Expression Of MARK2 In Hepatocellular Carcinoma And Its Effect On Migration Of HepG2 Cells

Background:Primary liver cancer is one of the common malignant tumors in the digestive system,the second cause of malignant tumor death in China and second to lung cancer.Hepatocellular carcinoma(HCC)is the most common,accounting for 90% of primary liver cancer.The infection of Hepatitis B and C,alcohol abuse,long-term exposure to aflatoxin,drinking water pollution,metabolic disorders and age are risk factors for the development and progression of HCC.At present,surgical resection and liver transplantation are the most effective treatments for HCC.Many treatments include radiofrequency ablation,arterial embolization chemotherapy(TACE),radiotherapy,and sorafenib-targeted drug therapy,all both improve patient prognosis and 5-year survival rate.Although these treatments have made some progress in clinical efficacy,the results are still unsatisfactory.Therefore,actively seeking new therapeutic approaches and exploring new targets has become the key to treating HCC and improving patient outcomes.Microtubule affinity regulating kinase 2(MARK2)is a serine/threonine protein kinase involved in the phosphorylation of microtubule-associated proteins,such as the Tau protein,a phosphatase involved in cell cycle regulation(CDC25),as well as class IIa histone deacetylases(such as HDAC7).In mammals,the MARK family consists of four members(MARK1-MARK4).Matenia et al.found that MARK2 has multiple functions,including roles in neural differentiation,neurodegeneration,cell polarity,intracellular transport,and cell migration,some of which are deregulated in cancer cells.Hubaux et al.found that in lung cancer cell lines,MARK2 is highly frequently disrupted at the DNA and RNA levels,and these changes result in cell lines gaining carcinogenic properties such as increased viability and anchor-independent growth.However,studies have shown that using the inducible lentiviral expression system to express MARK2 in LKB1-deficient cervical cancer HeLa cells,the epithelial-mesenchymal transition(EMT)can be reversed by the AMPK pathway to inhibit HeLa cell growth.The expression of MARK2 in liver cancer and its clinical significance have not been reported.Based on the above background,this study made a preliminary study on the expression of MARK2 in liver cancer,aiming to provide new targets and ideas for the clinical treatment of HCC.Objective:To investigate the difference of expression level of MARK2 in hepatocarcinoma tissues and adjacent tissues;to study the effect of MARK2 overexpression on the migration ability of HepG2 cells.Method:1.Tissue chip(HLivH030PG03-M-250)was purchased from Shanghai Core Biotechnology Co.,Ltd.,containing 65 cases of liver cancer tissue and its corresponding paracancerous tissue specimens.Immunohistochemistry was used to detect the expression of MARK2 in hepatocarcinoma tissues and adjacent tissues.2.Using UALCAN(http://ualcan.path.uab.edu/)to analyze the difference of MARK2 expression in liver cancer tissues and normal liver tissues in cancer gene map(TCGA)database,and its relationship with clinicopathological features of liver cancer patients The online survival analysis software Kaplan-Meier plotter was used to analyze the effect of MARK2 expression on survival and prognosis of patients with liver cancer.3.MARK2 overexpression plasmid(MARK2 transfection group)and empty vector negative control plasmid(Negative control,NC group)were stably transfected into hepatoma HepG2 cells,and the MARK2 eukaryotic expression vector stably transfected hepatoma cell line was screened and cultured.The negative control group cell line up-regulated the expression of MARK2 in HepG2 cells,and the expression of MARK2 in the experimental group and the negative control group by Western blot.4.Scratch test and Transwell assay were used to detect the effect of MARK2 overexpression on the migration ability of HepG2 cells.Result:1.UALCAN analysis of TCGA database results: Compared with normal liver tissue,the expression of MARK2 in liver cancer tissues was significantly increased(P<0.001);the high expression of MARK2 in liver cancer tissues was significantly correlated with TNM stage and pathological grade(P<0.001).Immunohistochemistry results: MARK2 expression in liver cancer tissues was significantly higher than that in adjacent tissues.2.Kaplan-Meier plotter survival analysis results: the 5-year survival rate of patients with high MARK2 expression was significantly lower than that of MARK2 low expression group(P<0.01).3.Western blot analysis results: After transfection of MARK2 overexpression plasmid,the expression level of MARK2 in the experimental group was significantly higher than that in the NC group.The results showed that the expression level of MARK2 was significantly increased in HepG2 cells transfected with MARK2 overexpression plasmid.4.Transwell results: Compared with the NC group,the number of HepG2 cells that migrated to the lower chamber through the filter MARK2 overexpression plasmid group was significantly increased,and the difference was statistically significant(226±10.017 vs 98±10.536,t = 15.290,P<0.05).It is indicated that MARK2 overexpression can promote the migration of HepG2 cells.5.Scratch test results: After scratching for 48 h,the scratch healing rate of HepG2 cells transfected with MARK2 was 53.8%,while the scratch healing rate of negative control group was 32.3%.The difference was statistically significant(P<0.05),indicating that the migration ability of HepG2 cells is enhanced after overexpression of MARK2.Conclusion:1.The expression of MARK2 in hepatocarcinoma tissues was significantly higher than that in adjacent tissues.The expression of MARK2 in liver cancer tissues was significantly correlated with TNM staging and pathological grade.The high survival time of MARK2 patients with high expression of liver cancer was short and the prognosis was poor.2.MARK2 can promote the migration of liver cancer cells,and may play a role in promoting cancer in liver cancer.