The Effects Of S100A6 On Proliferation,apoptosis And Migration Of Cervical Cancer Cell And Its Mechanism Research

Background and objectivesCervical cancer is the second most common gynecological cancer worldwide and remains as one of the leading causes of cancer-related death among women.With the popularity of cytology screening and the use of vaccines,cervical cancer in the early diagnosis and prevention has made considerable progress,but its metastasis and recurrence remain the main cause of death.Therefore,they are necessary to elucidate the underlying mechanisms and to explore remarkable biomarkers and drug targets to develop new options for early diagnosis and treatment of cervical cancer.S100A6 is a member of the S100 calcium-binding protein family and it participates in a variety of biological functions.The role of S100A6 on tumor development attracted much attention recently.Researches have demonstrated that S100A6 plays an important role in development and progression of human colorectal cancer cells,gastric cancer cells,hepatocellular carcinoma cells,osteosarcoma cells and so on.The protein level of S100A6 was significantly up-regulated in the tissues of cervical squamous cell carcinoma,its expression had positive correlation with the degree of cervical lesion and lymph node metastasis.But there is no report about definite effect of S100A6 on proliferation and migration of cervical cancer cells.In this study,we aimed to investigate the effect of S100A6 on the proliferation,apoptosis and migration of cervical cancer cells and its underlying mechanisms,to accumulate experimental basis for elucidating the mechanism of cervical cancer progression,and to provide new targets and theoretical basis for the diagnosis and treatment of cervical cancer.Methods1.The endogenous expression of S100A6 in cervical cancer cell linesS100A6 expression in these cells was detected by quantity polymerase chain reaction(q PCR).2.Preparation and verification of interventions for S100A6 expression(1)Recombinant adenovirus amplification and intervention validationThe recombinant adenoviruses Ad S100A6,Adsi S100A6,which carries S100A6 gene or S100A6-si RNA coding sequence respectively,and their control adenoviruses Ad GFP,Ad RFP were infected into HEK293 to amplify.Then these adenoviruses were used to infect cervical cancer cells and finally the total protein of these cells was extracted.Western Blot was used to verify the effect of these adenoviruses on S100A6 gene expression.(2)Preparation and verification of recombinant GST-S100A6 proteinE.coli BL21 was transformed by p GST-Moluc-S100A6 and then cultured in LB medium.IPTG was used to induce the expression of recombinant protein.After 6-8 h bacteria were collected and broken up by ultrasound on ice and the freed recombinant protein was purified by GS4 B beads.SDS-PAGE,Coomassie blue staining and Western blot was used to identify the GST-S100A6 protein.3.Effects of S100A6 on proliferation,apoptosis and migration of cervical cancer cell linesThe effects of S100A6 on proliferation and apoptosis were detected by MTT assay and Hoechst staining;the effect of S100A6 on migration was measured by wound healing assay and Transwell assay.4.The mchanism of S100A6-enhanced proliferation and migration of cervical cancer cell lines(1)Western blot was used to detect the changes of PI3K/Akt signaling pathway after S100A6 treatment,and the target gene m RNA level of β-catenin pathway was detected by PCR.(2)After He La cells were treated by S100A6 and LY2940002,Western Blot was used to detect the alteration of PI3K/Akt signaling pathway,MTT and Transwell assay were used to measure the alteration of proliferation and migration of the cells.