| Objective: Colorectal Cancer(CRC)is one of the most common malignant carcinomas of digestive system,which is harmful to human health.The occurrence and development of CRC is closely related to the genetic,lifestyle and precancerous lesions.According to the 2010 statistics,CRC is the third most common cancer in men and the second most common tumor in women.In our country,with the improvement of living standards and dietary changes,CRC has been increasing gradually year by year.With the change of dietary habits and structure,the morbidity and mortality were significantly increased.And younger,familial aggregation,genetic susceptibility are the characteristics of the disease in our country.CRC,like other tumor types,is a disease full of complex genes and multiple factors.At present,in order to improve the specificity and sensitivity of clinical treatment,the occurrence mechanism and related biomarkers of CRC have been research hotspot.Non coding RNA,especially the discovery of microRNA can regulate gene expression,which provides a new perspective for the study of CRC pathogenesis and targeted therapy.MicroRNA(miRNA)is a class of small molecule RNA size of about22 nucleotides,which can combine to the 3'untranslated region(3'UTR)of target genes and regulate gene expression.In general,a miRNA can be applied to the same of different target genes,play the same or different function,while multiple miRNAs can also regulate the expression of the same gene.Studies had shown that miRNAs may involve in the regulation of three fifths of human genes,including cell growth,differentiation,proliferation and apoptosis and so on.In recent years,a great number of studies had shown that miRNA was closely related to the evolution of various tumors.Depending on the role,miRNA can play a role in tumor suppressor genes or oncogenes.MiR-133 b was first thought to be a cardiac specific miRNA.Hu found that miR-133 b was down-regulated in CRC tissues and cells and correlated with the malignant degree of the tumor.However,the mechanism of miR-133 b in CRC was not clear,which need further investigation.MiR-204 was one of the most common abnormal expressions of miRNAs in malignant tumors.The miR-204 p recursor of ripening process was formed two single stranded small molecules: miR-204-3p and miR-204-5p.Most studies had found that miR-204-5p can significantly inhibit the proliferation of tumor,and was closely related to tumor grade.However,there are no reports about miR-204-5p in CRC.Our previous experiment results showed that(1)The expressions of miR-133 b and miR-204-5p in CRC tissues were significantly lower than adjacent normal tissues.(2)The expressions of miR-133 b and miR-204-5p in HT29 cells were higher than other CRC cells.(3)The target gene combined with miR-133 b and miR-204-5p was TGFBR1 through the online site(RegRNA,MiRBase,miRanda,targetScan).Based on the previous research results,(1)we established CRC cells expressed highly or inhibited of miR-133 b and miR-204-5p by transient transfection in HT29 cells.(2)Compared with the control group,we observed the effects of the proliferation and cell cycle of HT29 cells by highly expressing or inhibiting of miR-133 b and miR-204-5p.(3)We tested the target genes of miR-133 b and miR-204-5p by the dual luciferase assay,fluorescent quantitative PCR and Western-blot.Methods:(1)Using cationic liposome method,miR-133b/204-5p mimics and miR-133b/204-5p inhibitors were transiently transfected into HT29 cells.The cell proliferation and cell cycle of cells in vitro were evaluated using CCK8 method,cell cycle detection.(2)Four commonly used databases including RegRNA,MiRBase,miRanda and targetScan were used to forecast the target genes with the search terms of miR-133 b and miR-204-5p.The target gene was validated by the dual luciferase assay,fluorescent quantitative PCR and Western-blot.(3)SPSS16.0 was used to analyze the data.The CCK8 results were analyzed by variance analysis and single factor variance analysis of factorial design.The cell cycle assay,dual luciferase activity assay and fluorescent quantitative PCR results were analyzed by two Independent Samples T test.Approximate T test would be used when data missed variance.P<0.05 was statistically significant.Results:(1)It was successful to establish CRC cells expressed highly or inhibited of miR-133b/204-5p.(2)Compared with miRNA mimics NC group,the proliferation ability of miR-133 b mimics group and miR-204-5p mimics group were both decreased by CCK8 assay(P<0.0001,P <0.001);however,compared with miRNA inhibitors NC group,the proliferation ability of miR-133 b inhibitors group and miR-204-5p inhibitors group were both enhanced(P<0.001,P < 0.001).(3)Compared with miRNA mimics NC group,miR-133 b over-expression in HT29 cells was increased after the proportion of G1 phase(t=-11.24,P<0.001)and the proportion of S cells was decreased(t=6.117,P<0.001)by flow cytometry analysis.Mi R-204-5p over-expression in HT29 was increased after cells proportion of G1(t=-4.03,P<0.05)and the proportion of S cells was decreased(t=2.855,P <0.05).Compared with miRNA inhibitorsNC group,miR-133 b inhibition in HT29 cells was decreased after the proportion of G1 phase(t=4.506,P<0.05)and the proportion of S cells was increased(t=-11.66,P<0.001).MiR-204-5p inhibition in HT29 was decreased after the proportion of G1 phase(t=4.358,P<0.05),and the proportion of S cells was decreased(t=-9.76,P<0.001).(4)Pzex-FR02/TGFBR1 3?UTR vector and its mutation carries were constructed.Pzex-FR02/MUT-3?UTR and miR-133 b mimics,and Pzex-FR02/MUT-3?UTR and miRNA mimics NC were co-transfected into HEK293 cells,respectively.Luciferase activity was used to detect.The results have been showed that there were no changed in two groups(P=0.876).There were the same results in Pzex-FR02/MUT-3?UTR and miR-204-5p mimics group and Pzex-FR02/MUT-3?UTR and miRNA mimics NC group(P=0.638).Pzex-FR02/WT-3?UTR and miR-133 b mimics,and Pzex-FR02/WT-3?UTR and miRNA mimics NC were co-transfected into HEK293 cells,respectively.Luciferase activity was used to detect.The statistical analysis the differences were statistically significant(P<0.05).There were the same results in Pzex-FR02/WT-3?UTR and miR-204-5p mimics group and Pzex-FR02/ WT-3?UTR and miRNA mimics NC group(P<0.05).(5)The fluorescence quantitative PCR results showed that compared with miRNA mimics NC group,the expression of mRNA in TGFBR1 was significantly decreased in miR-133 b mimics group(t=9.78,P=0.001).Compared with miRNA mimics NC group,the expression of mRNA in TGFBR1 was significantly decreased in miR-204-5p mimics group(t=27.478,P<0.001).Compared with miRNA inhibitors NC group,the expression of mRNA in TGFBR1 was significantly increased in miR-133 b inhibitors group(t=-7.644,P<0.01).Compared with miRNA inhibitors NC group,the expression of mRNA in TGFBR1 was significantly increased in miR-204-5p inhibitorsgroup(t=-17.605,P<0.01).(6)Western-blot results showed that compared to miRNA mimics NC group,the expression level of TGFBR1 protein decreased after transfecting by has-miR-133b/204-5p mimics.Compared to miRNA inhibitors NC group,the expression level of TGFBR1 protein increased after transfecting by has-miR-133b/204-5p inhibitors.Conclusions:(1)It was effected the proliferation and cell cycle by overexpressing or inhibiting the expression of miR-133 b and miR-204-5p in HT29 cells.(2)It was proved that TGFBR1 was a downstream target gene of miR-133 b and miR-204-5p by dual luciferase assay,fluorescent quantitative PCR and Western-blot. |
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