h, the corresponding drug-containing medium was added according to the group
setting, and a blank control group was established. Before staining, the xing solution was washed with PBS; then, 100μL
RNase A was added in a 37 water bath for 30min, followed by the addition of 400μL PI for staining. The cells were
protected from light at 4 for 30min. Finally, a ow cytometer (FACSCalibur, Becton–Dickinson, New Jersey, USA) was
used to record the red uorescence at an excitation wavelength of 488nm. The experiment was repeated three times,
and the dierences in cell cycle between the three groups were compared.
2.8 Establishment ofCRC‑bearing mouse model andORF3a high expression animal model
2.8.1 Ethics statement
Animal experiment has passed the animal ethics audit of Zhejiang University Laboratory Animal Center. The ethics
number is 20617.
The purpose of this experiment was to analyze the eect of ORF3a on the colorectal tumor volume. 12 nude mice were
equally divided into a control group and an ORF3a high expression group. SW480 cell suspensions of each group were
inoculated subcutaneously into the right axilla of nude mice at a concentration of 1 × 107 cells/mL. The diameter of the
transplanted tumor was measured with a Vernier caliper. After 21days of inoculation, the tumor grew to 80–100 mm3.
Tumor diameter was measured to observe the growth of subcutaneously transplanted tumors in nude mice. At the end
of the experiment, the nude mice were sacriced, and the tumor mass was removed and weighed. Tumor volume (TV),
relative tumor volume (RTV), and the evaluation index of anti-tumor activity: tumor growth inhibition rate (%) were
calculated according to Eqs.(1), (2), and (3) to analyze the eect of ORF3A on the TV of transplanted nude mice.
where a and b represent the length and width, respectively.
where V0 is the tumor volume measured in caged administration (i.e. d0), and Vt is the tumor volume at each
measurement.
2.9 Hematoxylin–eosin staining
Hematoxylin–eosin (HE) staining was performed to analyze the eect of ORF3a on the morphology of CRC cells. First,
paran sections were prepared, dewaxed, and hydrated according to the conventional method. The sections were soaked
in xylene for 5min, then soaked in xylene again for another 5min. The slices were then soaked in anhydrous ethanol, 95%
ethanol, 85% ethanol, and 70% ethanol for 5min each. After washing with PBS, the sections were immersed in HE dye
solution of the kit (KGA224, Jiangsu Kaiji Biotechnology Company, Ltd., Jiangsu, China) for 3–5min in the dye cylinder,
washed with water for approximately 30–60s, and immersed in the dichromatic solution I in the hematoxylin–eosin dye
(1)
TV =12×a×b2,
(2)
RTV =VtV0,
(3)
Tumor growth inhibition rate (%)
=(average tumor weight of the model group average tumor weight of the administration group
)
average tumor weight of the model group ×100%.
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
solution kit for approximately 20s. After washing, the slices were immersed in tricolor II for approximately 40s. After
washing, the slices were stained with reagent 4 of the kit for 2min. The excess dye was washed with Reagent 5 Color
Enhancing Solution, washed twice, dried, and sealed. A digital pathological scanner (BX43, Olympus, Japan) was used
to scan the sections, observe the characteristics, including tumor cell morphology, degree of necrosis, interstitial blood
vessels, bleeding, and inammatory cell inltration, in the control group and the ORF3a high expression group, and
score the tumor.
2.10 Analysis ofapoptosis using theTUNEL assay
The TUNEL assay was used to detect and analyze the eect of ORF3a on apoptosis in CRC tissues. First, paran sections
were prepared using the method described above. Next, 1% Triton-100 was added to the samples, and the samples
were placed at 25 for 10min and soaked in PBS for 3min × 3 times. Then, 50µL 1xProteinaseK was dropped onto
each sample and incubated at 37 for 30min. After that, the sections were washed with PBS for 3min × 3 times. The
samples were treated with 3% H2O2-methanol solution for 15min and washed with PBS for 3min × 3 times to inactivate
the enzyme, and then 50µL TDT enzyme reaction solution was added to each sample and wetted in the dark at 37 for
1h. Next, the samples were soaked in PBS for 3min × 3 times for the TUNEL reaction. The sections were incubated with
50µL Streptavadin-HRP at 37°C for 30min in the dark and washed with PBS for 3min × 3 times. Two drops of freshly
prepared DAB solution were added to each sample for the DAB color reaction; the depth of dyeing was observed under
a microscope, stopped immediately after dyeing, gently rinsed with tap water for 15min, and the color reaction was
terminated with distilled water. Next, the slices were placed in hematoxylin dye solution, stained for 10min, washed
with distilled water, placed in hydrochloric acid methanol solution, and immediately washed with distilled water. The
slices were then soaked in 70, 85, and 95% ethanol for 5min each and immersed in anhydrous ethanol for 5min. After
soaking in xylene for 10min, xylene was replaced and soaked again for 10min. Neutral gum was added to the sections
covered with coverslips. Finally, a light microscope was used to observe and take photos of the three high-expression
regions for preservation.
