ABSTRACT: ObjectiveTo investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference
targeting at IER3IP1 gene.
MethodsThe shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected
by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope
and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid
differentiation correlated genes Gfi-1B, GPA and γ-globin were studied after being exposed to 0.2 μmol/L imatinib for two
ResultsThe shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited
with an inhibition efficiency of 76% (P<0.01). Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group (P<0.01). The proliferation ability was enhanced (P<0.01) and the proportion of cells at G0/G1 phase decreased but S phase increased (P<0.05) in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased.
There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining
positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L
STI571 for two days in K562/shRNA-IER3IP1 group (P<0.01).
ConclusionIER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may
play a role in erythroid differentiation and proliferation of K562 cells.
Chinese Journal of Cancer Research 04/2012; 21(3):163-170. · 0.18 Impact Factor
ABSTRACT: ObjectiveTo investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference (RNAi) on matrine-treated
MethodsThree oligonucleotides targeting CGI-100 gene and a pair of negative control containing the same nucleotide composition with
a different sequence were devised and chemically synthesized. The inhibition efficiency of CGI-100 expression by shRNA-CGI-100
in K562 cells was determined using semiquantitative RT-PCR and dot blot hybridization. The effect of CGI-100 RNAi on the growth
of K562 cells was examined using MTT assay and cell differentiation was measured by distinct approaches including flow cytometry,
benzidine staining and electron microscope. After CGI-100-konckdown K562 cells were incubated with 0.2 mg/ml of matrine or
30 μmol/L of hemin for 48 h, the expression levels of Glycophorin A(GPA)(CD235a) and Growth factor independence-1B mRNA(Gfi-1B
mRNA) were measured by RT-PCR and the protein levels of GPA, CD14 and CD15 were detected by flow cytometry.
ResultsThe eukaryotic expression vectors of CGI-100 RNAi were successfully constructed. The K562/shRNA-CGI-100 cell line was established
in which the inhibition efficiency of CGI-100 gene expression by shRNA-CGI-100 was 54%. CGI-100-knockdown inhibited the proliferation
and induced erythroid differentiation in K562 cells. Compared with the control K562 cells, the K562/shRNA-CGI-100 cells showed
decreased absorbance value detected by MTT assay, decreased enchromation, increased heterochromation, increased percentage
of G0/G1 phase cells, decreased population of S phase cells, decreased PI (proliferation index of cells), and elevated percentage
of benzidine-positive cells. Moreover, the sensitivity of K562/shRNA-CGI-100 cells to either matrine or hemin was enhanced
and the sensitivity of these cells to matrine was higher than that to hemin. Compared with the control K562 cells, matrine
treatment in K562/shRNA-CGI-100 cells resulted in increased inhibitory rate of proliferation, elevated percentage of benzidine-positive
cells, obviously up-regulated mRNA expressions of GPA and Gfi-1B, and increased mean fluorescence intensity (MFI) of GPA.
No CD14 expression was detected and no statistical significance was found for the detected CD15. Finally, the MFI of GPA increased
in K562/shRNA-CGI-100 cells treated with hemin and was 1.7 times less than that in cells exposed to matrine.
ConclusionThese results suggest that the function of CGI-100 gene is correlated with the deregulated proliferation and the block of
erythroid differentiation in K562 cells and may also be involved in matrine-induced erythroid differentiation in K562 cells.
Key wordsCGI-100-RNA interference-Leukemia-Erythroid differentiation-Matrine
Chinese Journal of Cancer Research 04/2012; 22(2):126-134. · 0.18 Impact Factor
ABSTRACT: To investigate the effect of IER3IIP1 gene silence by RNA interference on erythroid differentiation and proliferation of K562 cells.
The shRNA eukaryotic expression vectors targeting IER3IP1 gene were constructed. Silence effect was detected by semiquantitative RT-PCR after the vectors being transfected into K562 cells. The impact on K562 cells was studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, flow cytometry and RT-PCR, respectively. The expression of erythroid differentiation-related genes Gfi-1B, GPA and gamma-globin and GPA protein expressed on these cells were studied after being exposed to 0.2 micromol/L imatinib for 48 hours.
