Effect And Mechanism Of HAND1 On Endometrial Carcinoma

I.Background and purpose: Endometrial cancer is a common malignant tumor of the female reproductive system.Its global incidence has been reported to be on the rise and is predicted to be the the most common second malignant neoplasm of female reproductive system.HAND1(heart and neural crest derivatives expressed 1)is a protein expressed by cardiac nerve crest derivative,which plays a important role in trophoblast differentiation and cardiac development.At the same time,many articles have shown that HAND1 is involved in the occurrence and development of a variety of human cancers,and its expression level is related to the occurrence and prognosis of many cancers.It can regulate the expression of many genes,and it is speculated that HAND1 may be a indicator for tumor detection,diagnosis and prognosis.However,its physiological functionand mechanism in endometrial carcinoma have not been defined so far.In this study,we first detected the differential expression of HAND1 in human endometrial carcinoma,and studied the effect of HAND1 on the proliferation,migration and invasion in Ishikawa cells and RL95-2 cells.Secondly,the downstream gene c-MET(cellular-mesenchymal epithelial transition factor)of HAND1 was identified by RNA-seq.Whether HAND1 can regulated the proliferation,migration and invasion of cells through c-MET was confirmed in endometrial cancer cell lines Ishikawa and RL95-2.The regulation of HAND1/c-MET axis on EMT and signal pathway was detected also.Finally,nude mice tumorigenesis in vivo confirmed the effect of HAND1/c-MET on endometrial cancer.II.Methods:1.The expression of HAND1 in endometrial carcinoma was detected by immunohistochemistry.2.Western Blot and q PCR experiments to identified that whether the ostable cell lines of over-HAND1 and interfer-HAND1 were constructed using lentivirus system.3.The effects of HAND1 on proliferation,migration and invasion of endometrial cancer cells were detected by CCK8,clone formation,Transwell and cell scratch experiments in the overexpressed and interfered HAND1 stable cell lines.4.The downstream gene of HAND1 is c-Met which is analyzed and screened by RNAsequnce in overexpression stable cell line.5.Western Blot and q PCR were used to verify the relationship between HAND1 and cMET in stable HAND1 cell lines.6.HAND1 regulates the effect of c-MET on endometrial carcinoma(1)Immunohistochemistry to detect the expression of c-MET in endometrial carcinoma.(2)c-MET was interfered in Ishikawa cell and overexpressed with c-MET in RL95-2 cell.CCK8,clone formation,Transwell and cell scratch tests were used to confirm whether HAND1 can affect the proliferation,migration and invasion of endometrial cancer cells through c-MET.(3)Western Blot experiment confirmed the effect of HAND1 on cell proliferation and the expression of EMT and PI3K/AKT/m TOR pathway-related proteins by regulating c-MET.(4)the effect of HAND1/c-MET axis on endometrial carcinoma was verified by tumor formation experiment in nude mice,and the expression of related proteins in tumor in control group and treatment group was detected by Western Blot test.III.Results:1.Immunohistochemistry revealed that endometrial cancer had a low expression of HAND1 in comparison to the control group.2.Overexpression of HAND1 in Ishikawa and RL95-2 cells decreased the ability of cell proliferation,migration and invasion,and interference with HAND1 enhanced the ability of cell proliferation,migration and invasion.3.Overexpression of HAND1 in Ishikawa and RL95-2 cells decreased c-MET expression,and interference with HAND1 enhanced c-MET expression.Confirmation that HAND1 regulates c-MET expression changes.It can be confirmed that HAND1 regulates c-MET expression.4.The results of bioinformatics analysis showed that the expression of c-MET in endometrial carcinoma was higher than that in adjacent tissues,which was confirmed by immunohistochemistry.5.HAND1 inhibits the proliferation,migration and invasion of endometrial cancer cells by regulating c-MET and affects the expression of PI3K/AKT/m TOR pathway proteins.IV.Conclusions:1.The expression of HAND1 is low in endometrial carcinoma and can affects the proliferation,migration and invasion of endometrial cancer cells.2.The downstream gene c-MET was screened by transcriptome sequencing,which was highly expressed in endometrial carcinoma.3.HAND1 can affect the proliferation,migration and invasion of endometrial cancer cells and the expression of PI3K/AKT/m TOR pathway protein by regulating c-MET which can effect the progression of endometrial cancer.