| Background and purpose:Liver cancer is one of the major cancers threatening human health in the world.The incidence rate and mortality rate of liver cancer ranks sixth and fourth in all malignant tumors worldwidely.Hepatocellular carcinoma(HCC)accounts for 75%-85% of primary liver cancer,and leading 750 thousand patients die each year in the world.The incidence rate of HCC has great regional heterogeneity.About 85% of HCC occurs in developing and underdeveloped regions,and 50% of them occur in China.The prognosis of HCC at early stage is good,but most patients are at the advanced stage at the time of first diagnosis,and the incidence to mortality ratio of these patients are close to 100%.Although great progress has been made in the treatment of HCC in recent years,the prognosis is still unsatisfactory due to the high recurrence and metastasis rate.Therefore,it is an important research field to study the inducing factors of HCC and the process of HCC invasion and metastasis,and then to find the prognostic biomarkers and potential therapeutic targets of HCC.MicroRNA(miRNA)is a kind of short and highly conserved endogenous small RNA,which is about 21-25 nucleotides in length.It is single stranded and cannot encode protein.It is widely expressed in prokaryotic and eukaryotic organisms.miRNA mainly plays a regulatory role in vivo.Through base pairing with the 3’untranslated region of m RNA,the corresponding target m RNA is degraded or the translation is inhibited,so to regulate gene expression at the post transcriptional level.Since the first miRNA was discovered and reported,many miRNAs have been studied.Although many miRNAs have been found,it is still the tip of the iceberg for understanding the mechanism of tumorigenesis and development.Many studies have shown that miRNAs are closely related to the occurrence,invasion,metastasis and prognosis of HCC.Studies have shown that some miRNAs are highly expressed in HCC and play the role of oncogenes,while some miRNAs are lowly expressed in HCC and play the role of tumor suppressor genes.miR-557 is a newly discovered miRNA in recent years.It was first screened from microarray data of gastric cancer tissue.Existing studies have found that miR-557 is low expressed in pancreatic cancer,pancreatic ductal carcinoma,triple negative breast cancer and lung cancer.As a tumor suppressor gene,miR-557 participates in the regulation of tumor invasion,metastasis and other biological processes.However,the biological function of miR-557 in HCC remains unclear.We found that miR-557 was low expressed in HCC through biological information.Therefore,we speculate that miR-557 plays an important role in the occurrence and development of HCC,and its function and molecular mechanism need to be further explored.Based on this,we first detected the expression of miR-557 in liver cancer tissues and different liver cancer cell lines,and analyzed the relationship between the expression level and the clinicopathological characteristics and survival of HCC patients;then we used in vivo and in vitro experiments to study the effect of miR-557 on the biological behavior of liver cancer cell lines;and then we predicted and verified the expression of miR-557 by targeting RAB10 It affects the proliferation,migration,invasion,epithelial mesenchymal transition(EMT)and clone formation of hepatoma cells,as well as cell cycle distribution;Wnt / β-catenin signaling pathway is a very important pathway in the occurrence and development of HCC.Studies have shown that many miRNAs affect the progress of HCC through Wnt / β-catenin signaling.Finally,we preliminarily discussed whether miR-557 can regulate Wnt / β-catenin signaling pathway.This study includes the following three parts: Part one: the expression of mir-557 in HCC and its relationship with clinicopathological features and prognosis;Part two: To investigate the effect of mir-557 on the biological behavior of hepatoma cells in vitro and in vivo;Part three: mir-557 inhibits Wnt by targeting rab10/ β-The research of catenin signal.Part one: expression of miR-557 in hepatocellular carcinoma and its relationship with clinicopathological features and prognosis Aim:To investigate the expression of mir-557 in hepatocellular carcinoma and its relationship with clinicopathological features and prognosis.Methods:1.The expression of miR-557 was analyzed by bioinformatics.2.Real time quantitative PCR(RT-qPCR)was used to detect the expression of miR-557 in HCC tissues and corresponding adjacent non tumor tissues.3.The relationship between the expression of miR-557 and the clinicopathological data and survival of HCC patients was analyzed.4.RT-qPCR was used to detect the expression of miR-557 in HCC cell line and human normal liver cell line L02.Results:1.Bioinformatics analysis of GSE108724 data set showed that miR-557 was low expressed in HCC compared with adjacent non tumor tissues;analysis of GSE26323 data set showed that the expression level of miR-557 in lung metastatic HCC was lower than in situ HCC.2.The expression level of miR-557 in HCC was significantly lower than that in matched adjacent non tumor tissues.3.In 34 cases of HCC tissue samples,the expression level of miR-557 in poorly differentiated patients was lower than that in moderately well differentiated patients,and the expression level of miR-557 in HCC patients > 5cm was lower than that in HCC patients ≤ 5cm The expression level of miR-557 in HCC patients with stage III /IV is lower than that in HCC patients with stage I / II,and the expression level of miR-557 in poorly differentiated HCC patients is lower than that in HCC patients with high differentiation;the overall survival time of HCC patients with low expression of miR-557 is shorter.4.Compared with normal liver cells L02,miR-557 was down regulated in HCC lines MHCC-97 H,MHCC-97 L,Huh7,Hep G2 and SMMC-7721.The expression of miR-557 was the lowest in MHCC-97 H cells and