| Background and ObjectiveEsophageal squamous cell carcinoma(ESCC)comprises the majority of esophageal cancer with both high morbidity and high mortality worldwide,especially in China.Although ESCC always originates and is located in the narrow esophageal mucosa with relatively simple pathological types,the general outcome is still poor due to obscure symptom,late presentation and early metastasis.Therefore,further exploration of molecular mechanisms of ESCC progression and metastasis,screening new biomarkers for diagnosis and prognosis is necessary in ESCC to provide guidance for clinical individual therapy.MicroRNAs(miRNAs)are a class of small noncoding RNAs that function as key regulators of gene expression at the post-transcriptional level.Accumulating evidences have revealed that miRNAs play important roles in various human biological and pathological processes,including cell differentiation,proliferation,apoptosis,and carcinogenesis.The purpose of this study was to investigate the effect of miRNAs on ESCC progression and development via in vitro and in vivo models and to elucidate the biological roles of miR-143-3p on ESCC progression through aspects such as cell proliferation,apoptosis,invasion and metastasis,with the aim of better understanding the molecular mechanisms of ESCC and laying a foundation for formulating novel therapeutic target and prognosis factor for treatment of ESCC.Materials,Methods and ResultsPart Ⅰ.Screening and Identification of MiRNAs Profiles that Differentially Expressed in ESCC Tissues and CellsMaterials and Methods1、MiRNA microarray analysis was performed to investigate the difference of miRNA profiles between five pairs of ESCC tumor tissues and adjacent normal tissues.2、MiR-143-3p,miR-133b,miR-143-5p were selected for further study.Real-time RT-PCR analysis was performed to detect the expression of these miRNAs in a panel of human ESCC cell lines(Kyse30,Kyse70,Eca109,and Ec9706)and the human normal esophageal epithelial cell line HEEC.3、MiR-143-3p was selected for further study.Real-time RT-PCR analysis was performed to detect the expression of miR-143-3p in 80 primary ESCC tissues and corresponding normal tissues.4、The correlations between miR-143-3p expression levels and the clinicopathological factors as well as prognosis were analysed in the whole 80 ESCC clinical tissues.Results1、Based on miRNA microarray data,28 differently expressed miRNAs were detected in ESCC tumor tissues compared with their normal tissues with the fifty fold change cut-off point,including 21 highly expressed miRNAs and 7 lowly expressed miRNAs.2、Consistent with the microarray results,miR-143-3p exhibited the largest absolute foldchange in ESCC cell lines(p<0.01).3、It was observed that miR-143-3p expression level was significantly downregulated in ESCC tissues in comparison with corresponding normal tissues(p<0.01).4、In 80 cases of paired ESCC tissues,miR-143-3p expression level was associated with clinicopathological characteristics including TNM((p=0.026).)stage and lymph node metastasis(p=0.038).The Kaplan-Meier survival analysis indicated that the reduced expression of miR-143-3p was significantly correlated with shorter OS in ESCC patients.The univariate and multivariate analysis revealed that miR-143-3p expression functioned as an independent prognostic factor for ESCC patients.Part Ⅱ.The Function of MiR-143-3p on Biological Behaviors of ESCC Cells Materials and Methods1、The plasmids of miR-143-3p,anti-miR-143-3p and their corresponding controls were constructed and introduced into ESCC cell lines through stable transfection.MTT assay,colony formation assay were performed to detect the in vitro cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis status.Wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities.2、The plasmid of miR-143-3p and the corresponding control were stably transfected and inoculated subcutaneously into nude mice.Tumor diameters were examined every other day.Tumor volule was calculated using the equation:V=A×B2/2(mm3).The primary tumors were excised and analyzed by immunohistochemistry and TUNEL staining.Results1、MiR-143-3p inhibited proliferation,migration and invasion in ESCC cells.Conversely,downregulation of miR-143-3p could promote ESCC cell proliferation,inhibit apoptosis,and enhance the capacity of cell migration and invasion in vitro.2、In the animal model,miR-143-3p significantly reduced tumor growth rate,tumor volume,and average tumor weight in mice.TUNEL staining revealed that miR-143-3p expression led to an increased apoptotic rate in the tissues of tumor-bearing mice.Part Ⅲ.The Prediction and Biological Behaviors Identification of the Target Gene for miR-143-3pMaterials and Methods1、Four different online miRNA databases(TargetScanHuman 6.0,PicTar,Microrna.org,and PITA)were employed for prediction of miR-143-3p target genes.2、QKI gene was chosen as a preferred candidate target gene of miR-143-3p.Real-time RT-PCR analysis was performed to detect the expression of QKI mRNA in a panel of human ESCC cell lines(Kyse30,Kyse70,Eca109,and Ec9706)and the human normal esophageal epithelial cell line HEEC.3、Real-time RT-PCR analysis and immunohistochemistry were performed to detect the expression of QKI mRNA and QKI protein in 80 primary ESCC tissues and corresponding normal tissues.The correlation between QKI expression level and miR-143-3p expression level was analyzed.4、Dual luciferase activity assay was used to further confirm that QKI gene was the direct target