| Part 1 The expression of ciRS-7 in esophageal squamous cell carcinoma tissues and cells and the effect on its biological behaviorObjective: To investigate the expression of circular RNA ciRS-7 in esophageal squamous cell carcinoma(ESCC)tissues and cells and its effects on proliferation,migration and invasion of ESCC cells.Methods:1.The expression of ciRS-7 in ESCC tissues and cells was detected by RT-PCR and qRT-PCR.2.Sanger sequencing was used to identify the junction site of ciRS-7.3.The RNase R assay was used to verify the characteristics of ciRS-7 in ESCC cells.4.The effects of over-expression or knockdown of ciRS-7 on the proliferation,migration and invasion of ESCC cells were examined by CCK-8 assay,plate colony formation assay,wound healing assay and transwell assay.Results:1.RT-PCR results showed that the cDNA and gDNA of ESCC tissues and cells were used as templates,and the circular transcript could only be amplified in cDNA by divergent primers,while the linear transcript could be amplified in both cDNA and gDNA by convergent primers.qRT-PCR results showed that the expression level of ciRS-7 was significantly up-regulated in ESCC tissues compared with adjacent normal tissues(P<0.05).2.Sanger sequencing results of the PCR product verified the junction site of ciRS-7.3.RNase R results showed that ciRS-7 was more tolerant to RNase R digestion than its linear transcript(P<0.05).4.CCK-8 assay and plate colony formation assay showed that overexpression of ciRS-7 enhanced proliferation of TE13 cells(P<0.05),while knockdown of ciRS-7 inhibited proliferation of TE1 cells(P<0.05).The results of wound healing assay and transwell assay showed that overexpression of ciRS-7 enhanced migration and invasion of TE13 cells(P<0.05),while knockdown of ciRS-7 inhibited migration and invasion of TE1 cells(P<0.05).Summary:1.The expression of ciRS-7 in tumor tissues of ESCC patients was significantly higher than that in adjacent normal tissues,and the expression level of ciRS-7 was significantly correlated with pathological grade and clinical stage of ESCC patients.2.Over-expression of ciRS-7 could enhance proliferation,migration and invasion of ESCC cells;knockdown of ciRS-7 could inhibit proliferation,migration and invasion of ESCC cells.Part 2 Study on the regulation mechanism of ciRS-7 on tumor antigen MAGE-A family in esophageal squamous cell carcinomaObjective: To investigate the potential mechanism by which ciRS-7 acts as a miRNA sponge to regulate the expression of its downstream target gene MAGE-A family and then promoting cancer progression in ESCC.Method:1.StarBase and CircNet online databases were used to predict miRNAs that might interact with ciRS-7 and their binding sites.2.RIP and dual luciferase reporter gene assay were used to verify whether ciRS-7 could interact with miR-876-5p.3.The effects of over-expression or knockdown of miR-876-5p on the proliferation,migration and invasion of ESCC cells were examined by CCK-8 assay,plate colony formation assay,wound healing assay and transwell assay.4.The effects of over-expression of ciRS-7 and miR-876-5p on ESCC cell proliferation,migration and invasion were examined by rescue experiments.5.TargetScan online database was used to predict target genes of miR-876-5p.6.Dual luciferase reporter gene assay was used to verify whether miR-876-5p could interact with MAGE-A family.7.qRT-PCR was used to detect the expression of MAGE-A mRNA after over-expression of miR-876-5p and ciRS-7 in ESCC cells.8.Western blot was used to detect the expression of MAGE-A protein in over-expressed miR-876-5p and ciRS-7 in ESCC cells.9.The effects of knockdown of MAGE-A gene on proliferation,migration and invasion of TE1 cells were detected by CCK-8 assay,plate colony formation assay,wound healing assay and transwell assay.10.Immunohistochemical staining was used to detect the expression of MAGE-A protein in 112 cases of primary ESCC tissues,244 cases of lymph node tissues and 26 cases of distant metastatic tissues.Results:1.StarBase online database analysis showed that there are multiple miR-876-5p binding sites in ciRS-7.CircNet online database prediction results showed that ciRS-7 may act as a miR-876-5p sponge.2.RIP and dual luciferase reporter gene assay results showed that ciRS-7 could interact with miR-876-5p in ESCC cells(P<0.05).3.CCK-8 assay and plate colony formation assay showed that overexpression of miR-876-5p inhibited proliferation of TE1 cells(P<0.05);knockdown of miR-876-5p enhanced proliferation of Eca109 cells(P<0.05).The results of wound healing assay and transwell assay showed that overexpression of miR-876-5p inhibited migration and invasion of TE1 cells(P<0.05).Knockdown of miR-876-5p enhanced migration and invasion of Eca109 cells(P<0.05).4.Rescue experiments showed that over-expression of ciRS-7 enhanced proliferation,migration and invasion of ESCC cells(P<0.05),while supplementation with miR-876-5p partially attenuated increased proliferation,migration and invasion capacity of ESCC cells induced by over-expression of ciRS-7(P<0.05).5.TargetScan online database prediction results showed that miR-876-5p habors binding sites with the 3'UTR of multiple members of tumor antigen MAGE-A family.6.Dual luciferase reporter gene assay showed that miR-876-5p could interact with multiple members of MAGE-A family(P<0.05).7.qRT-PCR results showed that the expression level of MAGE-A mRNA in TE1 and KYSE30 cells was significantly decreased after over-expression of miR-876-5p(P<0.05),while the expression of MAGE-A mRNA was significantly recovered after over-expression of miR-876-5p and ciRS-7 simultaneously(P<0.05).8.Western blot analysis showed that the