The expression of IER3IP1 gene was significantly inhibited with a inhibition rate of 76% (P<0.01). Compared with the control group, bcr-abl mRNA level was significantly increased in K562-shRNA-IER3IP1 group (P<0.05). The proliferation capacity was significantly enhanced (P<0.01) and the proportion of cells at G0/G1 phase significantly decreased while at S phase significantly increased (P <0.05) in K562-shRNA-IER3IP1 group. Under the electron microscopy observation, the number of euchromatin was increased while heterochromatin decreased. There were structural abnormalities in endocytoplasmic reticulum and clusters of vesicular. The proportion of benzidine staining positive cells decreased from (44.7 +/- 2.5)% in control group to (22.7 +/- 1.5)% in tested group (P<0.01). mRNA expression of Gfi-1B, GPA and gamma-globin gene and the expression of GPA protein on cell surface were all significantly decreased after being exposed to 0.2 micromol/L imatinib for 48 hours in K562-shRNA-IER3IP1 group (P<0.01). During the erythroid differentiation induced by imatinib, mRNA expression of IER3IP1 obviously increased at the 3 h, and decreased from 6 h to 48 h.
Inhibition of expression of IER3IP1 gene can inhibit erythroid differentiation and elevate the proliferation of K562 cells. The IER3IP1 gene may play a role on erythroid differentiation and proliferation of K562 cells.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2009; 30(6):409-13.
ABSTRACT: This study was aimed to explore the expression level of the unknown cgi-100 gene in human leukemia K562 cells treated with matrine, and to investigate effect of cgi-100 on proliferation of K562 cells. The expression level of cgi-100 was detected by RT-PCR in K562 cells before and after being treated with matrine; pIRES2-EGFP/cgi-100 eukaryotic expression vector was constructed by DNA recombinant technique and was introduced into K562 cells by liposome-mediated DNA transfection. The cgi-100 gene expression level, growth-curve, and cell cycle of the modified K562-cgi-100 cells were detected by RT-PCR, Trypan blue staining and FCM. Morphological changes were observed under the optical and electron microscopes. The results indicated that the expression level of cgi-100 decreased in K562 cells treated with matrine. Heterochromatin decreased, euchromatin and the proportion of S phase in K562-cgi-100 cells increased, and cell proliferation enhanced. It is concluded that the expression of cgi-100 mRNA decreased in a dose- and time-dependent manner in the K562 cells treated with matrine and over-expression of cgi-100 elevates the proliferation and the immaturity level of K562-cgi-100 cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2008; 16(3):525-30.
ABSTRACT: To investigate the expression status of IER3IP1 gene during matrine induced K562 cell differentiation, and to figure out the function of IER3IP1 gene in K562 cell line.
Trypan-blue staining was used to analyze the growth inhibitory effect of matrine on K562 cells. Semi-quantitative RT-PCR was employed to investigate the expression status of IER3IP1 gene treated with different time and dosage of matrine. The alteration of cellular morphology, cellular proliferation and ultra-microstructure were observed and the cell cycle was detected on the recombinant IER3IP1 gene eukaryotic expression vector eYFP-IER3IP1 plasmid transfected K562 cells (K562/eYFP-IER3IPl).
Matrine inhibited the growth of K562 cells and reduced the expression of IER3IP1 gene. The expression level of IER3IP1 gene was transiently increased to three-to-four times in a dose-dependent manner after treated with matrine for 2 - 3 hours. Then, in 6-48 hours it maintained at a low level as compared with the control group. The proliferation rate of the K562/eYFP-IER3IP1 cells significantly slowed down with more cells blocked in G0-G1 phase (P < 0.05). The number of erythroid blast cells began to increase after 24 hours of matrine treatment. At the same time, differentiated erythroid cells could be observed.
Matrine can inhibit the growth of K562 cells, and transiently increase the expression level of IER3IP1 gene in a dose-dependent manner. The sensitivity of K562 cells to matrine maybe increased after being transfected by the eYFP-IER3IP1 plasmid, indicating a possible involvement of the IER3IP1 gene in the early response of the cells to matrine and its possible role in the erythroid cell differentiation.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 12/2007; 28(12):823-7.