| According to the data of the National Cancer Center,there were more than 550,000 new colorectal cancer(CRC)patients in China in 2020,most of them were advanced cancer.CRC has become a malignant disease that seriously threatens the lives and health of Chinese residents,causing a serious social burden.In recent years,with the progress of medical treatment and technology,although the prognosis of some patients has been significantly improved,the morbidity and mortality of CRC remain high and tend to be younger.Therefore,it is still an urgent need in the field of CRC treatment to explore and clarify the specific mechanisms of CRC occurrence and development,and then to find novel targets for diagnosis,treatment and prognosis prediction.Long non-coding RNAs(LncRNAs)are less conserved than proteincoding genes,but their expression patterns show high tissue specificity,it has become a new medium of tumorigenesis by virtue of its own multiple functions and multiple different mechanisms induced by its own.They play key roles in a range of complex cellular processes,including gene expression regulation,imprinting,chromatin modification,transcription,post-transcriptional and post-translational processing.Recent studies have shown that lncRNAs can be involved in CRC progression by coordinating with miRNAs and protein-coding mRNAs.LncRNAs act as competing endogenous RNAs(ceRNAs)by competitively occupying the shared binding sites of miRNAs,thereby sponging miRNAs and altering the expression of their downstream target genes.The ceRNA axis formed by lncRNA/miRNA/mRNA interactions has been found in multiple biological processes in CRC.However,the ceRNA axis of many lncRNAs still remain undiscovered.Insulin-like growth factor 2(IGFL2),as a secreted protein family member,has the most extensive expression pattern among the IGFL genes,which shares structural homology with the insulin growth factor(IGF)family.Other IGF family members are known to participate in important biological processes:metabolic control,growth and reproduction.Given the structural similarity between IGFL2 and IGF proteins,IGFL proteins may also act as growth regulators,and they are also expressed in many cancers.A recent study showed that lncRNAIGFL2-AS1,the antisense transcript of IGFL2,is highly expressed in a variety of cancers,but the function and mechanism of lncRNAIGFL2-AS1 in CRC are still unclear.This study explored the expression,function and mechanism of lncRNAIGFL2-AS1 in CRC through the following three parts.Part One Expression of lncRNA IGFL2-AS1 in colorectal cancer tissues and cell linesObjective:By detecting the expression level of lncRNA IGFL2-AS1 in CRC tissues and cell lines(SW837,LS1034,LS513,HCT116)and paired adjacent normal tissues and human normal colon epithelial cell lines(CCD841CoN)to analyze whether there is a difference,and then analyze whether it is related to clinicopathological characteristics.Methods:1.HE staining was performed on tumor tissue from 63 CRC patients and normal mucosal tissue located more than 10cm away from the tumor tissue for differentiation.2.Design and entrust biological companies to chemically synthesize LncRNA IGFL2-AS1 primers.3.Starbase database analyses the expression of LncRNA IGFL2-AS1.4.Total RNA was extracted from CRC tissue,SW837,LS1034,LS513,HCT116 cell lines and paired adjacent normal tissues,CCD-841CoN cell line,and detect the difference in the expression of LncRNA IGFL2-AS1 by reverse transcription,qRT-PCR and other methods.5.The correlation between the expression difference of LncRNA IGFL2AS1 and clinicopathological characteristics was analyzed statistically.Results:1.LncRNA IGFL2-AS1 is abundantly expressed in CRC according to Starbase database.2.LncRNA IGFL2-AS1 is abundantly expressed in CRC tissues and cell lines.The results of qRT-PCR showed that the expression of lncRNA IGFL2AS1 in CRC tissue and SW837,LS1034,LS513,and HCT116 cell lines was abundantly expressed compared with paired adjacent normal tissues and CCD841CoN cell lines,and the expression differences were statistically significant.It is of scientific significance(P<0.05),and it is consistent with the analysation results of the Starbase database.3.The expression level of lncRNA IGFL2-AS1 is positively correlated with lymph node metastasis and advanced TNM stage.The relationship between the expression of LncRNA IGFL2-AS1 and the clinicopathological characteristics of CRC patients was statistically analyzed.It was found that the expression level of LncRNA IGFL2-AS1 was positively correlated with lymph node metastasis and advanced TNM stage(P<0.05),but had no significant correlation with the patient’s gender,age,tumor location,depth of invasion,and tumor size.Conclusion:LncRNAIGFL2-AS 1 is abundantly expressed in CRC tissues and cells,and is positively correlated with lymph node metastasis and highgrade TNM stage.Part Two Effects of lncRNA IGFL2-AS1 on the proliferation of colorectal cancer cell lines and its subcellular localizationObjective:To explore the effects of knockdown and overexpression of lncRNA IGFL2-AS1 on the proliferation ability of CRC cell