M~6A Methylation Modifies The Expression Of RP11-150O12.3 In Colon Cancer And Its Biological Function On Colon Cancer

Part one Study on m⁶A modification regulating the expression of RP11-150O12.3 in colon cancer cellsObjective:lncRNA（DElncRNAs）associated with m⁶A differentially expressed in colon cancer were screened by TCGA database to study the effect of m⁶A methylation modification on the expression mechanism of RP11-150O12.3 in colon cancer.Methods:1.The EdgeR package was used to identify DElncRNAs in colon cancer from the TCGA database.Spearman correlation analysis was used to screen m⁶A-related DElncRNAs（|r|>0.4,P<0.001）.Obtaining prognostic-related lncRNAs based on univariate Cox regression analysis.The relationship between RP11-150O12.3 gene expression and overall survival was analyzed by Kaplan-Meier method.2.Univariate and multivariate Cox regression were used to analyze independent influencing factors related to the prognosis of colon cancer.3.Fluorescence probe in situ hybridization（FISH）was used to detect the subcellular localization of RP11-150O12.3 gene in NCM460 and SW480cells.4.The expression of RP11-150O12.3 gene in colon cancer cells and tissues were detected by qRT-PCR.5.The colorimetric method of microplate reader was used to detect the overall level of m⁶A in colon cancer cell lines.6.MeRIP-qPCR assay was used to determine whether RP11-150O12.3was modified by m⁶A methylation in colon cancer cells and normal colon epithelial cells.Results:1.The bioinformatics results showed that EdgeR was used to identify 2428 DElncRNAs between colon cancer and adjacent tissues from the TCGA database,and 704 m⁶A-related lncRNAs were screened out through Spearman correlation analysis.Combining prognostic information and performing univariate Cox regression,we found that there are 36 m⁶A-related lncRNAs.We use RP11-150O12.3 as the target gene for follow-up studies;Through Kaplan-Meier survival analysis,the results showed that patients with low expression of RP11-150O12.3 gene and those with higher expression had poorer overall survival（P=0.007）;2.Univariate Cox regression analysis results showed that T stage（HR=3.3243,95%CI:1.3432-8.2274）,N stage（HR=2.4482,95%CI:1.5881-3.7741）,TNM stage（HR=2.8718,95%CI:1.8417-4.4780）and RP11-150O12.3 gene expression（HR=0.9995,95%CI:0.9991-1.0000）affect the survival time of colon cancer patients.Multivariate Cox regression results showed that TNM staging（HR=8.7148,95%CI:3.0262-25.0967）,N staging（HR=0.3133,95%CI:0.1121-0.8756）and RP11-150O12.3 gene expression（HR=0.9995,95%CI:0.9991-1.0000）are independent prognostic factors that affect the survival time of patients;3.The results of FISH experiments showed that the expression of RP11-150O12.3 in colon cancer cells SW480 and normal colon epithelial cells NCM460 were mainly located in the cytoplasm;4.The results of qRT-PCR experiments showed that the expression of RP11-150O12.3 in SW480 and SW620 colon cancer cell lines was significantly lower than that of NCM460 normal colon epithelial cells(SW480 vs.NCM460:t=17.327,P=6.6×10^-5;SW620 vs.NCM460:t=20.705,P=3.2×10^-5).In 19 pairs of fresh colon cancer The expression of RP11-150O12.3 was detected in tissues and adjacent tissues,and the results showed that its expression in colon cancer tissues was significantly lower than that in adjacent tisues（Z=-2.133,P=0.033）;5.The overall level of m⁶A test showed that the overall level of m⁶A in colon cancer cell line SW480 was higher than that in normal colonic epithelial cells NCM460（t=-5.176,P=0.007）;6.The results of MeRIP-qPCR showed that RP11-150O12.3 had m⁶A methylation modification in colon cancer cells,and the degree of modification was higher than that of normal colon epithelial cells（t=-2.883,P=0.045）.Conclusions:1.The expression of RP11-150O12.3 in SW480 and SW620 colon cancer cells is lower than that in normal colonic epithelial cells NCM460,and the expression in colon cancer tissues is lower than its corresponding paracancerous tissues.The subcellular locations