| Objective:Hepatocellular carcinoma has become the one of the highest world's incidence tumors.And hepatocellular carcinoma (HCC) accounted for about 80 to 90 percent among them. With the development of research, some potential signaling pathways ,which were associated with hepatocellular carcinoma,had known through various mRNA modulation ways gradually. There is a class of small non-coding RNA molecules--microRNAs (miRNAs) have been found in recent years, which are involved in post-transcriptional regulation of gene expression in plants and animals.MicroRNAs contain 19-25 nucleotides,which can connect with target mRNA at the 3 'untranslated region (untranslated regions, UTR),and the connection can led to the degradation or inhibition of the target molecule's function. MiRNA involved in a series of important life processes, including cell proliferation, apoptosis, and differentiation. With the development of miRNA isoforms research,a variety of abnormalities in miRNA expression in human malignant tumors were revealed. It is suggest that miRNA may represent a new class of oncogenes or tumor suppressor genes.A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs , the hepato-specific miR-122 was found down-regulated in 70% of HCCs and in all HCC-derived cell lines. One as a tumor marker, miR-122 is involved in metastasis,which has a dramatic reduction.PSMD10, also known as Gankyrin, which is a 226 amino acid protein molecular weight of about 25KD. PSMD10 showed a high level expression in HCC. PSMD10 is an anti-apoptosis cancer protein, experiments have confirmed that overexpressed PSMD10 can improve the resistance ability to apoptosis of tumor cells in vitro and in vivo.To further determine whether miR-122 was expressed by regulating the PSMD10, thus affecting the apoptosis of hepatoma cells, and ultimately affected the sensitivity of chemotherapy drugs, we used proteomics methods on miR-122 inhibitor-transfected Huh7 cells as a model in this study, in order to find the role of miR-122 regulation to PSMD10,then futher explore the miR-122 regulation of apoptosis, the chemotherapy sensitivity of the channel, and the role of PSMD10 in such way.Methods:1 Construction of low expression of miR-122 cell model of hepatocellular carcinomaIn this study, we transiently transfected Huh7 cells with miR-122 inhibitor and control RNA. The suppressive effect of miR-122 inhibitor was demonstrated by TaqMan real-time PCR.2 Determine the reduction in miR-122 influence on PSMD10We used western blot assay to identify PSMD10 proteins that were differentially expressed between the miR-122 inhibitor-transfected Huh7 cells and control RNA-transfected Huh7 cells at 48h after transfected.3 Construction of plasmidThe synthesized CDK4 3'UTR fragment was connected to the pGL3-Control vector cloning vector ,and then the pMIR-3'UTR luciferase reporter plasmids was constructed.4 Identification of miR-122 target geneHuh7 cells were transfected with the reporter construct plasmid containing the 3'UTR of CDK4 and then co-transfected miR-122 inhibitor,miR-122 mimics or negative control.Used Dual-luciferase reporter gene assay and western blot.5 Identified the Link of miR-122 increased PSMD10We carried out the study from three areas,such as: the mRNA level, protein synthesis, Proteasome. In the experiment of mRNA level : comparing the transfection of miR-122 inhibitor and the control RNA in Huh7 cells, miR-122 expression at the mRNA level; treated miR-122-repressed Huh7 cells with the protein synthesis inhibitor cycloheximide (CHX) or the proteasome inhibitor MG-132. 