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ABSTRACT: Cisplatin is a classic chemotherapy agent used for treating human non-small cell lung cancer (NSCLC). However, cisplatin resistance is a challenge against successful clinical use. Glutathione S-transferase P1 (GSTP1) has been reported to contribute to cisplatin resistance in many studies. MicroRNAs (miRNAs) are short non-coding RNAs that are 21-25 nucleotides in length. They play a role in post-transcriptional gene regulation by inducing repression and/or mRNA degradation. Recent studies have shown that miRNAs are responsible for cisplatin resistance. This study aims to determine whether deregulated miRNAs can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1. Real-time RT-PCR revealed that GSTP1 mRNA expression was 2.7 ± 0.38 folds (p=0.039) upregulated in A549/CDDP cells, compared with the parental A549 cells, while miR-513a-3p expression was 0.34 ± 0.03 folds (p=0.023) downregulated. Luciferase activity assay proved that GSTP1 was a target gene of miR-513a-3p, which was confirmed by Western blot analysis. Furthermore, CCK-8 assay showed that overexpression of miR-513a-3p could enhance cisplatin-induced apoptosis in human lung adenocarcinoma cell lines, A549/CDDP and SPC-A-1. In conclusion, our data demonstrated that miR-513a-3p can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1.
Lung cancer (Amsterdam, Netherlands) 06/2012; 77(3):488-94. · 3.14 Impact Factor
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ABSTRACT: Ionizing radiation (IR) causes severe cellular damage both directly and indirectly and disrupts RNA integrity. RNA strand
breaks are the most frequent type of damage caused by IR. RNA damage is involved in the development of degenerative diseases,
including Alzheimer’s disease and Parkinson’s disease. However, the mechanism of mRNA damage and any resulting pathophysiological
outcomes are poorly understood. This is partly because there is a lack of sensitive tools to monitor damage randomly occurring
in RNA, especially RNA strand break damage in a given RNA. In this work, a method using the reverse transcription polymerase
chain reaction (RT-PCR) after poly(A) addition to 3′-end of RNA to determine RNA strand break damage in a specific RNA by
poly(A) polymerase has been developed. The levels of damage in specific mRNAs, including ABL1, TP53, GADD45A and ATR from IR-treated HeLa cells were examined. Strand breaks were detected in all mRNAs examined. The study provides a novel and
sensitive method based on 3′-end poly(A)-tailing RT-PCR to monitor RNA strand break damage.
KeywordsRNA damage–RNA strand break–ionizing radiation–poly(A) polymerase–RT-PCR
Chinese Science Bulletin 04/2012; 56(30):3172-3177. · 1.32 Impact Factor
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Shuai Li,
Juanjuan Zhu, Hanjiang Fu,
Jing Wan,
Zheng Hu,
Shanshan Liu,
Jie Li,
Yi Tie,
Ruiyun Xing,
Jie Zhu,
Zhixian Sun,
Xiaofei Zheng
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ABSTRACT: microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.
Nucleic Acids Research 09/2011; 40(2):884-91. · 8.03 Impact Factor
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ABSTRACT: Recently, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. However, the functions of these miRNAs in HCC remain largely undefined.
The expression profiles of miR-193b were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was also be used to screen the potential target genes of miR-193b. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-193b in hepatoma cells was examined further.
miR-193b was significantly down-regulated in most of the HCC tissues compared to the matching non-tumoural liver tissues. Furthermore, ectopic expression of miR-193b dramatically suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumours in nude mice. CCND1 and ETS1 were revealed to be regulated by miR-193b directly. By regulating the expressions of these oncogenes, miR-193b induced cell cycle arrest and inhibited the invasion and migration of hepatoma cells.
miR-193b may function as a tumour suppressor in the development of HCC by acting on multiple tumourigenic pathways.
European journal of cancer (Oxford, England: 1990) 10/2010; 46(15):2828-36. · 4.12 Impact Factor
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Jianhua Xiong,
Dianke Yu,
Na Wei, Hanjiang Fu,
Tianjing Cai,
Yuanyu Huang,
Chen Wu,
Xiaofei Zheng,
Quan Du,
Dongxin Lin,
Zicai Liang
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ABSTRACT: Previous studies have suggested that microRNAs (miRNAs) may play important roles in tumorigenesis, but little is known about the functions of most miRNAs in cancer development. In the present study, we set up a cell-based screen using a luciferase reporter plasmid carrying the whole approximately 4.7 kb 3'-UTR of estrogen receptor alpha (ERalpha) mRNA cotransfected with a synthetic miRNA expression library to identify potential ERalpha-targeting miRNAs. Among all the miRNAs, miR-22 was found to repress robustly the luciferase signal in both HEK-293T and ERalpha-positive MCF-7 cells. Mutation of the target site was found to abrogate this repression effect of miR-22, whereas antagonism of endogenous miR-22 in MDA-MB-231 cells resulted in elevated reporter signals. We assessed the miR-22 expression patterns in five breast cancer cell lines and 23 clinical biopsies and revealed that there is a significant inverse association between the miR-22 levels and ERalpha protein expression. To evaluate the potential of miR-22 as a potential therapeutic intervention, we found that reduction of endogenous ERalpha protein levels and suppression of cancer cell growth could be achieved in MCF-7 cells by miR-22 overexpression in a way that can be recapitulated by the introduction of specific small interfering RNA against ERalpha. The phenomena can be rescued by the reintroduction of ERalpha. Taken together, our data indicate that miR-22 was frequently downregulated in ERalpha-positive human breast cancer cell lines and clinical samples. Direct involvement in the regulation of ERalpha may be one of the mechanisms through which miR-22 could play a pivotal role in the pathogenesis of breast cancer.
