| Background and Objective:According to the World Health Organization,Colorectal cancer ranks third in incidence rate and second in mortality rate of malignancy worldwide,seriously threatens human health and lives.Its early symptoms are not evident.Most patients are diagnosed at already the advanced metastatic stages,thus,with an extremely low5-year survival rate.The occurrence and development of colorectal cancer is a multi-factor,multi-stage complex process affected by differentially expressed genes and altered pathways.Compared with m RNA,accumulated studies have revealed that circ RNA has a stronger enrichment across species.In eukaryotes,they were proven stable and conserved molecules,abundantly expressed in tissue/developmental specific patterns,with more potential as ideal diagnostic and prognostic biomarkers and promising therapeutic targets for cancer in clinical practice.They are involved in the expression regulation of genes in cancer and are associated with oncogenic mechanisms and pathways.Most circ RNAs harbor various types and amounts of miRNA binding sites,which specifically bind miRNA to reduce miRNA activity and up-regulate miRNA target gene expression.More than half of human m RNAs are estimated to be conserved miRNA targets.Thus,circ RNA is deemed to play an extended role by regulating gene expression.We collated and analyzed bioinformatic data for preliminary molecular characterization.Results of the pre-study revealed that hsa＿circ＿0062682 was upregulated in colorectal cancer tissue and hsa＿circ＿0062682 knockdown suppressed proliferation in colorectal cancer cells.Therefore,we selected hsa＿circ＿0062682 for this study.hsa＿circ＿0062682,from chr22:26936754-26937684,is 930 bp in length and formed by reverse splicing of exon 2 of the parental gene TPST2 into a loop.Recent studies have shown that hsa＿circ＿0062682 can promote the proliferation,migration,and invasion of many types of cancer.However,it is still unclear whether hsa＿circ＿0062682 is involved in the development of colorectal cancer by a ce RNA mechanism.This study will provide an in-depth analysis of the correlation between hsa＿circ＿0062682 and colorectal cancer,explore its role in the development of colorectal cancer and its molecular mechanism,and provide an important experimental basis for accurate diagnostic biomarkers and valuable therapeutic targets.Part I.Expression and clinical relevance of hsa＿circ＿0062682 in colorectal cancerMethods:1.RT-qPCR detected the expression of hsa＿circ＿0062682 in normal intestinal and colorectal cancer tissues.2.Based on the median value of hsa＿circ＿0062682 expression in colorectal cancer tissues,we divided all patients into the hsa＿circ＿0062682 high expression group or hsa＿circ＿0062682 low expression group and clarified whether the expression of hsa＿circ＿0062682 was associated with the development of colorectal cancer in conjunction with the clinicopathological characteristics of patients.Results:1.RT-qPCR showed that the expression of hsa＿circ＿0062682 was significantly up-regulated in colorectal cancer tissues compared with normal intestinal tissues.2.Patients in the hsa＿circ＿0062682 high expression group had a more advanced stage and were more likely to develop lymph node metastases and distant metastases than those in the hsa＿circ＿0062682 low expression group.Conclusions:1.hsa＿circ＿0062682 expression was significantly upregulated in colorectal cancer tissues.2.hsa＿circ＿0062682 expression correlated with the stage,lymph node metastases,and distant metastasis of colorectal cancer patients.Part II Biological characteristics of hsa＿circ＿0062682 and its effect on colorectal cancer cellsMethods:1.RT-qPCR detected the expression of hsa＿circ＿0062682 in normal intestinal epithelial cells(NCM460)and colorectal cancer cell lines(HCT116,LOVO,DLD1,SW620,SW480).2.Sanger sequencing,Actinomycin D,Rnase R,and nucleoplasmic isolation experiments analyzed the biological characteristics of hsa＿circ＿0062682.3.Si-hsa＿circ＿0062682 or hsa＿circ＿0062682 overexpression plasmids were transfected into SW480 and SW620,and the effect of hsa＿circ＿0062682 on the biological behavior of colorectal cancer cells was examined using CCK-8,Ed U,wound healing and Transwell assays.Results:1.RT-qPCR results showed that hsa＿circ＿0062682 was significantly more highly expressed in colorectal cancer cell lines compared to normal intestinal epithelial cells.2.Sanger sequencing verified that hsa＿circ＿0062682 had a head-tail spliced loop structure.Actinomycin D and Rnase R experiments demonstrated the stability of hsa＿circ＿0062682,and the nucleoplasmic separation experiment clarified the enrichment of hsa＿circ＿0062682 in the cytoplasm.3.Knockdown of hsa＿circ＿0062682 reduced the proliferation,migration,and invasion of colorectal cancer cells.In contrast,overexpression of hsa＿circ＿0062682significantly enhanced cell proliferation,migration,and invasion.Conclusions:1.hsa＿circ＿0062682 was highly expressed in colorectal cancer cells.2.hsa＿circ＿0062682 had the biological properties of a circular RNA.3.hsa＿circ＿0062682 promoted the proliferation,migration,and invasive ability of colorectal cancer cells.Part III.hsa＿circ＿0062682 promots colorectal cancer progression through miR-497-5p/Axin2Methods:1.According to bioinformatics,dual luciferase reporter gene assay,and RT-qPCR analysis,miR-497-5p was selected as a downstream miRNA molecule of hsa＿circ＿0062682.2.miR-497-5p mimics,miR-497-5p inhibitor were transfected in colorectal cancer cells and the effect of miR-497-5p on cell proliferation,migration and invasion ability was examined using