The Study Of MiR-let-7g/i Inducing Apoptosis In Hepatocellular Carcinoma Cell

Background:MicroRNAs (miRNAs) are small, non-coding RNAs of18-25nucleotides in length and have been evolutionarily conserved from worms to humans. MiRNAs inhibit translation or induce mRNA degradation in general by binding to the3’untranslational region (3’UTR) of target mRNAs, which have an important function in gene regulation.A recent study showed that about50%of annotated human miRNAs are located in areas of the genome, known as fragile sites that are associated with cancer. This indicates that miRNAs might have a crucial function in cancer progression. The full-scale analysis of miRNomes indicates that, only0.9%miRNAs were expressed abundantly in human normal liver, but these miRNAs accounted for88.2%of all miRNAs in liver, and four of the first nine miRNAs belong to the miR-let-7family.Lethal-7(let-7), a founding member of the miRNA family, is required for timing of cell fate determination in C. elegans. Let-7is conserved across diverse animal taxa, including worms, flies and humans. In humans, various let-7genes have been reported to map to regions deleted in human cancers, and let-7is poorly expressed in some cancers, suggesting that let-7miRNAs may be tumor suppressors. According to reports, overexpression of let-7inhibited cell growth of lung cancer cell in vitro by regulating RAS. The human genome contains11loci that encode let-7miRNA families Stem-loops:hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsalet-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-let-7g and hsa-let-7i, which are processed into seventeen mature sequence: hsa-let-7a-5p, hsa-let-7a-3p, hsa-let-7a-2-3p, hsa-let-7b-5p, hsa-let-7b-3p, hsa-let-7c, hsa-let-7d-5p, hsa-let-7d-3p, hsa-let-7e-5p, hsa-let-7e-3p, hsa-let-7f-5p, hsa-let-7f-1-3p, hsa-let-7f-2-3p, hsa-let-7g-5p, hsa-let-7g-3p, hsa-let-7i-5p and hsa-let-7i-3p, refering to " miRBase v.18.0"(http://www.mirbase.org/search.shtml). These families share complete sequence of eight consecutive nucleotides at their5’ends, but the differences at their3’ends leave open the possibility that let-7families are not functionally equivalent in all respects. Sequence differences in the3’end of the let-7family microRNAs may direct the repression of some distinct sets of targets, the repression of which could function coordinately to regulate biological behavior of tumor.. Many studies have also prompted that the complementary probability between3’UTR base and target gene is the structural basis of the same family miRNAs playing different functions, and can even enhance the combination strength and efficiency with3’mRNA. Our pre-experiment indicated that the expression levels of let-7family members in hepatocellular carcinoma are not consistent (data unpublished). The microarray data of another research also confirms this. Therefore, we hypothesized that let-7family members may function differently because of the base sequence differences at their3’UTR. So far, no research to detect single let-7family members has been reportd.A number of natural miRNA hairpins exist in clusters of multiple identical or different copies. As a single miRNA can bind to multiple targets whereas single gene may be regulated by multiple miRNAs. So, the combination of miRNAs can cooperate to regulate one or several pathways.Objective:To investigate the apoptosis of hepatoma cells induced by miR-let-7g/i and explore whether there was cooperation between the miR-let-7g and miR-let-7i, provide a theoretical basis for treating hepatocellular carcinoma with miR-let-7.Methods and Results:In this study, the special methylated and cholesterol labeled miR-let-7g/i agomir were employed to up-regulate the expression of hsa-let-7g and hsa-let-7i in hepatoma cells, to detect their effect on the abilities of proliferation and apoptosis. The specific steps are described as follows1The expression level of miR-let-7g/i in hepatocellular carcinoma cell linesThe hepatoma cell lines and normal liver cell line are cultured, and the RNA and protein were extracted. The expression level of miR-let-7g/i was quantified by using SYBGreen real-time RT-PCR.reverse transcriptase. The Western Blot analysis was performed to detect the cell expression of Bcl-xL. The results show that the miR-let-7g/1expression in hepatoma cells was decrease, when normalized to normal liver cells L-02; while the expression of its target gene Bcl-xL was increased.2Influence of up-regulation of miR-let-7g/i expression on the apoptosis of hepatoma cell2.1TransfectionThe miR-let-7g/i agomir and miR-let-7agomir Negative Control were transfected into human hepatoma cell BEL-7402by transfection reagent LipofectamineTM2000. There are Negative Control group "Ctrl", miR-let-7g agomir100nM group "let-7g", miR-let-7i agomir100nM group "let-7i" and co-transfection of miR-let-7g50nM and the7i agomir50nM group "let-7g+7i". The expression levels of miR-let-7g/i were quantified by using SYBGreen real-time RT-PCR.reverse transcriptase after transfection for24hours. The results showed that the expression of let-7g in the "let-7g" and "let-7g+7i" is2073,441times respectively that of the Ctrl. The expression of let-7i in the "let-7i" and "let-7g+7i" is1303,605times respectively that of the Ctrl after transfection. This shows that the cell model was successfully established.2.2EdU retention assaysBEL-7402cells were seeded in96-well plates and transfection was performed as above2.1. The cells were marked by EdU after transfection for48h, then fixed, the Apollo stained, DNA stained, observed by fluorescence microscopy and taken photographs. Compared with the control group, the Hoechst staining of the experimental group nuclei were dense stained, or chunky dense stained. Cell count was analyzed by Image-Pro Plus6.0software, the statistical results show that, compared to the control group, the DNA replication activity of "let-7g" and "let-7i" decreased. Compared to let-7i group, the DNA replication activity in the let-7g+7i group is decreased, P<0.05.2.3Flow cytometry for quantitative analysis of apoptosisBEL-7402cells were seeded in96-well plates and transfection was performed as above2.1. Cells were collected, after transfection for48h, and then resuspended in binding buffer, mixed with Annexin V-FIFC and propidium Iodide. Flow cytometry was applied and found that, the rate of apoptosis in the "let-7g","let-7i" and "let-7g+7i" is14.3%,7.7%和14.1%respectively, which formed a significant difference with the control group of2.2%. There are significant differences of the rate of apoptosis between let-7i group and let-7g+7i group, P<0.05.2.4Detection of the expression of the mitochondrial apoptotic pathway proteins.BEL-7402cells were seeded in96-well plates and transfection was performed as above2.1. After transfection for48h, add lysis buffer, collect the protein, and perform Western Blot to detect the expression of Bcl-xL, Bak and Bax. The results showed that the anti-apoptotic protein Bcl-xL expression was inhibited by the cooperation of let-7g and let-7i, P<0.05, and pro-apoptotic proteins Bak and Bax are upregulated.Conclusion:The miR-let-7g and miR-let-7i could inhibit the hepatoma cell DNA replication activity and proliferation, and induce apoptosis of hepatoma cells by regulating the apoptosis-related proteins. MiR-let-7g can enhance the ability of miR-let-7i inducing apoptosis of hepatoma cells.