48 hous after transfection , cells were collected and construtived to rotein samples, western blotdetection assay.6 Determine the role of CDK4 in the process of miR-122 regulatingthe stability of PSMD10We performed western blot to examine the protein level of PSMD10 in miR-122-repressed Huh7 cells treated with small interfering RNAs (siRNAs) against CDK4.7 Determine the effect of apoptosis of hepatoma cells by the reduced expression of miR-122Huh7 cells were transfected with the miR-122 inhibitor and the control RNA. At 8 hours after transfection, used PBS washed the cells and digested with trypsin. And then cells were grown in 96 well plates. At 24 hours after Huh7 cells were transfected with miR-122 inhibitor or control, Huh7 cells were incubated with cisplatin ( levels were : 0,2,8,12.5,20μg/ml ) for 24 hours. Relative live cell number was determined by CCK-8 assay under 450nm at two hous after stimulation. Western blot detection of active caspase-3 of cells stimulated with cisplatin (8μg/ml) for another 24 h after transfection with miR-122 inhibitor, siRNA against PSMD10 or respective control.8 Explored the role of apoptosis to PSMD10 miR-122 regulation in Hepatoma cellsFor this experiment, Huh7 cells were incubated with different concentrations( 0, 2, 8, 12.5, 20μg/ml, each concentration repeated six holes, 100μl MEM each hole,cell-free medium as control ) of cisplatin for another 24 hours after transfection with miR-122 inhibitor, siRNA against PSMD10 or respective control. In the process,cells were washed with PBS and planted into two 96-well plates at 24 hours after first transfection. Relative live cell number was determined by CCK-8 assay. Results:1 In Huh7 cells transfected with miR-122 inhibitor, the level of miR-122 expression of the mRNA decreased(Fig. 1A).2 Reduced expression of miR-122 caused Huh7 cells PSMD10 protein increase(Fig. 1B,C).3 Dual-luciferase reporter gene assay: Compared with the control group,luciferase activity of Huh7 cells transfected with miR-122 mimics was reducted significantly(Fig. 2B). However,luciferase activity of the Huh7cells transfected with miR-122 inhibitor was increased dramaticly (Fig. 2C). Western blot assay: expression of CDK4 increased in the miR-122 inhibitor groups, and reduced in the miR-122 mimics groups (Fig. 2D) . The difference between them is significant(p<0.05)(Fig. 2E).Therefore, CDK4 can be identified as the target genes of miR122.4 In the CHX (protein synthesis inhibitor) group:Compared with the control group,the expression of PSDM10 in cells tranfected with miR-122 inhibitor had been still increased. Oppsitely, MG-132 abolished the up-regulation of PSMD10 protein levels in miR-122 inhibitor-treated Huh7 cells.It can determine miR-122 inhibitor decreased protein degradation,which increases the expression of PSMD10 as a rusuilt(Fig. 3).5 Contrast to negative control group,the expression of PSMD10 cells transfected with interfering RNAs (siRNAs) against CDK4 and miR-122 inhibitor was decreased.6 By CCK8 assay,we found the number of surviving of cells transfected with miR122 inhibitor is always higher than that of negative RNA-transfected cells. Huh7 produced significant drug resistance by miR-122 inhibitors(concentration cisplatin : 8μg/ml, 12.5μg/ml) (Fig.5A). The expression of C-caspase3 was reduced in Huh7 cells transfected with miR-122 inhibitor (Fig. 5B).7 Down-regulated PSMD10 increased apoptosis of Huh7 cells (Fig. 6). The results show that PSMD10 can regulat anti-apoptosis of Huh7 cells. Then we can draw a conclusion that miR-122 regulates the PSMD10 furthermore effect apoptosis of hepatoma cells.Conclusions:1 MiR122 play an important role in the process of regulating cell apoptosis and treatment of liver cancer. Reduced expression of miR-122 can enhance the anti-apoptotic capacity of cells, also increased resistance to chemotherapy liver cancer, can reduce the sensitivity to chemotherapeutic drugs.It may provide us a new idea of treatment to hepatic carcinoma with low expression of miR-122.2 PSMD10 plays an important role in proapoptotic regulation of miR-122.3 CDK4 was identified miR-122 target genes. By dual luciferase reporter gene assay, compared with the control group, the increas of miR-122 inhibitor caused by the relative fluorescence was significantly increased (p= 0.003), while the change caused by miR-122 mimics was reduced in the relative fluorescence value (p<0.05).4 A pathway proapoptotic regulation of miR122 was established: miR-122 inhibitor decreased protein degradation,which increases the expression of PSMD10(which related with the decreased expression of CDK4), thereby increasing the of apoptosis of liver cancer cells, that is, improving the sensitivity to the chemotherapy drugs. |
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