Effects Of Gain-of-Function Mutation SHP-2Tyrosine Phosphatase On Malignant Biological Behaviors Of Liver Cancer Cells And Its Molecular Mechanism Study

BackgroundLiver cancer is one of the world’s three largest cause of cancer deaths, approximately80%of liver cancer patients is caused by the hepatitis virus, risk of hepatitis B viruscarriers suffering from liver cancer is5to15times higher than normal. About aquarter of hepatitis B virus chronic infection occur cirrhosis, each year there is3%to8%of patients with cirrhosis caused by the hepatitis B virus will deteriorate into livercancer. More and more researches reported that Gain-of-Function Mutation(GOF) andoverexpression of SHP-2play important roles in solid tumors occurrence andmetastasis. But, the relationship of SHP-2to malignant progression of liver cancerand the role of the SHP-2pathway in the biology of human liver cancer cells remainunknown.SHP-2is a protein tyrosine phosphatase, widely expressed in various tissues and cells,highly expressed in a variety of hematopoietic cells. SHP-2in the N-terminus containstwo SH2(Src homology2) domains, the C-terminus contains a PTP catalytic domainand two tyrosine residues (Tyr542and Tyr580), and a proline-rich motif (Pro). EachSH2domain has independent phosphotyrosine binding sites. Physiological state of theN-terminal SH2domains and PTP domain specific binding inhibition of the catalyticactivity of SHP-2, however, after stimulation of the various growth factors, interferonand insulin, etc. the tyrosine phosphorylation of the protein substrate and SHP-2SH2domain binding, or SHP-2C-side two tyrosine phosphorylation sites combined withits own SH2domain, PTP catalytic domain, is exposed, thereby SHP2proteindephosphorylation activity is restored, play a tyrosine phosphatase and the adapter protein function. In recent years, SHP-2activation mutation in blood disease studieshas yielded encouraging results, another SHP-2gene role in the occurrence of solidtumors growing cause for concern. SHP-2were also able to detect high levels ofexpression in the part of the human liver cancer cell lines; Suggesting that SHP-2mayplay a crucial role in the incidence and development process of liver cancer. However,little has been reported about the SHP-2associated with liver cancer.Methods(1)We used successfull constructed SHP-2eukaryotic expression vector transfectedinto HepG2cells,then screening and identification of HepG2cell lines that stablyexpressing SHP-2；(2)The detected the effect of SHP-2on cell proliferation, cloned forming ability,apoptosis, invasive and migration ability；(3)Analyze the possible mechanism of SHP-2effect on the malignant biologicalbehaviorn of Liver cancer..Results(1)The recombinant plasmid pcDNA3.1SHP-2-WT and pcDNA3.1SHP-2D61G/+eukaryotic expression vector were transfected into HepG2cells successfully, pickedmonoclonal cells, screening and identification stably expression of SHP-2of theHepG2monoclonal cell clones by Western blot.(2)The effects of SHP-2GOF and over-expression on HepG2cell proliferation andcycle: MTT results show the untransfected HepG2and vector groups have similarcell proliferation speed and GOF and over expression group have a supernal speed.;Plate clone and soft agar clone forming assay shows GOF and over-expression groupnot only the number of clone cell has more than control and vector groups, but alsomore than larger.; Cell cycle analysis showed GOF and over expression group of Sphase cell proliferation was significantly faster than the control group and vectorgroup cells. Above all, SHP-2transfection may increase cell growth ability.(3)The effects of SHP-2on liver cancer cell adhesion and apoptosis: Cell adhesion assay in vitro shows the rate of cell adhesion in GOF and over expression group ishigher than control and vector groups.; Annexin-V/EGFP Kit to detect apoptosisresults shows that the rate of apoptosis in GOF and over-expression group is alsolower than control and vector groups. Above all, SHP-2may accrescence capability ofcell adhesion and Anti-apoptosis.(4)The effects of SHP-2on the ability of migration and invasion in vitro of livercancer cell: Wound healing assay and Transwell chambers to detect cell migrationfound that cell migration rate was higher in SHP-2GOF and over-expression groupcells; Transwell chambers assay showed that invasive rate were more higher in SHP-2GOF and over-expression cells. Above all, SHP-2may increase the cell migration andinvasive ability.(5)CD133expression in transfected SHP-2mutant and wild-type eukaryoticexpression vector: The flow cytometry found SHP-2activating mutations andoverexpression cell of CD133expression was significantly higher than the emptyvector group and control group, description transfected SHP-2can promote theformation of cancer stem cells.(6)The mechanisms of SHP-2’s effects on HepG2cell biological behaviour:Western blot assay showed that the level of phospho-AKT/ERK and P38was higherin SHP-2GOF and overexpression cells. Showed that transfection of SHP-2promotesactivation of PI3K/AKT and RAS-RAF-MAPK pathway.Conclusion(1)Successfully transfected with pcDNA3.1SHP-2-WTand pcDNA3.1SHP-2D61G/+eukaryotic expression vector of SHP-2, identification and receive stable cell lines;(2)GOF and overexpression of SHP-2can promote HepG2cell adhesion,proliferation, colony formation, migration, invasive ability and the ability of the cellsto anti-apoptotic;(3)The effects of SHP-2on HepG2biological phenotype are relateed with theactivation of RAS-RAF-MAPK/PI3K-AKT signal pathway.