CCK-8,Ed U,wound healing and Transwell assays.3.According to bioinformatics,dual luciferase reporter gene assay,and RT-qPCR analysis,Axin2 was selected as a downstream m RNA molecule of miR-497-5p.4.After knocking down the expression of Axin2,the effect of Axin2 on cell proliferation,migration and invasion ability was examined using CCK-8,Ed U,wound healing and Transwell assays.5.Rescue experiments further validated that hsa＿circ＿0062682 targeted miR-497-5p to regulate the proliferation and metastasis of colorectal cancer cells.6.Rescue experiments further validated that miR-497-5p targeted Axin2 to regulate the proliferation and metastasis of colorectal cancer cells.Results:1.miR-497-5p was a downstream target of hsa＿circ＿0062682.It was lowly expressed in colorectal cancer cells and was regulated by hsa＿circ＿0062682.2.The proliferation,migration,and invasion of colorectal cancer cells under the effect of miR-497-5p mimics were significantly inhibited.In contrast,colorectal cancer cells proliferation,migration,and invasion of colorectal cancer cells were significantly enhanced in the presence of miR-497-5p inhibitor.3.Axin2 was a downstream target gene of miR-497-5p,and its expression was regulated by miR-497-5p.4.Silencing the expression of Axin2,the proliferation,migration and invasion of colorectal cancer cells were attenuated.5.hsa＿circ＿0062682 attenuated the miR-497-5p mediated inhibition of proliferation,migration,and invasion ability of colorectal cancer cells.On the other hand,silencing of hsa＿circ＿0062682 also reversed the miR-497-5p inhibitor mediated promotion of proliferation,migration,and invasion ability of colorectal cancer cells.6.Axin2 attenuated miR-497-5p mediated inhibition of proliferation,migration,and invasion ability of colorectal cancer cells.On the other hand,silencing of Axin2 also reversed the miR-497-5p inhibitor mediated promotion of proliferation,migration,and invasion ability of colorectal cancer cells.Conclusions:1.hsa＿circ＿0062682 targeted and regulated miR-497-5p expression in colorectal cancer cells.2.miR-497-5p inhibited the proliferation,migration,and invasive ability of colorectal cancer cells.3.miR-497-5p targeted and regulateed the expression of Axin2 in colorectal cancer cells.4.Axin2 promoted the proliferation,migration,and invasive ability of colorectal cancer cells.5.hsa＿circ＿0062682 could promote the malignant progression of colorectal cancer cells by attenuating the tumor suppressor activity of miR-497-5p.6.miR-497-5p inhibited the progression of colorectal cancer cells by targeting Axin2.Part IV hsa＿circ＿0062682 promotes colorectal cancer progression through the Wnt/β-catenin/EMT pathwayMethods:1.Western-blot detected Wnt/β-catenin/EMT pathway-related protein expression in colorectal cancer cells after knockdown of hsa＿circ＿006268.2.Rescue experiments validated that hsa＿circ＿0062682 targeted miR-497-5p to regulate the expression of Wnt/β-catenin/EMT pathway-related proteins in colorectal cancer cells.3.Rescue experiments validated that miR-497-5p targeted Axin2 to regulate the expression of Wnt/β-catenin/EMT pathway-related proteins in colorectal cancer cells.4.Colorectal cancer cells stably transfected with lentivirus sh-hsa＿circ＿0062682 were inculated under the skin of nude mice to construct a nude mouse colorectal cancer transplant tumor model to observe the effect of hsa＿circ＿0062682 on the growth of transplant tumors.RT-qPCR was performed to detect the expression of hsa＿circ＿0062682 and miR-497-5p in tumor tissues.5.Immunohistochemical analysis detected the expression of Axin2,and EMT-related proteins in colorectal cancer transplant tumor tissue.Results:1.After silencing hsa＿circ＿0062682,the expression of Axin2,β-catenin,N-cadherin and Vimentin were all down-regulated,and E-cadherin expression was increased in colorectal cancer cells.2.Co-transfection with hsa＿circ＿0062682 counteracted the miR-497-5p mediated inhibition of Wnt/β-catenin/EMT pathway-related proteins to some extent.On the other hand,down-regulation of hsa＿circ＿0062682 also reversed the miR-497-5p inhibitor mediated promotion of Wnt/β-catenin/EMT pathway-related proteins in colorectal cancer cells.3.The increased Axin2 partially counteracted miR-497-5p mediated inhibition of Wnt/β-catenin/EMT pathway-related proteins.On the other hand,miR-497-5p inhibitor mediated the promotion of Wnt/β-catenin/EMT pathway-related proteins in colorectal cancer cells were also attenuated after cotransfection with Si-Axin2.4.The volume and weight of transplanted tumors were reduced in the sh-hsa＿circ＿0062682 group of nude mice compared to the control group.RT-qPCR showed that the expression of hsa＿circ＿0062682 was knocked down in tumor tissue and the expression of miR-497-5p was elevated.5.Immunohistochemical analysis showed that the expression of Axin2,Vimentin,and N-cadherin was significantly reduced in the transplanted tumor tissues of the sh-hsa＿circ＿0062682 group,while the expression of E-cadherin was increased compared to the control group.Conclusions:1.Silencing of hsa＿circ＿0062682 inhibited the expression Wnt/β-catenin/EMT pathway-related proteins.2.hsa＿circ＿0062682 targeted miR-497-5p to regulate the expression of Wnt/β-catenin/EMT pathway-related proteins.3.miR-497-5p targeted Axin2 to regulate the expression of Wnt/β-catenin/EMT pathway-related proteins.4.Silencing of hsa＿circ＿0062682 inhibited the growth of colorectal cancer transplant tumors in nude mice.5.Silencing of hsa＿circ＿0062682 inhibited the expression of EMT-related proteins. |
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