The Study Of The Effects Of CYB5D2 Gene On Proliferation, Invasion And Metastasis Of Human Cervical Cancer Cell Hela And Its Mechanisms

Objective: To study the expression of CYB5D2 in cervical tissues,we analyzed its the relationship between the clinicopathological parameters and survival prognosis of cervical cancer,and its correlation with the expression of E-cadherin,a representative EMT marker,and preliminarily explored the clinical significance of CYB5D2 in the process of invasion and metastasis of cervical cancer.We further investigated the role of CYB5D2 gene overexpression in human cervical cancer cell line HeLa in cell proliferation,invasion,and metastasis and its possible molecular mechanism to provide a new target site for the clinical exploration of cervical cancer gene therapy.Methods:(1)Immunofluorescence assay was used to detect the expression of CYB5D2 and E-cadherin in normal cervical tissue,cervical intraepithelial neoplasia,and cervical cancer tissue.The clinical significance of them was analyzed.(2)Western blot and qRT-PCR assay were used to detect the expression of CYB5D2 protein and mRNA in overexpression of CYB5D2 HeLa cells;(3)MTS and plate clone formation assay were used to detect the cell proliferation in overexpression of CYB5D2 HeLa cells(4)Flow cytometry was used to detect the effect of CYB5D2 on cell cycle;(5)Cell scratch assay was used to detect the effect of cell migration ability in overexpression of CYB5D2 HeLa cells;(6)Transwell invasion assay was used to detect the effect of cell invasion ability in overexpression of CYB5D2 HeLa cells;(7)ELISA assay was used to detect the secretion of MMP2 and MMP9 in overexpressing CYB5D2 HeLa cells;(8)Western blot test detected the changes of EMT-related proteins E-cadherin,β-catenin,N-cadherin,Snail,Twist and Zeb2 in overexpressing CYB5D2 HeLa cells.Results:(1)The immunofluorescence results showed that,compared with 19 cases normal cervical tissues and 23 cases CIN tissues,the immunofluorescence intensity of CYB5D2 and E-cadherin was significantly reduced(P <0.05)in 99 cases cervical cancer tissues;the expression of two in different types was positively correlated(P<0.05);the expression of CYB5D2 was related to the tumor clinical FIGO stage(P<0.05),the expression of E-cadherin was related to the depth of tumor invasion,grade and the clinical FIGO stage(P<0.05);Kaplan-Meier survival analysis showed that the OS and RFS of thehigh expression of CYB5D2 mRNA cervical cancer patients were significantly increased(P<0.05);(2)Western blot and qRT-PCR results showed that the expression levels of CYB5D2 protein and mRNA in HeLa cells in the experimental group were significantly higher than those in the no-load control group(P<0.05);(3)MTS results showed that in the over-expressed CYB5D2 experimental group,the number of HeLa cells gradually significantly decreased(P<0.05);The results of the plate clone formation test showed that the colony number and size of the group that overexpressed CYB5D2 gradually decreased(P<0.05);(4)Flow cytometry results showed that in the over-expressed CYB5D2 experimental group,the cells ratio for G0 / G1,S,G2 / M phase cells was not significantly changed(P> 0.05);(5)Cell scratch test results showed that in the over-expressed CYB5D2 experimental group,the rate of cell migration was significantly reduced(P<0.05);(6)Transwell test results showed that in the over-expressed CYB5D2 experimental group,the number of cells invaded was significantly reduced(P<0.05);(7)ELISA test results showed that,in the over-expressed CYB5D2 experimental group,the cell secretion of MMP2 and MMP9 was significantly reduced(P<0.05);(8)Western blot results showed that,in the over-expressed CYB5D2 experimental group,the expression of E-cadherin,β-catenin protein gradually increased and the expression of N-cadherin,Snail,Twist,and Zeb2 protein gradually decreased(P <0.05).Conclusion:(1)In cervical cancer,both CYB5D2 and E-cadherin were down-regulated,showing a significant positive correlation and suggesting that CYB5D2 may reverse EMT by up-regulating E-cadherin and thus inhibit the occurrence of invasion and metastasis of cervical cancer;(2)CYB5D2 can inhibit proliferation,migration,and invasion of human cervical cancer HeLa cells;(3)The possible inhibition mechanism of HeLa cells may reverse the occurrence of the EMT response by regulating the expression of proteins such as E-cadherin,β-catenin,N-cadherin,Snail,Twist,and Zeb2 and the secretion of metastasis-related factors,MMP2 and MMP9,thereby exerting potential biological effects as a tumor suppressor gene in cervical cancer.