2.11 Statistical analysis
The data are expressed as the mean ± SD. Statistically signicant dierences between two groups were analyzed using
Student’s t-test, and multiple comparisons were performed using one-way analysis of variance (ANOVA). SPSS software
(version 13.0) was used for all statistical analyses. Statistical signicance was accepted at P < 0.05.
3 Results
3.1 Transfection ofORF3a intoSW480 cells
To examine the role of ORF3a in CRC, we inserted the ORF3a target gene into the expression vector to establish the
pCDH-CMV-MCS-EF1-Puro vector (ORF3a overexpression vector). After its introduction into SW480 cells by lentivirus
infection, ORF3a was signicantly overexpressed (P < 0.01), as measured using qPCR (Supplemental Figure1). A cell
model with high ORF3a expression was thus generated (Fig.1). The overexpressed ORF3a protein was localized to the
cytoplasm of SW480 cells (Fig.2).
3.2 ORF3a inhibits theviability andcell cycle ofSW480 cells
ORF3a inhibited the proliferation of CRC cells. Moreover, the inhibitory eect of ORF3a on cell proliferation was enhanced
with increase in time (Fig.3A). Increased expression of ORF3a resulted in a sharp reduction in the number of cells migrat-
ing through the Transwell membrane, demonstrating the inhibitory eect of ORF3a on the invasion ability of CRC cells
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
Fig. 1 ORF3a was successfully expressed in SW480 cells. A Screening results for stable strain of the control virus. The control virus was pack-
aged with a vector driving expression of GFP, and the overexpressing ORF3a vector did not drive the expression of GFP; hence, there was no
uorescence image. B qPCR results showing overexpression of ORF3a. C CCK8 assay results are presented as the mean ± SD values. Two-way
ANOVA was used for statistical analysis; *P < 0.05 and **P < 0.01 were used as the screening criteria for signicant dierences and very sig-
nicant dierences, respectively
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
(Fig.3B, C). ORF3a signicantly inhibited the migration ability of CRC cells (Fig.3D, E). Further research into the eect of
ORF3a on the cell cycle of CRC found that ORF3a blocked SW480 CRC cells in the G1 phase (Fig.3F, G).
3.3 ORF3a inhibits thegrowth oftumor
The growth of subcutaneous tumors overexpressing ORF3a was examined in nude mice. ORF3a reduced the colorectal TV
and weight (Fig.4A, B). In the control group (NC), the tumor tissues had clear margins, compact texture, and no necrotic
foci. No inammatory cell inltration or local hemorrhage was observed in the tumor tissues, and no neovasculariza-
tion was observed in the stroma. In the ORF3a high expression group (OE), the margins of the tumor tissues were not
clear, and focal necrosis was observed. No inammatory cell inltration or local hemorrhage was observed in the tumor
tissues, and no neovascularization was observed in the stroma. ORF3a exerts anti-tumor eects by inducing apoptosis
in CRC cells (Fig.4C).
3.4 ORF3a promotes apoptosis ofSW480 cells
The apoptosis of CRC cells was examined at 24, 48, and 72h by using ow cytometry. The apoptosis rate of cells with
high expression of ORF3a was higher than that of the blank and control groups (P < 0.001), and increased continuously
as a function of time (Fig.5A, B). Invivo experiments and pathological and morphological analyses of tumor tissues also
revealed that ORF3a induced the apoptosis of tumor cells (Fig.5C).
