| Esophageal carcinoma?EC?is one of the most frequently occurring malignances worldwide.EC is divided into two main histological types,including esophageal squamous cell carcinoma?ESCC?and esophageal adenocarcinoma?ECA?.ESCC as a main histological type is more common in Africa,Iran and North China.Despite tremendous advances being made in therapeutic strategies,the 5-year survival rate for patients with ESCC remains markedly poor.At present,chemotherapy is an effective therapeutic approach for ESCC patients;however,the development of drug resistance or toxicity is the main cause of treatment failure in patients with ESCC.Therefore,it is imperative to elucidate the mechanisms responsible for drug resistance and to seek for novel diagnostic and prognostic markers for ESCC patients.MicroRNAs?mi RNAs or mi Rs?are a class of small non-coding RNAs comprising of 19-25 nucleotides in length,which regulate gene expression by targeting related genes.Ample evidence has revealed that mi RNAs are involved in a number of cellular processes,such as cell apoptosis,cell cycle,cell invasion and metastasis,the regulation of signaling networks and drug resistance,etc.Most notably,mi RNAs are tightly associated with tumor initiation,development and progression in a variety of tumors via the modulation of their target gene levels;hence,mi RNAs may function as either oncogenes or tumor suppressors in different types of tumors, and some mi RNAs have been verified to be early diagnostic marker for a myriad of tumors.Therefore,it is imperative to interpret the function of mi RNAs in the occurrence and development of a large number of tumors,which will seek for new molecular target for many tumors and display gigantic clinic value.MiRNA-125a-5p?mi R-125a-5p?,a type of newly discovered mi RNA molecule,has been verified to be involved in the development and progression of a number of tumors,including laryngeal cancer,hepatocellular carcinoma,lung cancer and prostate carcinoma.Besides,the tumor suppressive function of mi R-125a-5p has also been supported by a number of investigations on a variety of tumor types.Recent investigation has revealed that mi R-125 a has been shown to enhance the sensitivity of paclitaxel-resistant colon cancer cells to paclitaxel,suggesting that mi R-125 a may prove to be a novel ancillary drug for use in chemotherapy for patients with tumors.These data imply that mi R-125a-5p has tremendous potential for use in the diagnosis,treatment and prognosis of a wide range of tumors.However,the roles of mi R-125a-5p in occurrence and development of ESCC remain unclear.Therefore,in the current study,we firstly examined mi RNA-125a-5p expression in ESCC tissues and investigated its associations of its expression with clinicopathological features and prognosis.Further investigation elucidated the effects of mi R-125a-5p upregulation or downregulation on cell proliferation,cell cycle,apoptosis,migration and invasion abilities in vitro in ESCC cells,and examined epithelial?mesenchymal transition?EMT?-related protein expressions.Finally,the effects of mi R-125a-5p combined with cisplatin on cell proliferation,apoptosis,migration and invasion were detected and the targeted gene regulated by mi R-125a-5p was confirmed by the Dual Luciferase Reporter Experiment,which will preliminarily clarify the possible molecular mechanisms.The current study will lay a foundation for the therapy and chemotherapy of ESCC patients using mi R-125a-5p as molecular target.Chapter I Expression of micro RNA-125a-5p in esophageal squamous cell carcinoma tissues and its association with prognosisMethods 1.Real-time quantitative PCR was used to detect the expression of mi R-125a-5p in 56 cases of ESCC tissues and matched normal tissues.2.Statistical assay: Data were expressed as meansąSD,which were at least from three times independently repeats.Statistical assay was performed using Graph Pad Prism 6.0 software.The comparisons of two groups were determined using t test,and comparisons of three groups or above were investigated using One way ANOVA.Survival curve was made using Kaplan-Meier,and the statistical difference was investigated by Log-rank