| Objectives: Micro RNAs(mi RNAs) are commonly dysregulated in a number of human cancers, for example, hepatocellular carcinoma(HCC), but the precise mechanism of dysregulation has not been extensively studied. Our previous studies showed that HCC cells are resistant to endoplasmic reticulum(ER) stress-induced apoptosis, as compared with normal human liver cells. Little is known about the relationship between micro RNAs and ER stress-mediated apoptosis resistance. In this study, we use micro RNA-chromatin immunoprecipitation(CHIP) microarray to test the mi RNA expression profile of Hep G2 cells, and found that the expression level of mi R-663 was significantly upregulated after co-incubated with tunicamycin, an ER stress inducer. The bioinformatics analysis also showed that mi R- 663 involved in ER stress and some tumor biological behavior such as proliferation, apoptosis and so on. On this basis, quantitative real-time polymerase chain reaction(q RT-PCR) assay was used to validate this result. To investigate the potential involvement of mi R-663 in HCC cell apoptosis and proliferation under ER stress, Hep G2 cells were transfected with mimics or inhibitors of mi R-663. Subsequently, we apply target gene analysis software to further predicte the target gene of mi R-663, which could affect the HCC cells apoptosis. Accordingly,this study was designed to explore the influence of ER stress on the mi R-663 and the possible mechanism of mi R-663 on resistant to ER stress-induced apoptosis in HCC. These achievements might bring a hopeful strategy to overcome apoptosis resistance on HCC.Method: 1. A concentration of 3 μM TM was used to induce ER stress in Hep G2 cells. Using mi RNAs-CHIP to detect mi RNAs expression changes after with and without TM treatment, and analyze mi RNAs expression spectrum in Hep G2 cell lines.2. ER stress was induced by 3 μM TM in HCC cell lines( Hep G2ã€Be L7402 and SMMC7721 cells). The mi R-663 expression level in HCC cell lines after without TM treatment and treated with TM for 24 h was measured by q RT-PCR.3. Mi R-663 mimics and mi R-663 inhibitors were transfected into Hep G2 cells using Lipofectamine RNAi MAX. The transfection efficiency was tested by q RT-PCR 24 h post-transfection.4. The influence on HepG2 cells proliferation was detected by CCK-8 assay when the mi R-663 expression level was upregulated or downregulated under ER stress.5. The influence on Hep G2 cells apoptosis was determind by Annexin V-FITC / PI when mi R-663 expression level was downregulated under ER stress.6. Application of target gene prediction software to analyze the target gene of mi R-663.7. Using qRT-PCR and ELISA assay to detect the effect on TGFB1 when upregulated or downregulated mi R-663 expression, to test the TGFB1 expression level changes after without TM treatment and treating with TM for 24 h and after transfecting TGFB1 si RNA.8. The influence on Hep G2 cell apoptosis was tested by Annexin V-FITC / PI when the expression level of TGFB1 was downregulated.Results: 1. Mi RNAs-CHIP microarray analysis showed that mi R-663 was the most obviously upregulated after TM treated among seventy differentially expressed mi RNAs, especially at 24 hours.2. q RT-PCR results consistent with the mi RNA-CHIP result, the relative mi R-663expression level was 1.93±0.16-fold upregulated in Hep G2 cells after exposure to 3 μM TM for 24 hours. The expression level of mi R-633 was also increased in Bel-7402 cells and SMMC-7721 cells by 1.88±0.24 and 2.15±0.21 times compared with the untreated group, respectively.3. qRT-PCR results showed that the expression of mi R-663 in cells treated with mi R-663 mimics showed a 1,128.7±74.16-fold change over the mimics negative control group(NC), while expression of mi R-663 in cells treated with mi R-633 inhibitors showed only a 0.27±0.13-fold change compared with the inhibitor negative control group(INC).4. CCK-8 assay demonstrated that inhibition of mi R-663 using mi R-663 inhibitors combined with TM led to a remarkable decline in Hep G2 cell proliferation compared with the TM only treated cells. On the contrary, the rate of cell proliferation in cells treated with TM combined with mi R-663 mimics was significantly higher than cells treated with TM alone.5. Annexin V-FITC/PI apoptosis assay showed that inhibition of mi R-663 combined with TM treatment significantly enhanced cell apoptosis compared with untransfected cells.6. Target gene prediction analysis showed that TGFB1 is a potential target gene of mi R-663.7. q RT-PCR and ELISA assay indicated that transfection of the mi R-663 inhibitors resulted in a significant increase both in TGFB1 m RNA and protein expression, while transfection of mi R-663 mimics significantly reduced TGFB1 m RNA and protein levels. Moreover, TGFB1 was markedly decreased at both the m RNA and protein level after treating Hep G2 cells with TM. TGFB1 expression was decreasing at both the m RNA and protein levels after transfecting with TGFB1 si RNA.8. Annexin V-FITC/PI apoptosis assay showed that the ratio of apoptotic cells wassignificantly reduced after transfection with si RNA against TGFB1 for 48 hours in Hep G2 cells.Conclusion:1. The expression level of mi R-663 was upregulated in HCC cells under ER stress.2. The expression level of mi R-663 was upregulated and downregulated by mi R-663 mimics and mi R-663 inhibitors effectively.3. Downregulation of mi R-663 could relatively not only inhibit cell proliferation but also promote cell apoptosis of Hep G2 cells under ER stress.4. TGFB1 expression level was markedly decreased in Hep G2 cells under ER stress.5. Decreasing mi R-633 expression led to an upregulation of the TGFB1 m RNA and protein levels, while increasing the mi R-663 expression resulted in a downregulation of the TGFB1 m RNA and protein levels in HCC. TGFB1 is a potential target gene of mi R-663.6. Interference of TGFB1 m RNA could reduce apoptosis in HCC.7. Mi R-663 plays a pivotal role in ER stress-mediated apoptosis resistance in HCC cells by downregulating TGFB1. |
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