Fig. 2 The localization of
ORF3a in SW480 cells. The
immunouorescence was
used to stain ORF3a protein in
the blank, control, and ORF3a
high expression groups. The
rst row represents protein
staining in the cytoplasm
(blue), the third row rep-
resents protein staining in
the nucleus (red), and the
middle row represents protein
expression after the combina-
tion of cytoplasm and nucleus
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
4 Discussion
At present, COVID-19, characterized by severe acute respiratory syndrome, has developed into a global health threat
[24, 25]. Viral infection is an important factor predisposing for cancer, and related reports have shown that coronavi-
ruses are associated with certain tumors. It has been confirmed that the virus exists in the intestines of patients with
COVID-19 [17, 2628], and approximately 15% of patients with COVID-19 have gastrointestinal symptoms along with
respiratory symptoms [29]. However, the relationship between coronavirus and CRC is not yet known. To explore
the relationship between SARS-CoV-2 and CRC, we analyzed the RNA sequence of COVID-19 and found that its gene
ORF3a might be a proto-oncogene that induces or inhibits CRC. Further study revealed that ORF3a of COVID-19 could
act on CRC cells and have an impact on the occurrence, invasion, and migration of CRC.
First, we constructed a high-expression lentiviral vector of ORF3a of COVID-19 and successfully infected SW480
cells. Then, through invitro cell experiments, we examined the effects of COVID-19 and ORF3a on the occurrence,
invasion, migration, and apoptosis of CRC cells and on the differential expression of proteins in SW480 cells. Animal
experiments and histopathological analysis confirmed that ORF3a can inhibit CRC tumor growth and induce apop-
tosis of CRC cells as well as tumor death.
Fig. 3 ORF3a inhibited cell viability and blocked cell cycle progression. A Cell proliferation of the blank group, control group, and ORF3a
high expression group was compared. B, C Invasion of CRC cells. There was no signicant dierence between the blank and control groups
(P > 0.05). There were statistically signicant dierences between the high expression group and the blank group and between the high
expression group and the control group (**P < 0.001). D, E The migration length of cancer cells in the high expression ORF3a group was
shorter (**P < 0.001), while the migration length of cancer cells in the blank group and the control group was longer (P > 0.05). F, G Cell cycle
was detected using PI staining
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
Our study also verified that ORF3a inhibited SW480 cell cycle progression invitro. ORF3a blocked SW480 cells in
the G1 phase. All mammalian cells, including tumor cells, produce RNA and proteins rapidly during the period from
mitosis to pre-DNA replication (G1 phase), and DNA replication in the next phase (S phase) is ready for material and
energy. After the G1-S transition, cells undergo intense proliferation and DNA replication. As one of the most impor-
tant stages of the cell cycle, the G1 phase is the limiting point for mammalian cell division and proliferation [30]. The
G1 phase is characterized by complex and active molecular changes, which are easily affected by environmental
conditions. In cancer cells, the control of the G1 phase limit point is lost for various reasons, resulting in uncontrolled
proliferation, and the cells release a large number of wrong cell division signals, leading to the occurrence of cancer
[31]. Therefore, ORF3a can block CRC cells at the G1 phase and inhibit their entry into the cell cycle and their division,
thereby preventing proliferation, migration, and proliferation.
Fig. 4 ORF3a inhibited tumor growth and promoted tumor death. A, B Comparison of tumor volume, body weight, and tumor weight
between the control group and the ORF3a high expression group. B Comparison of tumor histological morphology between the control
group and the ORF3a high expression group. C Comparison of tumor tissues between the control group and the ORF3a high expression
group
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
Ottaiano etal. [32] reported that (a) the tumor burden of three patients with metastatic colon cancer decreased
unexpectedly following 2019-nCoV infection, (b) one patient with liver metastasis showed complete remission on
CT scan 1month after 2019-nCoV infection, and (c) two other patients infected with 2019-nCoV showed an unex-
pected decrease in their metastases one to three months later. These studies showed that 2019-nCoV has an inhibi-
tory effect on colon cancer, and ORF3a may be the key responsible factor, which is consistent with our results. The
specific mechanism of the correlation between 2019-nCoV and colon cancer needs to be further studied and verified.