(3)PCR and Western Blot were used to detect the effect of S100A6 on EMT of these cells.Results1.S100A6 was expressed at low level in He La cells and at much higher in Si Ha cells and Ca Ski cells.The m RNA level of S100A6 in Si Ha and Ca Ski cells was 6.92 times and 7.54 times of those of He La cells,respectively.2.Western Blot result showed that Ad S100A6 and Adsi S100A6 significantly increased and decreased the level of S100A6,respectively,suggesting that the two adenoviruses can effectively interfere with the expression of the gene.SDS-PAGE and Coomassie blue staining result showed that the relative molecular mass of GST-S100A6 was about 36 k D,which was in line with our expectation for its relative molecular mass;Western Blot result showed that GST-S100A6 was specifically recognized by anti-S100A6 monoclonal antibody.All suggested that the prepared recombinant protein was GST-S100A6.3.Effects of S100A6 on proliferation,apoptosis and migration of cervical cancer cell lines(1)S100A6 promoted the proliferation of cervical cancer cell lines.The proliferation ability of He La and Ca Ski cells after Ad S100A6 infection was significantly increased.Their OD492 values at 72 h were 1.2 times(P<0.01)and 1.3 times(P<0.005)of that of the control groups,respectively.The proliferation ability of Si Ha and Ca Ski cells infected with Adsi S100A6 was significantly decreased.The OD492 values at 72 h were only 81.9 % and 81.3 %(P <0.05)of that of the control groups,respectively.(2)S100A6 had no significant effect on the apoptosis of cervical cancer cell lines.Compared with the control group,there was no significant difference in the number of apoptotic cells in He La and Ca Ski cells which was treated by Ad S100A6;there was also no significant difference in the number of apoptotic cells in Si Ha and Ca Ski cells which was treated by Adsi S100A6.(3)S100A6 promoted the migration of cervical cancer cell lines.The wound healing rate in Ad S100A6 groups at 72 h was 2.1 fold(P<0.05)and 3.6 fold(P<0.01)of that in Ad GFP groups of He La cells and Ca Ski cells,respectively.Consistently,the wound healing rate decreased significantly after Adsi S100A6 treatment.The wound healing rate at 72 h of Si Ha cells and Ca Ski cells were 59 %(P<0.05)and 56 %(P<0.05)of that in their Ad RFP groups,respectively.The number of transmembrane cells at 72 h in Ad S100A6 groups was 5 times(P<0.005)and 2 times(P<0.01)of that in Ad GFP groups of He La cells and Ca Ski cells,respectively.The number of transmembrane cells at 72 h in Si Ha and Ca Ski cells treated with Adsi S100A6 were only 34 %(P<0.005)and 35 %(P<0.01)of that in their Ad RFP groups.The results was consistent with the wound healing assay.All suggested that S100A6 can promote the migration of cervical cancer cells.4.The mechanism of S100A6-enhanced proliferation and migration of cervical cancer cell lines(1)S100A6 activated PI3K/Akt signaling pathway in cervical cancer cells.The phosphorylation levels of Akt(p-Akt)and GSK3β(p-GSK3β),and β-catenin level were increased in Ad S100A6 group of He La cells;the m RNA levels of c-myc,MMP7 and cyclin D1 were increased in Ad S100A6 group of He La cells.(2)The promotive effect of S100A6 on the proliferation and migration of cervical cancer cells was mediated by the activation of PI3K/Akt signaling pathway.(1)PI3K inhibitor LY294002 down-regulated p-Akt significantly.The optimal concentration was 10 μM.(2)LY294002 treatment inhibited the up-regulation of p-Akt,p-GSK3β and β-catenin which were induced by S100A6.(3)LY294002 impaired the S100A6-enhanced proliferation of He La cells partially.The OD492 value was 60 %(P<0.05)at 48 h and 49.9 %(P<0.005)at 72 h of that in their control groups.(4)LY294002 impaired the S100A6-enhanced migration of He La cells partially,the number of transmembrane cells at 72 h was 57.14 %(P<0.005)of that in the control group.(3)S100A6 promoted the EMT of cervical cancer cells.Up-regulating S100A6 in He La cells increased the protein level of N-cadherin and m RNA levels of vimentin,Snail and Twist.On the contrary,down-regulating S100A6 in Si Ha cells increased the protein level of E-cadherin and decreased the protein level of N-cadherin,the m RNA levels of vimentin,Snail and Twist.ConclusionsS100A6 promotes the proliferation,migration and EMT of cervical cancer cell lines.The promotive effect of S100A6 on the proliferation and migration of cervical cancer cells is related to the activation of PI3K/Akt signaling pathway.S100A6 may be a potential target for cervical cancer diagnosis and treatment.