FEBS Journal 02/2010; 277(7):1684-94. · 3.79 Impact Factor
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ABSTRACT: In recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined.
The expression profiles of miR-183 were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was used to screen the potential target genes of miR-183. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-183 in hepatoma cells was examined.
Among the 25 HCC samples analyzed, microRNA-183 was significantly up-regulated (twofold to 367-fold) in 17 samples compared with the matching nontumoral liver tissues. Programmed cell death 4 (PDCD4) was identified as the target gene of miR-183. Moreover, PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-beta1.
We conclude that miR-183 can inhibit apoptosis in human HCC cells by repressing the PDCD4 expression, and miR-183 may play an important role in HCC development.
BMC Cancer 01/2010; 10:354. · 3.01 Impact Factor
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ABSTRACT: It has been known for decades that ionizing radiation (IR) promotes carcinogenesis and high-linear energy transfer (LET) IR has a higher risk than low-LET IR for carcinogenesis; however, the mechanism remains unclear. MicroRNAs (miRNAs) have a critical effect on carcinogenesis through post-transcriptional modification. In this study, our purpose is to explore whether miRNAs are involved in IR-(especially high-LET IR) promoted liver carcinogenesis. We showed here that among several hundred miRNAs, miR-21 was the only one that increased 6 folds in high-LET IR-promoted mouse liver tumors when compared with that in the non-irradiated liver tissues. We also showed that miR-21 was up-regulated in human or mouse hepatocytes after exposure to IR, as well as in liver tissues derived from whole body irradiated mice. The increased level of miR-21 was more significant in high-LET irradiated cells or liver tissues. After the non-irradiated, low-LET or high-LET irradiated human hepatocytes were over-expressed with miR-21, these cells became tumorigenesis in nude mice. The tumors derived from high-LET-irradiated-cells were largest, and accompanied by more significant changes in the miR-21-targets: PTEN and RECK. In addition, we showed that IR-induced up-regulation of miR-21 depended on the up-regulation/activation of AP-1 (at an earlier time, within 2 h) and the ErbB/Stat3 pathway (at a later time, more than 2 h), which was also IR dose dependent. Taken together, we conclude that IR-induced up-regulation of miR-21 plays an important role in IR (especially high-LET IR)-promoted liver carcinogenesis.
International journal of clinical and experimental medicine 01/2010; 3(3):211-22.
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ABSTRACT: miRNAs are a class of small RNA molecules with regulatory function, and play an important role in tumor development and progression. It has been demonstrated that tumor-derived miRNAs exist in the circulating nucleic acids of cancer patients. This phenomenon implies that detection of the circulating miRNA may be an effective method for non-invasive diagnosis of cancer. In this review, we summarize the applications of the circulating miRNA as biomarkers in cancer diagnosis, as well as the latest research progress in this area.
Science in China Series C Life Sciences 12/2009; 52(12):1117-22. · 1.61 Impact Factor
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ABSTRACT: tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.
FEBS letters 01/2009; 583(2):437-42. · 3.54 Impact Factor
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ABSTRACT: Several studies have shown that miR-34a represses the expression of many genes and induces G1 arrest, apoptosis, and senescence. In the present study, we identified the role of miR-34a in the regulation of tumor cell scattering, migration, and invasion. Down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues and associated with the metastasis and invasion of tumors. Furthermore, resected normal/tumor tissues of 25 HCC patients demonstrated an inverse correlation between miR-34a and c-Met-protein. In HepG2 cells, ectopic expression of miR-34a potently inhibited tumor cell migration and invasion in a c-Met-dependent manner. miR-34a directly targeted c-Met and reduced both mRNA and protein levels of c-Met; thus, decreased c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Taken together, these results provide evidence to show the suppression role of miR-34a in tumor migration and invasion through modulation of the c-Met signaling pathway.