line HCT116 and its subcellular localization.Methods:1.Entrusted a biochemical synthesis company to design and synthesize siRNA and overexpression plasmids.2.The HCT116 cell line was further transfected with Lipofectamine 2000 transfection reagent,and the transfection efficiency was detected by qRT-PCR.3.MTT and colony formation assays were used to detect cell viability and cloning ability.4.The lncLocator database predicts the subcellular localization of lncRNA IGFL2-AS1 and further validates it with Nuclear/Cytosol fraction-ation assay.Results:1.Successfully knockdown and overexpression of lncRNA IGFL2-AS1 in HCT116 cell line.HCT116 cells transfected with si-RNA and overexpression plasmid were detected respectively,and it was found that compared with the control group,the expression of lncRNA IGFL2-AS1 in the knockdown group was significantly decreased(P<0.05),and the expression of si-IGFL2-AS1-2 had the highest efficiency(P<0.05);the expression of lncRNA IGFL2-AS1 was significantly increased in the overexpression group(P<0.05).2.Knockdown lncRNA IGFL2-AS1 can inhibit the viability and cloning ability in HCT116 cells.The results of MTT and colony formation assay showed that compared with the control group,after knocking down lncRNA IGFL2-AS 1,the viability and cloning ability of HCT116 cells were significantly inhibited(P<0.05).3.Overexpression of lncRNA IGFL2-AS1 can promote the viability and cloning ability of HCT116 cells.The results of MTT and colony formation assay showed that compared with the control group,the viability and cloning ability of HCT116 cells was significantly improved after overexpression of lncRNA IGFL2-AS1(P<0.05).4.The lncLocator database predicts that lncRNA IGFL2-AS1 is mainly located in the cytoplasm,and the Nuclear/Cytosol fractionation assay confirmed that it is mainly expressed in the cytoplasm of HCT116 cells.Conclusion:Knockdown lncRNA IGFL2-AS1 can significantly inhibit the viability and cloning ability of HCT116 cells;overexpression lncRNA IGFL2-AS1 can significantly promote the viability and cloning ability of HCT116 cells;LncRNA IGFL2-AS1 is mainly located in the cytoplasm of HCT116 cells.Part Three Molecular mechanism of lncRNA IGFL2-AS1 promoting proliferation of colorectal cancer cellsObjective:To explore the molecular mechanism of lncRNA IGFL2-AS1 promoting proliferation of colorectal cancer cells.Methods:1.Starbase database screened the downstream target genes of lncRNA IGFL2-AS1 and their expression level,the binding relationship between lncRNA IGFL2-AS1 and its downstream target gene was further testified via the RIP and dual-luciferase reporter gene assays.2.Detection of downstream target gene expression in CRC tissues and cell lines by qRT-PCR。3.Starbase and TargetScan databases predicted the downstream target genes of miR-433-3p,the binding relationship between miR-433-3p and its downstream target gene was further testified via the dual-luciferase reporter gene assay.Its expression was detected by qRT-PCR.4.Pearson Correlation Coefficient analysis was used for correlation analysis between lncRNA IGFL2-AS1,miR-433-3p and PAK4.5.The effects of inhibitor-433-3p and overexpression of PAK4 on the proliferation of HCT116 cells were verified by MTT and colony formation assay,respectively.Results:1.Starbase database screened the downstream target gene of lncRNA IGFL2-AS1 as miR-433-3p,and the RIP and dual-luciferase reporter gene assays both confirmed that they can combine.2.The expression of miR-433-3p was significantly decreased in CRC tissues and cell lines.The results of qRT-PCR showed that the expression of miR-433-3p in CRC tissue and SW837,LS1034,LS513,and HCT116 cell lines was significantly decreased compared with paired adjacent normal tissues and CCD-841CoN cell lines,and the expression differences were statistically significant.It is of scientific significance(P<0.05),and it is consistent with the analysation results of the Starbase database.3.Starbase and TargetScan databases predicted the downstream target gene of miR-433-3p as PAK4,and dual-luciferase reporter gene assays confirmed that they can combine.The results of qRT-PCR showed that the expression of PAK4 in CRC tissue and cell lines was abundantly expressed,it is of scientific significance(P<0.05).4.Pearson correlation coefficient analysis showed that lncRNA IGFL2AS1 was negatively correlated with miR-433-3p;PAK4 was negatively correlated with miR-433-3p and positively correlated with lncRNA IGFL2-AS1.5.Both inhibitor-433-3p and overexpression of PAK4 could partially reverse the inhibition effect of si-IGFL2-AS1-2 on HCT116 cell proliferation.MTT and colony formation assay showed that compared with the control group,the proliferation ability of HCT116 cells in the si-IGFL2-AS1-2+inhibitor-433-3p group and the si-IGFL2-AS1-2+oe-PAK4 group were both partly reversed.(P<0.05)Conclusion:MiR-433-3p is the downstream target gene of lncRNA IGFL2-AS1,PAK4 is the downstream target gene of miR-433-3p,lncRNA IGFL2-AS1 may promote HCT116 cell proliferation through miR-4333p/PAK4 axis. |
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