| objective: The expression of miR-451 and c-myc in cervical squamous cell carcinoma,cervical intraepithelial neoplasia and normal cervical epithelium and the expression of E-cadherin and Vimentin in cervical squamous cell carcinoma were detected,furthermore,the effect of miR-451 and c-myc on the biological characteristics and biological behavior of SiHa cell lines was investigated,to explore the role of miR-451 and c-myc in cervical squamous cell carcinoma epithelial-mesenchymal transition.Methods:1.To explore the role of miR-451 and c-myc in EMT of cervical squamous cell carcinoma by histological methods.Cervical squamous cell carcinoma,cervical intraepithelial neoplasia and normal cervical tissue were gathered.In situ hybridization was used to detect the expression of miR-451 in each group,and immunohistochemistry was used to detect the expression of c-myc in each group and the expression of E-cadherin and Vimentin in cervical squamous cell carcinoma tissues.The relationship between miR-451,c-myc,E-cadherin,Vimentin and the clinicopathological features of cervical squamous cells was analyzed respectively.2.Effect of miR-451 and c-myc on biological characteristics and biological behavior of SiHa Cell Line.(1)MiRNA mimichsa-mir-451 a was transfect into SiHa cells to increase the miR-451 expression.The miRNA hsq-mir-451 a inhibitor was transfected into SiHa cells to decrease the expression of miR-451.The siRNA-c-myc fragment was transfected into SiHa cells to establish c-myc silencing model.(2)The change of c-myc mRNA after changing the expression of miR-451 was analyzed by RT-PCR.Meanwhile,the expression of epithelial mesenchymal markers(E-cadherin and Vimentin)and c-myc were detected by western blot.(3)The proliferation ability of SiHa cells was detected by CCK8 assay and plate clone formation assay,the migration ability was detected by scratch assay,and the invasion ability was examined by transwell invasion assay.Results:1.Histological test results:(1)The positive rates of miR-451 and c-myc in cervical squamous cell carcinoma were 16.4% and 88.5%,respectively.The positive rates of miR-451 and c-myc in cervical intraepithelial neoplasia were 36.7% and 60.0%,respectively.The positive expression rates in the normal tissues were 80% and 10%,respectively.There was significant difference in the expressions of the two in each group(P <0.05).The expression of mi R-451 was correlated with pathological grade and lymph node metastasis of cervical cancer(P <0.05),but not with tumor stage and age(P> 0.05).The expression of c-myc was correlated with clinical stage and patient's age(P <0.05),but not with tumor size,lymph node metastasis and tumor pathological grade(P> 0.05).Spearman's correlation analysis showed that the expression of miR-451 and c-myc in cervical cancer was negatively correlated(r =-0.257,P = 0.044).(2)The positive expression rate of E-cadherin and Vimentin in cervical squamous cell carcinoma was 55.7% and 67.2% respectively.Both of them were correlated with the stage of cervical cancer and lymph node metastasis(P <0.05),but not with the pathological type and age(P> 0.05).(3)There was a positive correlation between the expression of miR-451 and E-cadherin in cervical cancer(r = 0.305,P = 0.042),and a negative correlation between the expression of miR-451 and Vimentin in cervical cancer(r=-0.351,P= 0.018).(4)There was a negative correlation between the expression of c-myc and E-cadherin in cervical cancer(r =-0.321,P= 0.036),and a positive correlation between c-myc and Vimentin in cervical cancer(r = 0.406,P = 0.006).2.Effect of miR-451 and c-myc on the Biological Characteristics and Biological Behavior of SiHa Cells:(1)After up-regulating the expression of miR-451,the mRNA and protein expression of c-myc both decreased in SiHa cells.Meanwhile,the protein expression of E-cadherin increased and Vimentin decreased.CCK8 experiment showed that the growth of SiHa cells was inhibited and the inhibition effect increased with time,plate cloning experiments showed that the rate of cell clone formation decreased,scratch test results showed that the migration distance of 48 h cells in experimental group was decreased,transwell invasion assay showed that the number of cells penetrating matrigel in experimental group was less than that in control group at 24 hours of culture,all with statistical significance(P<0.05).(2)After down-regulating the expression of miR-451,the mRNA and protein expression of c-myc were both increased in SiHa cells,the expression of E-cadherin protein decreased,and Vimentin protein increased.CCK8 experiment showed that the growth of SiHa cells was promoted and the promotion effect increased with time,plate cloning experiments showed that the rate of cell clone formation increased,scratch test results showed that the migration distance of 48 h cells in experimental group was increased,transwell invasion assay showed that the number of cells penetrating matrigel in experimental group was more than that in control group at 24 hours of culture,all with statistical significance(P<0.05).(3)After silencing the expression of c-myc by siRNA,the expression of c-myc mRNA decreased in experimental group(P<0.05),and the protein expression of E-cadherin increased,but Vimentin decreased.CCK8 experiment showed that the growth of SiHa cells was inhibited and the inhibition effect increased with time,plate cloning experiments showed that the rate of cell clone formation decreased,scratch test results showed that the migration distance of 48 h cells in experimental group was decreased,transwell invasion assay showed that the number of cells penetrating matrigel in experimental group was less than that in control group at 24 hours of culture,all with statistical significance(P<0.05).Conclusion:1.The expression of miR-451 and c-myc are closely related to the accurance and development of cervical squamous cell carcinoma.The decrease of miR-451 expression and the increase of c-myc expression can promote the invasion and metastasis of cervical squamous cell carcinoma.2.Detection of miR-451 and c-myc expression has certain guidance value for the diagnosis and treatment of the cervical squamous cell carcinoma.3.miR-451 may be one of the upstream regulators of c-myc and negatively regulate epithelial-mesenchymal transition of cervical squamous cell carcinoma by inhibiting the expression of c-myc,thus inhibit the invasion and metastasis of cervical squamous cell carcinoma.4.miR-451 and c-myc may be new therapeutic targets for cervical squamous cell carcinoma. |
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