Study On The Mechanism Of WTX Gene About Regulation Of Proliferation And Metastasis In Hepatocellular Carcinoma Cells

BackgroundPrimary liver cancer is a common cancer in the world,with a high degree of malignancy and poor prognosis.The morbidity and mortality were higher in our country,and the mortality was second in all malignant tumors.Most of the patients had been in the middle and late stage of treatment,the rate of surgical resection was less than 20%.Although the application of the comprehensive means such as the progress of surgery and radiotherapy and chemotherapy,the treatment effect of liver cancer is improved,but the long-term effect is still poor.Postoperative recurrence and metastasis is also an important factor affecting the prognosis.Therefore,it is of great significance to explore the molecular mechanism of HCC development and metastasis,and to seek new therapeutic targets for improving the therapeutic effect and prognosis of HCC.WTX is located on the X chromosome,its function is mainly to suppress the tumor,the single mutation of WTX gene is likely to cause the occurrence of tumor.The results showed that the WTX gene was found in Wilms tumor,leukemia,gastric cancer and colon cancer,and had an effect on the invasion and metastasis of the tumor.But the relationship between WTX gene and liver cancer and its mechanism is still not clear.In this study,the relationship between WTX and hepatocellular carcinoma and its possible mechanism were studied.Object1 To detect the expression of WTX gene in the tissue samples of patients with HCC,and analysis different expression in liver cancer tissue and paracancerous tissue.To clarify the relationship between WTX gene and the clinical features of liver cancer.To find the different expression between normal liver cell and liver cancer cell.2 To analysis the effect of WTX on the ability of hepatocellular carcinoma cells by up-regulating WTX gene and inhibiting WTX with si RNA.3 To research targeting pathways of WTX gene about biological behavior by bioinformatics analysis.In order to demonstrate the WTX gene as a predictive marker of prognosis of hepatocellular carcinoma,and to provide evidence for clinical treatment.Method1 At first,the liver tissue of 49 cases of liver cancer and paracancerous tissue were collected from Jiangxi People's Hospital.All patients had no history of other malignant tumor before surgery,and did not accept chemotherapy and other treatment measures.What's more,the paracancerous tissue were selected from the edge of the lesion more than 2cm.All tissues were diagnosed by pathological section.Secondly,the expression of WTX protein in HCC were detected by immunohistochemistry.At last,we collected the clinical data,such as tumor stage,size,prognosis and other data.In addition,liver cell line L02,MHCC97 L,SMMC-7721,MHCC97 H,and HepG2 were collected,and the expression of WTX gene was detected by Blot Western and fluorescence quantitative PCR method.2 Hepatocellular carcinoma cell lines with over expression of WTX was established by transfected with plasmids via lipofection method,and inhibition of WTX gene in hepatocellular carcinoma cells was performed by siRNA.Western Blot and realtime PCR was applied to detect the expression of WTX in HCC cell lines.The effects WTX on hepatocellular carcinoma cells were described by means of CCK8,clone formation test,scratch test and transwell assay.3 Database,such as TCGA?GEO,was used to analysis the expression pattern of WTX and the targeting pathway.Western Blot was used to show the genes changing with overexpression of WTX.Result1 The result of immunohistochemical assay showed that the expression of WTX in primary hepatocellular carcinoma was significantly lower than that in the corresponding paracancerous tissue(P<0.05).The expression level of WTX gene was significantly correlated with the clinical TNM and stage of liver cancer(P<0.05).Survival data demonstrated that patients with WTX gene upregulation had better prognosis than those with low expression.2 WTX was up-regulated by infected with plasmids in HCC cell lines.the expression of WTX in HCC cells was significantly higher than that of control group by Western Blot and quantitative PCR.CCK8 experiment showed that WTX inhibited proliferation and Clone formation assay also proved HCC cells with WTXoverexpression owned a weaker clone formation ability.Would-healing assay demonstrated that WTX decreased invasion and metastasis ability.And we got the similar results via the transwell assay.Subcutaneous transplantation tumor model also show that WTX could inhibit tumorigenic ability.Down-regulating WTX was performed via siRNA in MHCC97 L cell line.The results of CCK8 showed that the low expression of WTX in HCC cell line could significantly enhance the proliferation of MHCC97 L cell line.Would-healing assay and trasnswell test showed that low expression of WTX can significantly enhance the invasion and migration of hepatocellular carcinoma cells.3 WTX was significantly lower In HCC tissues than in normal liver tissues from GEO database.It was found that there were 25 overlapping genes between the WTX related genes and the Wnt pathway by analysis the TCGA database and the KEGG pathway database,which suggested that WTX may take part in hepatocellular carcinoma.High expression of WTX caused decreasing of ?-catenin on HCC cells,which was verified by the means of western blot.Luciferase reporter assay showed that the transcriptional activity of TCF was significantly decreased with over-expression of WTX,which also proved that the Wnt pathway was inhibited.Conclusion1 WTX gene was low expression in hepatocellular carcinoma,and was significantly correlated with TNM stage.The patients with high expression of WTX gene could own better prognosis.2 Over expression of WTX in HCC could inhibit the malignant biological behavior of HCC.Low expression of WTX can significantly enhance the proliferation,invasion and migration in hepatocellular carcinoma cells.3WTX can inhibit Wnt pathway by reducing the expression of ?-catenin,decreasing transcriptional activity of TCF and then play the ability to inhibit the malignant biological behavior.The effect of WTX regulating the transcription of the TCF family was dependent on the expression of ?-catenin.