Expression And Functional Analysis Of OTC In Primary Liver Cancer

ObjectiveTo investgate the Ornithine transcarbamylase（OTC）expression in hepatocellular carcinoma（HCC）and analyze the associations between OTC expression and clinicopathological parameters as well as clinical outcome of HCC patients.To discover the role of OTC in the proliferation of liver cancer cells and analyze the molecular mechanism,thus providing a new clue for clinical therapy of HCC.Methods1.The level of OTC mRNA in HCC and its association with clinical outcome of HCC patients was analyzed based on datasets from the GEO（Gene Expression Omnibus database）and TCGA（The Cancer Genome Atlas）database.2.Immunohistochemistry,Western blotting assay and RT-qPCR were used to detect the levels of OTC in 42 HCC tissues and their adjacent non-tumor livers.3.The associations between OTC expression and clinicopathological parameters of HCC patients were analyzed by Pearson’sχ² test or Fisher’s exact test.4.RT-qPCR and Western blotting assay were used to detect the OTC levels in primary human hepatocytes（PHH）,immortalized hepatocyte cell（MIHA）and liver cancer cells.5.The effect of OTC expression on liver cancer cells proliferation was measured using trypan blue exclusion assay and CCK-8 assay;DNA synthesis was measured using bromodeoxyuridine assay;and the ability of colony forming was determined by colony-formation assay.6.The proliferation of liver cancer cells transfected with siOTC was measured using trypan blue exclusion assay and CCK-8 assay;DNA synthesis was measured using bromodeoxyuridine assay;and the ability of colony forming was determined by colony-formation assay.7.The effect of OTC overexpression on the apoptosis of liver cancer cells was determined using flow cytometry and western blotting assay.8.The content of NH₄⁺in OTC overexpressed or OTC gene silenced cells was detected by spectrophotometric method;The content of NH₄⁺in5 HCC tissues with OTC high expression OTC expressed and in 5 HCC tissues with OTC low expression was detected by spectrophotometric method.9.Apoptosis-related genes were screened by RT-qPCR and then verified using Western blotting assay.The effect of Bax silencing on apoptosis induced by OTC overexpression was determined by flow cytometry and Western blotting,and the growth rate was examined by trypan blue exclusion assay.Results1.Data from GEO and TCGA dataset revealed a significantly decreased OTC mRNA expression in the HCC tissues;OTC mRNA levels in patients with early stage tumors（stage I）was higher than those with stage II or stage III tumors;HCC patients with low OTC expression exhibited significantly shorter disease-free survival and 5-year overall survival rates compared with those with high OTC level.2.OTC mRNA and protein expression were significantly lower in the liver cancer cells compared with those in PHH.3.Significantly lower OTC positive rate was observed in HCC tissues compared with non-tumor tissues as detected by immunohistochemistry;Both OTC mRNA and protein were downregulated on average in the HCC tissues compared with their adjacent non-tumor samples as determined by RT-qPCR and western blotting assay.4.Lower OTC levels were associated with a larger tumor size and higher tumor grade.5.OTC overexpression significantly decreased proliferation rate of SK-Hep-1 and Huh-7 cells;EdU staining also indicated that the exogenous overexpressed OTC dramatically inhibited DNA synthesis;Decreased numbers and sizes of SK-Hep-1 and Huh-7 cell colonies was observed in the OTC overexpression groups compared with the control groups.6.The loss of OTC significantly increased proliferation rate of SK-Hep-1 and Huh-7 cells.EdU staining indicated that OTC depletion promoted DNA synthesis;OTC knockdown increased numbers and sizes of SK-Hep-1 and Huh-7 cell colonies compared with the control groups.7.OTC overexpression promoted the apoptosis of SK-Hep-1 and Huh-7 cells.8.The content of NH₄⁺in liver cancer cells was not affected by OTC,and there was no difference between the concentration of NH₄⁺in OTC high expressed tumor tissues and OTC high expressed tumor tissues;9.OTC promoted the apoptosis of liver cancer cells by increasing the expression of Bax;Silencing of Bax antagonizes the apoptosis and growth inhibition of liver cancer cells induced by OTC overexpression.ConclusionOTC is downregulated in HCC where it is associated with poor prognosis in hepatocellular carcinoma.Moreover,OTC was functioned as a tumor suppressor in liver cancer by promoting apoptosis of liver cancer cells through increasing Bax expression and inducing Bax-dependent apoptotic pathway.Thus,OTC may serve as a new potential therapeutic target for liver cancer treatment.