the highest in Huh7 cells.Conclusion: The expression of miR-557 is low in HCC tissues and cell lines,and it is significantly correlated with malignant biological characteristics and worse prognosis.Part two:discusses the effect of miR-557 on the biological behavior ofhepatoma cells in vitro and in vivo Aim:To investigate whether interfering or promoting the expression of mir-557 affects the biological behavior of hepatoma cells.Methods:1.MHCC-97 H cells were transfected with miR-557 mimics / mimic NC or lentivirus Lv-miR-557 / LV vector in vitro,and Huh7 cells were transfected with inhibitors / inhibitor NC or Lv-sponge-miR-557 / Lv-sponge-NC.The transfection efficiency was verified by RT-qPCR.2.CCK-8 assay and cell clone formation assay were used to detect the effect of miR-557 on the proliferation potential of HCC cells;scratch assay and Transwell assay were used to detect the effect of miR-557 on the migration and invasion of HCC cells.3.RT-qPCR and Western blot were used to detect the effect of miR-557 on epithelial mesenchymal transition(EMT)of HCC cells.4.Flow cytometry was used to detect the effect of miR-557 on cell cycle distribution of HCC.5.The effect of miR-557 on the tumorigenicity of HCC cells in vivo was detected by subcutaneous tumorigenesis assay in nude mice.Results:1.Mi R-557 mimics or Lv-miR-557 can significantly increase the expression level of miR-557 in MHCC-97 H cells,while inhibitors or LV sponge-miR-557 can significantly reduce the expression level of miR-557 in Huh7 cells,which can be used in the next experiment.2.CCK-8 assay,cell clone formation assay,scratch assay and Transwell migration assay showed that up-regulation of miR-557 expression could inhibit the proliferation,migration and invasion of MHCC-97 H cells,and down-regulation of miR-557 expression could promote the proliferation,migration and invasion of Huh7 cells.3.Compared with NC group,up-regulation of miR-557 increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in MHCC-97 H cells,while down-regulation of miR-557 in Huh7 cells resulted in the opposite results.4.Cell cycle experiments showed that up-regulation of miR-557 could block the cell cycle of MHCC-97 H cells in G0 / G1 phase;down regulation of miR-557 could promote the cell cycle transition from G0 / G1 to G2 / m.5.In vivo experiments showed that up regulation of miR-557 inhibited the subcutaneous tumorigenicity of MHCC-97 H cells in nude mice;down regulation of miR-557 further enhanced the subcutaneous tumorigenicity of Huh7 cells in nude mice.Conclusion: Up regulation of miR-557 expression can inhibit the proliferation,migration,invasion,epithelial mesenchymal transition,growth in vivo and induce cell cycle arrest in G0 / G1 phase.Inhibition of miR-557 expression showed the opposite trend.Part three: miR-557 inhibits Wnt / β-catenin signaling by targeting RAB10Aim:To explore the potential mechanism and downstream signaling pathway of mir-557 on hepatocellular carcinoma.Methods:1.The target genes of miR-557 were predicted by online bioinformatics software Targetscan,miRDB,mi Walk and miRTarbase.2.The double luciferase gene report experiment verified the targeted regulation relationship between miR-557 and RAB10.3.In MHCC-97 H cells with up-regulated miR-557 expression and Huh7 cells with down regulated miR-557 expression,the m RNA and protein expression of RAB10 were detected by RT-qPCR and WB assay.4.Immunohistochemistry(IHC)was used to detect the expression of RAB10 protein in HCC tissues and adjacent tissues.RT-qPCR was used to detect the expression of RAB10 m RNA in 34 HCC tissues and matched adjacent non tumor tissues,and the correlation with miR-557 was analyzed.5.Co-transfection of miR-557 mimics + pc DNA3.1-RAB10 or miR-557 inhibitors + si RAB10 was used to analyze whether promoting / interfering the expression of RAB10 could reverse the effect of miR-557 mimics / inhibitors on the biological behavior of HCC cells.6.In MHCC-97 H cells with up-regulated miR-557 expression and Huh7 cells with down regulated miR-557 expression,WB assay was used to detect the changes of important molecules GSK-3 β,p-gsk-3 β,β-Catenin and non-p-β-Catenin in Wnt / β-catenin signaling pathway.7.After CO transfection of miR-557 mimics + pcdna3.1-RAB10 or miR-557 inhibitors + si RAB10,the changes of GSK-3β,p-GSK-3β,β-Catenin and non-p-β-Catenin in Wnt / β-catenin signaling pathway were detected by WB assay.Results:1.The target gene of miR-557 was predicted to be RAB10 by bioinformatics software Targetscan,miRDB,mi Walk and miRTarbase.2.The dual luciferase gene reporter assay confirmed that miR-557 could inhibit RAB10 expression3.RT-qPCR was used to detect the changes of potential target genes in MHCC-97 H cells with up-regulated miR-557 expression and Huh7 cells with down regulated miR-557 expression,among which RAB10 changed significantly,and WB assay verified the changes of RAB10.4.IHC detected the expression level of RAB10 in 34 HCC patients’ cancer tissues and matched adjacent non tumor tissues,the results showed that the expression level of RAB10 in cancer tissues was higher than that in adjacent non tumor tissues;RT-qPCR showed the same result,and was negatively correlated with miR-557.5.The effect of miR-557 mimics / inhibitors on the biological behavior of HCC cells can be partially reversed by promoting / interfering with RAB10 expression.6.Up regulation of miR-557 expression can reduce GSK-3 β phosphorylation,increase β-Catenin phosphorylation and decrease non-p-β-Catenin incorporation,thus inhibiting Wnt / β-Catenin signal;down regulation of miR-557 expression has the opposite effect;promoting / interfering with RAB10 expression can partially reverse the effect of miR-557 mimics / inhibitors on Wnt / β-Catenin signal in HCC.Conclusion: miR-557 can regulate the activity of Wnt / β-Catenin signaling pathway by inhibiting rab10 expression. |
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