Fig. 5 ORF3a induced apoptosis of CRC cells. A, B Apoptosis was detected by ow cytometry. The apoptosis rate in the high expression
ORF3a group was higher than that in the blank and control groups (P < 0.001). There was no signicant dierence between the blank and
control groups (P > 0.01). C Tissue staining of the mice showed that the apoptosis cells with high expression of ORF3a was more than that of
the control group
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
In addition, studies have shown that patients with active 2019-nCoV gastrointestinal infections experience changes
in their intestinal ora. After the 2019-nCoV respiratory tract infection recovery, even if there are no gastrointestinal
symptoms, long-term "Latent" gastrointestinal infection signs will still be present [33]. This indicates that 2019-nCoV
infection causes long-term eects and aects the gastrointestinal tract. Therefore, it is necessary to study the relation-
ship between 2019-nCoV and CRC, and ORF3a was proven to be a very good starting point.
The eects of ORF3a on CRC are rst reported. The expression of virus or non-virus associated ORF protein has been
reported to be related to tumor. Overexpression of ORF48 on chromosome 8 can reduce the proliferation, migration and
invasion of CRC cells, and play an inhibitory role in CRC by inhibiting MAPK signaling pathway [34]. Alternative T-Antigens,
a tumor-associated antigen encoded by ORF5 in human polyomavirus, is thought to disrupt the signaling pathway
of tumor cell proliferation [35]. When expressed alone, adenovirus ORF4 protein induces an evolutionarily conserved,
Caspase-independent, cancer-selective cell death [36].
Although the role of COVID-19 infection and its functional gene ORF3a in the occurrence and migration of CRC was
partially claried through cell and animal experiments, further studies on the molecular mechanism are still needed. The
molecular pathway and the factors involved in the inhibitory eect of ORF3a on CRC need to be claried. At the same
time, under eective and strict epidemic prevention and control policies, the number of patients infected with COVID-19
complicated with CRC is very small, and it is dicult to carry out clinical validation studies. The changes in the prevalence
of CRC, tumor progression, lung metastases from CRC, and treatment sensitivity among patients with COVID-19 should
be evaluated. In addition, the results of this study were obtained on a single cell line and animal model, so it is necessary
to verify the results through multiple cell lines or animal models. Elucidation of the specic function of ORF3a in CRC is
expected to provide a scientic basis for a new anti-tumor strategy for CRC.
5 Conclusion
In the present study, we demonstrated for the rst time the inhibitory eect of the novel coronavirus ORF3a on the inu-
ence of CRC. Compared with CRC without ORF3a expression, cell viability, cell division and replication, and tumor growth
were inhibited when ORF3a was overexpressed. Moreover, the expression of nuclear and cytoplasmic proteins in CRC
was aected by ORF3a. These ndings can contribute to a better understanding of the mechanism through which the
virus aects the occurrence and progression of CRC and provide a new direction for the treatment of CRC.
Acknowledgements The authors gratefully acknowledge the database available to us for this study.
Authors’ contributions All authors participated in the conception and design of the study. Conceived and drafted the manuscript: WW and
YX. Wrote the paper: MJ, HX and CG. Analysed the data: HS and WY. Drew gures: MJ, HS and WY. All authors read and approved the nal
manuscript.
Funding This research was supported by Huzhou Science and Technology Department of Key Research and Development Project
(2020ZDT2015).
Data availability The datasets generated during the current study are not publicly available but obtained from corresponding authors on
reasonable request.
Declarations
Ethics approval and consent to participate Notapplicable.
Consent for publication Notapplicable.
Competing interests The authors declare that no conicts of interest exist.
Vol:.(1234567890)
Research Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6
1 3
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: GLOBOCAN estimates of inci-
dence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49.
2. Song M, Chan AT, Sun J. Inuence of the gut microbiome, diet, and environment on risk of colorectal cancer. Gastroenterology.
2020;158(2):322–40.
3. Reis SAD, da Conceição LL, Peluzio M. Intestinal microbiota and colorectal cancer: changes in the intestinal microenvironment and their
relation to the disease. J Med Microbiol. 2019;68(10):1391–407.
4. Camarillo-Guerrero LF, Almeida A, Rangel-Pineros G, Finn RD, Lawley TD. Massive expansion of human gut bacteriophage diversity. Cell.
