| Hepatocellular carcinoma(HCC)is one of the most common malignancies,with a 5-year survival rate of less than 20%.HCC is characterized by strong invasion and metastasis,lack of effective tumor biomarkers,so many patients have been found to be advanced stage with poor prognosis.Although treatment methods and levels are constantly improving,the results are still unsatisfactory,so it is very important to find new HCC biomarkers and accurate and effective treatment options.MicroRNAs(miRNAs)are a class of highly conserved,non-coding small-molecule RNAs with a length of about 21-25 nucleotides(nt).About 50%of human miRNAs are located at tumor-related sites,which means that miRNAs are inseparably related to tumors.MiR-133a is one of the most studied and characteristic miRNAs so far.MiR-133a plays an important role in many tumors.Although it has been reported that miR-133a can inhibit the migration and invasion of HCC cells through the downstream target gene IGF-1R,its specific molecular mechanism,potential functions and related signaling pathways are still unclear.In view of the importance of miR-133a in the development and progression of tumors,we conducted in-depth studies on the relationship between miR-133a and HCC,and found potential target and signaling pathway closely related to it.Our study verified that miR-133a may become a new therapeutic target for HCC.Part One:The expression and biological function of miR-133a in HCCPurposeTo investigate the correlation between miR-133a and clinical prognostic characteristics of HCC patients,and to explore the effects of miR-133a on the development and metastasis of HCC cells.Methods1.We downloaded microRNA HCC expression profile information from TCGA(http://cancergenome.nih.gov/)and GEO(https://www.ncbi.nlm.nih.gov/geo/)public databases,analyzed the expression of miR-133a in HCC and normal tissues.2.Thirty-four specimens of HCC and non-cancerous tissues were collected,and the expression of miR,133a was detected by RT-PCR.Simultaneously,COX regression analysis and Kaplan-Meier survival analysis were used to study the relationship between miR-133a and prognosis of HCC patients.3.RT-PCR was used to detect the expression of miR-133a in human normal liver epithelial cell line and HCC cell lines.4.MiR-133a mimics and miR-133a inhibitors were transfected into HCC cells respectively.The effect of miR-133a on the biological behaviors of HCC was detected by CCK-8,EdU,colony formation,wound healing,transwell assays and flow cytometry.Results1.According to TCGA sand GEO data analysis,miR-133a was down-regulated in HCC tissues,the difference was statistically significant(P<0.05).2.The results of HCC samples showed that the expression of miR-133a was significantly decreased in HCC tissues compared with adjacent non-cancerous tissues(P<0.001).The expression of miR-133a was closely related to tumor size(P=0.017),TNM stage(P=0.035)and metastasis(P=0.032).In addition,patients with low miR-133a expression had shorter survival time(P<0.05).3.The expression of miR-133a in six HCC cell lines were lower than normal liver cell line(P<0.05).4.Over expression of miR-133a inhibited the proliferation,migration and invasion of HCC cells,and promoted cell apoptosis.Down regulation of miR-133a promoted the proliferation,migration and invasion of HCC cells,and inhibited cell apoptosis.ConclusionMiR-133a was low expressed in HCC,inhibited the proliferation and metastasis of HCC cells,and miR-133a was closely related to the prognosis of HCC patients.Part Two:Study on the targeting relationship between mir-133a and FOSL2PurposeTo predict the possible target gene of miR-133a and study the role and mechanism of target gene in the progression of HCC.Methods1.TargetScan,starBase,miRTarbase and miRWalk were used to predict potential target genes of miR-133a,then FOSL2 was selected as the target gene of miR-133a by RT-PCR.2.The luciferase experiment verified the targeted regulatory relationship between miR-133a and FOSL2.3.RT-PCR was used to detect the expression of FOSL2 in HCC tissues and cells.The relationship between FOSL2 and clinical characteristics or patient prognosis was also analyzed.4.MiR-133a mimics and FOSL2 overexpression plasmid were co-transfected into SMCC-7721 and Huh7 cells.Afterwards,the proliferation,migration and