| Part One Expression and clinical significance of circ＿0005231 in esophageal squamous cell carcinoma(ESCC)Objective: To investigate the expression of circ＿0005231 in blood samples and tissue samples of patients with ESCC and its significance in the diagnosis and prognosis of ESCC.Methods:1.The differentially expressed circ RNAs in ESCC were screened by analyzing the ESCC-related circ RNA chip data(GSE112496 and GSE131969)in the GEO database.2.Real-time quantitative real-time PCR(q RT-PCR)experiments were used to detect the expression of the screened circ RNAs in ESCC tumor tissues and adjacent normal tissues,and further clarify the target circ RNAs.3.The expression of circ＿0005231 in the blood samples of 22 healthy volunteers and 40 patients with ESCC,as well as the tumor tissues and adjacent normal tissues of 40 patients with ESCC,were detected by q RT-PCR.4.The diagnostic value of blood cir C＿0005231 expression in patients with ESCC was analyzed by receiver operating characteristic(ROC)curve.5.Kaplan-Meier(K-M)survival curve analysis of cir C＿0005231expression on the prognosis of patients with ESCC.Results:1.Analysis of ESCC-related circ RNA microarray data(GSE112496 and GSE131969)in the GEO database showed that circ＿0005939,circ＿0000219,circ＿0005231,circ＿0005379 and circ＿0026512 were differentially expressed in ESCC blood and tumor tissues.2.q RT-PCR detected the expression of the above five differentially expressed circ RNAs in ESCC tumor tissues and adjacent normal tissues,and the results showed that circ＿0005231 was most highly expressed in ESCC tumor tissues.3.The expression of circ＿0005231 in blood samples and tissue samples was detected by q RT-PCR,and the results showed that circ＿0005231 was highly expressed in both blood samples and tumor tissue samples of ESCC patients.4.The ROC curve analysis showed that the area under the curve AUC of circ＿0005231 expression in blood to diagnose ESCC was 0.870(95%CI:0.782-0.958).5.K-M survival curve analysis showed that high expression of circ＿0005231 was associated with poorer survival in ESCC patients.Summarys:1.circ＿0005231 was highly expressed in the blood and tumor tissues of ESCC patients.2.circ＿0005231 might be a potential biomarker for ESCC diagnosis and prognosis prediction.Part Two Knockdown of circ＿0005231 inhibits the proliferation,migra-tion and invasion of ESCC cellsObjective: To investigate the effect of circ＿0005231 expression loss on the proliferation,migration and invasion of ESCC cells.Methods:1.ESCC cells TE-1 and Eca-109 were digested with RNase R,and the expression of circ＿0005231 was detected by q RT-PCR.2.Cytoplasmic and nuclear RNAs of TE-1 and Eca-109 cells were isolated,and the expression of circ＿0005231 was detected by q RT-PCR.3.ESCC cells TE-1 and Eca-109 were transfected with sh RNA targeting circ＿0005231(sh-circ＿0005231)and nonsense sh RNA(sh-NC),respectively,and the expression of circ＿0005231 was detected by q RT-PCR.4.The effect of circ＿0005231 knockdown on the proliferation of ESCC cells was detected by colony formation and Ed U staining experiments.5.The effect of circ＿0005231 knockdown on the migration ability of ESCC cells was detected by scratch assay.6.Transwell assay was used to detect the effect of circ＿0005231knockdown on the invasion ability of ESCC cells.7.Western blot assay to detect the effect of circ＿0005231 knockdown on epithelial-mesenchymal transformation(EMT)of ESCC cells.Results:1.circ＿0005231 was formed by back splicing of exons 5-7 of the parental gene EIF3 H.2.After digestion with RNase R,the results of q RT-PCR showed that the expression of circ＿0005231 was not affected by RNase R.3.The results of q RT-PCR showed that the expression level of circ＿0005231 was decreased in TE-1 and Eca-109 cells transfected with sh-circ＿0005231.4.The results of clone formation experiments showed that the clone formation ability of TE-1 and Eca-109 cells decreased after knockdown of circ＿0005231.5.The results of Ed U staining experiments showed that the Ed U positive rate of TE-1 and Eca-109 cells decreased after knockdown of circ＿0005231.6.The results of scratch experiments showed that the migration ability of TE-1 and Eca-109 cells was reduced after