2021;184(4):1098–109.
5. Turkington CJR, Varadan AC, Grenier SF, Grasis JA. The viral janus: viruses as aetiological agents and treatment options in colorectal cancer.
Front Cell Infect Microbiol. 2020;10:601573.
6. Ibragimova MK, Tsyganov MM, Litviakov NV. Human papillomavirus and colorectal cancer. Med Oncol. 2018;35(11):140.
7. Bai B, Wang X, Chen E, Zhu H. Human cytomegalovirus infection and colorectal cancer risk: a meta-analysis. Oncotarget.
2016;7(47):76735–42.
8. Puronen CE, Ford ES, Uldrick TS. Immunotherapy in people with HIV and cancer. Front Immunol. 2019;10:2060.
9. Patel BB, Lipka S, Shen H, Davis-Yadley AH, Viswanathan P. Establishing the link between hepatitis B virus infection and colorectal adenoma.
J Gastrointest Oncol. 2015;6(5):492–7.
10. Zapatka M, Borozan I, Brewer DS, Iskar M, Grundho A, Alawi M, Desai N, Sültmann H, Moch H, Cooper CS, etal. The landscape of viral
associations in human cancers. Nat Genet. 2020;52(3):320–30.
11. Challenor S, Tucker D. SARS-CoV-2-induced remission of Hodgkin lymphoma. Br J Haematol. 2021;192(3):415.
12. Saini KS, Tagliamento M, Lambertini M, McNally R, Romano M, Leone M, Curigliano G, de Azambuja E. Mortality in patients with cancer
and coronavirus disease 2019: a systematic review and pooled analysis of 52 studies. Eur J Cancer. 2020;139:43–50.
13. Kuderer NM, Choueiri TK, Shah DP, Shyr Y, Rubinstein SM, Rivera DR, Shete S, Hsu CY, Desai A, de Lima Jr. LG, etal. Clinical impact of COVID-
19 on patients with cancer (CCC19): a cohort study. Lancet. 2020;395(10241):1907–18.
14. Kaźmierczak-Siedlecka K, Vitale E, Makarewicz W. COVID-19—gastrointestinal and gut microbiota-related aspects. Eur Rev Med Pharmacol
Sci. 2020;24(20):10853–9.
15. Wu A, Peng Y, Huang B, Ding X, Wang X, Niu P, Meng J, Zhu Z, Zhang Z, Wang J, etal. Genome composition and divergence of the novel
coronavirus (2019-nCoV) originating in China. Cell Host Microbe. 2020;27(3):325–8.
16. Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, Zhu H, Zhao W, Han Y, Qin C. From SARS to MERS, thrusting coronaviruses into the spotlight.
Viruses. 2019;11(1):59.
17 Issa E, Merhi G, Panossian B, Salloum T, Tokajian S. SARS-CoV-2 and ORF3a: nonsynonymous mutations, functional domains, and viral
pathogenesis. mSystems. 2020;5(3):e00266.
18. Xia H, Cao Z, Xie X, Zhang X, Chen JY, Wang H, Menachery VD, Rajsbaum R, Shi PY. Evasion of type I interferon by SARS-CoV-2. Cell Rep.
2020;33(1):108234.
19. Bianchi M, Borsetti A, Ciccozzi M, Pascarella S. SARS-Cov-2 ORF3a: mutability and function. Int J Biol Macromol. 2021;170:820–6.
20. Siu KL, Yuen KS, Castaño-Rodriguez C, Ye ZW, Yeung ML, Fung SY, Yuan S, Chan CP, Yuen KY, Enjuanes L, etal. Severe acute respiratory
syndrome coronavirus ORF3a protein activates the NLRP3 inammasome by promoting TRAF3-dependent ubiquitination of ASC. Faseb
J. 2019;33(8):8865–77.
21. Li J, Qu C, Li F, Chen Y, Zheng J, Xiao Y, Jin Q, Jin G, Huang X, Jin D. Inonotus obliquus polysaccharide ameliorates azoxymethane/dextran
sulfate sodium-induced colitis-associated cancer in mice via activation of the NLRP3 inammasome. Front Pharmacol. 2020;11:621835.