invasion of HCC cells in each group were detected.5.FOSL2 interference plasmid was transfected into SMCC-7721 and Huh7 cells for biological behavior researches.Then we observed whether the role of si-FOSL2 was consistent with miR-133a,and detected changes in mRNA and protein levels of the AP-1 family.Results1.MiR-133a could specifically bind to the 3'-UTR of FOSL2.2.FOSL2 was overexpressed in HCC tissues and cells(P<0.05).3.Spearman analysis showed that the expression of FOSL2 and miR-133a were negatively correlated(R=-0.77,p<0.001).The expression of FOSL2 was closely related with TNM stage(P=0.047)and metastasis(P=0.017).In addition,the survival prognosis of patients with high expression of FOSL2 was poor.4.Overexpression of FOSL2 could reverse the inhibitory effect of miR-133a on HCC cells and promoted the proliferation of HCC.While downregulation of FOSL2 showed a tumor suppressive effect,which was consistent with the role of miR-133a.5.Protein and mRNA levels of c-Jun,JunB,c-Fos,and FosB were decreased in HCC cells transfected with si-FOSL2(P<0.05),while no change was found of FOSL1 expression(P>0.05).ConclusionFOSL2 was a target gene of miR-133a.MiR-133a regulated the progression and metastasis of HCC by inhibiting the expression of FOSL2.Part Three:Study of TGF-?/Smad3 signaling pathway in miR-133a regulating the proliferation and metastasis of HCCPurposeTo demonstrate that TGF-?/Smad3 signaling pathway was involved in miR-133a regulation of HCC progression.Methods1.GSEA analysis predicted the signaling pathways involved in the regulation of HCC by FOSL2.2.The protein level of the treatment groups was detected by Western blot with TGF-? stimulation for 24 hours.3.The effect of TGF? on the biological behaviors of transfected HCC cells was detected.Results1.GSEA analysis predicted that TGF-? pathway was highly enriched in FOSL2 high expression group.2.After the stimulation of TGF-?,protein level of p-smad3 decreased significantly in miR-133a group while increased in FOSL2 group(P<0.05).3.MiR-133a inhibited the promoting ability of TGF-? in HCC cells.ConclusionMiR-133a regulated the proliferation and metastasis of HCC through the FOSL2/TGF-?/Smad3 axis.Part Four:MiR-133a inhibits the proliferation of xenograft tumors in nude micePurposeTo verify the anti-tumor effect of miR-133a in nude mice.Methods1.SMCC-7721 cells transfected with miR-NC and miR-133a mimics were injected subcutaneously into nude mice to construct nude mice xenograft model.2.Changes in tumor size were observed weekly through Caliper IVIS Lumina ?.3.The tumor size and weight in two groups were measured.4.RT-PCR was used to detect the expression of miR-133a and FOSL2 and to describe the correlation between them.Western blot was used to detect the protein level of FOSL2.5.The expression of caspase-3 in tumor tissues of nude mice was detected by Immunohistochemistry.Results1.MiR-133a inhibited tumor proliferation in nude mice.2.The expression of miR-133a in miR-133a mimics group increased significantly,and FOSL2 level was decreased(P<0.05).There was a negative correlation between miR-133a and FOSL2 expression(R=-0.94,P=0.017).3.The expression of caspase-3 was increased in miR-133a overexpression group.ConclusionIn nude mice xenograft model,miR-133a inhibited the proliferation rate of HCC cells and promoted cell apoptosis.The expression of FOSL2 was negatively correlated with miR-133a.Full text conclusion1.The expression of miR-133a was significantly downregulated in HCC and patients with low miR-133a expression had poor prognosis.2.MiR-133a inhibited the proliferation,migration and invasion of HCC cells.We inferred that miR-133a acted as a tumor suppressor gene in HCC.3.Both in vivo and in vitro experiments showed that FOSL2 was a target of miR-133a,and the expression of miR-133a and FOSL2 was negatively correlated,suggesting that FOSL2 was involved in the regulation of miR-133a on HCC.4.FOSL2 was highly expressed in HCC tissues.Patients with high FOSL2 expression had shorter survival and worse prognosis,indicating that FOSL2 was a sign of poor prognosis.5.MiR-133a targeted FOSL2 and inhibited TGF-?/Smad3 signaling pathway during the progression of HCC.This regulatory axis may become a new target for HCC therapy. |
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