NDRG1Subcellular Expression In Hepatocellular Carcinoma And Preliminary Study On Its Function

Background and objective:Liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death. In women, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death. Hepatocellular carcinoma (HCC) is the most common form of liver cancer, which accounts for70%-85%. HCC is a most devastating cancer with insidious early symptom, low early diagnosis and poor prognosis and the incidence of cancer metastasis and recurrence after tumors resection is high. NDRG1has been reported to be a multifunctional protein associated with carcinogenesis and tumor progression. Interestingly, NDRG1was given two different roles in HCC development. Our research has discussed the characteristics of NDRG1expression and subcellular localization in HCC and clarified the influence of NDRGl expression on the cancer cell biology function, and finally we verified the changes of some of the metastasis-related genes after NDRG1gene was knocked out using PCR array test.Methods:1. The expression of NDRG1protein was investigated in75cases of HCC tissue arrays by immunohistochemical assay. We focused on the analysis of the correlation between NDRG1subcellular localization and the clinic pathological features of HCC, trying to explore the expression profile of NDRG1protein in the progression of HCC. 2. NDRG1gene expression was knocked out by transfecting NDRG1-shRNA. Western blot and qPCR were employed to validate the effect of downregulation of NDRG1in transfected BEL7402cells.3. Cell migration assay was measured using the Boyden-transwell assay and wound-healing assay. Cell invasion assay was measured using the Boyden-transwell assay.4. Cell proliferation was assessed by cell counting kit-8(CCK8) assay according to the protocol.5. Flow cytometry was used to evaluate the level of cancer cell apoptosis.6. PCR array test was used to verify the changes of some of the metastasis-related genes after NDRG1gene was knocked out.Results:1. No NDRG1expression was observed in normal liver tissue and high expression had been observed in human HCC cells. NDRG1subcellular localization had a close correlation with the clinic pathological features of HCC. NDRG1was primarily expressed in the cytoplasm and there was a negative correlation between NDRG1cytoplasm staining and TNM stage (P<0.01). In other words, higher expression was observed in the tumor with the smaller size. NDRG1expression in the membrane was significantly higher in the primary liver neoplasms than in the adjacent tissues (P<0.01).2. NDRG1expression was significantly downregulated in BEL7402cell after NDRG1gene knockout, which had no influence on the cell shape.3. Boyden-transwell assay and wound-healing assay showed that downregulation of NDRG1significantly enhanced cell migration and invasion (P<0.01).4. CCK8results showed that downregulation of NDRG1resulted in significant increase in cell proliferation (P<0.01).5. Flow cytometry showed that downregulation of NDRG1significantly enhanced cell apoptosis (P<0.01). 6. PCR array test showed that downregulation of NDRG1resulted in increased expression of CTSL1, IL1B, ITGB3, MMP10and SERPINE1, and decreased expression of MMP13and TNFSF10.Conclusion:1. Our research clarified the characteristics of NDRG1expression in HCC and firstly discussed the correlation between NDRG1subcellular localization and the clinic pathological features of HCC. NDRG1expression was significantly higher in HCC than in the aberrant normal liver tissue. The analysis of NDRG1subcellular localization showed that there was a negative correlation between NDRG1cytoplasm expression and TNM stage and NDRG1membrane expression was significantly higher in HCC than in the adjacent normal tissues.2. Our research confirmed that the reduction of NDRG1showed an increase in cell proliferation, cell apoptosis, cell migration and cell invasion.3. PCR array test showed that NDRG1might inhibit HCC metastasis by modulating the expression of CTSL1、IL1B、ITGB3、MMP10、SERPINE1、MMP13、TNFSF10.