| Objective: To explore the mechanism of miR-3666 mediated invasion of cervical cancer cells and the mechanism of PTTG1 mediated miR-3666/ZEB1 pathway regulating the invasion of cervical cancer cells.PTTG1 can be mediated by miR-3666 to enhance the molecular mechanism of ZEB1 mediated CC cell invasion and regulation axis.Method: 1.We compared the expression level of ZEB1 and miR-3666 in non tumor cervical tissue and cervical cancer tissue by Western blot and RT-q PCR,and calculated the bivariate correlation by Spielman rank correlation coefficient.2.In the NCBI database,the ZEB1 3’-UTR sequence was found,and the binding site of miR-3666 and ZEB1 m RNA3’UTR was found according to the miRNA target prediction software.We used plasmid transfection,RT-q PCR and luciferase activity quantification to verify the mechanism of miR-3666 binding to ZEB1 m RNA 3’UTR to inhibit its translation in CC cells.3.The Transwell invasion test was used to demonstrate that miR-3666 mediated the invasion of cervical cancer cells by mediating ZEB1.4.The content of PTTG1 protein in cervical cancer tissue was detected by Western blot.The relationship between miR-3666 expression and PTTG1 regulation was detected by plasmid transfection,RT-q PCR and luciferase activity quantification.5.The Transwell invasion test was used to verify the study of PTTG1 through the miR-3666/ZEB1 pathway to regulate the invasion of cervical cancer.Result: 1.The level of ZEB1 in CC samples of patients compared with non tumor cervix tissue(NT)was significantly higher than that in 35 patients.The level of miR-3666 was significantly reduced by RT-q PCR.A strong negative correlation between CC and ZEB1(?=-0.65,P <0.0001,N =35).These data show that there is a causal relationship between miR-3666 and ZEB1 in CC.2.The relationship between miR-3666 and ZEB1 in CC cells indicated by experimental data was used to detect whether miR-3666 could be expressed against ZEB1.Bioinformatics analysis found that there is a miR-3666 binding site ranging from 500 to 506 bases in ZEB1 m RNA 3’UTR.The use of human CC cell lines Ca Ski and H3 to detect miR-3666 in CC cells can be used to regulate ZEB1 translation by base pairing.miR-3666 was expressed by transfection of plasmid overexpressed miR-3666.The expression of miR-3666 was inhibited by the plasmid carrying as-miR-3666.Ca Ski and H3 cells transfected the plasmid carrying the empty sequence as the control(null).The transfected plasmid cells were purified by flow cytometry to co express GFP.The overexpression or inhibition of miR-3666 in Ca Ski and H3 cells is verified by RT-q PCR.Then the miR-3666 modified Ca Ski and H3 cells were transfected with 1 micrograms of ZEB1 3’UTR luciferase plasmid.The luciferase activity was quantified in these cells,and the results showed that miR-3666 targeted the 3’UTR of ZEB1 m RNA and inhibited its expression.The change of miR-3666 levels in Ca Ski and H3 cells did not affect the m RNA level of ZEB1.However,Western Blot showed that over expression of miR-3666 in Ca Ski and H3 cells cells significantly decreased the expression level of ZEB1 protein,and inhibited miR-3666 expression and significantly increased ZEB1 protein expression in Ca Ski and H3 cells cells.It shows that miR-3666 inhibits the translation of ZEB1 protein in Ca Ski and H3 cells.3.Transwell cell migration assay showed that the overexpression of miR-3666 led to the weakening of the invasion of Ca Ski and H3 cells.Similarly,the depletion of miR-3666 leads to enhanced invasion of Ca Ski and H3 cells.In conclusion,these data suggest that miR-3666 regulates the invasion of cervical cancer cells by mediating ZEB1.4.Whether the level of miR-3666 in CC cells can be regulated by PTTG1.The expression of PTTG1(sh Pttg1)was inhibited by transfection of PTTG1 by transfection plasmid by cell transfection plasmid in CC cells,or transfected with RNA plasmid with short hairpin to transfect CC cells.Ca Ski and H3 cells also transfected plasmid carrying the scrambled sequence as a control(SCR).PTTG1 was expressed or suppressed in Ca Ski and H3 cells by RT-q PCR.Through RT-q PCR,PTTG1 overexpression in Ca Ski and H3 cells significantly decreased the level of miR-3666,while inhibition of PTTG1 significantly increased the level of miR-3666.The results showed that PTTG1 could inhibit the expression of miR-3666 in CC cells.5.Transwell cell migration assay revealed that PTTG1 depletion caused Ca Ski and H3 cell invasion to weaken.PTTG1 overexpression induced Ca Ski and H3 cell invasion enhancement,indicating PTTG1 increased the invasion of cervical cancer through miR-3666/ZEB1 pathway.Conclusion: 1.Compared with CC samples from non tumor tissues,the miR-3666 level in cervical cancer tissue decreased significantly,while the level of ZEB1 increased significantly,and miR-3666 was negatively correlated with ZEB1 level.2.Bioinformatics analysis found that there was a miR-3666 binding site at the base of 500 to 506 bases of ZEB1 m RNA 3’UTR.And in the Ca Ski and H3 cell lines of cervical cancer,the target of miR-3666 to bind to the 3’UTR of ZEB1 m RNA inhibits the translation of its protein.3.Transwell cell migration detection found that miR-3666 targeting ZEB1 in the Ca Ski and H3 cell lines of cervical cancer inhibited the invasiveness of cervical cancer cells.4.PTTG1 was significantly higher in the cervical cancer tissues,and the expression of miR-3666 was inhibited by PTTG1 in the Ca Ski and H3 cell lines of cervical cancer.5.Transwell cell migration detection found that PTTG1 enhanced the invasiveness of cervical cancer cells by inhibiting the expression of miR-3666 in the Ca Ski and H3 cell lines of cervical cancer.In this study,the PTTG1/miR-3666/ZEB1 regulation axis was proposed for the first time.In the regulation axis PTTG1 could inhibit the invasion of CC cells mediated by miR-3666 enhanced ZEB1.This discovery provides a new idea for the study of the molecular mechanism of PTTG1 oncogenes. |
PDF Full Text Request