The Mechanism Of MiR-133a On The Proliferation Of Human Hepatocellular Carcinoma

Background and aimHCC is the fifth most common malignant tumor in China and the third leading cause of death from cancer.Highly malignant with unobvious symptoms and high postoperative recurrence rate,HCC progresses rapidly and is difficult to treat.The pathogenesis of HCC is still unclear.Although progress has been made to achieve short-term effect and longer survival time of HCC in recent years,there is still no effective treatment with fewer side effects mainly because of its high recurrence rate and the poor long term effect of the treatment.Therefore,to improve the survival rate of HCC patients should strengthen the study of molecular pathological mechanism of HCC.Furthermor,it is important to find target genes associated with recurrence and metastasisand explore the new way of biological therapy.More and more studies have shown that the regulation of gene expression plays an important role in the pathogenesis of HCC.MicroRNAs refer to a special RNA family composed of 18-25 nt endogenous single stranded non-coding short RNAs,which widely exist in eukaryotic cells.After combining with the 3′ untranslated region of target mRNA,they negatively regulate the transcription and protranscription process of their target gene.They promote the degradation of target m RNA or terminate it’s translation at post-transcriptional level.They participate in the biological processes of proliferation,apoptosis,invasion,migration and cell cycle regulation of tumor cells.We used quantitative PCR to compare the expression profiles of tumor associated miRNA in normal liver cells and hepatocellular carcinoma cells,and found that miR-133 a expression was lower in HCC cells.MiR-133 a plays a negative regulatory role in multiple malignant tumors,but its function and mechanism in the occurrence and development of HCC has not been reported.This research aims to study the expressive difference of miR-133 a in HCC and the relationship with clinicopathological features,as well as the role of miR-133 a in the pathogenesis of HCC.Materials and Methods1.Detecting the expressive difference of miR-133 a in HCC tissues and adjacent tissues with qRT-PCR,and statistically analyzing the relationship between the expressive difference and the clinic pathological features of HCC.2.Detecting the expressive difference of mi R-133 a between HCC cell lines and normal liver cell lines with qRT-PCR;establishing HCC cell lines with miR-133 a over-expression by lentivirus technologyand studying the effect of miR-133 a on the proliferation and cell cycle of HCC cell lines using MTT and flow cytometry.3.Studying the migration ability of high miR-133 a expression in HCC cell lines using the cell scratch test.4.Studying the invasion ability using the Transwell test.5.Studying the effects of miR-133 a on HCC growth in vivo using tumor-bearing nude mice.6.Predicated the target genes of miR-133 a by using target prediction softwareand confirmed them by Westernblot and luciferase reporter assay.7.The expression of target gene in HCC tissues and adjacent tissues was detected by qRT-PCR,and the correlation between the expression of target gene and miR-133 a was analyzed.Results1.We found that miR-133 a was of low expression in HCC tissue and HCC cell lines,and was inversely related to the size of tumor and serum AFP level.2.We confirmed that miR-133 a was of lower expression in HepG2 and Huh7 than in LO2.In miR-133 a over-expression Huh7 cells,the proliferation of Huh7 was inhibited and the cell cycle was blocked in G1/G0.3.The cell scratch test showed that miR-133 a inhibited HCC cell migration.4.The Transwell test showed that the cell invasion was remaining unaffected.5.The HCC-bearing nude mice showed that miR-133 a inhibited HCC proliferation and growth.6.We predicted the target genes of miR-133 a may be CDK13;the target gene CDK13 were confirmed by westernblot analysis and the binding sites betweenmiR-133 a and target genes CDK13 were proved by the luciferase reporter system.We analyzed the mRNA expression level of CDK13 in HCC samples and found that a statistically significant inverse correlation between miR-133 a and CDK13 mRNA.ConclusionThis study found that miR-133 a was low expression in both HCC tissue and HCC cell lines,and was inversely related to the size of tumor and serum AFP level.This study also confirmed that miR-133 a inhibited proliferation and migration,blocked cell cycle in HCC,but did not have influence on HCC cell invasion.HCC-bearing nude mice confirmed that miR-133 a inhibited HCC proliferation in vivo.We predicted the target genes of miR-133 a may be CDK13.Further the target gene CDK13 was confirmed by westernblot analysis and the binding sites between miR-133 a and target genes CDK13 were proved by the luciferase reporter system.Finally,we analyzed the expression level of CDK13 in HCC samples and found that a inverse correlation between miR-133 a and CDK13 m RNA.All these revealed that miR-133 a play an important role in HCC especially tumor proliferation.