微小RNA-182靶向调控特定富含AT碱基DNA序列结合蛋白2基因对结肠癌细胞增殖和迁移的影响

MicroRNA-182 regulates colon cancer cell proliferation and migration by targeting special AT-rich sequence-binding protein 2 gene

目的观察微小RNA(miRNA,miR)-182通过靶向特定富含AT碱基DNA序列结合蛋白2(SATB2)基因对结直肠癌细胞增殖迁移的作用,探讨其对SATB2/烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nox4)通路的调节机制。方法对数期生长人结肠癌细胞(HT-29)细胞,采用脂质体转染法将si-miR-182、Control-si转至HT-29细胞,分别设为Si-miR-182组、N-miR-182组,另取不做任何处理细胞为对照组。测定3组细胞中miR-182基因表达、增殖和迁移、SATB2、nox4、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)mRNA和蛋白表达。重复计量资料比较采用重复测量方差分析,两两样本比较采用LSD-t检验。结果Si-miR-182组miR-182基因相对表达量(0.35±0.05)低于对照组(1.24±0.26)和N-miR-182组(1.20±0.25),差异均有统计学意义(t=7.517、7.455,P<0.05);Si-miR-182组培养24、48、72 h MTT试验吸光度(A)值均低于对照组和N-miR-182组,差异均有统计学意义(24 h:t=2.667、2.664;48 h:t=4.559、4.524;72 h:t=7.257、6.981;P<0.05);与培养24 h比较,3组培养48、72 h MTT试验A值均升高(48 h:t=5.507、5.092、3.741;72 h:t=11.330、10.637、9.229;P<0.05),且72 h MTT试验A值高于48 h,差异均有统计学意义(t=7.411、6.941、5.214,P<0.05)。Si-miR-182组细胞迁移率[(53.90±3.19)%]低于对照组[(81.66±5.92)%]和N-miR-182组[(80.35±5.40)%],差异均有统计学意义(t=9.231、9.430,P<0.05);Si-miR-182组细胞SATB2、E-cadherin mRNA和蛋白相对表达量高于对照组和N-miR-182组(SATB2:t=10.930、11.158;E-cadherin:t=9.288、9.369;P<0.05),nox4、Vimentin mRNA和蛋白相对表达量低于对照组和N-miR-182组,差异均有统计学意义(nox4:t=8.955、7.590;Vimentin:t=6.543、6.644;P<0.05)。结论沉默miR-182基因可显著抑制结直肠癌细胞增殖及迁移能力,可能通过激活SATB2、E-cadherin的表达、抑制nox4、Vimentin的表达、参与SATB2/nox4通路共同调控。

Objective To observe the effect of microRNA(miRNA,miR)-182 on the proliferation and migration of colorectal cancer cells by targeting special AT-rich sequence-binding protein 2(SATB2)gene,and to explore its effect on SATB2/icotinamide adenine dinucleotide phosphate oxidase 4(nox4)pathway.Methods HT-29 cells in logarithmic phase were transfected with Si-miR-182 and Control-si resectively,by liposome transfection,serving as Si-miR-182 group and N-miR-182 group,respectively.The expression of miR-182 gene,the cell proliferation and migration,the mRNA and protein expression of SATB2,nox4,E-cadherin and vimentin were examined.Results The relative expression of miR-182 gene in Si-miR-182 group(0.35±0.05)was lower than that in control group(1.24±0.26)and N-miR-182 group(1.20±0.25)(t=7.517,7.455,P<0.05).The A values of methyl thiazol tetrazolium(MTT)test in Si-miR-182 group were lower than those in control group and N-miR-182 group after 24,48 and 72 h of culture(24 h:t=2.667,2.664;48 h:t=4.559,4.524;72 h:t=7.257,6.981;P<0.05).Compared with 24-h culture,the A value of MTT test in 48 and 72 hours in three groups were increased(48 h:t=5.507,5.092,3.741;72 h:t=11.330,10.637,9.229;P<0.05),and the A value of MTT test in 72 hours were higher than those in 48 hours(t=7.411,6.941,5.214,P<0.05).The cell migration rate of Si-miR-182 group[(53.90±3.19)%]was lower than that of the control group[(81.66±5.92)%]and N-miR-182 group[(80.35±5.40)%](t=9.231,9.430,P<0.05).The relative expressions of SATB2,E-cadherin mRNA and protein in Si-miR-182 group were higher than those in control group and N-miR-182 group(SATB2:t=10.930,11.158;E-Cadherin:t=9.288,9.369;P<0.05),while the relative expressions of nox4,vimentin mRNA and protein in Si-miR-182 group were lower than those in control group and N-miR-182 group(nox4:t=8.955,7.590;Vimentin:t=6.543,6.644;P<0.05).Conclusion Silencing of miR-182 gene can significantly inhibit the proliferation and migration of colorectal cancer cells,possibly by activating SATB2,E-Cadherin expression,inhibiting the expression of nox4,Vimentin,and participating in the SATB2/nox4 pathway.