The Mechanism Of Circular RNA Circ-CCDC85a Affecting The Biological Behavior Of Breast Cancer Through Regulation Of IL6-R And STAT3

Part 1 The expression of circ-CCDC85 A in esophageal squamous cell carcinoma tissues and cells and the effect on its biological behaviorObjective:To investigate the expression of circular RNA circ-CCDC85 A in a breast cancer tissues and cells and its effects on proliferation,migration and invasion of breast cancer cells.Methods:1.The expression of circ-CCDC85 A in breast cancer tissues and cells were detected by RT-PCR and q RT-PCR.2.Sanger sequencing was used to identify the junction site of circ-CCDC85 A.3.The RNase R assay was used to verify the characteristics of circ-CCDC85 A in breast cancer cells.4.The effects of over-expression or knockdown of circ-CCDC85 A on the proliferation,migration and invasion of breast cancer 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 c DNA and g DNA of breast cancer tissues and cells were used as templates,and the circular transcript could only be amplified in c DNA by divergent primers,while the linear transcript could be amplified in both c DNA and g DNA by convergent primers.q RT-PCR results showed that the expression level of circ-CCDC85 A was significantly down-regulated in breast cancer tissues compared with adjacent normal tissues(P<0.05).2.Sanger sequencing results of the PCR product verified the junction site of circ-CCDC85 A.3.RNase R results showed that circ-CCDC85 A 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 over-expression of circ-CCDC85 A inhibited proliferation of MDA-MB-231 cells(P<0.05),while knockdown of circ-CCDC85 A enhanced proliferation of MCF-7 cells(P<0.05).The results of wound healing assay and transwell assay showed that over-expression of circ-CCDC85 A inhibited migration and invasion of MDA-MB-231 cells(P<0.05),while knockdown of circ-CCDC85 A enhanced migration and invasion of MCF-7 cells(P<0.05).Summary:1.The expression of circ-CCDC85 A in tumor tissues of breast cancer patients was significantly lower than that in adjacent normal tissues.2.Over-expression of circ-CCDC85 A could inhibit proliferation,migration and invasion of breast cancer cells;knockdown of circ-CCDC85 A could enhance proliferation,migration and invasion of breast cancer cells.Part 2 Study on the regulation mechanism of circ-CCDC85 A on IL-6R and STAT3 in breast cancerObjective: To investigate the potential mechanism by which circ-CCDC85 A acts as a mi RNA reservoir to regulate the expression of its downstream target gene IL-6R and STAT3 and then inhibiting cancer progression in breast cancer.Method:1.Miranda and Circ Net online databases were used to predict mi RNAs that might interact with circ-CCDC85 A and their binding sites.2.q RT-PCR was used to detect the expression of mi R-660-3p gene after over-expression or knockdown of circ-CCDC85 A in breast cancer cells.3.RIP and Actinomycin D assay were used to verify whether circ-CCDC85 A could interact with mi R-660-3p.4.The effects of over-expression or knockdown of mi R-660-3p on the proliferation,migration and invasion of breast cancer cells were examined by CCK-8 assay,plate colony formation assay,wound healing assay and transwell assay.5.The effects of over-expression of circ-CCDC85 A and knockdown of mi R-660-3p on breast cancer cell proliferation,migration and invasion were examined by rescue experiments.6.Target Scan online database was used to predict target genes of mi R-660-3p.7.Dual luciferase reporter gene assay was used to verify whether mi R-660-3p could interact with IL-6R and STAT3.8.q RT-PCR was used to detect the expression of IL-6R and STAT3 m RNA after over-expression of circ-CCDC85 A and knockdown of mi R-660-3p in breast cancer cells.9.Western blot was used to detect the expression of IL-6R and STAT3 protein in over-expression of circ-CCDC85 A and knockdown of mi R-660-3p in breast cancer cells.Results:1.Miranda online database analysis showed that there are two mi R-660-3p