Inhibitory Effects Of C-phycocyanin On Proliferation And Migration In Cervical Cancer HeLa Cells And Screening Of Its Interacting Proteins

Objective: To study the inhibitory effect and mechanism of C-phycocyanin(C-PC)on the proliferation and migration of HeLa cells induced by transforming growth factor beta 1(TGF-β1),and to screen the cell membrane proteins interacting with C-PC by human proteomics chip,so as to provide theoretical basis for further revealing the mechanism of C-PC entry.Methods: CCK-8 method was used to detect the effect of different concentrations(50,100,200,400,800,1600 μg/m L)of C-PC on the proliferation of HeLa cells after 24 h and 48 h.The follow-up experiment was divided into four groups: control group,TGF-β 1 group-β 1 treatment group,TGF-β 1 combined with C-PC treatment group,C-PC alone treatment group.The characteristics of HeLa cells in different experimental groups were detected: the morphological changes of HeLa cells were observed under microscope;Cell plate clone test was used to detect the colony forming ability of HeLa cells;The migration ability of HeLa cells was detected by wound healing assay and Transwell chamber assay;The m RNA expression of Slug,Snail,Zeb1 and Twist in HeLa cells was detected by RT-PCR;The expression levels of Vimentin,E-cadherin and N-cadherin in HeLa cells were detected by Western blot.Furthermore,human protein microarray was used to predict and screen the receptors on the cell membrane interacting with C-PC and analyze their functions.Immunofluorescence assay was used to identify the interaction protein and C-PC from HeLa cells to verify the prediction results of protein chip.Using pull-down experiment,through the C-PC with GST tag α And β The target protein was screened and verified from the total protein of HeLa cells..Results: 1.CCK-8 assay showed that HeLa cells treated with C-PC for 24 h and 48 h were significantly inhibited(P < 0.05 or P < 0.01)in a concentration dependent manner.The IC50 value of HeLa cells treated with C-PC for 24 h was 255.4 μg/m L and the IC50 of 48 h treatment was 387 μg/m L.Therefore,the working time of C-PC was 24 h and the working concentration was 200 μg/m L.2.The morphology of HeLa cells was observed under the microscope.HeLa cells in the control group showed tight polygonal connections.In the 10 ng/m L TGF-β1 treatment group,HeLa cells became long fusiform cells with strong migration ability and loose connections.In the 10 ng/m L TGF-β1+200 μg/m L C-PC treatment group,the proportion of HeLa cells in long spindle shape decreased,most of them were polygonal,w hich inhibited the morphological changes induced by TGF-β1.HeLa cells treated with 200 μg/m L C-PC alone showed decreased adherence ability,increased cell debris and dead cells.3.Compared with the control group,the number of colonies in TGF-β1 alone group was significantly increased,the number of colonies in TGF-β1 and C-PC CO treatment group was significantly decreased,and the number of colonies in 200 μg/m L C-PC alone group was significantly inhibited(all P<0.01).4.The results of wound healing te st and Transwell cell migration test showed that the migration ability of HeLa cells was significantly enhanced after TGF-β1 induction,while C-PC inhibited this phenomenon,and the migration ability of HeLa cells was significantly decreased after C-PC treatment alone.5.Compared with the control group,the m RNA expression levels of Slug,Twist,Snail and Zeb1 in HeLa cells treated with TGF-β1 were significantly increased,and the above changes could be reversed by the addition of C-PC(P<0.05 or P< 0.01),while the m RNA expression levels of Slug,Twist,Snail and Zeb1 in HeLa cells treated with C-PC alone were significantly inhibited.6.Western blot results showed that the expression levels of Vimentin,N-cadherin,ITPR3,ZDHHC5 and S yntenin in HeLa cells treated with TGF-β1 were significantly higher than those in the control group(P<0.05 or P<0.01).The expression levels of these proteins in the TGF-β1+C-PC CO treatment group were significantly lower than those in the TGF-β1 treatment group(P<0.05 or P<0.01).The protein expression levels in the C-PC treatment group were significantly lower than those in the control group(all P<0.05).The expression level of E-cadherin in TGF-β1 group was significantly lower than that in control group(P<0.01).Th e expression level of E-cadherin in TGF-β1+C-PC group was significantly higher than that in TGF-β1 group.Compared with control group,the expression level of E-cadherin in C-PC group was significantly increased(P<0.05).7.Three kinds of cell proteins(ITPR3,ZDHHC5,S yntenin)interacting with C-PC in HeLa cells were successfully screened by human proteomics chip.8.ZDHHC5 protein was extracted from the total protein of HeLa cells by pull-down assay,and the binding of C-PC to ZDHHC5 was confirmed by imm unofluorescence assay.Conclusion: C-PC can significantly inhibit the proliferation and migration of HeLa cells induced by TGF-β1.C-PC may be transported into HeLa cells by binding with ZDHHC5 protein.