Research On The Mechanism Of Hiwi In Hepatocarcinogenesis

Objective:Hiwi, also known as Piwill, is one of the member of human Piwi family. It has been reported to be up-regulated in various cancers such as esophageal squamous cell carcinoma, gastric carcinoma, pancreas adenocarcinoma and hepatocellular carcinoma, but its biological function remains largely unknown. In this thesis, we focus on the impacts of Hiwi on the proliferation and apoptosis of hepatocarcinoma cell, and conduct a preliminary investigation of the biological function of Hiwi.Methods:Firstly, recombinant plasmids expressing human Hiwi were constructed. Hiwi expression plasmids were transfected to HepG2 or mouse primary hepatocytes and the changes were observed constantly. Cell viability was assayed by MTT, cell cycle by propidium iodide staining, cell proliferation by EdU. Transwell was adopted to quantify the impact of Hiwi on the cell migration. The expression of genes involved in epithelial-mesenchymal transition (EMT) was then quantified using real-time PCR in cells over-expressing Hiwi.Secondly, the culture method of mouse primary hepatocytes was established, a recombinant adenovirus with GFP marker expression of human Hiwi was constructed, they were used to infect HepG2 and primary hepatocyte and periforsine or acetaminophen (APAP) was added to induce cell apoptosis, MTT method was adopted to detect cell activity, and TUNEL staining was conducted onto HepG2 cells treated by APAP to identify the cells apoptosis.Thirdly, a vivo model of liver over-expressed Hiwi was established through mice tail vein injection of adenovirus. The expression of Hiwi in liver tissue and the mRNA level of test apoptosis related gene and EMT related gene was detected after 5 days. After the mice tail vein injection of adenovirus, injection peritoneal of APAP for the establishment of acute liver injury model was conducted, and the impact of Hiwi expression on the acute liver injury in mice caused by APAP was detected, and the level of DNA methylation in mouse liver of Hiwi over-expression and primary hepatocytes was examined.Results:Firstly, human Hiwi gene was cloned and its recombinant plasmid was generated. With MTT, flow cytometry, and Edu incorporation assay, over-expression of Hiwi was found failing to promote the proliferation and migration of HepG2 cells or mice primary hepatocytes. Meanwhile, no detectable alteration was found in the migration of cells with over-expressing Hiwi and the expression of EMT.Secondly, Over-expression of Hiwi also failed to suppress apoptosis induced by APAP or periforsine.Thirdly, a vivo model of liver over-expressed Hiwi was established through mice tail vein injection of adenovirus. By real-time PCR analysis, no detectable alteration was observed in the mRNA levels of apoptosis related genes, i.e., BcL2, BAX, MCL1, p53 and EMT related genes, i.e., E-cadherin and N-cadherin. Besides,no significant influence of the Hiwi expression was observed on the APAP caused acute liver injury in mice, the level of DNA methylation in mouse liver of Hiwi over-expression and primary hepatocytes.Conclusions:The above results suggested that although Hiwi is expressed simultaneously with HCC, it is more likely to be induced by HCC instead of acting as an oncogene. Therefore, the function of Hiwi still remains to be illuminated.