| Part One The expression and clinical characteristics of mi R-424-5p in human colorectal cancer tissues.Objective:Real-time polymerase chain reaction was used to detect the expression of mi R-424-5p in colorectal tissues and non-tumor adjaceent tissues.The relationship brtween clinicopathological features and colorectal cancer was analyzed.Further analysis of mi R-424-5p expression and its relationship with clinicopathological features.Methods:1.From the TCGA database website(https://cancergenome.nih.gov)We downloaded the gene sequencing data and clinical data of 619 cases of colorectal cancer and 21 cases of normal colorectal tissue,obtained the micro-RNA sequencing date,analyzed them by R language,and constructed the mi-RNAs expression matrix aggregation.2.The expression level of mi R-424-5p in 59 cases of colorectal cancer and their matched normal tissues was analyzed by RT-q PCR.the results showed that the expression level of mi R-424-5p in colorectal cancer tissues was significantly higher that in adiacent normal tissues,which was consistent with the expression of mi R-424-5p in TCGA database.3.Total RNA was extracted from tissues by Trizol,and then c DNA was obtained by reverse transcription.Finally,the expression level of mi R-424-5p was determined by real-time quantitative PCR,the relative expression of mi R-424-5p in colorectal cancer was analyzed by 2-?CTwith U6 as internal reference.4.Statistical software SPSS 21.0 was used to analyze the data.Mann Whitney test was used to compare the two groups,in which p<0.05 was statistically significant.The data of non normal distribution is represented by P50(P25,p75),and the data of normal distribution is represented by meanąstandard deviation.Results:1.The expression level of mir-424-5p in colorectal cancer tissues was higher than that in adjacent normal tissues in TCGA database.2.The expression level of mi R-424-5p in colorectal cancer tissues was higher than that in adjacent normal tissues.RT q PCR was used to analyze the expression level of mi R-424-5p in 59 colorectal cancer tissues and matched adjacent normal tissues.The results showed that the expression level of mi R-424-5p in colorectal cancer tissues was significantly higher than that in adjac-ent normal tissues(median=6.12,P25=1.87,p75=65.12)and adjacent normal tissues(median=3.29,P25=0.51,p75=19.84),which was consiste-nt with the expression of mi R-424-5p in TCGA database.3.The expression level of mi R-424-5p in colorectal cancer samples is closely related to the clinicopathological characteristics.The results showed that the expression level of mi R-424-5p in Dukes stage C and D[2.695(1.518,43.650)]was higher than that in Dukes stage A and B,[2.695(1.518,43.650)],(P=0.0159).and mi R-424-5p expression in T3 and T4 stage samples[33.650(2.191,117.400)]was higher than that in T1 and T2 stage samples[3.770(1.470,14.670)],(P=0.045).mi R-424-5p expression in adenocarcinoma[12.380(1.905,170.100)]was higher than that in mucinous carcinoma[4.848(1.641,26.720)],(P=0.0451).The expression of mi R-424-5p was no significant correlation with age,gender,tumor location,distant metastasis and tumor differentiation.Conclusions:mi R-424-5p is highly expressed in colorectal cancer tissue,and was related to Dukes stage,T stage and pathological type of colorectal cancer tissue.Part Two Function of mi R-424-5p in human colorectal cancer cellsObjective:To study the biological functions of mi R-424-5p in SW480colorectal cancer cells and explore the possible mechanism of mi R-424-5p in the occurrence and development of colorectal cancer.Methods:1.The SW480 cells were transfected with mi R-424-5p mimic,mimic Control and mi R-424-5p inhibitor and inhibitor control by lipofectamine 2000.after 48 hours,RNA was extracted by Trizol methods,and the relative expression of mi R-424-5p was determined by RT-q PCR.2.After transfection of mimics or inhibitors,CCK-8 kit was used to detect cell proliferation,the OD values of 24h,48h,72h and 96h were measured and the growth curve of cells was drawn.The effects of mi R-424-5p on the proliferation of colorectal cancer cells were compared.After transfection,the number of cell clones was detected by cell clone experiment at 10-14 days.According to the results of the two experiments,we analyzed the cell proliferation status and explored the effect of mi R-424-5p on the proliferation of colorectal cancer cells.3.After transfection of mimics or inhibitors,cell scratch repair test,migration and invasion chamber test were used to detect the migration and invasion ability of cells,and to explored the effect of mi R-424-5p on the metastasis of colorectal cancer cells.Results:1.After transfection of mi R-424-5p