The Effects Of CRKⅡ On Proliferation,migration And Invasion Capacities Of Murine Hepatocarcinoma Hca-P Cells

Background: Hepatocarcinoma is known as one of the most leading causes of cancer-related deaths worldwide due to its high recurrence,high metastasis and poor prognosis.The effective treatments are still urgently required for hepatocarcinoma.Lymphatic metastasis(LNM)is a major mechanism of tumor metastasis.It is an early step of cancer malignancy.LNM is associated with a 50% reduction in a favorable prognosis of cancer patients.The murine hepatocarcinoma cells Hca-P is a subcloned cell line derived from hepatocellular carcinoma cells by Dai Lian Medical University.Hca-P is characterized with low lymphatic metastatic potential(LNM rate is ~25%)and metastasize only to lymph node without dissemination to other organs.Therefore,the Hca-P cell line is an ideal experimental subject for the study of LNM of hepatocellular carcinoma.CRK(CT10 regulator of kinase)was originally discovered as oncogene product of v-Crk in a CT10 chicken retrovirus.CRK family consists of three members: CRKI/Ⅱ and CRKL.c-CRKI and c-CRKⅡ are encoded by a same gene;c-Crk L is encoded by another gene.CRK lacks catalytic activity,and participate in a wide range of intracellular signaling pathways of signal transduction,via its SH2 and SH3 domain connected to different signal molecules which is no direct interaction between the role of signal molecules.CRKⅡ as an important member of CRK family,studies have found that is associated with a variety of human cancers,but the relationship between CRKⅡ and hepatocellular carcinoma but lack of research.Objective: 1.The pGPU6/GFP/Neo-sh RNA-CRKⅡinterference plasmid was constructed and stably transfected into Hca-P,and the monoclonal cell lines of CRK Ⅱ stably down-regulated were obtained;2.The effect of CRKⅡ knockdown on the proliferation,migration,invasion and lymph node adhesion capacities of Hca-P was evaluated.3.The eukaryotic expression vector of pc DNA3.1/V5-His B-CRKⅡwas constructed and transiently transfected to Hca-P cells,CRKⅡ was up-regulated in Hca-P cells;4.The effect of CRKⅡ overexpression on the proliferation,migration and invasion capacities of Hca-P cells was assessed.5.The effect of CRKⅡ level on the signaling pathway of p130cas/Dock180/Rac1 was evaluated.Methods: 1.Based on the CrkⅡSH3C of CRKⅡ,specific sh RNA and unrelated sequence(NC)were designed,p GPU6/GFP/Neo-sh RNA-CRKⅡ and-NC was constructed and stably transfected into Hca-P cells using Lipofectamine? 2000,the monoclonal cell lines of CRK Ⅱ stably down-regulated were obtained using limited dilution method against G418 screening,and CRKⅡ expression level was evaluated using WB.2.The primers were designed according to the CDs of CRKⅡ as well as the vector of pc DNA3.1-V5/His B.PCR was performed to amplify CDs of CRKⅡ,the purified products were cloned into p EASY-blunt simple cloning vector to obtain prokaryotic cloning plasmid p EASY-blunt simple-CRKⅡ,then products were identified by digestion and sequence.The prokaryotic cloning plasmid,pc DNA3.1-V5/His B empty vectors were performed double digestion,purified target fragments were ligated to obtain pc DNA3.1/V5-His B-CRKⅡ eukaryotic expression vector.3.pc DNA3.1/V5-His B-CRKⅡ and pc DNA3.1/V5-His B empty vectors were transfected into Hca-P cells by Lipofectamine? 2000,the expression level of CRKⅡwas detected by Western blot after 24 h transfection.4.The effect of CRKⅡ knock-down on the in vitro proliferation of Hca-P cells was accessed using trypan blue dye exclusion assay.The effect of CRKⅡ overexpression on the in vitro proliferation of Hca-P cells was accessed using CCK-8 assay.5.The effect of CRKⅡ knock-down /overexpression on the in vitro colony formation ability of Hca-P cells was accessed using soft agar colony formation assay.6.The effect of CRK Ⅱ knock-down /overexpression on the in vitro migriation and invasion ability of Hca-P cells was accessed using transwell chamber assay.7.The effect of CRKⅡ knock-down on the in vitro adhesive capability of Hca-P cells was accessed using lymph node adherence assay.8.The effect of CRK Ⅱ level on the signaling pathway of p130 cas /CRK/Dock180/Rac1 was evaluated by Western blot.Results: 1.Monoclonal Hca-P cell lines with stable CRKⅡ knockdown were obtained and named as sh CRKⅡ-894-Hca-P and sh CRKⅡ-852-Hca-P,CRKII expression levels in which were decreased by 52% and 42%.2.Enzymatic digestion and sequencing detection indicated that the p EASY-blunt simple-CRKⅡcloning plasmid and pc DNA3.1/V5-His B-CRKⅡeukaryotic expression vectors were successfully constructed.3.Compared with sh CRKⅡ-NC-Hca-P,the proliferation capacities of sh CRKⅡ-894-Hca-P at 48,72,96 h and sh CRKⅡ-852-Hca-P at 72,96 h were dramatically decreased;The colony forming,migration,invasion and lymph node adhesion capacities of sh CRKⅡ-894-Hca-P decreased by 76.1%,71.2%,79.9%,71.1%;The colony forming,migration,invasion and lymph node adhesion capacities of sh CRKⅡ-852-Hca-P decreased by 74.6%、71.4%、80.2%、55.2%.4.Compared with pc DNA3.1/V5-His B-Hca-P cells,CRKⅡ expression levels in pc DNA3.1/V5-His B-CRK Ⅱ-Hca-P cells were increased by ~1.85-fold.5.Compared with pc DNA3.1/V5-His B-Hca-P cells,the proliferation capacities of pc DNA3.1/V5-His BCRKⅡ-Hca-P were dramatically increased at 72 and 96h;The colony forming,migration and invasion capacities of pc DNA3.1/V5-His B-CRKⅡ-Hca-P were increased by 1.71,1.79,1.56 fold.6.The effect of CRKⅡ level on the signaling pathway of p130cas/CRK/Dock180/Rac1 was no statistically difference.Conclusion: 1.Monoclonal sh CRKⅡ-894-Hca-P and sh CRKⅡ-852-Hca-P with stable CRKⅡ knockdown were obtained;2.CRKⅡ down-regulation inhibits in vitro proliferation,migration,invasion and lymph node adhesion capacities of Hca-P cells 3.The pc DNA3.1/V5-His B-CRKⅡeukaryotic expression vectors were successfully constructed and CRKⅡ expression levels in pc DNA3.1/V5-His B-CRKⅡ-Hca-P cells were increased by ~1.85-fold.4.CRKⅡ overexpression enhances in vitro proliferation,migration and invasion capacities of murine hepatocarcinoma Hca-P cells.5.The effect of CRKⅡ level on the signaling pathway of p130cas/CRK/ Dock180/Rac1 was no statistically difference.