The CircRNA Hsa＿circ＿0008274 Co-Upregates The Target Gene SERPINE1 By Means Of CeRNA And Binding RBP To Promote The Progression Of Colorectal Cancer And Its Mechanism

Objective: As one of the most deadly malignant tumors in the world,colorectal cancer(CRC)ranks the third and second among all tumors,respectively.The early symptoms of colorectal cancer are relatively hidden,mostly showing no obvious symptoms or only abdominal pain,dyspepsia,changes in bowel habits and other non-specific symptoms,which are easy to be ignored by patients.As a result,a large proportion of diagnosed patients are first diagnosed in the middle and late stages,which seriously affects the therapeutic effect.Despite advances in the management of colorectal cancer,such as endoscopy,the initial clinical presentation of metastatic colorectal cancer(m CRC)still occurs in approximately 20% of patients,and up to 50% of patients with local disease will eventually develop metastasis,which has a very adverse impact on the prognosis of patients with colorectal cancer.There are many genes and complex signaling pathways involved in the occurrence and development of colorectal cancer.Therefore,it is particularly important to conduct in-depth research on the occurrence and development mechanism of colorectal cancer in order to search for early diagnosis and treatment markers.circular RNAs(cir RNAs),a special type of non-coding RNA,form a covalently closed continuous ring by reverse splicing of the 5 ’to 3’ ends.This special structure makes circ RNA more stable than linear RNA and not easily degraded by RNA exonuclease.In the past decade,circ RNA has emerged as a large class of major non-coding RNA molecules,many of which play key roles in the development and progression of cancer through different mechanisms of action.Studies have shown that circ RNA is rich in micro RNA(mi RNA)binding sites and can play the role of mi RNA sponge in cells,which indirectly promotes the expression of target genes of mi RNA.In addition,circ RNA can interact with a variety of RNA-binding proteins(RBPs),affecting the structure,localization and other characteristics of RBP,thus exerting biological functions.Although circ RNA mostly function as non-coding RNA,some circ RNA with internal ribosome entry sites(IRES)have the ability to act as translation templates,producing peptides or proteins with biological functions that influence the development of tumors.The circ RNA hsa＿circ＿0008274(hsa＿circ UGGT2＿065)is the circ RNA from gene UGGT2.In lung cancer and liver cancer,there are relevant studies on the effect of hsa＿circ＿0008274 on tumor development,but there are no relevant reports in the field of colon cancer.Micro RNA mi R-140-3p is involved in the progression of a variety of malignant tumors,including gastric cancer,liver cancer and bladder cancer.In colon cancer,mi R-140-3p can bind to PDL1,BCL2 and other genes,promote the proliferation and migration of colon cancer and inhibit the apoptosis of tumor cells.As an important RNA-binding protein,HuR(ELAVL1)is involved in post-transcriptional gene regulation and is overexpressed in most human cancers.In the cytoplasm,abnormally increased HuR proteins can promote the translation of a variety of cancer-promoting messenger RNAs(m RNAs)and affect the biological behaviors of tumor cell growth,proliferation,immune escape,invasion and metastasis.SERPINE1(PAI-1),a member of the serine protease inhibitor superfamily,is an inhibitor of fibrinolysis.This gene has been shown to be highly expressed in various types of tumor tissue or plasma.In colon cancer,SERPINE1 has been shown to increase tumor cell proliferation and significantly promote colon cancer invasion and migration.At the same time,this gene is also associated with EMT,VEGF,JAK-STAT and other important signaling pathways.Through bioinformatics and sequencing analysis,we demonstrated that hsa＿circ＿0008274 is highly expressed in colon cancer and can affect SERPINE1 m RNA levels.Based on the analysis