22. Ren Y, Shu T, Wu D, Mu J, Wang C, Huang M, Han Y, Zhang XY, Zhou W, Qiu Y, etal. The ORF3a protein of SARS-CoV-2 induces apoptosis in
cells. Cell Mol Immunol. 2020;17(8):881–3.
23. Kern DM, Sorum B, Mali SS, Hoel CM, Sridharan S, Remis JP, Toso DB, Kotecha A, Bautista DM, Brohawn SG. Cryo-EM structure of SARS-
CoV-2 ORF3a in lipid nanodiscs. Nat Struct Mol Biol. 2021;28(7):573–82.
24. Benvenuto D, Giovanetti M, Salemi M, Prosperi M, De Flora C, Junior Alcantara LC, Angeletti S, Ciccozzi M. The global spread of 2019-nCoV:
a molecular evolutionary analysis. Pathog Glob Health. 2020;114(2):64–7.
25. Zhang YZ, Holmes EC. A genomic perspective on the origin and emergence of SARS-CoV-2. Cell. 2020;181(2):223–7.
26. Gupta S, Parker J, Smits S, Underwood J, Dolwani S. Persistent viral shedding of SARS-CoV-2 in faeces—a rapid review. Colorectal Dis.
2020;22(6):611–20.
27. Qian Q, Fan L, Liu W, Li J, Yue J, Wang M, Ke X, Yin Y, Chen Q, Jiang C. Direct evidence of active SARS-CoV-2 replication in the intestine. Clin
Infect Dis. 2020;73(3):361–6.
Vol.:(0123456789)
Discover Oncology (2022) 13:14 | https://doi.org/10.1007/s12672-022-00473-6 Research
1 3
28. Wu Y, Guo C, Tang L, Hong Z, Zhou J, Dong X, Yin H, Xiao Q, Tang Y, Qu X, etal. Prolonged presence of SARS-CoV-2 viral RNA in faecal
samples. Lancet Gastroenterol Hepatol. 2020;5(5):434–5.
29. Yang L, Tu L. Implications of gastrointestinal manifestations of COVID-19. Lancet Gastroenterol Hepatol. 2020;5(7):629–30.
30. Kar S. Unraveling cell-cycle dynamics in cancer. Cell Syst. 2016;2(1):8–10.
31. Massagué J. G1 cell-cycle control and cancer. Nature. 2004;432(7015):298–306.
32. Ottaiano A, Scala S, D’Alterio C, Trotta A, Bello A, Rea G, Picone C, Santorsola M, Petrillo A, Nasti G. Unexpected tumor reduction in meta-
static colorectal cancer patients during SARS-Cov-2 infection. Ther Adv Med Oncol. 2021;13:17588359211011456.
33. Zuo T, Liu Q, Zhang F, Lui GC, Tso EY, Yeoh YK, Chen Z, Boon SS, Chan FK, Chan PK, etal. Depicting SARS-CoV-2 faecal viral activity in
association with gut microbiota composition in patients with COVID-19. Gut. 2021;70(2):276–84.
34. Lei L, An GY, Zhu ZQ, Liu SZ, Fu YT, Zeng XN, Cao Q, Yan BB, etal. C8orf48 inhibits the tumorigenesis of colorectal cancer by regulating
the MAPK signaling pathway. Life Sci. 2021;266:118872.
35. van der Meijden E, Feltkamp M. The human polyomavirus middle and alternative T-antigens; thoughts on roles and relevance to cancer.
Front Microbiol. 2018;9:398.
36. Kleinberger T. Biology of the adenovirus E4orf4 protein: from virus infection to cancer cell death. FEBS Lett. 2020;594(12):1891–917.
Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aliations.
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca²⁺-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.
Article
Full-text available
Herein, we describe three patients affected by metastatic colorectal cancer (mCRC) experiencing infection by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) and reduction of disease burden during coronavirus disease 2019 (COVID-19) course. Insights into tumor-associated angiotensin-converting enzyme (ACE)-2 expression and lymphocyte function suggest a correlation between host/SARS-Cov-2 infection and tumor burden reduction. This may shed new light into (a) the infection mechanism of SARS-CoV-2 virus and (b) the multiple aspects of a composite antiviral immune response with potential paradoxical and unexpected applications.