knockdown of circ＿0005231.7.The results of Transwell laboratory experiments showed that the invasive ability of TE-1 and Eca-109 cells was reduced after knockdown of circ＿0005231.8.The results of Western blot experiments showed that the expression of E-cadherin was decreased in TE-1 and Eca-109 cells after knockdown of circ＿0005231,while the expression of N-cadherin was increased.Summarys:1.circ＿0005231 was not affected by RNase R and was stably expressed in ESCC cells.2.Knockdown of circ＿0005231 could inhibit the proliferation,migration,invasion and EMT of ESCC cells,suggesting that circ＿0005231 may be an important regulator in ESCC progression.Part Three Circ＿0005231 regulates the malignant biological behavior of ESCC cells by targeting mi R-383-5p/KIAA0101 axisObjective: To explore the potential molecular mechanism of circ＿0005231 regulating the malignant biological behavior of ESCC cells.Methods:1.mi RNAs with targeted complementary binding sites to circ＿0005231were analyzed using the online bioinformatics databases circbank,starbase and circinteractome.2.Ago2 RIP,mi R-383-5p pull down experiments and dual luciferase reporter gene experiments were used to analyze the targeting relationship between circ＿0005231 and mi R-383-5p.3.The expression of mi R-383-5p in ESCC tumor tissue and adjacent non-tumor tissue,as well as in ESCC cells,were detected by q RT-PCR.4.TE-1 and Eca-109 cells were co-transfected with mi R-383-5p inhibitor or mi RNA inhibitor with sh-NC or sh-circ＿0005231,respectively.and the proliferation,migration,invasion and EMT of cells were detected by colony formation,Ed U staining,scratching,Transwell chamber and Western blot experiments.5.The online bioinformatics database Targetscan was used to analyze the potential downstream target genes of mi R-383-5p,and intersected with the esophageal cancer-related top250 genes in the UALCAN database;the mi R-383-5p pull down experiment was used to preliminarily screen the target genes,and the dual-luciferase reporter gene experiment verified the targeting relationship between mi R-383-5p and KIAA0101.6.Western blot assay was used to detect the effect of mi R-383-5p overexpression on the expression of KIAA0101 in TE-1 and Eca-109 cells.The expression of KIAA0101 in TE-1 and Eca-109 cells co-transfected with sh-NC or sh-circ＿0005231 and mi RNA inhibitor or mi R-383-5p inhibitor was also detected.7.q RT-PCR and Western blot experiments were used to detect the expression of KIAA0101 in ESCC tumor tissues and adjacent normal tissues,as well as in ESCC cells.8.TE-1 and Eca-109 cells were co-transfected with KIAA0101 overexpression plasmid(pc DNA3.1-KIAA0101,KIAA0101)or pc DNA3.1empty plasmid(pc DNA3.1)with sh-NC or sh-circ＿0005231,respectively.and the proliferation,migration,invasion and EMT of cells were detected by colony formation,Ed U staining,scratching,Transwell chamber and Western blot experiments.Results:1.The circbank,starbase and circinteractome databases all predicted that circ＿0005231 and mi R-383-5p have complementary binding sites;Ago2 RIP,mi R-383-5p pull down experiments and dual luciferase reporter gene experiments showed that circ＿0005231 could bind to mi R-383-5p targeted binding.2.The results of q RT-PCR showed that mi R-383-5p was lowly expressed in ESCC tumor tissues and cancer cells.3.After co-transfection of mi R-383-5p inhibitor and sh-circ＿0005231into TE-1 and Eca-109 cells,the cell proliferation,migration and invasion abilities were enhanced,the expression level of E-cadherin was decreased,and the expression level of N-cadherin was increased.4.Targetscan predicted that mi R-383-5p had a complementary binding site with the 3’UTR of KIAA0101,and the UALCAN database data showed that KIAA0101 was highly expressed in esophageal cancer.Mi R-383-5p pull down experiment and dual luciferase reporter gene results showed that mi R-383-5p can target binding to KIAA0101.5.Western blot assay detected the expression of KIAA0101 in TE-1 and Eca-109 cells transfected with mi R-383-5p mimics.The results showed that compared with the control,KIAA0101 in TE-1 and Eca-109 cells was overexpressed with mi R-383-5p.In addition,Western blot experiments detected the expression of