| objective:Cervical cancer is the second most common malignant tumor of women,preceded only by breast cancer.invasion and metastasis are main reasons of relapse after treatments and the cause of death in patients.In recent years,the relationship between mesenchymal transition and tumor invasion and EMT become a hot topic.FMNL2(formins like 2)gene is one of formin families.As the most effective actin nucleation factor,formins involved in numerous actin based processes and also express disorders on tumor differentiation,invasion and metastasis in pathological cases.The study focuse on the expressions of FMNL2 and relationships between FMNL2 gene and cervical cancer's EMT.In order to reveal new ways with FMNL2 gene involved in cancer metastasis,and provide new clues for the study of the mechanism of tumor metastasis.Methods:l.Immunohistochemical detected the FMNL2,E-cadherin,Vimentin expressions in 73 cases of cervical squamous cell carcinoma(29 cases of cervical lymph node metastatic squamous carci-noma),39 cases of cervical adenocarcinoma(12 cases of cervical lymph node metastatic adenocarcinoma)and 30 cases of uterine myomectomy patients with normal cervical tissues.2.Culture in vitro human cervical adenocarcinoma Hela cells,squamous carcinoma Siha cells with the experimental groups containing concentrations 100nmol/L of siRNA transfection medium reagents were incub-ated 72h and the control groups with none siRNA medium were incubated 72h.Detect:(1)Semi-quantitative RT-PCR assayed the mRNA expression of FMNL2 gene and epithelial mesenchymal transition related markers Vimentin,E-cad-herin in experimental groups and control groups,and analyzed their relevan-ce;(2)Cell immunohistochemistry detected the protein expression of FMNL2 and epithelial mesenchymal transition related markers Vimentin,E-cadherin in experimental groups and control groups,and analyze their relevance;(3)CCK8 detected the cell proliferation of the experimental groups and the control groups;(4)Healing assay detected changes of cell migration in experimental groups and control groups.Results:1.Cervical cancer tissues immunohistoche-mistry showed(1)The expressions of FMNL2,Vimentin,E-cadherin in cervical squamous epithelium,cervical squamous cell carcinoma,squamous cell carcin-oma of cervical lymph node metastatic tissues were FMNL2:6.67%,67.12%,51.72%,Vimentin:0,30.14%,34.48%,E-cadherin:100%,72.60%,68.97%and in the cervical glands,cervical adenocarcinoma,cervical adenocarcinoma metast-atic lymph node tissue were FMNL2:13.33%,66.67%,58.33%,Vimentin:0,41.03%,41.67%,E-cadherin:100%,64.10%,66.67%.(2)The expression of FM-NL2 in cervical cancer was significantly higher than normal cervical tissue,the difference was statistically significant(SCC ?2=31.088,p=0.000,adenocarc-inoma ?2=19.627,p=0.000);Vimentin expression in cervical carcinoma signif-icantly higher than normal cervical tissue,the differences were statistically significant(SCC p=0.OOO;adenocarcinoma,p=0.000);E-cadherin expression in cervical cancer was significantly lower than that of normal cervical tissue,the difference was statistically significant(SCC p=0.000;adenocarcinoma,p=0.000).The expression of FMNL2 was positively correlated with Vimentin expr-ession(SCC r=0.269,p=0.029;adenocarcinoma r=0.369,p=0.037),and was neg-atively correlated with E-cadherin expression(SCC r=-0.299,p=0.011;adenoc-arcinoma r=-0.332,p=0.049)in cervical carcinoma(3)FMNL2,Vimentin,E-cadherin expression differences in cervical cancer and cervical lymph node metastasis(FMNL2:squamous ?2=2.105,p=0.147,adenocarcinom-a ?2=0.279,p=0.597;Vimentin:squamous ?2=0.182,p=0.670;adenocarcinoma?2=0.002,p=0.969;E-cadherin:squamous ?2=0.135,p=0.713,adenocarcinoma?2=0.026,p=0.871),differential expression in cervical squamous cell carcinomaand adenocarcinoma(FMNL2:?2=0.002,p=0.961;Vimentin:?2=1,344,p=0.246;E-ca dherin:?2=0.869,p=0.351)and lymph node metastatic cervical