Article
Full-text available
Bacteriophages drive evolutionary change in bacterial communities by creating gene flow networks that fuel ecological adaptions. However, the extent of viral diversity and its prevalence in the human gut remains largely unknown. Here, we introduce the Gut Phage Database, a collection of ∼142,000 non-redundant viral genomes (>10 kb) obtained by mining a dataset of 28,060 globally distributed human gut metagenomes and 2,898 reference genomes of cultured gut bacteria. Host assignment revealed that viral diversity is highest in the Firmicutes phyla and that ∼36% of viral clusters (VCs) are not restricted to a single species, creating gene flow networks across phylogenetically distinct bacterial species. Epidemiological analysis uncovered 280 globally distributed VCs found in at least 5 continents and a highly prevalent phage clade with features reminiscent of p-crAssphage. This high-quality, large-scale catalog of phage genomes will improve future virome studies and enable ecological and evolutionary analysis of human gut bacteriophages.
Article
Full-text available
Inonotus obliquus polysaccharide (IOP), the primary constituent of the parasitic fungus Inonotus obliquus , has anti-tumor, anti-inflammatory, anti-oxidation effects. However, the roles of IOP on colitis-associated cancer (CAC) are still unclear. Herein, we tested the efficacy of IOP using a mouse model of CAC induced by azoxymethane and dextran sulfate sodium (AOM/DSS). We confirmed that intragastric administration of IOP decreased CAC-induced body weight loss, colon tissue damage, colon shortening, and expression of proinflammatory mediators. Meanwhile, IOP treatment increased in expression of the NLRP3 inflammasome, IL-1β, and IL-18 in the colon of CAC mice. Moreover, in vitro , IOP inhibited the proliferation of SW620 colorectal cancer cells. Finally, overexpression of NLRP3 with plasmid transfection could further enhance the activation of NLRP3 inflammasome by IOP. Taken together, these results suggest that IOP suppresses the development of CAC, possibly by activation of the NLRP3 inflammasome, and reveal that IOP may be a therapeutic drug candidate for CAC.
Article
Full-text available
In recent years, our understanding of the importance of microorganisms on and within our bodies has been revolutionized by the ability to characterize entire microbial communities. No more so is this true than in cases of disease. Community studies have revealed strong associations between microbial populations and disease states where such concomitance was previously absent from aetiology: including in cancers. The study of viruses, in particular, has benefited from the development of new community profiling techniques and we are now realising that their prominence within our physiology is nearly as broad as the diversity of the organisms themselves. Here, we examine the relationship between viruses and colorectal cancer (CRC), the leading cause of gastrointestinal cancer-related death worldwide. In CRC, viruses have been suggested to be involved in oncogenesis both directly, through infection of our cells, and indirectly, through modulating the composition of bacterial communities. Interestingly though, these characteristics have also led to their examination from another perspective—as options for treatment. Advances in our understanding of molecular and viral biology have caused many to look at viruses as potential modular biotherapeutics, where deleterious characteristics can be tamed and desirable characteristics exploited. In this article, we will explore both of these perspectives, covering how viral infections and involvement in microbiome dynamics may contribute to CRC, and examine ways in which viruses themselves could be harnessed to treat the very condition their contemporaries may have had a hand in creating.
Article
This article provides an update on the global cancer burden using the GLOBOCAN 2020 estimates of cancer incidence and mortality produced by the International Agency for Research on Cancer. Worldwide, an estimated 19.3 million new cancer cases (18.1 million excluding nonmelanoma skin cancer) and almost 10.0 million cancer deaths (9.9 million excluding nonmelanoma skin cancer) occurred in 2020. Female breast cancer has surpassed lung cancer as the most commonly diagnosed cancer, with an estimated 2.3 million new cases (11.7%), followed by lung (11.4%), colorectal (10.0 %), prostate (7.3%), and stomach (5.6%) cancers. Lung cancer remained the leading cause of cancer death, with an estimated 1.8 million deaths (18%), followed by colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast (6.9%) cancers. Overall incidence was from 2-fold to 3-fold higher in transitioned versus transitioning countries for both sexes, whereas mortality varied <2-fold for men and little for women. Death rates for female breast and cervical cancers, however, were considerably higher in transitioning versus transitioned countries (15.0 vs 12.8 per 100,000 and 12.4 vs 5.2 per 100,000, respectively). The global cancer burden is expected to be 28.4 million cases in 2040, a 47% rise from 2020, with a larger increase in transitioning (64% to 95%) versus transitioned (32% to 56%) countries due to demographic changes, although this may be further exacerbated by increasing risk factors associated with globalization and a growing economy. Efforts to build a sustainable infrastructure for the dissemination of cancer prevention measures and provision of cancer care in transitioning countries is critical for global cancer control.