of miR-143-3p.5、Real-time RT-PCR analysis was performed to identify the alternative spliced QKI protein(QKI-5,QKI-6,and QKI-7)that was important for miR-143-3p regulation effect.6、The plasmids of pLenti QKI-5,pLenti siQKI-5 and their corresponding controls were constructed and introduced into ESCC cell lines through stable transfection.MTT assay,colony formation assay were performed to detect cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis status.Wound healing assays and transwell assays with or without Matrigel were performed to detect the migration and invasion abilities in vitro.7、The above stably transfected cells were inoculated subcutaneously into nude mice.Tumor diameters were examined every other day.Tumor volume was calculated using the equation:V=A×B2/2(mm3).The primary tumors were excised and analyzed by immunohistochemistry and TUNEL staining.Results1、The QKI gene was bioinformatically chosen as a preferred candidate target gene of miR-143-3p.2、In ESCC cell lines,QKI mRNA were obviously upregulated compared with the human normal esophageal epithelial cell line HEEC.3、In clinical tissues samples,it was observed that the relative levels of QKI mRNA and protein were significantly upregulted,Through linear regression analysis,it was observed that there was a significant inverse association between the expression of miR-143-3p and that of QKI mRNA.4、The dual luciferase activity assay confirmed that QKI was the direct target of miR-143-3p.5、It was found that miR-143-3p overexpression markedly downregulated QKI-5 mRNA expression,whereas miR-143-3p downregulation increased QKI-5 mRNA expression,which indicating that the tumor suppressive effect of miR-143-3p was via suppressing QKI-5 expression.6、Downregulation of QKI-5 inhibited proliferation,migration and invasion in ESCC cells.Conversely,overexpression of QKI-5 could promote ESCC cell proliferation,inhibit apoptosis,and enhance the capacity of cell migration and invasion in vitro.7、In the animal model,downregulation of QKI-5 significantly reduced tumor growth rate,tumor volume in mice,whereas overexpression of QKI-5 expression significantly increased tumor growth rate and tumor volume.TUNEL staining revealed that downregulation of QKI-5 expression increased apoptotic rate in transfected tumor-bearing mice,while overexpression of QKI-5 expression revealed the opposite results.Part Ⅳ.The Analysis of Molecular Mechanisms of miR-143-3p in Regulating Proliferation,Invasion and Metastasis of ESCCMaterials and Methods1、Kyse70 cell stably transfected with miR-143-3p was transfected with pLenti QKI-5 to restore the expression of QKI-5.MTT assay,colony formation assay were performed to detect cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis rate.Wound healing assays and transwell assays were performed to detect the migration and invasion abilities in vitro.2、The above stably transfected cells were inoculated subcutaneously into nude mice.Tumor diameters were examined every other day.Tumor volume was calculated using the equation:V=A×B2/2(mm3).The primary tumors were excised and analyzed by immunohistochemistry and TUNEL staining.3、Immunohistochemical staining and Western blotting assays were performed to analyze the effect of miR-143-3p on the expression of epithelial and mesenchymal molecular makers in ESCC cells and tumor tissues from the xenograft tumors.Results1、Ectopic QKI-5 expression could rescue the decreased expression of QKI-5 in Kyse70 cell transfected with miR-143-3p.The proliferation ability of miR-143-3p cells was partially rescued after transfection of pLenti QKI-5.Meanwhile,the migratory and invasive capacity of miR-143-3p cells was also partially rescued.2、Restoration of QKI-5 significantly reversed the suppression of tumor growth induced by miR-143-3p in nude mice models.TUNEL analyses also displayed attenuated apoptosis in the group enforced the expression QKI-5 of miR-143-3p mice.3、MiR-143-3p could significantly increase the expression of epithelial markers(E-cadherin and β-catenin)and decrease the expression of mesenchymal markers(N-cadherin and Vimentin).Meanwhile,knockdown the expression of QKI-5 could mimic the effects of miR-143-3p overexpression on epithelial and mesenchymal markers transition.It was suggested that reexpression of QKI-5 could partially rescue miR-143-3p-mediated EMT transition in ESCC cells.Conclusion1、MiR-143-3p was significantly downregulated in ESCC tissues and cell lines.Downregulation of miR-143-3p was correlated with TNM stage,lymph node metastasis and overall sruvival in ESCC patients.2、MiR-143-3p functioned as a tumor suppressor in ESCC cells,causing cell proliferation suppression,apoptosis enhancement,invasion and metastasis inhibition and EMT reversion in ESCC cells.3、QKI gene is the potential target of miR-143-3p,and the overexpression of QKI in ESCC tissues exhibited a significant inverse association with the expression of miR-143-3p.4、QKI-5 could increase cell proliferation,inhibit cell apoptosis,promote cell invasion and EMT transition.In conclusion,this study is for the first to show the dysregulation of miR-143-3p/QKI-5 axis in ESCC and its effects on tumor proliferation,invasion,metastasis and EMT process.This study uncovers the mechanisms of miR-143-3p dysregulation in ESCC at the post-transcriptional level,indicating the posiblity of targeting miR-143-3p/QKI-5 axis to prevent and reverse the ESCC progression and metastasis in clinic. |
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