Regulation And Molecular Mechanism Of SIK2 On Malignant Biological Behavior Of Triple Negative Breast Cancer Cells

Objective:1.To analyze and verify the expression of salt-inducible kinase 2(SIK2)in triple-negative breast cancer(TNBC).2.To explore the effect of SIK2 on the invasion and migration ability of TNBC cell lines.3.To explore the effect of SIK2 on the epithelial-mesenchymal transition of TNBC cell lines.4.To explore the molecular mechanism of SIK2 affecting the migration,invasion and EMT of TNBC cell lines.Method:1.The Cancer Genome Atlas(TCGA)and Clinical Proteomics Tumor Analysis Consortium(CPTAC)databases were analyzed using the Ualcan online analysis tool to explore the expression of SIK2 in TNBC.2.The collected 8 pairs of TNBC tissues and their corresponding paracancerous tissue specimens were used to verify the expression of SIK2 in TNBC cancer tissues by quantitative real time PCR(qRT-PCR)technology.3.A recombinant SIK2 overexpression plasmid was constructed to initially verify the function of SIK2 in TNBC cell lines.4.Two SIK2 stable overexpression TNBC cell lines,MDA-MB-231 and HCC-1937,were constructed by lentivirus infection SIK2 overexpression;and the overexpression was detected by Western blotting.5.Analysis of the regulatory effect of SIK2 on TGF-β/Smad signaling pathway by Western blotResult:1.Ualcan website analysis of TCGA and CPTAC database results showed that the SIK2 mRNA and protein levels in TNBC patients were significantly lower than those in their normal controls.The collected TNBC tissues and their corresponding paracancerous tissue samples were used for qRT-PCR verification,and the results were also confirmed that the expression of SIK2 mRNA in TNBC tissues was significantly lower than that of its corresponding paracancerous tissues,which was consistent with the analysis results from the Ualcan website.Kaplan-Meier Plotter survival analysis also confirmed that triple-negative breast cancer patients with low expression of SIK2 had worse prognosis.2.After bacteria PCR,double enzyme digestion,agarose electrophoresis and sequencing verification by Shanghai Sangon,the results showed that the constructed SIK2 overexpression recombinant vector was completely correct.The constructed recombinant plasmid was used to preliminarily verify that the cell function was as expected.3.The established stable SIK2 overexpression MDA-MB-231 and HCC-1937,two TNBC cell lines,were verified by Western blotting,and the results showed that SIK2 was significantly overexpressed,confirming that the negative control and SIK2 stable overexpression of TNBC cell lines MDA-MB-231 and HCC-1937 had been successfully constructed.4.Wound healing test and Transwell assay showed that the migration and invasion abilities of TNBC cells overexpressing SIK2 were significantly reduced.5.The influence of SIK2 overexpression on the expression levels of E-cadherin,Vimentin,snail,and slug proteins was analyzed by Western blotting.The results showed that the expression level of E-cadherin in SIK2 overexpressing group was significantly higher than that in their controls.The expression levels of Vimentin,snail,and slug were significantly lower than that in their controls.It is suggested that the overexpression of SIK2 inhibits the EMT process,and then inhibits the migration and invasion of TNBC cells.6.The protein expression levels of p-Smad2,p-Smad3,TGFβRI,and Smad2/3 in TNBC cell lines after overexpression of SIK2 were detected by Western blot,and the results showed that the protein expression levels of p-Smad2,p-Smad3,and TGFβRI in the SIK2 overexpression group significantly lower than that in its control group,but Smad2/3 protein expression was not significantly different between SIK2 overexpression and its control groups.Conclusion:1.The expression level of SIK2 is higher in triple-negative breast cancer tissues,and in patients with triple-negative breast cancer,the low expression of SIK2 is related to the prognosis of patients.2.SIK2 overexpression inhibits the migration and invasion of triple-negative breast cancer cell lines.3.SIK2 overexpression inhibits the epithelial-mesenchymal transition of triple-negative breast cancer cell lines.4.The overexpression of SIK2 may inhibit the epithelial-mesenchymal transition process by inhibiting the TGF-β/Smad signaling pathway,thus inhibiting the.migration and invasion of triple-negative breast cancer cell lines.