Role And Mechanism Of SHP2 Induced Proliferation In Human Breast Cancer Cells

Objective SHP2(SH2 domain-containing protein-tyrosine phophatase-2)is a non-receptor tyrosine phosphatase encoded by the Ptpn11 gene and is widely present in eukaryotic cells.SHP2 has been shown to be related in breast cancer,leukemia,lung cancer,liver cancer,gastric cancer,and oral cancer.It also plays a critical role in a variety of tumor-related life processes,such as cell invasion,migration,apoptosis,DNA damage,cell proliferation,cell cycle and drug resistance.The abnormal proliferation ability of tumor cells is one of the main reasons for the difficulty of tumor curing and high mortality.It is confirmed in many tumors that the proliferation ability of cancer cells is significantly stronger than normal cells.Meanwhile,SHP2 is the first oncogene which has phosphatase activity.The effect of phosphatase activity on tumors is also one of the focuses of the academic community.The purpose of this study was to determine the effect of SHP2 and its phosphatase activity on the proliferation of breast cancer cells in vitro and to explore the molecular mechanisms,thus providing some new clues for in-depth mechanism research and breast cancer treatment.Methods 1.SHP2 was knockdown in MDA-MB-231 cell lines by si RNA and sh RNA,and was knockdown in BT549 cell lines by si RNA.The effect of SHP2 knockdown was determined by Western blot.2.The effect of SHP2 knockdown on breast cancer proliferation was determined by CCK8 assay,colony formation assay and flow cytometry.3.The effect of SHP2 knockdown on the expression of cyclin B1 and Cyclin D1 proteins was determined by Western blot.4.The effect of SHP2 knockdown on the m RNA levels of cyclin B1 and Cyclin D1 was determined by real-time PCR.5.Western blot was used to determine the half-life and degradation mechanism of Cyclin B1 and Cyclin D1,and explore the mechanism through which SHP2 regulated the expression of Cyclin B1 and Cyclin D1.6.A series of mutant recombinant plasmid of SHP2 was construted by nucleotide point mutations.The recombinant plasmid was imported by lentivirus infection in the SHP2 stable knockdown cell lines,to construct a negative control group(sh SHP2-p CDH),a wild-type SHP2 expressing group(sh SHP2-SHP2),and phosphatase activity of SHP2 activating mutation of N308 D and E76K(sh SHP2-N308 D,sh SHP2-E76K)and phosphatase activity of SHP2 inactivating mutation of(sh SHP2-T468M).7.The effect of SHP2 phosphatase activity on the proliferation in breast cancer cells was determined by CCK8 assay,colony formation assay and flow cytometry 8.The effect of SHP2 phosphatase activity on Cyclin B1 and Cyclin D1 expression was determined by Western blot.Results 1.Downregulation of SHP2 in breast cancer cells decreased cell proliferation and colony formation capacity.2.Downregulation of SHP2 in breast cancer cells resulted in a significant increase in the proportion of cells in the G2/M phase.3.Knockdown of SHP2 resulted in decreased expression of Cyclin B1 and Cyclin D1 in breast cancer cells.4.Downregulation of SHP2 had no significant effect on the m RNA levels of cyclin B1,Cyclin D1 in breast cancer cells.5.Knockdown of SHP2 decreased the expression of Cyclin B1,Cyclin D1 probably through proteasome-ubiquitination mechanism in breast cancer cells.6.phosphatase activity of SHP2 activating mutation enhanced the proliferation of breast cancer cells,while phosphatase activity of SHP2 inactivating mutation reduced the proliferation of breast cancer cells.7.phosphatase activity of SHP2 activating mutation upregulated the expression of Cyclin B1 and Cyclin D1,while phosphatase activity of SHP2 inactivating mutation odownregulated the expression of Cyclin B1 and Cyclin D1.Conclusion: In this study,we demonstrated that SHP2 enhanced the proliferation capability of breast cancer cells.Knockdown of SHP2 decreased the expression of Cyclin B1 and Cyclin D1 probably through ubiquitin-proteasome system.Phosphatase active of SHP2 increased the proliferation in breast cancer cells by increasing the expression of Cyclin B1 and Cyclin D1,whlie phosphatase inactive of SHP2 reduced the proliferation of breast cancer cells by decreasing the expression of Cyclin B1 and Cyclin D1.