expression of MAGE-A protein in TE1 and KYSE30 cells was significantly decreased after over-expression of miR-876-5p(P<0.05),while the expression of MAGE-A protein was significantly recovered after over-expression of miR-876-5p and ciRS-7 simultaneously(P<0.05).9.CCK-8 assay and plate colony formation assay showed that knockdown of MAGE-A gene inhibited proliferation of TE1 cells(P<0.05).Wound healing assay and transwell assay showed that knockdown of MAGEA gene inhibited migration and invasion of TE1 cells(P<0.05).10.Immunohistochemical staining results showed that MAGE-A protein was highly expressed in primary ESCC tissues,metastatic lymph node tissues and distant metastatic tissues,and its expression level was significantly associated with high pathological grade,lymph node metastasis,distant metastasis or recurrence,and poor prognosis of ESCC patients(P<0.05).Summary:1.ciRS-7 binds to miR-876-5p in TE1 cells,and miR-876-5p inhibits the luciferase activity of ciRS-7.2.Over-expression of miR-876-5p can inhibit proliferation,migration and invasion of ESCC cells;knockdown of miR-876-5p can enhance proliferation,migration and invasion of ESCC cells.Furthermore,over-expression of ciRS-7 antagonized the inhibitory effect of miR-876-5p on ESCC cell proliferation,migration and invasion.3.Tumor antigen MAGE-A family is highly expressed in ESCC tissues,metastatic lymph node tissues and distant metastatic tissues,and its expression level is associated with high pathological grade,lymph node metastasis,distant metastasis or recurrence,and poor prognosis in ESCC patients.Knockdown of MAGE-A gene in TE1 cells inhibits proliferation,migration and invasion.4.miR-876-5p directly targets tumor antigen MAGE-A family and controls proliferation,migration and invasion of ESCC cells,at least in part,by inhibiting the expression of MAGE-A family.5.ciRS-7 can effectively reverse the down-regulation of miR-876-5pmediated tumor antigen MAGE-A family expression at the mRNA and protein levels.Moreover,ciRS-7 reversed the inhibitory effect of miR-876-5p on MAGE-A gene in ESCC cells in a miR-876-5p dose-dependent manner.Part 3 In vivo study on the mechanism of ciRS-7 function in esophageal squamous cell carcinomaObjective: To verify the mechanism of ciRS-7 function in esophageal squamous cell carcinoma in vivo.Methods:1.The expression correlation of ciRS-7,miR-876-5p and MAGE-A family in 86 cases of ESCC tissues was analyzed by qRT-PCR.2.TE1 cell line stably over-expressing ciRS-7 was constructed and subcutaneously inoculated after co-transfection of miR-876-5p agomir to establish a nude mouse xenograft model.After the tumor grew,intratumoral injection of miR-876-5p agomir was performed.The growth of transplanted tumors in each group was observed.3.Immunohistochemical staining was used to detect the expression of MAGE-A,Ki67,PCNA,MMP2 and MMP9 proteins in transplanted tumor tissues of nude mice.Results:1.qRT-PCR results showed that the expression level of ciRS-7 in ESCC tissues was significantly negatively correlated with the expression level of miR-876-5p(P<0.05);the expression level of miR-876-5p was significantly negatively correlated with the expression level of MAGE-A gene(P<0.05);the expression level of ciRS-7 was significantly positively correlated with the expression level of MAGE-A gene(P<0.05).2.Compared with control group,the volume and weight of subcutaneous xenografts in ciRS-7 over-expressing group were significantly increased(P<0.05),and the volume and weight of subcutaneous xenografts in miR-876-5p over-expressing group were significantly decreased(P<0.05),while the volume and weight of subcutaneous xenografts in over-expressing ciRS-7 and miR-876-5p simultaneously group were significantly restored.3.Immunohistochemical staining results showed that the expression levels of MAGE-A,Ki67,PCNA,MMP2 and MMP9 proteins in transplanted tumor tissues of ciRS-7 over-expressing group were significantly higher than those in control group(P<0.05),and the expression levels of MAGE-A,Ki67,PCNA,MMP2 and MMP9 proteins in subcutaneous xenografts of miR-876-5p over-expressing group were significantly lower than those in control group(P<0.05),while the expression levels of MAGE-A,Ki67,PCNA,MMP2 and MMP9 proteins in subcutaneous xenografts of over-expressing ciRS-7 and miR-876-5p simultaneously group were significantly restored.Summary:1.The expression level of ciRS-7 in ESCC tissues was significantly negatively correlated with the expression level of miR-876-5p.The expression level of miR-876-5p was significantly negatively correlated with the expression level of MAGE-A gene.The expression level of ciRS-7 was significantly positively correlated with the expression level of MAGE-A gene.2.ciRS-7 can promote the growth and metastasis of xenograft tumors in nude mice and the expression of tumor antigen MAGE-A family.miR-876-5p can inhibit the growth and metastasis of xenograft tumors in nude mice and the expression of tumor antigen MAGE-A family.Moreover,over-expression of ciRS-7 partially reversed the inhibitory effect of miR-876-5p on xenograft tumor growth and metastasis,and tumor antigen MAGE-A family expression in nude mice.Conclusion: The circular RNA ciRS-7 is up-regulated in ESCC tissues and promotes the proliferation,migration and invasion ability of ESCC cells.In addition,ciRS-7 could inhibit the targeting regulation of miR-876-5p to tumor antigen MAGE-A family through acting as a miR-876-5p sponge,thereby exerting its oncogenic function in ESCC.Our study provides new evidence for ciRS-7 as a “miRNA sponge” and a novel potential therapeutic target for ESCC. |
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