KIAA0101 in TE-1 and Eca-109 cells co-transfected with sh-NC or sh-circ＿0005231 and mi RNA inhibitor or mi R-383-5p inhibitor.The expression of KIAA0101 in cells was inhibited after sh-circ＿0005231,while the expression of KIAA0101 in cells was increased after co-transfection of sh-circ＿0005231 and mi R-383-5p inhibitor.6.The expression of KIAA0101 m RNA in 40 ESCC tumor tissues and adjacent normal tissues was detected by q RT-PCR.The results showed that compared with the adjacent normal tissues,the KIAA0101 m RNA expression level in tumor tissues was increased.Western blot was used to detect the expression level of KIAA0101 protein in ESCC tissues and cells,the results showed that compared with the control,the expression of KIAA0101 protein in ESCC tumor tissue and cancer cells was increased.7.After co-transfection of KIAA0101 and sh-circ＿0005231 into TE-1 and Eca-109 cells,the cell proliferation,migration and invasion abilities were enhanced,the expression level of E-cadherin was decreased,and the expression level of N-cadherin was increased in the cells.Summarys:1.Mi R-383-5p was the target mi RNA of circ＿0005231,and inhibiting mi R-383-5p could attenuate the inhibitory effect of circ＿0005231 silencing on ESCC cell proliferation,migration,invasion and EMT.It is suggested that circ＿0005231 exerts its biological function by targeting mi R-383-5p.2.KIAA0101 was a target gene of mi R-383-5p,and overexpression of mi R-383-5p in ESCC cells could inhibit the expression of KIAA0101 in cells.In addition,knockdown of circ＿0005231 in ESCC cells could inhibit the expression of KIAA0101 in cells,which this inhibitory effect was reversed after co-transfection of mi R-383-5p inhibitor.It suggested that circ＿0005231could regulate the expression of KIAA0101 by acting as a mi R-383-5p sponge.3.Overexpression of KIAA0101 could reverse the inhibitory effects of circ＿0005231 silencing on ESCC cell proliferation,migration,invasion and EMT.It is suggested that knockdown of circ＿0005231 may play a tumor suppressor role in ESCC cells by targeting mi R-383-5p and inhibiting the expression of its target gene KIAA0101.Part Four Study on the function of cir C＿0005231 in vivoObjective: To reveal the effect of circ＿0005231 loss-of-function on ESCC tumor growth in vivo.Methods:1.The xenograft tumor mouse model was established by subcutaneous injection of Eca-109 cells stably transfected with sh-NC or sh-circ＿0005231.the tumor volume was measured every 3 days from the day of injection,the mice were sacrificed on the 27 th day,and the tumor was removed Organize,photograph and weigh.2.The expression of circ＿0005231 in xenograft tumors was detected by q RT-PCR.3.Immunohistochemical staining(IHC)was used to detect the expressions of KIAA0101,Ki-67,E-cadherin and N-cadherin in the xenograft tumors.Results:1.The results of the in vivo xenograft tumor experiments showed that the volume growth of the xenograft tumors in the circ＿0005231 knockdown group was slowed down,and the tumor weight was significantly lower than that of the control group.2.The results of q RT-PCR showed that the expression of circ＿0005231 in the xenograft tumors of the circ＿0005231 knockdown group was significantly decreased.3.The results of IHC experiments showed that the positive expression rates of KIAA0101,Ki-67 and N-cadherin in the xenograft tumors of the circ＿0005231 knockdown group were significantly decreased,while the positive expression rate of E-cadherin was significantly increased.Summarys:1.Silencing circ＿0005231 could inhibit ESCC tumor growth in vivo,confirming that circ＿0005231 may serve as a potential target for ESCC targeted therapy.2.Silencing circ＿0005231 might inhibit ESCC tumor in vivo by inhibiting the EMT of tumor cells.Conclusions:The high expression of circ＿0005231 in tissues and blood is associated with poor prognosis of ESCC patients.Circ＿0005231 may be an important regulator in the progression of ESCC.Knockdown of circ＿0005231may play a tumor suppressor role in ESCC cells by targeting mi R-383-5p and inhibiting the expression of its target gene KIAA 0101.Circ＿0005231 may be a potential target for ESCC targeted therapy. |
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