| ObjectiveLncRNAZEB1-AS1 is a kind of LncRNArelated to tumor progression,but at present,there are still few studies on the role of LncRNAZEB1-AS1 in colorectal cancer(CRC),and its regulatory mechanism needs further study.In the study,the purpose of this study is to study the relationship between LncRNAZEB1-AS1 and the clinical characteristics and immune microenvironment of colorectal cancer through bioinformatics and clinical samples.At the same time,the effect of LncRNAZEB1-AS1 on the proliferation and migration of colorectal cancer and its specific molecular mechanism were further clarified.Finally,the cause of abnormal expression of LncRNAZEB1-AS1 in colorectal cancer was clarified from the angle of m6 A modification.Methods1.The sequencing data sets of colorectal cancer and normal tissues adjacent to cancer were collected through TCGA database.The expression level of LncRNAZEB1-AS1 in colorectal cancer was analyzed,and the influence of LncRNAZEB1-AS1 on prognosis was analyzed by Kaplan-Meier curve.The potential regulatory pathway of ZEB1-AS1 was studied by gene set enrichment analysis(GSEA).For screening the risk genes for the prognosis of colorectal cancer,the LASSO Cox model was used to construct its clinical prognosis model,and the clinical prognosis prediction model was constructed by establishing and testing the predictive nomogram.Finally,functional enrichment analysis was used to predict the immunotherapy response with LncRNAZEB1-AS1 as the risk score.2.The results of bioinformatics are further verified through experimental research,80 fresh CRC tissues and matched normal colon tissues adjacent to cancer were collected,and the expression level of LncRNAZEB1-AS1 in tumor and normal colon tissues adjacent to cancer was detected by RT-QCPR,and the relationship between LncRNAZEB1-AS1 expression and clinical features and prognosis was analyzed.Lovo and HT29 cells were used to knock down the expression of LncRNAZEB1-AS1,CCK8,clone formation,Transwell invasion and migration experiments were used to evaluate the effect of LncRNAZEB1-AS1 on the proliferation and migration of colorectal cancer,and the potential regulatory targets and mi RNA of LncRNAZEB1-AS1 were further analyzed.In this study,the results showed that there was a significant correlation between lncRNAZEB1-AS1 and YAP1.Star Base website was used to predict the potential adsorbed mi RNAs of lncRNAZEB1-AS1,,the interacting mi RNAs of lncRNAZEB1-AS1 were found in the database,and at the same time,the interacting mirnas of YAP1 were intersected,and the mi R-5590-3p with the highest score was selected.,and the LncRNAZEB1-AS1 was verified by luciferase report test and phenotype rescue experiment.Finally,nude mice model of tumorigenesis and orthotopic liver metastasis model of colorectal cancer was used to evaluate the effect of LncRNAZEB1-AS1 knock-down on tumor proliferation and metastasis.3.The potential m6A modification site of LncRNAZEB1-AS1 was predicted by SRAMP online software.GEPIA was used to analyze the correlation between LncRNAZEB1-AS1 and m6 A modification related factors.Among them,the expression of LncRNAZEB1-AS1 highly correlates with YTHDC1.Immunohistochemistry was used to detect the relationship between the expression of YTHDC1 in colorectal cancer and its clinical features,and to analyze the correlation between YTHDC1 expression and LncRNAZEB1-AS1.RNA interference knocks down the expression of LncRNAZEB1-AS1 in HT29 and Lovo cells.M6 A RNA immunoprecipitation(Me RIP)YTHDC1 and Me RIP-q PCR were used to detect the level of RNA m6 A modification of LncRNAZEB1-AS1,and RNA half-life test was used to determine the regulation of m RNA stability of LncRNAZEB1-AS1 after knocking down YTHDC1.Finally,after point mutation of the key m6 A modification site of LncRNAZEB1-AS1,luciferase report experiment verified the specific m6 A modification site of LncRNAZEB1-AS1.Results1.In TCGA database,the expression of LncRNAZEB1-AS1 is significantly upregulated,and high expression of LncRNAZEB1-AS1 is related to the poor prognosis of patients.Univariate and multivariate Cox regression analysis showed that LncRNAZEB1-AS1 could be used as an independent prognostic factor for colorectal cancer treatment(P <0.05).LncRNAZEB1-AS1 is related to the production of interleukin-8,the positive regulation of cell aging,the positive regulation of tissue remodeling,the production of vascular endothelial growth factor and the binding of chemokine receptor(CCR)through GESA.The Risk score of clinical prognosis model was constructed for genes(METTL14,VIRMA,EIF5 B,NUDT3,KIFC1,TPT1,CIITA)co-expressed in LncRNAZEB1-AS1.ROC analysis showed that the accuracy of risk score in predicting prognosis in the training set was: 1 year AUC = 0.650,3 years AUC = 0.706;and 