CRKL Overexpression Suppresses In Vitro Proliferation, Invasion And Migration Abilities Of Murine Hepatocarcinoma Hca-P Cells

Background: Metastasis is the major biological characteristic of malignant neoplasms, which involves the degradation of extracellular matrix and cell migration, is the major task and difficulty of tumor prevention. The lymph node is always the initial organ of tumor metastasis, the early lymphatic metastasis is a main cause for the high mortality and poor prognosis of malignant tumor patients, which is a complex process involving multiple genes and procedure, including the biology process of tumor cells proliferation, migration and invasion, yet the metastatic mechanism is poorly understood.The mouse hepatocarcinoma cells Hca-P and Hca-F are a pair of synogenetic hepatocellular carcinoma ascites cell lines, which share the same genetic background and different potential of lymph node metastasis(LNM). The Hca-P cells with low metastatic potential(25%) and Hca-F cells with high metastatic potential(75%) are the ideal experimental subjects for the study of lymph node metastasis.The signal adaptor protein CRKL is ubiquitously expressed and conserved across eukaryotic organisms as a member of CRK family, which involves in various cancer-related signaling pathways. CRKL is closely related to clinic pathologic features and prognosis of several cancers, and is becoming hotspot molecules for oncological research. Our previous Western blot results found that the protein expression level of CRKL in Hca-P is 3.05-fold higher than that of CRKL in Hca-F cells(P<0.05), and down-regulation of CRKL by RNA interfering could significantly enhance lymphatic metastatic potential of Hca-P, implying the expression level of CRKL is closely related to tumor LNM and CRKL may be the potential target molecule for inhibiting LNM of hepatocellular carcinoma.Objective: 1. To construct pc DNA3.1-V5/His B-CRKL and p EGFP-N1-CRKL eukaryotic expression plasmid. 2. To localize CRKL-GFP fusion protein by laser scanning confocal after transiently transfected p EGFP-N1-CRKL plasmid to mouse hepatocarcinoma Hca-P cells. 3. To detect overexpression of CRKL by Western blot after transiently transfected pc DNA3.1-V5/His B-CRKL plasmid to mouse hepatocarcinoma Hca-P cells. 4. To investigate the effect of CRKL overexpression on proliferation, migration, invasion and colony formation abilities of Hca-P cells after transiently transfected pc DNA3.1-V5/His B-CRKL plasmid, which provide a solid base for further studying the lymphatic metastatic mechanism.Methods: 1. The primer pair-1 and primer pair-2 were designed according to the CDS of CRKL as well as the vector of pc DNA3.1-V5/His B and p EGFP-N1. 2. RT-PCR was performed to amplify CDS of mouse CRKL, the purified products were cloned into p EASY-blunt simple cloning vector to obtain prokaryotic cloning plasmid p EASY-blunt simple-CRKL-1 and p EASY-blunt simple-CRKL-2, then products were identified by digestion and sequence. The prokaryotic cloning plasmid, pc DNA3.1-V5/His B and p EGFP-N1 empty vectors were performed double digestion, purified target fragments were ligated to obtain pc DNA3.1/V5-His B-CRKL and p EGFP-N1-CRKL eukaryotic expression vector. 3. p EGFP-N1-CRKL and p EGFP-N1 empty vectors were transfected into Hca-P cells by Lipofectamine® 2000, the transfection efficiency and CRKL-GFP fusion protein were investigate by inverted fluorescence microscope and Laser Scanning Confocal Microscope(LCSM), respectively. 4. pc DNA3.1/V5-His B-CRKL and pc DNA3.1/V5-His B empty vectors were transfected into Hca-P cells by Lipofectamine® 2000, the expression level of CRKL was detected by Western blot after 24 h transfection. 5. CCK-8 method measured the influence of CRKL up-regulation on Hca-P cell proliferation ability, soft agar colony formation method detect the influence of CRKL up-regulation on Hca-Pcell colony formation ability, Transwell chamber assay was used to detect the influence of CRKL up-regulation on Hca-P cell migriation and invasion ability.Results: 1. The enzyme digestion and sequencing results showed that the cloning plasmid p EASY-CRKL-1 and p EASY-CRKL-2, the eukaryotic expression plasmid pc DNA3.1/V5-His B-CRKL and p EGFP-N1-CRKL were successfully constructed, the CDS of CRKL was exactly correct. 2. The laser scanning confocal result showed that CRKL protein was mainly expressed in cytoplasm of Hca-P cells. 3. Western blot results showed that the total expression level of CRKL and CRKL-His in pc DNA3.1/V5-His B-CRKL-Hca-P cells was 1.39-fold and 1.69-fold of those in Hca-P and pc DNA3.1/V5-His B-Hca-P cells, respectively, and the CRKL- His Tag was detected in pc DNA3.1/V5-His B-CRKL-Hca-P cells only. 4. CCK-8 result indicated that the proliferation rate of pc DNA3.1/V5-His B-CRKL-Hca-P cells were significantly inhibited than controls. 5. Soft agar colony formation result indicated that the colony formation abilities of pc DNA3.1/V5-His B-CRKL-Hca-P cells were significantly inhibited than controls. 6. Transwell assays indicated that the Hca-P cell migration and invasion capacities were apparently reduced following CRKL overexpression.Conclusion: 1. The cloning plasmid p EASY-CRKL-1 and p EASY-CRKL-2 were constructed successfully, eukaryotic expression plasmid pc DNA3.1/V5-His B-CRKL and p EGFP-N1-CRKL were constructed successfully. 2. The CRKL protein was mainly expressed in cytoplasm of Hca-P cells. 3. CRKL up-regulation resulted in significant inhibition in proliferation ability of Hca-P cells. 4. CRKL up-regulation resulted in significant inhibition in colony formation ability of Hca-P cells. 5. CRKL up-regulation resulted in significant inhibition in migration and invasion capacities of Hca-P cells.