| BACKGROUD AND PURPOSECervical cancer is the most common malignancy carcinoma of women’s reproductive systems. In recent years, It is reported that the incidence of cervical cancer has declined in the general population, but in developing countries especially in China, due to the increase of HPV infection, confusion of screening programs, the incidence and mortality of cervical cancer has actually increased and the young tendency. Although the integrated treatment of screening and surgical has been effective in improving the prognosis of early cervical cancer, but cervical cancer that occur metastasis and recurrence is difficult to achieve cure completely, which were also a major risk factor of death in patients. Thus, strengthening basic research of cervical cancer in invasion and elucidating the molecular mechanisms of cancer metastasis, there was important scientific significance to predict metastasis early, diagnosis and prognosis of cervical cancer as well as to improve the cure rate and reduce mortality. It was reported that there was an abnormal expression of miRNAs in human cancers, more than50%of miRNAs were located in human cancer-related gene region, e.g. fragile sites, the loss of heterozygosity areas or near oncogenes area, and tumor suppressor gene site, those miRNAs occur loss, amplification or translocation easily. Thus, miRNAs play an important role in regulating by targeting specific oncogenes or tumor suppressor genes in tumor development and progression. Meanwhile, miRNAs are also regulated by the upstream transcription factors, so that complex regulatory network regulate tumor development, the report of relationship between miRNAs and cervical cancer gradually increased in recent years, miRNAs have an important role in the process of invasion and metastasis of cervical cancer.In this paper, we use the miRNA chip in the stable cell lines (siha-twist2and siha-shtwist2) to screen miRNA221which has the relationship with twist2that was a factor about invasion and metastasis in cervical cancer, thus, we can make clear the role of miRNA221in cervical cancer invision and metastasis. The purpose of this paper is as follows:1. Screening and identification of miRNA correlated with invasion and metastasis of cervical cancer;2. The role of miR221in the invasion and metastasis of cervical cancer by ARID1A and wnt/β-catenin signaling pathway;3. Identification of interaction between miR221and the upstream transcription factor twist2.RESEARCH METHODS1. Screening and identification of miRNA correlated with invasion and metastasisof cervical cancer 1.1Using stable cell lines (siha-twist2and siha-shtwist2) and miRNA chip for screening miRNA related to invasion and metastasis factor Twist2and verification through qPCR.1.2Testing the effects of miRNA221on the invasion and metastasis of cervical cancer cells by qPCR, westernblot, the scratch test and Borden chamber.2. The role of miR221in the invasion and metastasis of cervical cancer by ARID1A and wnt/β-catenin signaling pathway.2.1Using10miRNA target gene prediction software (DIANAmT、miRanda、 miRDB、miRWalk、RNAhybrid.PICTAR4、PICTAR5、PITA、RNA22、Targetscan) were selected to forecast the target of miRNA221, and using the software DAVID6.7provided by NIAID/NIH to analyse signal pathway about the target of miR221.2.2Using the westernblot, vector construction and dual luciferase reporter analysis experiment to verify the regulation mechanism among miR221, ARID1A and wnt/p-catenin signal pathway.3. Identification of interaction between miR221and the upstream transcription factor twist2.3.1Testing the effects of twist2regulation miR221on the invasion and metastasis of cervical cancer cells by qPCR, westernblot, the scratch test and Borden chamber.3.2Using the vector construction and luciferase reporter assay to verify the action way between the upstream transcription factor Twist2and miRNA221RESULTS1. Screening and identification of miRNA correlated with invasion and metastasis of cervical cancer 1.1Screening of miRNA related to invasion and metastasis in cervical cancerUsing the paired cell lines siha, siha-tw2, siha-shtw2to test on chip, the results suggest that there were8differentially expressed microRNAs, including miRNA221;Using the real-time PCR to test the8differentially expressed microRNAs about result of chip in paired cell