The Effect Of Male Germ Cell-associated Kinase On Hepatocellular Carcinoma And Its Correlation With Prognostic

Background and Aims Hepatocellular carcinoma(HCC)is the sixth most common malignant tumor worldwide,which is characterized by rapid progression and high mortality rate.HCC is the third most frequent cause of cancer-related death,China is also a high incidence of primary liver cancer,which the mortality rate is located the second place in the mortality of malignant tumors in China.However,it is worth noting that in the epidemiological investigation has shown that the morbidity of males with HCC is significantly higher than that of females,the ratio of morbidity in males to that in females is 2-4:1.According to worldwide statistics,the liver cancer in males is the fifth most common malignant tumors,and the second frequent cause of cancer-related mortality.In females,the liver cancer is the seventh commonly diagnosed cancer and the sixth leading cause of cancer mortality.Recently,many researches had confirmed that androgen receptor(AR)is an important factor leading to gender differences in liver cancer.It is found that the high expression of AR can be detected in HCC cell lines and tissues,and it could regulate the expression and activity of molecules which are associated with cell proliferation,tumor growth or metastasis in HCC.In the early stage of HCC,AR exhibits marked oncogenic properties,but functions as a suppressor of metastasis in late-stage HCC.In addition,study indicated that the expression of AR can leads to abnormal expression of Wnt signaling pathway,as well as increase the expression of ?-catenin,thus promoting the development of HCC.Cycle-related kinase cell(CCRK)is a member of the cyclin dependent kinase family(CDK20),is expressed in a variety of human cancers.By analyzing the clinical samples of patients with HCC,the result showed that there was a certain correlation with CCRK and AR,and it was correlated with the poor prognosis of patients.CCRK is an important downstream regulation regions of AR,which can promote transcription by ligand-activated AR,and promote the occurrence of HCC by mediating by activation of ?-catenin pathway.Male germ cell associated kinase(MAK)is a protein kinase,which is closely relate to serine / threonine protein kinase.The earliest studies found that MAK was highly expressed in adult testis germ cells and the sperm formation in meiotic division stage,the high expression of MAK was also detected in prostate?retina?respiratory tract?choroid plexus?kidney and other tissues,but in liver tissues had no significant expression.In recent years,researches have confirmed that MAK is a direct transcriptional target and a coactivator protein of androgen receptor(AR),which can activate the transactivation of AR In addition,some scholars have proposed that there is a certain correlation between MAK and CCRK;MAK as a downstream modulator of CCRK signaling,when CCRK is overexpressed,it can promote the dual phosphorylation of MAK.In the malignant glioblastoma,CCRK can be recovered by activating the MAK,thereby inhibiting the development of tumor.In summary,MAK serves as the downstream modulator of AR and CCRK,which plays an important role in development of malignant tumor.Although,there are few studies about the role of MAK in the progress of HCC,we speculate that the expression of MAK may related to the development of HCC.Therefore,this is requires us to further explore.In this study,we analyzed the expression of MAK in HCC cells and tissues by Real-time RCR ? Western blot and immunohistochemistry.And we proceeded from the following two aspects to analyze the role of MAK in HCC:(1)To analyze the expression of MAK in HCC cell lines and tissues,and investigated the correlation between MAK expression and prognosis of patients with HCC.(2)To construction the HCC cells with the low and high expression of MAK,then to investigate the role on the development of HCC and the potential mechanism.Methods and Results Part1 To analyze the expression of MAK and its correlation with prognosis of HCC patients.In part1,the four different human HCC cells lines(LM3,SMMC-7721,Huh-7,Hep-G2)and the normal liver cells(LO2)were detected the m RNA level and the protein expression of MAK by the RT-PCR and Western-blot,respectively.The results showed that the m RNA level of MAK in HCC cells were higher than the normal liver cells(LO2),the m RNA expression of MAK in LM3 cell was the highest,while the Hep-G2 cell is lower.The Western-blot also confirmed that the expression of MAK was higher in HCC cells,and the protein expression of MAK in LM3 was higher than other HCC cells lines.Then,we performed immunohistochemical staining on the paraffin section of HCC and normal specimens to detect the expression of MAK in tumor and normal tissues.We found that MAK expression was positive in the cell nucleus,while MAK in normal liver tissues was negative.In further experiment,we collected the clinical data of 141 patients who underwent hepatectomy in our hospital.The expression of MAK in tumor tissues were detected by the RT-PCR.And combined with postoperative follow-up data to explore the survival of patients.Take Kaplan-Meier and Cox multivariate analysis of prognosis of patients.Results: in 141 cases of HCC,70 cases(49.6%)were low expression of MAK,71 cases(50.4%)were high expression of MAK.In positive expression of MAK,57 cases of male,the median survival time for 25 months;14 cases of females,the median survival time was17 months;univariate and multivariate Cox analysis showed that MAK expression is a prognostic independent risk,in MAK expression positive patients without tumor survival and overall survival deviation from MAK negative expression patients.Therefore,we hypothesized that the expression of MAK may have an impact on the development of HCC,while the specific mechanisms need to be further explored.Part 2 To explore the effects of male germ cell associated kinase on epithelial mesenchymal transition and migration and invasion in hepatocellular carcinoma In part2,we mainly discuss the role of MAK in the development of HCC and to explore possible mechanism.First,we use lentiviral transfection to construct stable low expression of HCC in Lm3 cell(sh RNA-MAK)and high expression of MAK in SMMC-7721 cells(High-MAK).Then,the scratch assay and Transwell assay were used to detect the effect of the different expression of MAK on the invasion of LM3 cells and SMMC-7721 cells.The experimental result indicates that the invasion and metastasis were increased on the over expression of MAK group.The invasion and metastasis ability of sh RNA-MAK group was decreased.And the cell immunofluorescence,Western blot and RT-PCR were performed to detect the protein expression change of in epithelial marker E-cadherin,stromal cells mark matter of N-cadherin,vimentin and EMT related transcription factor Snail in treated group and the control group,respectively.The experimental results show that: The high expression of MAK can promote EMT occurrence,and use the sh RNA to interference the expression of MAK could inhibit EMT on HCC cells.Conclusion In summary,the combined results of two parts,the following conclusions can be drawn:1 MAK were highly expressed in HCC cell lines and tumor tissues.The expression of MAK was correlated with the prognosis of the patients.In patients with high expression of MAK,the survival prognosis was worse than that of patients with low expression of MAK.2 The expression of MAK can enhance the migration ability of metastasis and invasion of hepatocellular carcinoma cells,And it can promote the occurrence of EMT in HCC cells.