lymph node metastatic squamous cell carcinoma and adenocarcinoma of the cervix differe-ntial expression(FMNL2:?2=0.149,p=0.699;Vimentin:?2=0.189:p=0.664;E-cadherin:?2=0.021,p=0.886)were not statistically significant.2.RT-PCR results showed that:(1)The relative gray value of FMNL2's mRNA in experimental groups were lower than control groups,the differences were statistically significant(Hela cells t=-10.568,p=0.000;Siha cells t=-5.766,p=0.004).In control groups compared to the two cell lines relative gray value of FMNL2's mRNA in Hela cells were higher than Siha cells,the differences were statistically significant(t=3.755,p=0.020).(2)The relative gray value of Vimentin's mRNA in experimental groups was lower than control groups,the differences were statistically significant(Hela cells t=-7.056,p=0.002;Siha cells t=-2.800,p= 0.049).In control groups compared to the two cell lines relative gray value of Vimentin's mRNA in Hela cells were higher than Siha cells,the differences were statistically significant(t=16.768,p=0.000).(3)The relative gray value of E-cadherin's mRNA in experimental groups were higher than control groups,the difference was statistically significant(Hela cells t=7.545,p=0.002;Siha cells t=10.066,p=0.000).In control groups compared to the two cell lines relative gray value of Vimentin's mRNA in Hela cells were higher than Siha cells,the differences were statistically significant(t=-4.311,p=0.012).(4)Silen-cing FMNL2 in the two cell lines,the mRNA expression of Vimentin were down,and the mRNA expression of E-cadherin were raised.3.cell immunoh-istochemistry results showed:(1)The average optical density of FMNL2's protein in experimental groups were lower than the control group,the differences were statistically significant(Hela cells t=-28.000,p=0.000;Siha cells t=-12.886,p=0.000).In the control group compared to the two cell lines relative gray value of FMNL2's protein in Hela cells were higher than Siha cells,the differences were statistically significant(t=3.273,p=0.017).(2)The average optical density of Vimentin's protein in experimental groups were lower than control groups,the differences was statistically signif-icant(Hela cells t=-16.047,p=0.000;Siha cells t=-11.887,p=0.000).In control groups compared to the two cell lines relative gray value of FMNL2's protein in Hela cells were higher than Siha cells,the differences were statistically significant(t=23.669,p=0.000).(3)The average optical density of E-cadherin's protein in experimental groups were higher than control groups,the differences were statistically significant(Hela cells t=5.539,p=0.002;Siha cells t=7.498,p=0.000).In control groups compared to the two cell lines relative gray value of FMNL2's protein in Hela cells were higher than Siha cells,the differences were statistically significant(t=-27.001,p=0.000).(4)silencing FMNL2 in two cell lines,expression of Vimentin's protein expression were reduced,expression of E-cadherin's protein were increased.4.Proliferation changes:CCK-8 assay showed that silencing FMNL2 in the two cell lines,experimental groups and control groups was not statistically significant inhibition rate difference(p>0.05).5.Migration changes:scratch test results showed that the experimental groups of Hela and,Siha cells healing rates of 24h,48h were respectively 45%,53%and 42%,47%,and the control groups were 89%,97%and 83%,91%.Compared with the control groups,the migration of experimental groups were significantly reduced(p<0.05).Conclusions:1.FMNL2 gene may promote the development,invasion and metastasis of cervical cancer;2.FMNL2 may stimulate the cervix EMT to enhanced invasion and metastasis of cervical cancer;3.Cervical cancer Hela,Siha cell lines exist EMT,Hela cell lines more obvious;4.FMNL2 gene silencing may have a certain extent in EMT of cervical cancer;5.FMNL2 gene silencing may inhibit migrateon of cancer cell lines. |
PDF Full Text Request