Study Of Knock-down Of Expression Of JNK Gene By ShRNA In Mouse Hepatocellular Carcinoma Cell Lines And Its Correlation With Lymphatic Metastasis

Background: The most important difference between malignant and benign tumors is metastasis. Metastasis is the fundamental cause of high mortality and poor prognosis of the patients who suffer from malignant tumors. The mechanism is not clear until now and it is an important project for discussion. To epithelial malignant tumors, the early metastatic mode is lymphatic metastasis which is an important factor which affects prognosis of the patients. Therefore, it is very important to investigate the molecular mechanism of lymphatic metastasis and find new approaches of intervention . Primary hepatocellular carcinoma is one of the most common malignant tumor with high incidence of a disease in our country. Although a mass of researches have been done, its mechanism is not clear.Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines which has the different rates of lymphatic metastasis . Hca-F is the cell lines whose metastatic rate is more than 70% and Hca-P less than 30%. They are the ideal models for the researches of mechanism of lymphatic metastasis .Our experimental group has engaged in the research work for the mechanism of lymphatic metastasis. We have screened out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines by using suppressive subtractive hybridization and gene chip assays respectively and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. The expressing level of JNK was much higher in Hca-F than that in Hca-P cell lines in both gene and protein levels which showed that JNK maybe play an important role in lymphatic metastasis of mouse hepatocarcinoma. In the tissue of human being, there is JNK protein of the same origin with mouse.So it is very important to study the relationship between JNK and lymphatic metastasis of mouse hepatocarcinoma.c-Jun N-terminal kinase ( JNK )is the kinase which can phosphates c-Jun in a specific way. INK is an important member of mitogen-activated protein kinase pathway( MAPK ) and is located in focal adhesion which is the important cellular signal transduction. MAPK is a Ser/Thr kinase which is widely present in mammal cells. It has a close relation with tumors. JNK is necessary to growth, morphogenesis and differentiation of cells. JNK signal transduction participates in many physiological process. The recent studies showed that JNK signal transduction also has a close relation with certain diseases and tumors. And it is regarded as a potential target for treatment in clinical practice.In this study, JNK was being as the subject to discuss the role it played in process of lymphatic metastasis of Hca-F celllines. There was not such reports home and abroad by retrieval.Objective: 1.To observe the expression levels of p-JNK in mouse hepatocarcinoma ascites cell lines Hca-F and Hca-P for further study of its function and mechanism in lymphatic metastasis of hepatocarcinoma. 2.To build expression vector of pSilencer-shRNA and obtain cellline Hca-F of markedly decreased expression of JNK by stable transfection. 3. To study the influence on proliferation, migration and invasion of mouse hepatocellular carcinoma Hca-F celllines after inhibition of JNK expression by shRNA and discuss the correlation between expressing level of JNK and lymphatic metastasis of mouse hepatocarcinoma.Methods: 1. The expression of p-JNK protein was detected in Hca-F and Hca-P cells by Western Blot. 2. Three shRNAs (shRNA-1,shRNA-2,shRNA-3) and unrelated sequence were designed according to the gene sequence of JNK ( NM₀16700.3 ). pSilencer? 3.1-H1 neo vector was obtained and BamH I and EcoR I enzymes were used to digest.The annealing of shRNA compositive sequence was performed and T4 DNA ligase was used to connect the pSilencer vector with three shRNAs and unrelated sequence after annealing. The connecting products was named pSilencer-shRNA-1,pSilencer-shRNA-2, pSilencer-shRNA-3 and pSilencer- shRNA-unrelated sequence. The expressing vector was obtained by extraction of vectors. The sequence of expressing vectors was identified by sequencing and the result was compared to that in gene bank. The three pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively and the most effective pSilencer-shRNA vector was selected according to the results of RT-PCR and Western blotting. The unrelated shRNA transfected Hca-F celllines and normal Hca-F celllines were negative control group and positive control group respectively. 3. The cell viability was evaluated by CCK-8. The cell migration and invasion capability was evaluated by transwell assays.Results: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P (P<0.01). 2. The expression vector of pSilencer-shRNA was built and transfected to Hca-F cells successfully. The sequence of three shRNAs in expressing vectors was identified by sequencing and the result was identical to that in gene bank.The most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. The expressions of mRNA and protein of JNK in Hca-F cells after transfection of shRNA-2 were markedly decreased compared with the other groups ( P<0.01, P<0.05 ). pSilencer-shRNA-2 was chosen as the cellline for further study. 3. The result of CCK-8 showed that JNK could promote cell proliferation. Ttranswell assays showed that migration capability was decreased after knock-down of JNK expression in Hca-F cell lines (17.89±1.65 vs 35.10±1.27, 31.83±1.69, P<0.05 ). Invasion capability was decreased after knock-down of JNK expression in Hca-F cell lines(10.03±2.81 vs19.73±1.60 ,19.23±1.24,P<0.05 ).Conclusions: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P . 2. The Hca-F cell lines of markedly knock-down of JNK expression by stable transfection was obtained and the most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. And pSilencer-shRNA-2 was chosen as the cellline for further study which provided a solid foundation for further studies of relationship between JNK and lymphatic metastasis of hepatocellular carcinoma. 3. Being as the different protein between Hca-F and Hca-P cell lines, the knock-down of JNK expression could inhibit proliferation, decrease migration and invasion capability of mouse hepatocellular carcinoma celllines. JNK maybe play an important role in lymphatic metastasis of hepatocellular carcinoma.The possible mechanism is that JNK located in focal adhesion can regulate paxillin, c-Jun, AP-1 and the genes in connection with tumor metastasis regulated by AP-1.