test.P<0.05 was considered significant.Results1.The expression of mi R-125a-5p in ESCC tissues was dramatically lower than that in normal tissues,which exhibited significantly statistical difference?P<0.0001?.2.The expression of mi R-125a-5p in ESCC tissues harboring III and IV staging was significantly lower than that in ESCC tissues having I and II staging,and there was statistical difference?P<0.01?;and the expression of mi R-125a-5p in ESCC patients with lymph node metastasis was markedly lower than that in ESCC patients without lymph node metastasis,and statistical difference was found?P<0.001?.3.The survival time of ESCC patients with high mi R-125a-5p expression was obviously longer than that of ESCC patients with low mi R-125a-5p expression,and there was statistical difference?P<0.05?.4.The expression of mi R-125a-5p was tightly correlated with TNM staging,histological grading and lymph node metastasis of ESCC patients?P<0.05?,but not related to the patients' age and gender?P>0.05?.5.The results of cox proportional hazards model revealed that clinical staging,lymph node metastasis and mi R-125a-5p were independent prognostic factors for ESCC patients.Chapter II The effects of micro RNA-125a-5p on cell biological behavior in esophageal squamous cell carcinoma and its molecular mechanismsMethods1.Real-time quantitative PCR was used to examine the expression of mi R-125a-5p in ESCC cells?Eca109,EC9706,EC1,TE1,KYSE450 and KYSE70?and normal esophageal epithelial cell Het-1A.2.NC,mi R-125a-5p mimic and inhibitor were employed to transfect into ESCC EC1 and TE1 cells,respectively,which was divided into 4 groups: control group,NC group,mi R-125a-5p mimic group and mi R-125a-5p inhibitor group.3.CCK-8 was used to detect the proliferation ability of different groups at 24 h,48h,72 h and 96 h,and Flow cytometry was utilized to investigate the cell cycle and apoptosis in different groups,respectively.4.Would healing assay and Transwell chamber were exploited to cell migration and invasion abilities in various groups,respectively.5.Western blot was used to detect EMT-related proteins E-cadherin,N-cadherin and Vimentin expressions.6.Statistical assay: All data was performed using Graph Pad Prism 6.0 software.Data were expressed as meansąSD.The comparisons of two groups were determined using t test,and comparisons of three groups or above were investigated using one way ANOVA.P<0.05 was considered significant.Results1.The expression of mi R-125a-5p in ESCC cells?Eca109,EC9706,EC1,TE1,KYSE450 and KYSE70?was markedly lower than that in normal esophageal epithelial cell Het-1A,in which EC1 and TE1 exhibited lowest level.2.Compared with control group and NC group,the expression of miR-125a-5p in ESCC EC1 and TE1 cells in mi R-125a-5p mimic group was significantly increased,and there was statistical significance?P<0.05?.However,the expression of mi R-125a-5p in mi R-125a-5p inhibitor group was obviously downregulated,and there was statistical significance?P<0.05?.3.Compared with control group and NC group,the proliferation of ESCC EC1 and TE1 cells in mi R-125a-5p mimic group was significantly suppressed,and statistical difference was found?P<0.05?.Conversely,the proliferation of ESCC EC1 and TE1 cells in mi R-125a-5p inhibitor group was obviously promoted,and there was statistical significance?P<0.05?.4.Compared with control group and NC group,cell number in G0/G1 phase or S phase in mi R-125a-5p mimic group was significantly increased or decreased in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.In contrast,cell number in G0/G1 phase or S phase in mi R-125a-5p inhibitor group was significantly reduced or increased in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.5.Compared with control group and NC group,apoptotic cell numbers in mi R-125a-5p mimic group were significantly enhanced in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.In contrast,apoptotic cell numbers in mi R-125a-5p inhibitor group were significantly reduced in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.6.Compared with control group and NC group,cell migration ability in mi R-125a-5p mimic group was significantly inhibited