lines, the paired T test and single factor variance analysis to2-△△CT showed that the most obvious difference expression is miRNA221(F=64.64,P<0.001), further testing the expression of miRNA221in cervical cancer tissue, the result showed that the expression of miRNA221was positive correlation to the expression of twist2, which corresponded to the result of chip(r=0.70,F=24.75, P<0.001).1.2Effect of miRNA221on the invasion and metastasis of cervical cancer cells(1) Using the real-time PCR to test the expression of miRNA221and marker related to EMT (E-CAD、N-CAD、Vimentin) in cervical cancer cell lines siha, siha-nc(m), siha-nc(i), siha-m, siha-i. With the blank group as the control, the single factor analysis of variance test showed that there were significant differences among5groups(F=34.1, P<0.001; F=27.026, P<0.001; F=17, P<0.001), further comparison two groups in the5groups, the result showed that the expression of miRNA221in siha-m group was significant higher than in siha and siha-nc(m) group, the expression of E-CAD was obviously low, but the expression of N-CAD and vimentin, were significantly high; The expression of miRNA221in siha-i group was significant lower than in siha and siha-nc(i) group, the expression of E-CAD was obviously high, but the expression of N-CAD and vimentin were significantly low. (2) Using the westernblot to test the expression of marker related to EMT (E-CAD、N-CAD、Vimentin) in cervical cancer cell lines siha, siha-nc(m), siha-nc(i), siha-m, siha-i. The result showed that the expression of E-CAD in siha-m group was significant lower than in siha and siha-nc(m) group, but the N-CAD and Vimentin was significantly high; contrarily, the expression of E-CAD in siha-i group was significant higher than in siha and siha-nc(i) group, but the N-CAD was significantly low.(3) Using the scratch experiment to test the migration ability of siha, siha-nc(m), siha-nc(i), siha-m, siha-i, the result showed that the migration ability in siha-mi group was higer than siha and siha-nc(m) group, but the migration ability in siha-in group was lower than siha and siha-nc(i) group.(4) Using the Borden chamber to test the invasive ability in vitro of cervical cancer cell lines siha, siha-nc(m), siha-nc(i), siha-m, siha-i, the result showed that the invasive ability in vitro among5groups had significantly difference.the cells through the Borden chamber in siha-m group were much more than siha, siha-nc(m), but in siha-i group, the cells through the Borden chamber were less than siha, siha-nc(i).2. The role of miR221in the invasion and metastasis of cervical cancer by ARID1A and wnt/p-catenin signaling pathway2.1Prediction and verification of the target and signal pathway about miRNA221(1) Biological information forecasting related to target of miRNA22110commonly used miRNA target gene prediction software (DIANAmT、miRanda、 miRDB、miRWalk、RNAhybrid、PICTAR4、PICTAR5、PITA、RNA22、Targetscan) were selected to forecast the target of miRNA221, prediction of ARID1A gene was the highest,thus we choose the ARIDIA as the target of miRNA221, and identify the role of ARID1A in experiment.(2) Biological information forecasting related to signal pathway of miR221Using the DAVID6.7provided by NIAID/NIH (http://david.abcc.ncifcrf.gov/tools.jsp), Biocarta pathway and KEGG pathway to analysis the target of miRNA221, the results show that the prediction of wnt/β-catenin signal pathway was the highest.2.2Regulation of miR221on the downstream target genes of ARIDIA and wnt/β-catenin signal pathway(1) Using the westernblot to test the expression of ARID1A and P-catenin in cervical cancer cell lines siha, siha-nc(m), siha-nc(i), siha-m, siha-i. the results show that the expression of ARIDIA in siha-m group was significantly lower than siha and siha-nc(m); but the expression of ARIDIA in siha-i group was significantly lower than siha and siha-nc(i); the expression of phosphorylated P-catenin in siha-m group was obviously lower than siha and siha-nc(m); however, the expression of phosphorylated β-catenin in siha-i group was obviously lower than siha and siha-nc(i).(2) Transfecting the plasmid pscicheck2/ARID1A and miR-221mimic to the siha cell, testing the luciferase activity in cells of each group using the dual luciferase report analysis, the results show that the luciferase activity in miR221mi-luc group was obviously lower than siha and miR221NC-luc(F=635.4, P<0.001), the difference between siha and miR221NC-luc was not significant. 