are mainly located in the cytoplasm.2.Low expression of RP11-150O12.3 gene may be related to poor prognosis of colon cancer3.The overall level of m⁶A modification in colon cancer cell lines is higher than that in normal epithelial cells.The m⁶A methylation modification level of RP11-150O12.3 in colon cancer cells is higher than that in normal colon epithelial cells.Part two Effect of RP11-150O12.3 gene on the biological function of colon cancer cellsObjective:SW480 colon cancer cells with stable overexpression and knockdown of RP11-150O12.3 gene were constructed to study the effect of RP11-150O12.3 gene on the biological function of colon cancer cells.Methods:1.The SW480 colon cancer cell line with stable overexpression and knockdown was constructed by packaging lentivirus.The positive rate of green fluorescent protein（GFP）was detected by flow cytometry,and the expression of RP11-150O12.3 gene was detected by qRT-PCR.2.Clone formation test and MTT proliferation test detect the change of cell proliferation ability.3.The Transwell chamber test detects changes in the invasion and migration ability of colon cancer cells.4.Flow cytometry detects changes in cell cycle and apoptosis.Results:1.SW480 colon cancer cell line with overexpression and knockdown of RP11-150O12.3 was successfully constructed.The results of flow cytometry showed that the positive ratio of GFP in the overexpression group and the knockdown group was more than 80%.qRT-PCR results showed that compared with the control group,the expression of RP11-150O12.3 was significantly increased in the overexpression group(t=-27.025,P=1.1×10^-5)and significantly decreased in the knockdown group（t=3.332,P=0.029）;2.MTT assay and clone formation assay were used to detect the proliferation ability of colon cancer cells.MTT assay showed that the proliferation ability of colon cancer cells was decreased after overexpression of RP11-150O12.3 gene（t=7.715,P=0.002）.On the contrary,compared with the control group,the proliferation ability of colon cancer cells in the knockdown RP11-150O12.3 group was significantly improved（t=-2.879,P=0.045）.The results of clone formation experiment showed that the relative clone rate of SW480 cells overexpressing RP11-150O12.3 was significantly lower than that of control group（t=9.654,P=0.001）,but the clone ability of knockdown group was higher than that of control group（t=-7.043,P=0.002）;3.Transwell compartment assay was used to detect the invasion and migration ability of colon cancer cells.The results showed that the migration（t=6.564,P=0.003）and invasion（t=6.012,P=0.004）ability of RP11-150O12.3 overexpression group was significantly lower than that of control group,and the migration（t=-4.031,P=0.016）and invasion（t=-5.691,P=0.005）ability of RP11-150O12.3 knockdown group was higher than that of control group;4.Cell cycle was detected by flow cytometry.The results showed that compared with the control group,the proportion of cells in G0/G1phase（t=-9.188,P=0.001）was increased in the over-expression group of RP11-150O12.3,while the proportion of cells in S phase（t=9.318,P=0.001）was decreased compared with the control group,the proportion of cells in G0/G1 phase（Z=-1.993,P=0.046）decreased and the proportion of cells in S phase（t=-6.337,P=0.003）increased in RP11-150O12.3 knockdown group;5.Cell apoptosis was detected by flow cytometry,and the results showed that the apoptosis rate of the overexpressed RP11-150O12.3 group was higher than that of the control group,but the difference was not statistically significant（t=-0.334,P=0.755）.Compared with the control group,the apoptosis rate of RP11-150O12.3 group was decreased,and the difference was statistically significant（t=3.492,P=0.025）.Conclusions:1.The successful construction of RP11-150O12.3 gene overexpression and knockdown SW480 cell line was verified by flow cytometry and qRT-qPCR.2.RP11-150O12.3 gene can inhibit the proliferation,invasion,migration,cell cycle and promote cell apoptosis of colon cancer cell line SW480.