binding sites in circ-CCDC85 A.Circ Net online database prediction results showed that circ-CCDC85 A may interact with mi R-660-3p.2.q RT-PCR results showed that the expression level of mi R-660-3p gene in MDA-MB-231 cells was significantly elevated after over-expression of circ-CCDC85A(P<0.05),while the expression level of mi R-660-3p gene in MCF-7 cells was significantly decreased after knockdown of circ-CCDC85A(P<0.05).3.RIP assay results showed that circ-CCDC85 A could interact with mi R-660-3p in breast cancer cells(P<0.05).4.Actinomycin D assay results showed that circ-CCDC85 A could affect the stability of mi R-660-3p gene in breast cancer cells(P<0.05).5.CCK-8 assay and plate colony formation assay showed that over-expression of mi R-660-3p inhibited proliferation of MDA-MB-231 cells(P<0.05);knockdown of mi R-660-3p enhanced proliferation of BT-549 cells(P<0.05).The results of wound healing assay and transwell assay showed that over-expression of mi R-660-3p inhibited migration and invasion of MDA-MB-231 cells(P<0.05).Knockdown of mi R-660-3p enhanced migration and invasion of BT-549 cells(P<0.05).6.Rescue experiments showed that over-expression of circ-CCDC85 A inhibited proliferation,migration and invasion of breast cancer cells(P<0.05),while supplementation with knockdown of mi R-660-3p partially attenuated reduced proliferation,migration and invasion capacity of breast cancer cells induced by over-expression of circ-CCDC85A(P<0.05).7.Target Scan online database prediction results showed that mi R-660-3p habors binding sites with the 3'UTR of IL-6R and STAT3.8.Dual luciferase reporter gene assay showed that mi R-660-3p could affect the activity of IL-6R and STAT3 gene(P<0.05).9.q RT-PCR results showed that the expression level of IL-6R and STAT3 m RNA in MDA-MB-231 cells was not significantly changed after over-expression of circ-CCDC85A(P>0.05)or knockdown of mi R-660-3p(P>0.05),while the expression of IL-6R and STAT3 m RNA was not significantly changed after over-expression of circ-CCDC85 A and knockdown of mi R-660-3p simultaneously(P>0.05).10.Western blot analysis showed that the expression of IL-6R and STAT3 protein in MDA-MB-231 cells was significantly decreased after over-expression of circ-CCDC85A(P<0.05),and the expression of IL-6R and STAT3 protein in MDA-MB-231 cells was significantly increased after knockdown of mi R-660-3p(P<0.05),while the expression of IL-6R and STAT3 protein was significantly recovered after over-expression of circ-CCDC85 A and knockdown of mi R-660-3p simultaneously(P<0.05).Summary:1.circ-CCDC85 A binds to mi R-660-3p in breast cancer cells,and Over-expression of circ-CCDC85 A can promoted the expression of mi R-660-3p gene,and knockdown of circ-CCDC85 A can inhibited the expression of mi R-660-3p.2.Over-expression of mi R-660-3p can inhibit proliferation,migration and invasion of breast cancer cells;knockdown of mi R-660-3p can enhance proliferation,migration and invasion of breast cancer cells.Furthermore,over-expression of mi R-660-3p antagonized the promoted effect of knockdown of circ-CCDC85 A on breast cancer cell proliferation,migration and invasion.3.mi R-660-3p directly targets IL-6R and STAT3,and at least partially inhibit the expression of interleukin 6 receptor and STAT3 to exert cancer suppressive effect.4.over-expression of mi R-660-3p can effectively reverse the up-regulation of IL-6R and STAT3 expression mediated by knockdown of circ-CCDC85 A at the protein levels.Conclusion:The circular RNA circ-CCDC85 A is down-regulated in breast cancer tissues and inhibits the proliferation,migration and invasion ability of breast cancer cells.In addition,circ-CCDC85 A could promote the targeting regulation of mi R-660-3p to IL-6R and STAT3 through acting as a mi R-660-3p reservoir,thereby exerting its Inhibitory function in breast cancer.Our study provides new evidence for circ-CCDC85 A as a “mi RNA reservoir” and a novel potential biomarkers and therapeutic target for breast cancer.