The Study Of The Expression And Function Of Protocadherin CDHR2 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common clinical malignant tumor.Its incidence increased year by year,and the mortality of liver cancer continues to rise.In2012,the global number of deaths due to hepatocellular carcinoma reached 745000 people,is an important cause of malignant tumor death.In China,hepatocellular carcinoma is the second highest mortality rate in cancer mortality.Because of insidious onset of the disease,the liver cancer was always found in the advanced period.Current treatment methods of HCC include surgery,local ablation,hepatic artery embolization chemotherapy,radiotherapy,systemic chemotherapy and traditional Chinese medicine treatment,but the effect of these treatments is poor.It is urgent that the pathogenesis of hepatocellular carcinoma be clarified,and biological targets for early diagnosis and treatment be mined.CDHR2 belongs to the protocadherin family,a subfamily of the cadherin superfamily.The gene was predominantly expressed in liver,kidney and colon tissues,which was designated Protocadherin LKC initially.CDHR2 protein composes of 1310 amino acids,including 7 cadherin repeat domain,a transmembrane domain and a PDZ-binding domain.Protocadherin is a kind of transmembrane protein with the same antigen-binding activity,which are mainly composed of several to 110 cadherin extracellular domain consisting of repeated sequences.Previous studies showed that the protocadherin expression levels are closely related with cell proliferation.The expression levels and alterative functions of protocadherin caused by gene mutation,may lead to tumorigenesis,acceleration of the tumor invasion and metastasis,indicating that protocadherin was closely related with the occurrence of tumorigensis and tumor development.As a member of protocadherin superfamily,CDHR2 also has the same important features.It has been reported that CDHR2 was involved in the cell contact inhibition mediated by alterative localization of?-catenin,resulting in tumor cell growth arrest and proliferation inhibition in colorectal cancer cells,which suggested that CDHR2 play an important role in the development of colorectal cancer.However,the expression levels and function of CDHR2 in HCC has not been reported yet.In our previous study,we found that CDHR2 was significantly downregulated in HCC tissues,compared with adjacent tissues by cDNA microarray.In this study we analyzed the expression levels and regulation mechanism of CDHR2 in HCC,and explored the molecular mechanisms and biological function of CDHR2 in HCC.Firstly,we detected the mRNA and protein expression levels of CDHR2 in HCC cell,by semi-quantitative PCR method and Western-blot,respectively.Then we cloned human CDHR2 gene and constructed the inducible lentviral expression system carrying CDHR2 gene.Infection the cells which were HCC cell lines with low CDHR2 expression level or deficiency were infected with lentivirus,before the cell proliferation,clone forming and anchorage independence ability were analyzed by doubling time assay,EdU incorporation assay,Colony assay,and soft agar assay,respectively.And the vivo tumorigenicity was also evaluated in nude mice.We performed high-throughput protein microarray technique to explore cell signaling pathways alteration induced by CDHR2 overexpression in HCC cells,which were validated preliminarily.Whether low expression of CDHR2 was related with epigenetic alteration was detected by methylation level analysis.CDHR2 specific polypeptides were designed and synthesized for rabbit polyclonal antibody preparation used to Western blot and immunohistochemistry.HCC clinical tissue samples were collected for semi-quantitative PCR and immunohistochemistry to detect the expression levels of CDHR2 in HCC.Our study found that CDHR2 downregulation or silence in a variety of HCC cell lines.The aberrant expression of CDHR2 in smmc-7721 and PLC/PRF/5 inhibited the proliferation,colony formation and anchorage independent growth capability of liver cancer cell,and the in vivo tumorigenicity in nude mice.Then we found that CDHR2 inhibit Akt phosphorylation,and decrease the expression of COX-2,through which CDHR2 inhibited cell proliferation.The transfection of constitutively activated AKT1,myr-Akt,reversed the effect of down-regulation of COX-2 expression and cell proliferation inhibition by CDHR2.The low expression of CDHR2 was related partly with the hypermethylation of CpG island in its promoter region.clinical liver cancer tissue specimens CDHR2 in mRNA and protein levels in HCC tissues showed Finally,downregulated expression of CDHR2 mRNA and proteins were detected in clinical liver cancer tissue specimens.Our results showed that CDHR2 gene was downregulated or silenced in HCC.Overexpression of CDHR2 inhibits hepatocellular carcinoma cell proliferation mediated by Akt signal pathway,revealing a new regulatory mechanism for HCC development.