results of prediction software,both mi R-140-3p and HuR had binding sites of hsa＿circ＿0008274 and SERPINE1.Based on the above results,we hypothesized that hsa＿circ＿0008274 can promote the expression of target gene SERPINE1 by binding mi R-140-3p and HuR,thus promoting the occurrence and development of colon cancer.This project intended to study the expression of circrnas hsa＿circ＿0008274 in colon cancer tissues and cell lines,and analyze its correlation with pathological materials of patients.In addition,through the in vitro and in vivo experiments,we further study the influence of circ RNAs on biological behavior of colon cancer cells and verify the interaction between hsa＿circ＿0008274,mi R-140-3p,HuR,and SERPINE1.Methods: 1.Circ RNA primer design and stability verification: q PCR primers across splicing sites were designed for the special structure of circ RNA,and sanger sequencing was used to verify whether the q PCR products were from circ RNA.Total RNA was treated with RNase R,and the difference of UGGT2 and circr RNA stability before and after treatment was detected by q PCR.2.Differences of hsa＿circ＿0008274 in colon cancer tissues and cell lines: Human colon cancer cell lines HCT116,HT29,RKO,SW480 and normal intestinal epithelia cell line NCM460 were cultured.q PCR was used to detect the difference in the expression level of hsa＿circ＿0008274 between colon cancer cell lines and normal intestinal epithelia.Tumor and adjacent normal tissues of colon cancer patients from the First Hospital of China Medical University were collected.RNA was extracted and paraffin sections were prepared.The expression difference of hsa＿circ＿0008274 in tumor and normal intestinal tissues was analyzed by q PCR and in situ hybridization,and the relationship between the expression level and pathological data of patients was analyzed.3.To verify the effect of hsa＿circ＿0008274 on the biological behavior of colon cancer: HCT116 and SW480 colon cancer cell lines were transfected with plasmid and lentivirus,and hsa＿circ＿0008274 stable overexpression and knockout cell lines were screened.The effects of hsa＿circ＿0008274 on invasion and migration of colon cancer cells were tested by transwell assay.At the same time,CCK-8and plate cloning experiments were used to detect the effect of circ RNA on the proliferation of tumor cells.The effect of hsa＿circ＿0008274 on cell apoptosis was determined by flow cytometry.Finally,in order to identify the target gene of hsa＿circ＿0008274,we sequenced SW480 cell lines transfected with hsa＿circ＿0008274overexpressed plasmid and empty control(NC)plasmid,and screened out the target gene SERPINE1.4.Construct and verify the hsa＿circ＿0008274/mi R-140-3p/SERPINE1 regulatory network: Fluorescence in situ hybridization(FISH)was used to verify the localization of hsa＿circ＿0008274 in cells,and prediction software was used to predict the mirnas that can bind to hsa＿circ＿0008274 and SERPINE1.The mi R-140-3p levels in hsa＿circ＿0008274 cell lines were detected by q PCR.The co-localization relationship between hsa＿circ＿0008274 and mi R-140-3p was verified by FISH experiment,and the binding relationship between hsa＿circ＿0008274 and mi R-140-3p and mi R-140-3p and SERPINE1 was verified by double luciferase reporter gene experiment.In addition,the binding relationship of hsa＿circ＿0008274/mi R-140-3p/SERPINE1 was verified by RNA immunocoprecipitation(RIP)assay targeting Ago2 protein.5.Function reversal experiment verified the regulation axis of hsa＿circ＿0008274/mi R-140-3p/SERPINE1:Function reversal experiments between hsa＿circ＿0008274,mi R-140-3p and SERPINE1 were performed by CCK8,transwell and plate cloning experiments.Meanwhile,the influence of the regulatory relationship between hsa＿circ＿0008274 and mi R-140-3p on the protein level of the target gene SERPINE1 was detected by western-blot(WB)assay.6.Build and verify the hsa＿circ＿0008274/HuR/SERPINE1 regulation axis: The co-binding proteins of hsa＿circ＿0008274 and SERPINE1 were predicted by the prediction