5 years AUC = 0.Among them,patients with high risk score have higher infiltration levels of B cells,macrophages and Tfh cells,and patients with a high risk score have lower response to immunotherapy than patients with low risk score.2.the expression level of lncRNAZEB1-AS1 in CRC was significantly higher than that in normal tissues adjacent to cancer,in which the high expression level of lncRNAZEB1-AS1 was closely related to the tumor size(≥5cm)and higher TNM stage(Ⅲ/Ⅳ stage)of CRC(P<0.05),while the expression level of lncRNAZEB1-AS1 was related to the age,sex and tumor differentiation of CRC patients.The overall survival rate of patients with high expression of lncRNAZEB1-AS1 in colorectal cancer was significantly correlated(Logrank test=5.665,P=0.0173).3.The expression of lncRNAZEB1-AS1 in Lovo and HT-29 cells was knocked down,and the growth vigor and clonal formation ability of colorectal cancer cells were significantly lower than those in the control group,and the cell invasion and migration ability were significantly decreased,with statistical significance(P <0.05).After Lovo and HT29 cells knocked down the expression of ZEB1-AS1,western-blot analysis showed that the expression levels of Yap1,CTGF,Areg,Vimentin,Snail,Slug and Twist all decreased,while the expression level of E-cadherin increased.The expression of lncRNAZEB1-AS1 was positively correlated with the m RNA levels of YAP1,CTGF,VIM,SNAIl,SNAI2 and Twist.4.Mi R-5590-3p has a matching binding site with the 3’ end of YAP1,and the 3’ end of lncRNAZEB1-AS1 has a matching binding site with the 5’ end of mi R-5590-3p.Transfection of mi R-5590-3p mimetic can significantly inhibit the luciferase activity of ZEB1-AS1 reporter gene,and transfection of mi R-5590-3p mimetic can significantly inhibit the luciferase activity of YAP1 reporter gene.There was a significant negative correlation between the expression of lncRNAZEB1-AS1 and mi R-5590-3p in colorectal cancer tissues(r =-0.427,P < 0.05).YAP1 was negatively correlated with the expression of mi R-5590-3p(r =-0.472,P <0.05),and lncRNAZEB1-AS1 was positively correlated with YAP1(r=0.425,P <0.05).Lovo and HT29 colon cancer cells can down-regulate the expression of YAP1 after knocking down lncRNAZEB1-AS1,and the expression level of YAP1 can be restored after inhibiting the expression of mi R-5590-3p with mi RNA inhibitor.Overexpression of lncRNAZEB1-AS1 can increase the proliferation and migration ability of Lovo and HT29 cells,while knocking down the expression of YAP1 at the same time can decrease the proliferation and migration ability of Lovo and HT29 cells.5.The tumor growth ability of Lovo cell transplanted tumor model with 5.lncRNAZEB1-AS1 knockdown was significantly lower than that of the control group(P <0.05).At the same time,Lovo cells knocked down by lncRNAZEB1-AS1 were inoculated into the cecum of BALB/c nude mice.After knocking down lncRNAZEB1-AS1 of Lovo cells,the survival time of mouse cecal in situ tumor model was significantly prolonged(Figure 19C),and the number of liver metastatic nodules of colorectal cancer was significantly lower than that of the control group,with statistical significance(P <0.05).6.GPEIA database analyzed the correlation between LncRNAZEB1-AS1 and the m RNA expression of Writers and Readers.LncRNAZEB1-AS1 was positively correlated with the expressions of METTL14(R=0.27,P<0.001)and YTHDC1(R=0.35,P<0.001)in colorectal cancer.The high expression of YTHDC1 in colorectal cancer is closely related to the larger tumor size(≥5cm)and the later clinical stage(Ⅲ/Ⅳ)(P<0.05).After knocking down YTHDC1,the cells were treated with actinomycin,and the relative expression level of LncRNAZEB1-AS1 m RNA in Lovo and HT29 cells was significantly lower than that in the control group at 6 and 9 hours.YTHDC1 can directly bind LncRNAZEB1-AS1;in Lovo and HT29 cells;After knocking down METTTL 3/METTTL 14,the direct binding of YTHDC1 to LncRNAZEB1-AS1 in Lovo and HT29 cells was significantly reduced.The luciferase reporter gene system verified that knock-down METTTL 3/METTTL 14 did modify the 427 th adenine base of ZEB1-AS1.Conclusions1.High expression of lncRNAZEB1-AS1 in colorectal cancer is an important marker of tumor progression,prognosis and regulation of tumor immune microenvironment.2.Targeting lncRNAZEB1-AS1 can inhibit the proliferation and metastasis of colorectal cancer.3.LncRNAZEB1-AS1 regulates the expression of YAP1 in colorectal cancer through mi R-5590-3p,and promotes the proliferation and metastasis of colorectal cancer.4.YTHDC1 promotes the stability of the m RNA expression of lncRNAZEB1-AS1 in colorectal cancer through m6 A modification,and promotes the invasion and metastasis of colorectal cancer. |
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