mimics for 48 hours,the expression of mi R-424-5p in SW480 cells was significantly up-regulated compared with the control group,with an average up-regulated multiple of 22 times.After transfection of mi R-424-5p inhibitors for 48 hours,the expression mi R-424-5p in SW480 cells was significantly down regulated compared with the control group,with an average down regulated multiple of-10.5 times(P<0.05).conclusion:mi R-424-5p mimics and inhibitors have been successfully transfected.2.After transfection for 24h,48h,72h and 96h,the OD values of mi R-424-5p mimics,mi R-424-5p inhibitor experimental group and control group were calculated at each time point,and the value-added rate was calculated.The increase of mi R-424-5p level in colorectal cancer cells can promote the proliferation of colorectal cancer cells.However,the level of mi R-424-5p in colorectal cancer cells decreased,which could inhibit the proliferation of colorectal cancer cells.The colony formation assay also showed that mi R-424-5p overexpression could significantly promoted the colony forming ability of SW480 cells compared with the negative control group,and mi R-424-5p in SW480 cells significantly decreased and then inhibited the colony forming ability of SW480 cells.3.Compared with the negative control group,overexpression of mi R-424-5p enhanced the migration and ability of SW480 cells,However,compared with the control group,inhibition of mi R-424-5p weakened the migration ability of SW480 cells.Transwell invasion chamber experiment also showed that overexpression of mi R-424-5p significantly increased the number of SW480 cells in each high-power field compared with negative control group,and overexpression of mi R-424-5p significantly promoted the invasion ability of SW480 cells.Compared with the negative control group,inhibition of mi R-424-5p significantly reduced the number of SW480 cells in each high-power microscopic field,and inhibition of mi R-424-5p significantly inhibited the invasion ability of SW480 cells.Conclusions:Overexpression of mi R-424-5p can promoted the proliferation,migration and invasion of colorectal cancer cells.Knockdown of mi R-424-5p can inhibit the proliferation,migration and invasion of colorectal cancer cells.Part Three Molecular mechanism of mi R-424-5p in human color-ectal cancer cells.Objective:All target genes and signal pathways of mi R-424-5p were screened by transcriptome sequencing and bioinformatics gene enrichment analysis,which provided a reliable basis for comprehensive elucidation of the mechanism of mi R-424-5p.Methods:1.mi R-424-5p mimic,mimic control,mi R-424-5p inhibitor and inhibitor control were transfected into SW480 cells by Lipofectamine 2000.Total RNA was extracted 48 hours later.2.Transcriptome sequencing was used to screen differentially expressed gens.The volcano map of differentially expressed gens(DEG)was drawn by gglot program of R language,and functional annotation,GO analysis and pathway enriched(KEGG)analysis were performed by bioinformatics gene enrichment analysis method.3.In order to further study and predict the target genes of mi R-424-5p,we analyzed the down regulated target genes after mi R-424-5p mimics and the target genes predicted by Target Scan.Results:1.After sequencing of mi R-424-5p mimic(experimental group)and mi R-424-5p mimic control,2302 differential genes(DEGs)were found between experimental group and control group.of which 1322 were up-regulated genes and 980 were down regulated genes.2.Go enrichment analysis showed that DEGs were significantly enriched in serine hydrolase activity,serine peptidase,endopeptidase,guanosine triphosphatase,PDZ domain binding growth factor activity and other biological processes;and DEGs were also enriched in a variety of disease-related clusters.Such as skin diseases,infertility and colon diseases.2.KEGG metabolic pathway enrichment analysis was performed on DEG.The results showed that Wnt(Wnt signaling pathway)signaling pathway,wnt6,Wnt11,DVL1,Rac3,fzd9,Sfrp5 and fzd2,vascular endothelial growth factor(VEGF)signaling pathway and TGF beta signaling pathway were down regulated in DEG.Up regulated DEGs was significantly enriched MAPK signalling pathway,insulin signaling pathway.3.The results of down-regulated differential genes and the Wenn diagram using Target Scan to predict the target genes of mi R-424-5p showed that there were 30 overlapping genes.Conclusions:the potential mechanism of mi R-424-5p in colorectal cancer is through the regulation of Wnt signaling pathway,vascular endothelial growth factor signaling pathway,TGF beta signaling pathway and MAPK signaling pathway... |
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