website,and the hsa＿circ＿0008274 probe was constructed.The RBP binding to hsa＿circ＿0008274 was screened by RNA pull down assay combined with mass spectrometry.In addition,HuR antibody was used to perform RIP experiments to verify the binding relationship between HuR and hsa＿circ＿0008274 and SERPINE1.At the same time,nuclear-cytoplasmic isolation WB and immunofluorescence(IF)assay were used to verify that hsa＿circ＿0008274 could change the cell localization of HuR.WB assay was used to verify the effect of HuR on SERPINE1 protein level.Meanwhile,the effect of the regulatory relationship between hsa＿circ＿0008274 and HuR on the stability of SERPINE1 m RNA was verified by actinicin D assay.7.Effect of hsa＿circ＿0008274/HuR/SERPINE1 regulation axis on biological behavior of colon cancer: The function reversal experiments between hsa＿circ＿0008274,HuR and SERPINE1 were performed by CCK8,transwell,plate cloning experiments to verify the binding relationship between hsa＿circ＿0008274/HuR/SERPINE1.8.In vitro and in vivo experiments confirm that hsa＿circ＿0008274 plays biological functions by targeting SERPINE1: SERPINE1 was knocked down in overexpressed hsa＿circ＿0008274 cell lines and SERPINE1 was overexpressed in knocked down hsa＿circ＿0008274 cell lines.After the treatment,CCK8,transwell,plate cloning experiments were used to detect the differences of proliferation,invasion and migration ability among treated cell lines.Meanwhile,subcutaneous tumor-forming experiments were performed in Balb/c nude mice at 4-6 weeks to verify that SERPINE1 knockdown can reverse the effect of hsa＿circ＿0008274 overexpression on tumor cell proliferation in mice.Results: 1.Sanger sequencing verified that the q PCR product originated from hsa＿circ＿0008274 and spanned the splicing site,and q PCR also verified the stability of hsa＿circ＿0008274 as circrnas after RNase R treatment.2.Hsa＿circ＿0008274 was highly expressed in colon cancer cell lines HCT116,HT29,RKO and SW480 compared with normal cell lines NCM460,but the results of RKO cell line were not statistically significant.In patients with colon cancer,the expression level of hsa＿circ＿0008274 in tumor tissues was higher than that in adjacent normal tissues,and the expression level was correlated with T,N,stage and other indicators of patients.3.Transwell experiment demonstrated that overexpression of hsa＿circ＿0008274 could promote the invasion and migration ability of colon cancer cells,while knockdown hsa＿circ＿0008274 had an inhibitory effect.In addition,CCK8,plate cloning experiments showed that hsa＿circ＿0008274 could significantly promote the proliferation of colon cancer cells,and inhibiting its expression could reduce the proliferation of colon cancer cells.Flow cytometry showed that hsa＿circ＿0008274 could not significantly affect the anti-apoptotic ability of colon cancer cells.Based on the sequencing results and bioinformatics analysis,we found that SERPINE1 is a potential target gene for hsa＿circ＿0008274 to exert biological influence.Meanwhile,q PCR and WB experiments showed that overexpression of hsa＿circ＿0008274 could improve the m RNA and protein levels of SERPINE1,while knockdown of hsa＿circ＿0008274 showed the opposite effect.4.FISH experiments showed that hsa＿circ＿0008274 was mainly located in the cytoplasm and had the potential to adsorb mi RNA as an RNA sponge and further affect the biological behavior of colon cancer.According to the results of prediction software and TCGA database analysis,we found that mi R-140-3p had binding sites with hsa＿circ＿0008274 and SERPINE1.At the same time,overexpression of hsa＿circ＿0008274 can reduce the level of mi R-140-3p,otherwise,it can increase the level of mi R-140-3p.Fluorescence co-localization experiments also verified that both hsa＿circ＿0008274 and mi R-140-3p were located in the cytoplasm.Dual luciferase assay further verified the binding relationship between mi R-140-3p,hsa＿circ＿0008274 and SERPINE1.RIP experiment also confirmed that pull-down AGO2 protein