| Background and purposeHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world and the second largest cause of cancer death in the world.About 745,500 people die each year.HCC is highly invasive,has a high recurrence rate,and often has poor prognosis.HCC is prone to chemotherapy resistance.Conventional chemotherapy is not effective in patients with advanced HCC,Resistance to chemotherapy has become one of the main obstacles to the treatment of HCC.Therefore,to uncover the molecular mechanism of chemotherapeutic resistance of HCC,andfind ways to overcome the chemoresistance of HCC has become the key to successful treatment of HCC.Six2 is a member of the six homologue box family and participates in mesenchymal-epithelial transformation,has been shown to promote metanephric mesenchyme cell proliferation and inhibit cell apoptosis during kidney development.And Six2 deficiency leads to mesenchyme progenitors deletion,dysdifferentiation and severe kidney hypoplasia.Additionally,Six2 could mark and regulate a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.A previous study has shown that Six2 can promote breast cancer metastasis.KM-Plotter analysis showed that HCC patients with Six2 expression levels had negatively correlated with overall survival.And since gene expression programmes and cellular processes that are utilized in embryogenesis are often recovered in tumours,It is worth noting that cancer cell sternness could result in cells metastasis.and epithelial-mesenchymal transition(EMT)process could confer tumour cell stemness,we infer that Six2 misexpression is related to the HCC cell sternness.Both cancer cell sternness and EMT are associated with cancer chemotherapy resistance and metastasis.E-cadherin,the epithelial marker of EMT,acts as a tumour suppressor in tumour metastasis and stemnness;however,the mechanisms by which E-cadherin is regulated in HCC are still unclear.and the roles of Six2 on HCC chemotherapeutic sensitivity are still unclear.In order to clarify the role and the mechanismthe of Six2 in the prognosis and chemoresistance of HCC?we have carried out some researches as follows.Materials and methods1.The expression of Six2 protein in HCC tissues,matched normal tissues and HCC cell lines was detected by immunohistochemistry,real-time luorescence quantitative PCR and Western blot.KM plotter was used to analyze the influence of Six2 expression level on the overall survival of 364 HCC patients.2.Six2 gene was known down by interference technique,the apoptosis of HCC cells was induced by 5-FU,and then CCk8 assay and cell apoptosis assay were used to observe the changes of cell viabillity and cell apoptosis in HCC cells with six2 knockdown or not were treated by 5-FU.the apoptosis-related genes were also examined by Western blot.The expression of tumor sternness markers(ALDH1 and Nanog)was also detected in HCC cells with Six2 knockdown or not.And further,Cell spheroid formation ability and ALDH1 activity was also measured.3.Stable HCC cell lines with overexpression of Six2 and E-cadherin proteins were established.Western blot and real-time quantitative PCR were used to detect the expression of E-cadherin in HCC cells with Six2 overexpression or not.the apoptosis of HCC cells was induced by 5-FU,and then CCK8 and Western blot techniques were used to detect the activity of cells and the expression of apoptosis-related proteins to verify the relationship between E-cadherin expression and Six2-mediated 5-FU sensitivity.Spheroid assay,ALDH activity assay and stem cell marker gene mRNA level were used to verify the relationship between the expression of E-cadherin and the cells sternness of Six2-mediated HCC cells.4.Western blot and real-time quantitative PCR were used to detect the expression of E-cadherin in HCC cells with Six2 overexpression or not were treated with the methyltransferase inhibitor Decitabine.Methylation-Specific PCR was used to detect the methylation status of CpG island of E-cadherin promoter and the difference of E-cadherin methylation level in L02,HepG2 and Huh7 cell lines.Finally,the relative expression of Six2 and E-cadherin in HCC tissues was verified by fluorescence real-time quantitative PCR.Result1、Six2 expression is increased in HCC cells and tissues,and correlates with poor prognosisIn 39.0%HCC tissues showed positive expression,and the matched adjacent tissues of positive expression was 12.2%(P<0.01).The mRNA and protein levels of Six2 in HCC tissues were higher than those in normal tissues(P<0.01),Significant correlations were found between Six2 expression and the AFP.There were no statistically significant differences between Six2 expression and the other clinicopathological parameters,such as patient age,gender,HBsAg,tumor size,tumor differentiation and TNM stageand.and the expression of Six2 in HCC cells was higher than that in normal hepatocytes(L02 cell lines),especially in HepG2 and Huh7(P<0.01).The KM analysis showed that the HCC patients with high Six2 expression was 45.1%(164/364),and the overall survival rate was significantly lower than that of patients with low expression.The difference was statistically significant(P<0.05).2.Six2 negatively regulates 5-FU sensitivity and potentiates HCC cells sternness in HCC cellsCompared with the control group,Six2-shRNA treatment could increase the apoptosis rate induced by 5-FU,decrease the cell activity,and increase the expression of apoptosis-related proteins cleaved caspase-3 and cleaved PARP in HCC cell line after stable knockdown of Six2,(P<0.01).The effective knockdown of Six2 and 5-FU had synergistic effect.Six2 knockdown could significantly decrease the ability of Spheroid and the activity of ALDH1,(P<0.01).and down-regulate the expression of stem cell marker protein Nanog and ALDH1.3.Six2 regulates 5-FU sensitivity and cells stemness via regulating E-cadherin expressionE-cadherin expression was decreased or increased in cells with Six2 overexpression or knockdown,respectively,(P<0.01)..E-cadherin over-expressed virus was transfected into Six2-overexpressed HCC cells,and the expression of E-cadherin recovered.we observed that re-expression of E-cadherin rescued the 5-FU sensitivity in Six2 overexpressed cells,(P<0.01).and was sufficient to restore 5-FU-induced apoptosis in Six2 overexpressed cells.Additionally,restoration of E-cadherin expression attenuated Six2 overexpression-mediated upregulation of cells stemness,characterized as the decrease of stemness markers(ALDH1 and Nanog)expression,cells spheres size and numbers,and ALDH1 activity(all of P<0.01)..4.Six2 inhibits E-cadherin expression via stimulating promoter methylationThere was no significant change of the expression of E-cadherin in the HCC cells with Six2 overexpression were treated with 5μM dicitabine(P<0.01).However,the expression of E-cadherin in the control groups were not further increased(P>0.05).The methylation analysis of E-cadherin promoter showed that the expression of E-cadherin was partially increased in HCC cells.The methylation of E-cadherin CpG island in Six2-overexpressed HCC cells was significantly increased(P<0.01).High-level Six2 and low-level E-cadherin-expressing HCC cells were showed high-expression E-cadherin promoter methylation(P<0.01).L02,which expressed low level of Six2 and high level of E-cadherin,showed low expression of E-cadherin promoter methylation(P<0.01).There was a negative correlation between the expression of Six2 and E-cadherin in 41 cases of HCC(R2=0.4167,P<0.01).Conclusion1.Six2 may contribute to HCC progression.2.Six2 could attenuate chemotherapy sensitivity of HCC cells via potentiating HCC cells sternness.3.Six2 regulates 5-FU sensitivity and cells sternness via regulating E-cadherin expression.4.Six2 could repress E-cadherin expression via altering the methylation status of its promoter. |
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