in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.In contrast,cell migration ability in mi R-125a-5p inhibitor group was significantly enhanced in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.7.Compared with control group and NC group,cell invasion ability in mi R-125a-5p mimic group was significantly inhibited in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.In contrast,cell invasion ability in mi R-125a-5p inhibitor group was significantly enhanced in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.8.Compared with control group and NC group,E-cadherin expression cell invasion ability in mi R-125a-5p mimic group was significantly inhibited in ESCC EC1 and TE1 cells,and there was statistical significance?P<0.05?.In contrast,cell invasion ability in mi R-125a-5p inhibitor group was significantly enhanced in ESCCEC1 and TE1 cells,and there was statistical significance?P<0.05?.Chapter III Micro RNA-125a-5p enhanced the sensitivity of cisplatin on esophageal squamous cell carcinoma cells and its molecular mechanismsMethods1.CCK-8 was used to detect the proliferation of ESCC EC1 and TE1 cells after treatment with cisplatin combined with NC or mi R-125a-5p mimic.Flow cytometry,would healing experiment and Transwell chamber were exploited to investigate the cell apoptosis,cell migration and invasion abilities in NC group,mi R-125a-5p mimic alone group,cisplatin alone group and mi R-125a-5p combined with cisplatin group.2.Western blot was employed to detect the expressions of EMT-related proteins E-cadherin,N-cadherin and Vimentin in NC group,mi R-125a-5p mimic alone group,cisplatin alone group and miR-125a-5p combined with cisplatin group.3.The targeted genes of mi R-125a-5p were searched using online softwares Target Scan?http://www.targetscan.org/vert72/?,mi Randa?http://www.microrna.org/ microrna/home.do?and mi RDB?http://www.mirdb.org/?.Dual-luciferase reporter gene assay confirmed the direct targeted gene of mi R-125a-5p.Western blot was used to detect the expressions of t-STAT3,p-STAT3 and VEGF proteins of EC1 and TE1 cells in control group,NC group and mi R-125a-mimic group as well as NC group,mi R-125a-mimic alone group,cisplatin alone group and mi R-125a-5p combined with cisplatin group.4.Western blot was utilized to investigate the expressions of t-STAT3,p-STAT3 and VEGF proteins in NC group,mi R-125a-5p combined with cisplatin group and mi R-125a-5p/cisplatin/IL-6 group.5.CCK-8,Flow cytometry and Transwell chamber were used to investigate cell proliferation,apoptosis and invasion ability in NC group,mi R-125a-5p combined with cisplatin group and mi R-125a-5p/cisplatin/IL-6 group,respectively.6.Statistical assay: All data was performed using GraphPad Prism 6.0 software.Data were expressed as meansąSD.The comparisons of two groups were determined using t test,and comparisons of three groups or above were investigated using one way ANOVA.P<0.05 was considered significant.Results1.Compared with NC transfection group,cell proliferation was signicantly suppressed in mi R-125a-5p mimic group after treatment with different concentration cisplatin.2.Cell apoptotic ratios of ESCC EC1 and TE1 cells in miR-125a-5p mimic alone group and cisplatin alone group were markedly higher than those in NC group,and there were statistical differences?P<0.05?.However,apoptotic cell numbers in ESCC EC1 and TE1 cells dramatically increased in mi R-125a-5p mimic combined with cisplatin group,and there were statistical differences?P<0.05?.3.Migrated distances of ESCC EC1 and TE1 cells in miR-125a-5p mimic alone group and cisplatin alone group were markedly lower than those in NC group,and there were statistical differences?P<0.05?.However,migrated distances in ESCC EC1 and TE1 cells dramatically decreased in mi R-125a-5p mimic combined with cisplatin group,and there were statistical differences?P<0.01?.4.Invasive cell numbers of ESCC EC1 and TE1 cells in miR-125a-5p mimic alone group and cisplatin alone group were markedly lower than those in NC group,and there were statistical differences?P<0.05?.However,invasive cell numbers in ESCC EC1 and TE1 cells dramatically decreased in mi R-125a-5p mimic combined