3. Identification of interaction between miR221and the upstream transcription factor twist23.1Regulation of twist2on miR221promote cervical cancer invasion and metastasis(1) Using the real-time PCR to test the expression of marker related to EMT (E-CAD、N-CAD、Vimentin) in cervical cancer cell lines siha、sihatw2、sihashtw2、 sihatw2-i、sihashtw2-m. With the blank group as the control, the single factor analysis of variance test showed that there were significant differences among5groups(F=221.991, P<0.001; F=369.073, P<0.001; F=404.261, P<0.001), further comparison two groups in the5groups, the result showed that the expression of E-CAD in sihatw2-i(transfected with miR221inhibitor) group was significant higher than in sihatw2group, but the expression of N-CAD and vimentin, were significantly high; The expression of E-CAD in sihashtw2-m group was significant lower than in sihashtw2, but the expression of N-CAD and vimentin were significantly low.(2) Using the westernblot to test the expression of marker related to EMT (E-CAD、N-CAD、Vimentin) in cervical cancer cell linessih、sihatw2、sihashtw2、 sihatw2-i、sihashtw2-m. The result showed that the expression of E-CAD in sihashtw2-m group was significant lower than in sihashtw2, but the N-CAD and Vimentin was significantly high; contrarily, the expression of E-CAD in sihatw2-i group was significant higher than in sihatw2group, but the N-CAD and Vimentin was significantly low.(3) Using the scratch experiment to test the migration ability of siha、sihatw2、 sihashtw2、sihatw2-i、sihashtw2-m, the result showed that the migration ability in sihashtw2-m group was higer than sihashtw2group, but the migration ability in sihatw2-i group was lower than sihatw2group.(4) Using the Borden chamber to test the invasive ability in vitro of cervical cancer cell linessiha、sihatw2、sihashtw2、sihatw2-i、sihashtw2-m,the result showed that the invasive ability in vitro among5groups had significantly difference.the cells through the Borden chamber in sihashtw2-m group were much more than sihashtw2, but in sihatw2-i group, the cells through the Borden chamber were less than sihatw2.3.2Validation of the mode of action between the upstream transcription factor twist2and miRNA221(1) Through the database of Ensembl, acquiring the miR-221precursor sequences upstream of the2KB online as the miR-221promoter sequence.(2) Using the three software named Mapper, Consite and TRED to predict the transcription factor that can combined with the promoter of miRNA221, and comprehensive consideration of the study about invasion and metastasis of cervical cancer, we determined the twist2as the transcription factor.(3) Testing the luciferase activity between miR-221promoter sequence and twist2the transcription factor using the dual luciferase report analysis, the results show transfecting the PGL3-basic/221and pcDNA3.1/tw2at the same time, the luciferase activity in group tw2+221was significantly higher than221and Blank group, which illustrated that twist2can promote the transcriptional activity of miRNA221(F=130, P<0.001).CONCLUSIONS1Screening the8miRNAs related to twist2that was associated with invasion and metastasis of cervical cancer successfully, the differential expression of miRNA221was the most obvious using the cervical cancer cell lines siha, siha-tw2and siha-shtw2.2MiRNA221promotes the invasion and metastasis of cervical cancer through ARID1A.3Twist2promotes the expression of miRNA221in cervical cancer.4Proposed a new regulation mechanism, that is miRNA221regulated by transcriptional factor twist2promotes invasion and metastasis of cervical cancer through ARID1A and wnt/β-catenin signal pathway.THE INNOVATION OF THIS PROJECT1MiRNA221played an important role in multi-cancers, but there was no studies about the relationship between miRNA221and cervical cancer.2Proposes a new miR-221expression regulation mechanism, The topic has a very good original innovation and distinctive characteristics.3Clarify the mechanism of twist2/miR-221/ARID1A in the invasion and metastasis of cervical cancer, which will provide new gene and miRNA targets to the diagnosis, prognosis, and drug screening of cervical cancer. |
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