| Objective: To investigate the expression level of Wnt-1 induced secretion protein 2(WISP2)in cervical cancer tissues and cells and its relationship with HPV infection,and to explore the effect of WISP2 gene on the proliferation and migration of cervical cancer cells and its effect on Wnt/β-Catenin signaling pathway,analysis of β-Catenin gene inhibitor XAV-939 on the proliferation and migration of cervical cancer cells.Methods:(1)Constructing organizational chip analysis of WISP2 proteins in normal cervical tissues and cervical cancer tissues.(2)Western blot and qRT-PCR detection of protein and mRNA expression levels of WISP2 gene in cervical cells,design and construct and constructed two plasmids that expressed and interfere with WISP2 genes were transfected with cervical cancer SiHa,HeLa cells.The overexpression experiment group and the control group were overexpressed by WISP2 groups and general granules(NC groups);interfering WISP2 gene experiments were transfected with sh-WISP2-1120,sh-WISP2-1047 cell group,non-interference transfection cells(NC group)was a control group.At the same time,Western blot was used to analyze the expression of EMT molecular proteins related to cervical cancer metastasis.(3)The second generation hybrid capture system-2(HC-2)is used to detect HPV infection in cervical tissues.(4)CCK-8 experiment and plate cloning experiment to detect the changes in the proliferation ability of cervical cancer cell lines SiHa and HeLa after WISP2 gene overexpression,inhibition and XAV-939 inhibitor stimulation.(5)Transwell experiment and scratch experiment analyze the changes in the migration ability of cervical cancer cell lines SiHa and HeLa cells after WISP2 gene overexpression,inhibition and XAV-939 inhibitor stimulation.(6)Laser confocal microscope experiment observes the co-localization of WISP2 protein and β-Catenin protein after transfection of WISP2 gene overexpression.Results: The organized chip experiment found that in normal cervical tissue,WISP2 is mainly expressed in cytoplasmic and expression in the cellular nucleus.WISP2 is low in cytoplasm in CIN3 and cervical cancer tissues,high expression in cell nuclear nucleus(P<0.05);There is no statistical difference between WISP2 gene expression and the degree of age and differentiation of patients(P>0.05);Western blot and qRT-PCR method test the results of protein and mRNA expression in cervical cancer cells showed that the expression of WISP2 total protein in cervical cancer SiHa,HeLa and C33 A cell line significantly lower than HaCaT cells(a kind of immortalization stomatal formation cells).The amount of mRNA in the WISP2 gene in SiHa,HeLa cell line was significantly higher than that of HaCaT cells,and the expression level of the WISP2 gene in the C33 A cell line was significantly lower than that of the HaCaT cell line.Compared to HaCaT cells,WISP2 proteins were significantly reduced in SiHa,HeLa and C33 A cytoplasmic,and increased in the nucleus(P<0.05);Sample HC-2 detection results collected in the previous period,combined with immunohistochemistry results,the analysis found that WISP2 gene expression was associated with HPV infection(P<0.05);It can be seen from the laser confocal microscope that the WISP2 gene was expressed in cervical cancer SiHa and HeLa,the WISP2 gene was expressed,WISP2 andβ-Catenin were co-located,together into the nucleus And the amount of WISP2 protein was significantly increased during transfection;CCK-8 experiment,Tablet clone experiment and Western blot experiment showed that after expressing WISP2 gene,WISP2 increased more significant in the nucleus,and showed the role of β-Catenin signaling pathway,proliferating related protein c-Myc and Cyclin D1 have significant increased significantly(P<0.05),resulting in proliferative ability of cervical cancer SiHa and HeLa cells;After the β-Catenin inhibitor XAV-939 stimulated cervical cancer cells expressing WISP2 gene,the amount of β-Catenin protein expression decreased,and the expression of WISP2 entered the nucleus was significantly reduced,and the expression of c-Myc and Cyclin D1 protein was significantly reduced(P<0.05),ultimately reversed the proliferation capacity of cervical cancer cells caused by expressing WISP2;After knocking the WISP2 gene,WISP2 in the cytoplasm is reduced to the PLC-γ signaling pathway,resulting in an increase in β-Catenin expression to increase the expression of β-Catenin in the nucleus,which promoting the increase in proliferative protein C-Myc and Cyclin D1 increased(P<0.05),the proliferation capabilities of cervical cancer SiHa and HeLa cells were significantly increased;Transwell Experiments and Scratch Experiments show that after overexpressing WISP2 gene,EMT-related proteinβ-Catenin,Vimentin,Slug protein expression increase,Claudin-1 protein expression increased,which caused cervical cancer cell migration capability to be significantly enhanced;XAV-939 also inhibited EMT-related molecules Vimentin,Slug protein expression,increased Claudin-1 protein expression(P<0.05),thereby inhibiting cell migration capabilities;After knocking the WISP2 gene,the inhibition of WISP2 in the cellular pulp was weakened,which promoted the conversion of epithelial cells to mesophage cells,and cervical cancer cell migration capacity was significantly enhanced(P<0.05).Conclusion: WISP2 gene expression is correlated with HPV infection.WISP2 is highly expressed in the cytoplasm of normal cervical tissues and cells,but it is highly expressed in the nucleus of cervical cancer tissues and cells.Overexpression of WISP2 can promote the role of Wnt/β-Catenin signaling pathway,and increase the co-localization with β-Catenin in the nucleus,which promotes the proliferative and migration ability of cervical cancer.XAV-939 reversed the effects of overexpression of WISP2.Knockdown of WISP2 gene promotes the expression of β-Catenin in the nucleus through PLC-γ signaling pathway,and thus the role of Wnt/β-Catenin signaling pathway,resulting in proliferation and migration capability of cervical cancer cells. |
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