Cancer letters 12/2008; 275(1):44-53. · 4.86 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of various biological processes. Although there is emerging evidence that some microRNAs can function as oncogenes or tumor suppressors, the specific role of miRNA in human hepatocellular carcinoma (HCC) is unclear at this point. In this study, we examined the microRNA expression profiles in a set of 20 human HCC specimens by miRNA microarray and quantitative real-time polymerase chain reaction. The results showed that among the 20 HCC samples analyzed, microRNA-101 was significantly down-regulated twofold or more (twofold to 20-fold) in 16 samples compared with the matching nontumoral liver tissues. Using both a luciferase reporter assay and Western blot analysis, we showed that microRNA-101 repressed the expression of v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene, a key component of the activator protein-1 (AP-1) transcription factor. Moreover, using a luciferase expression vector (pAP-1-Luc) driven by seven copies of an AP-1 cis-element, we observed that microRNA-101 expression inhibited phorbol 12-myristate 13-acetate (PMA)-induced AP-1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced microRNA-101 expression inhibited the invasion and migration of cultured HCC cells, respectively. These findings suggest that microRNA-101 may play an important role in HCC. CONCLUSION: MicroRNA-101, which is aberrantly expressed in HCC, could repress the expression of the FOS oncogene.
Hepatology 12/2008; 49(4):1194-202. · 11.66 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to be involved in diverse biological processes by regulating gene expression. Numerous miRNAs have been identified in various species, and many more miRNAs remain to be detected. Generally, hundreds of mRNAs have been predicted to be potential targets of one miRNA, so it is a great challenge to identify the genuine miRNA targets. Here, we generated the cell lines depleted of Drosha protein and screened dozens of transcripts (including Cyclin D1) regulated potentially by miRNA-mediated RNA silencing pathway. On the basis of miRNA expressing library, we established a miRNA targets reverse screening method by using luciferase reporter assay. By this method, we found that the expression of Cyclin D1 (CCND1) was regulated by miR-16 family directly, and miR-16 induced G1 arrest in A549 cells partially by CCND1. Furthermore, several other cell cycle genes were revealed to be regulated by miR-16 family, including Cyclin D3 (CCND3), Cyclin E1 (CCNE1) and CDK6. Taken together, our data suggests that miR-16 family triggers an accumulation of cells in G0/G1 by silencing multiple cell cycle genes simultaneously, rather than the individual target.
Nucleic Acids Research 10/2008; 36(16):5391-404. · 8.03 Impact Factor
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ABSTRACT: miRNAs regulate gene expression by inhibiting translation or by targeting messenger RNA (mRNA) for degradation in a post-transcriptional fashion. In the present study, we show that ectopic expression of miR-34a reduces both mRNA and protein levels of cyclin D1 (CCND1) and cyclin-dependent kinase 6 (CDK6). We also demonstrate that miR-34a targets the 3'-untranslated mRNA region of CCND1 as well as CDK6, which in turn interferes with phosphorylation of retinoblastoma. In addition, we show that overexpression of miR-34a induces a significant G1 cell-cycle arrest in the A549 cell line. Taken together, our data suggest that the effects of miR-34a on G1 cell cycle arrest are through the down-regulation of CCND1 and CDK6, which is associated with other targets of miR-34a either additively or synergistically.
FEBS Letters 05/2008; 582(10):1564-8. · 3.54 Impact Factor
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ABSTRACT: The initiation and progression of tumor is regulated by multiple genes. Survivin belongs to the inhibitor of apoptosis protein (IAP) family and is overexpressed in most types of human tumors. Apoptin, originally identified from chicken anemia virus (CAV), can specifically induce apoptosis of human tumor cells rather than normal cells. In this study, survivin expression was silenced by microRNA (miRNA)-mediated RNA interference (RNAi); meanwhile, the engineered miRNA vector was also designed to express apoptin gene. The apoptosis and cell growth were then examined by flow cytometry and MTT assay. The miRNA-mediated knockdown of survivin in combination with apoptin overexpression significantly induced apoptosis and inhibited cell growth. Importantly, the combined strategy was more effective on inducing apoptosis and inhibiting cell growth than either survivin downregulation or apoptin overexpression alone. Taken together, the combined strategy offers potential advantages in control of tumorigenesis, and thus deserves further research as a preferred approach in cancer gene therapy.
Cancer biology & therapy 05/2008; 7(7):1053-60. · 2.64 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are short 20-25 nucleotides RNA molecules that have been shown to regulate gene expressions in a variety of eukaryotic systems. miRNAs are widespread in eukaryotes and several hundred of miRNAs have been identified, but still a lot of miRNAs have not been detected in various eukaryotic organisms. However, it is not an easy work to clone miRNAs by traditional methods. Here, we describe the identification of 27 miRNAs from a human fetal liver cDNA library by a novel cloning method. Low molecular weight RNA fraction (< or = 200 nt) from fetal liver tissue was extracted, and polyadenylated by poly(A) polymerase. A 5' RNA adaptor was ligated to poly(A)-tailed RNA using T4 RNA ligase. After reverse transcription, the cDNA was amplified by PCR with two adaptor primers. The PCR product with a size about 109 bp was recovered and cloned into T vector. After sequencing, database searching, and expression profiling, 5 novel miRNAs were discovered among other 22 known miRNAs in human fetal liver. These finding indicate that a large diverse population of miRNAs may function to regulate gene expression in hepatocyte.
FEBS Letters 07/2005; 579(17):3849-54. · 3.54 Impact Factor
