Effect of miRNA-933 on the apoptosis and proliferation of LX-2 cells and its molecular mechanism / 临床肝胆病杂志

ABSTRACT

ObjectiveTo investigate the regulatory effect of miRNA-933 on the apoptosis and proliferation of human hepatic stellate cell line LX-2 and its mechanism. MethodsFirstly， with human liver tissue for research， gene microarray technology was used to detect the differentially expressed genes in liver tissue between liver cirrhosis/chronic hepatitis B tissue and normal liver tissue， among which the significantly differentially expressed miRNAs were identified， and thus miRNA-933 was determined as the research object. Then， with the human hepatic stellate cell line LX-2 for research， miRNA-933 mimic and inhibitor （miRNA-933 siRNA） were used to construct the LX-2 models of overexpression and knockdown， and the cells transfected with mimic-NC （overexpression） or siRNA-NC （knockdown） were established as the negative control group. Quantitative real-time PCR and Western blot were used to measure the expression levels of miRNA-933 and activation biomarkers； techniques such as cell proliferation assay and flow cytometry were used to investigate the effect and mechanism of miRNA-933 on cell apoptosis， proliferation， and activation. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and Bonferroni correction was also performed. ResultsA total of 18 significantly differentially expressed miRNAs were obtained based on the results of gene microarray， among which miRNA-933 was significantly downregulated （P<0.05）. After LX-2 cells were transfected with miRNA-933 mimic or siRNA， compared with the negative control group， miRNA-933 siRNA significantly downregulated the expression of miRNA-933 （P=0.000 7）， while miRNA-933 mimic significantly upregulated the expression of miRNA-933 （P=0.000 3）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly upregulated the expression of collagen I and α-SMA （P<0.001）， while miRNA-933 mimic significantly inhibited the expression of collagen I and α-SMA （P<0.05）. Flow cytometry showed that compared with the negative control group， miRNA-933 siRNA significantly downregulated the apoptosis rate of LX-2 cells （P=0.031 9）， and miRNA-933 mimic significantly upregulated the apoptosis rate of LX-2 cells （P=0.005 5）. Western blot showed that compared with the negative control group， miRNA-933 siRNA could inhibit the expression of Caspase-3 （P=0.006 7） and poly（ADP-ribose） polymerase-1 （PARP-1） （P=0.003 0） and upregulate the expression of B-cell lymphoma-2 （Bcl-2） in LX-2 cells （P=0.002 0）， while miRNA-933 mimic could significantly upregulate the expression of Caspase-3 （P=0.011 8） and PARP-1 （P=0.049 5） and downregulated the expression of Bcl-2 （P=0.002 1）. Cell proliferation assay showed that compared with the negative control group， miRNA-933 siRNA could promote the proliferation of LX-2 cells （P=0.011 5）， while on the contrary， miRNA-933 mimic could inhibit the proliferation of LX-2 cells （P=0.001 2）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly inhibited the expression of Kruppel-like factor 6 （KLF6） and downregulated the expression of activating transcription factor 4 （ATF4）， activating transcription factor 3 （ATF3）， and C/EBP homologous protein （CHOP）， while miRNA-933 mimic promoted the expression of the above proteins （all P<0.05）. ConclusionThis study shows that miRNA-933 may promote cell apoptosis and inhibit cell activation and proliferation by promoting the activation of the KLF6/ATF4/ATF3/CHOP/Bcl-2 signal axis in LX-2 cells.