The Role And Mechanism Of Hsa＿circ＿0002465 In Colorectal Cancer Progression And Metastasis

Objective Metastasis is the leading cause of death in colorectal cancer(CRC)patients.Therefore,exploring the driving molecules of CRC growth,progression,and metastasis,and elucidating the disease mechanism that affects CRC can provide broader ideas for the targeted therapy of metastatic CRC.Circular RNA(circ RNA),as a closed-loop molecule,can regulate downstream molecules through the mechanism of competing endogenous RNA(ce RNA)to produce corresponding biological effects.Many studies have confirmed that circ RNAs are significantly abnormally expressed in CRC and are closely related to the prognosis and survival of patients.However,as ce RNA,in the development process of CRC,especially its invasive process,the interaction and regulation mode between circ RNA and possible regulated mi RNAs and target genes has not been fully clarified,and continuous and in-depth research is required.Method First,we performed circ RNA high-throughput sequencing analysis on CRC cell lines and obtained circ RNAs with the most significant differences in expression.By performing whole transcriptome high-throughput sequencing in6 colorectal cancer cell lines(high metastatic potential cell lines HCT116,SW620,LOVO,and low metastatic potential cell lines SW480,RKO,DLD1),the detection of ring-shaped in colorectal cell lines The expression of RNA was improved,so as to obtain circular RNAs with differential expression in the process of colorectal cancer metastasis.We performed quantitative polymerase chain reaction(q PCR)in 126 pairs of CRC fresh frozen tissue and colorectal cancer cell lines to validate the sequencing results.Next,we clarified the inherent characteristics of the hsa＿circ＿0002465 structure(such as stability,ring-forming properties,and cellular sublocalization)through RT-PCR,RNase R digestion,RNA FISH and other experiments.At the same time,we analyzed the expression of hsa＿circ＿0002465 and clinical baseline parameters in patients and prognostic indicators.Furthermore,we applied proliferation assays(CCK-8,cloning assays),invasion and migration(cell scratch and Transwell assays),cycle and apoptosis(cell flow assays).Colorectal cancer hsa＿circ＿0002465overexpressed and silenced stable transfected cell lines were inoculated into the back subcutaneous tissue of experimental nude mice,respectively,and the tumor volume and weight were recorded.The number,size and texture of liver metastases were recorded.Finally,we predicted the possible binding mi RNA and target gene of hsa＿circ＿0002465 through the online database,and verified the binding relationship between the target mi RNA and the target gene by dual-luciferase reporter experiment and RNA pulldown.Functional rescue experiments of cells reconfirmed the above results.Result The results of cell sequencing showed that there were 8536 circ RNAs with differential expression,among which 4502 were up-regulated and 134 were significantly up-regulated in high metastatic cell lines;4,034 were down-regulated and 81 were significantly down-regulated.Compared with adjacent normal tissues,hsa＿circ＿0002465,hsa＿circ＿0084927,and hsa＿circ＿0006814 were most significantly up-regulated in CRC tissues,and the expression in patients with distant metastasis was higher than that in patients without distant metastasis;the above three circ RNAs were highly expressed in CRC with high metastatic potential.It was significantly expressed in cells,and finally we selected hsa＿circ＿0002465 as the research molecule through functional experiments.hsa＿circ＿0002465 is significantly highly expressed in colorectal cancer tissues and cell lines,and its expression in colorectal cancer high metastatic potential cell lines is higher than that in low metastatic potential cell lines;analysis of clinical data shows that the expression level of hsa＿circ＿0002465 is related to clinical stage,The depth of invasion,distant metastasis and gross tumor type were significantly correlated.The ROC curve found that hsa＿circ＿0002465 could diagnose distant metastasis of colorectal cancer.Log-rank results showed that clinical stage,depth of invasion,distant metastasis,nerve invasion,and preoperative CEA were significantly correlated with the overall survival time of colorectal cancer patients.The results of multivariate analysis showed that the depth of tumor invasion,distant metastasis,nerve invasion,preoperative CEA,and hsa＿circ＿0002465 were independent high-risk factors for the prognosis of patients with colorectal cancer.Next,we overexpressed and silenced HCT116,LOVO and SW480 cell lines:overexpression of hsa＿circ＿0002465 significantly enhanced the proliferation of colorectal cancer SW480 cell lines in vitro and in vivo.In contrast,silencing of hsa＿circ＿0002465 significantly reduced the proliferation of colorectal cancer HCT116 and LOVO cell lines in vitro and in vivo.The results of biological function experiments showed that overexpression and silencing of hsa＿circ＿0002465 significantly promoted and inhibited the malignant biological behavior of colorectal cancer cells,respectively;however,it failed to produce significant differences in cell cycle and apoptosis.In the subcutaneous tumorigenesis experiment,the tumor growth(volume and weight)of the hsa＿circ＿0002465 overexpression group was significantly higher than that of the control group,and the tumor growth of the silence group was significantly lower than that of the control group;in the liver metastasis model,the overexpression group nude mice The number of metastatic nodules in the liver was significantly more than that in the control group,and the number of liver metastatic nodules in the nude mice in the silence group was significantly less than that in the control group.Finally,the mi RNAs and target genes bound downstream of hsa＿circ＿0002465 were determined by bioinformatics methods and dual-luciferase reporter experiments: mi R-216a-5p and TGFβ1,and TGFβ1 was the target gene regulated by mi R-216a-5p.Rescue recovery experiments confirmed that both mi R-216a-5p and TGFβ1 blocked the biological behavior changes mediated by hsa＿circ＿0002465.Conclusion Hsa＿circ＿0002465 was most significantly up-regulated in CRC tissues;and its expression level was significantly higher in CRC cell lines with high metastatic potential than in colorectal cancer cell lines with low metastatic potential.The expression level of hsa＿circ＿0002465 was significantly correlated with clinical stage,depth of invasion,distant metastasis and gross tumor type.It has a certain degree of diagnostic value and is an independent high-risk factor for the prognosis of colorectal cancer patients.hsa＿circ＿0002465 can also significantly inhibit the malignant process of CRC in vitro and in vivo,and hsa＿circ＿0002465 can act as a ce RNA to indirectly upregulate the expression of TGFβ1 by competitively binding to miR-216a-5p.