with cisplatin group,and there were statistical differences?P<0.001?.5.Compared with NC group,the expression of E-cadherin was markedly increased and the expressions of N-cadherin and Vimentin were significantly reduced in mi R-125a-5p mimic alone group and cisplatin alone group,and there was statistical significance?P<0.05?.Most notably,the expression changes of E-cadherin,N-cadherin and Vimentin were most notably in mi R-125a-5p combined with cisplatin group,compared with NC group,there was statistical difference?P<0.05?.6.Compared with NC group,mi R-125a-5p transfection can reduce luciferase activity in ESCC EC1 and TE1 cells in wide type STAT3-3'UTR,but didn't alter luciferase activity in ESCC EC1 and TE1 cells in mutation STAT3-3'UTR.Compared with control group and NC group,the expressions of t-STAT3,p-STAT3 and downstream gene VEGF proteins were significantly downregulated after transfection with mi R-125a-5p mimic,and the differences were statistically significant?P<0.05?.7.The expressions of t-STAT3,p-STAT3 and downstream gene VEGF proteins were significantly downregulated in mi R-125a-5p mimic alone group and cisplatin alone group,compared with NC group,the difference was statistically significant?P<0.05?.Most notably,the downregulation of t-STAT3,p-STAT3 and downstream gene VEGF proteins was most notably,compared with NC group,the difference was statistically significant?P<0.05?.8.Compared with NC group,the expressions of t-STAT3,p-STAT3 and VEGF proteins were obviously downregulated in mi R-125a-5p mimic combined with cisplatin group,and there was statistical difference?P<0.05?.However,compared with mi R-125a-5p mimic combined with cisplatin group,the expressions of t-STAT3,p-STAT3 and VEGF proteins were obviously upregulated in mi R-125a-5p/ cisplatin/IL-6 group,and there was statistical difference?P<0.05?.9.Compared with NC group,the proliferation of ESCC EC1 and TE1 cells were markedly suppressed.However,compared with mi R-125a-5p mimic combined with cisplatin group,the proliferation ability of ESCC EC1 and TE1 cells were obviously enhanced in mi R-125a-5p/cisplatin/IL-6 group.10.Compared with NC group,the apoptotic cell numbers of ESCC EC1 and TE1 cells were obviously increased in mi R-125a-5p mimic combined with cisplatin group,and there was statistical difference?P<0.05?.However,compared with mi R-125a-5p mimic combined with cisplatin group,the apoptotic cell numbers of ESCC EC1 and TE1 cells were evidently reduced in mi R-125a-5p/cisplatin/IL-6 group,and there was statistical difference?P<0.05?.11.Compared with NC group,the invasion ability of ESCC EC1 and TE1 cells were obviously inhibited in mi R-125a-5p mimic combined with cisplatin group,and there was statistical difference?P<0.05?.However,compared with mi R-125a-5p mimic combined with cisplatin group,the invasive ability of ESCC EC1 and TE1 cells were evidently recovered in mi R-125a-5p/cisplatin/IL-6 group,and there was statistical difference?P<0.05?.Conclusion1.Mi R-125a-5p at low level in ESCC is tightly associated with occurrence and development of ESCC,and may be a vital molecular marker of clinical staging,metastasis and prognosis judgement for ESCC patients.Clinical staging,lymph node metastasis and mi R-125a-5p is independent prognostic factors for ESCC patients.2.The alterations of mi R-125a-5p expression significantly change tumor-related biological feature including proliferation,cell cycle,apoptosis,migration and invasion,which may be tightly correlated with the changes of EMT-related signaling pathway.3.Mi R-125a-5p markedly enhances the suppressive role of cisplatin on ESCC cells,apoptosis and inhibition of migration and invasion,which is closely related to the suppression of EMT signaling pathway.4.STAT3 is a direct target gene of mi R-125a-5p,and mi R-125a-5p combined with cisplatin significantly inactivates STAT3 signaling pathway.IL-6 reverses the inactivation of STAT3 signaling pathway triggered by mi R-125a-5p combined with cisplatin.STAT3 reactivation evoked by IL-6 dramatically promotes cell proliferation,suppresses apoptosis and enhances invasion ability. |
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