Article
In this study, analysis of changes of SARS-CoV-2 ORF3a protein during pandemic is reported. ORF3a, a conserved coronavirus protein, is involved in virus replication and release. A set of 70,752 high-quality SARS-CoV-2 genomes available in GISAID databank at the end of August 2020 have been scanned. All ORF3a mutations in the virus genomes were grouped according to the collection date interval and over the entire data set. The considered intervals were: start of collection-February, March, April, May, June, July and August 2020. The top five most frequent variants were examined within each collection interval. Overall, seventeen variants have been isolated. Ten of the seventeen mutant sites occur within the transmembrane (TM) domain of ORF3a and are in contact with the central pore or side tunnels. The other variant sites are in different places of the ORF3a structure. Within the entire sample, the five most frequent mutations are V13L, Q57H, Q57H + A99V, G196V and G252V. The same analysis identified 28 sites identically conserved in all the genome isolates. These sites are possibly involved in stabilization of monomer, dimer, tetramerization and interaction with other cellular components. The results here reported can be helpful to understand virus biology and to design new therapeutic strategies.
Article
In this study, analysis of changes of SARS-CoV-2 ORF3a protein during pandemic is reported. ORF3a, a conserved coronavirus protein, is involved in virus replication and release. A set of 70,752 high-quality SARS-CoV-2 genomes available in GISAID databank at the end of August 2020 have been scanned. All ORF3a mutations in the virus genomes were grouped according to the collection date interval and over the entire data set. The considered intervals were: start of collection-February, March, April, May, June, July and August 2020. The top five most frequent variants were examined within each collection interval. Overall, seventeen variants have been isolated. Ten of the seventeen mutant sites occur within the transmembrane (TM) domain of ORF3a and are in contact with the central pore or side tunnels. The other variant sites are in different places of the ORF3a structure. Within the entire sample, the five most frequent mutations are V13L, Q57H, Q57H + A99V, G196V and G252V. The same analysis identified 28 sites identically conserved in all the genome isolates. These sites are possibly involved in stabilization of monomer, dimer, tetramerization and interaction with other cellular components. The results here reported can be helpful to understand virus biology and to design new therapeutic strategies.
Article
Aims Colorectal cancer (CRC) is a leading cause of cancer-related death globally. Thus, in this study, we aimed to investigate chromosome 8 open reading frame 48 (C8orf48) as a biomarker for early detection of CRC. Main methods RNA expression and methylation profiles were downloaded from The Cancer Genome Atlas (TCGA) database. Cell proliferation, migration and invasion assays were performed to confirm the function of C8orf48 in CRC cells. Dual-luciferase reporter assay was used to identify that C8orf48 was the direct target of miR-556. Genomics of Drug Sensitivity in Cancer (GDSC) database, gene set enrichment analysis (GSEA) and western blot analysis were performed to explore the mechanism of C8orf48. Key findings we found that C8orf48 is down-regulated in clinical samples of CRC tissues. Enrichment analysis showed that C8orf48 is associated with methylation biomarkers in CRC, and TCGA database confirmed that the methylation of C8orf48 is up-regulated in the early stage of CRC. We further revealed that the overexpression of C8orf48 decreased CRC cell proliferation, migration and invasion. Luciferase reporter indicated that C8orf48 was the direct target of the oncogene miR-556. Additionally, we used GDSC database, GSEA database and western blot analysis to demonstrate that C8orf48 plays a suppressor role in CRC by inhibiting MAPK signaling pathway. Significance C8orf48 was identified as a biomarker for early detection of CRC for the first time, and might provide novel information for CRC prediction and therapy.