can enrich hsa＿circ＿0008274,mi R-140-3p and m RNA of SERPINE1.Moreover,the levels of hsa＿circ＿0008274 and SERPINE1 enriched in the cells overexpressing mi R-140-3p were higher in the NC cell lines.5.Mi R-140-3p was overexpressed in hsa＿circ＿0008274 overexpressed cell lines,mi R-140-3p was knocked down in hsa＿circ＿0008274 knocked down cell lines.CCK8,plate cloning experiments showed that overexpression of mi R-140-3p could reverse the improvement of the proliferation ability of colon cancer cells by hsa＿circ＿0008274,and knockdown of mi R-140-3p could reverse the inhibitory effect of knockdown hsa＿circ＿0008274 on the proliferation of colon cancer cells.Transwell experiment also showed that regulating the level of mi R-140-3p could reverse the effect of hsa＿circ＿0008274 on the invasion and migration ability of colon cancer cells.Meanwhile,q PCR and WB experiments showed that overexpression of mi R-140-3p in hsa＿circ＿0008274 cell lines could reduce the enhancement effect of hsa＿circ＿0008274 on SERPINE1 m RNA and protein content.When mi R-140-3p was knocked down in hsa＿circ＿0008274 cell line,the m RNA and protein levels of SERPINE1 were increased.6.RNA Pull Down experiment combined with mass spectrometry detection was used to obtain the type of RBP binding to hsa＿circ＿0008274.The intersection of results and predicted website results was used to obtain RBP HuR.RIP experiments showed that hsa＿circ＿0008274 and SERPINE1 m RNA could be enriched by pull-down HuR.In addition,more SERPINE1 m RNA could be enriched in hsa＿circ＿0008274 overexpressed cell lines compared with NC cell lines.Nuclear-cytoplasmic isolation WB and IF experiments showed that HuR protein level in cytoplasm increased after hsa＿circ＿0008274 overexpression,and decreased otherwise.WB assay showed that overexpression or knockdown of HuR could increase or decrease SERPINE1 protein levels,respectively.Meanwhile,actinicin D experiment showed that overexpression of hsa＿circ＿0008274 could increase the stability of SERPINE1 m RNA,while knockdown of HuR could decrease the stability of SERPINE1 m RNA.Knockdown of hsa＿circ＿0008274 could decrease the stability of SERPINE1 m RNA.However,overexpression of HuR can inhibit this attenuating effect.7.HuR was knocked down in hsa＿circ＿0008274 knocked down cell lines and overexpressed in hsa＿circ＿0008274overexpressed cell lines.CCK8,plate cloning experiments showed that knockdown of HuR could reverse the improvement of the proliferation ability of colon cancer cells by hsa＿circ＿0008274,and overexpression of HuR could reverse the inhibitory effect of knockdown of hsa＿circ＿0008274 on the proliferation of colon cancer cells.Transwell experiment also showed that adjusting HuR level could reverse the effect of hsa＿circ＿0008274 on the invasion and migration ability of colon cancer cells.8.CCK8,plate cloning experiments showed that SERPINE1 knockdown could reverse the promoting effect of hsa＿circ＿0008274 on proliferation,and overexpression of SERPINE1 could reverse the inhibiting effect of hsa＿circ＿0008274 on proliferation.Meanwhile,transwell experiment also showed that SERPINE1 knockdown could reverse the promoting effect of hsa＿circ＿0008274 on invasion and migration,and overexpression of SERPINE1 could reverse the inhibiting effect of hsa＿circ＿0008274 knocken down on invasion and migration.Subcutaneous tumor formation in nude mice showed that overexpression of hsa＿circ＿0008274 could promote tumor growth,while deletion of SERPINE1 could reverse this effect.Conclusion: In this study,the role of hsa＿circ＿0008274 in the occurrence and development of colon cancer was reported for the first time,demonstrating that hsa＿circ＿0008274 is highly expressed in colorectal cancer,and is positively correlated with T,N and stage.At the same time,it was verified that hsa＿circ＿0008274 promoted the expression of target gene SERPINE1 by binding mi R-140-3p as ceRNA and binding HuR by RBP-dependent way,and promoted the proliferation,invasion and migration of colon cancer cells.