miR-4319通过靶向调控USP2抑制乳腺癌细胞侵袭

MiR-4319 inhibits breast cancer cell invasion by targeting USP2

目的探讨miR-4319与泛素特异性蛋白酶2(USP2)表达的相关性以及miR-4319靶向USP2通过核转录因子κB(NF-κB)信号通路对乳腺癌细胞侵袭的影响。方法实时荧光定量PCR(qRT-PCR)检测miR-4319在正常乳腺癌上皮细胞(MCF10A)、低侵袭性乳腺癌细胞(MCF7)和高侵袭性乳腺癌细胞(MDA-MB-231)中的表达量。将MCF10A、MCF7和MDA-MB-231细胞分为6组进行转染:(1)MDA-MB-231/NC组,瞬时转染插入一段乱码序列(scramble 1),即作为miR-4319过表达对照质粒;(2)MDA-MB-231/miR-4319组,瞬时转染插入目的片段miR-4319质粒过表达miR-4319,即miR-4319过表达;(3)MDA-MB-231/miR-4319+Con组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达对照质粒,即miR-4319过表达以及USP2过表达对照;(4)MDA-MB-231/miR-4319+USP2组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达质粒,即miR-4319过表达和USP2过表达;(5)MDA-MB-231/miR-4319inhibitor组,瞬时转染miR-4319的反义序列抑制miR-4319的表达,即抑制miR-4319的表达;(6)MDA-MB-231/miR-4319inhibitor NC组,瞬时转染插入一段乱码序列(scramble 2),即作为抑制miR-4319组的对照组。qRT-PCR检测miR-4319在MDA-MB-231细胞中的转染效率。荧光素酶实验检测miR-4319与USP2 mRNA是否存在结合位点。qRT-PCR检测miR-4319在乳腺癌细胞中过表达后的USP2 mRNA表达水平。蛋白质印迹法检测过表达miR-4319或抑制miR-4319表达后的USP2蛋白表达水平。Transwell侵袭实验检测过表达miR-4319或USP2后MDA-MB-231细胞侵袭能力。双荧光素酶实验检测miR-4319对NF-κB信号通路活性的影响以及过表达USP2后miR-4319对NF-κB信号通路活性的影响。结果 qRT-PCR结果显示,miR-4319相对表达量在细胞MDA-MB-231(t=14.860,P<0.001)和MCF7(t=12.770,P<0.001)中分别为0.330±0.075和0.570±0.082,均低于细胞MCF10A(1.012±0.051);miR-4319相对表达量MDA-MB-231/miR-4319组(3.980±0.083)高于MDA-MB-231/NC组(1.009±0.058),差异有统计学意义,t=102.90,P<0.001。荧光素酶实验结果显示,pGL3-USP2 3’-UTR-WT报告载体与miR-4319质粒共转染后的荧光素酶活性下降,差异有统计学意义,t=15.740,P<0.001。qRT-PCR结果显示,USP2 mRNA相对表达量MDA-MB-231/NC组(1.013±0.058)和MDA-MB-231/miR-4319组(0.988±0.062)基本没有变化,差异无统计学意义,t=1.201,P=0.296。而蛋白质印迹法结果显示,USP2蛋白相对表达量miR-4319组(0.371±0.083)低于miR-4319/NC组(1.003±0.064),miR-4319/inhibitor组(1.982±0.093)高于inhibitor/NC组(1.104±0.072),USP2蛋白相对表达量的组间比较差异有统计学意义,F=212.4,P<0.001。Transwell侵袭实验结果显示,穿过Matrigel细胞数MDA-MB-231/miR-4319组(58.337±5.972)低于MDA-MB-231/NC组(192.371±5.476),差异有统计学意义,t=29.720,P<0.001;穿过Matrigel细胞数MDA-MB-231/miR-4319+USP2组(167.197±8.292)高于MDA-MB-231/miR-4319+Con组(63.283±10.397),差异有统计学意义,t=14.150,P=0.001。双荧光素酶结果显示,miR-4319/NF-κB-luc组荧光素酶活性(0.324±0.06)低于NC/pRL-TK组(1.024±0.080)、NC/NF-κB-luc组(2.023±0.110)和miR-4319/pRL-TK组(1.109±0.050),组间比较差异有统计学意义,F=237.1,P<0.001;miR-4319+USP2/NF-κB-luc组荧光素酶活性(2.032±0.12)高于miR-4319+USP2/pRL-TK组(1.094±0.100)、miR-4319+Con/pRL-TK组(1.063±0.080)和miR-4319+Con/NF-κB-luc组(0.334±0.050),组间比较差异有统计学意义,F=164.2,P<0.001。结论 miR-4319在乳腺癌细胞中低表达,miR-4319靶向结合USP2,过表达USP2逆转了miR-4319对乳腺癌细胞侵袭能力的抑制作用和NF-κB转录活性的抑制作用。miR-4319通过靶向结合USP2抑制NF-κB信号通路,从而抑制乳腺癌细胞的侵袭。

Objective To investigate the correlation between miR-4319 and ubiquitin specific protease 2(USP2)expression and the effect of miR-4319 targeting USP2 on breast cancer cell invasion through the NF-κB signaling pathway.Methods QRT-PCR was used to detect the expression of miR-4319 in normal breast cancer epithelial cells(MCF10 A),low-invasive breast cancer cells(MCF7)and highly invasive breast cancer cells(MDA-MB-231).Divide MCF10 A,MCF7 and MDA-MB-231 cells into 6 groups for transfection:(1)MDA-MB-231/NC group,transiently transfected to insert a scramble sequence(scramble 1),which served as a miR-4319 overexpression control plasmid;(2)MDA-MB-231/miR-4319 group,transiently transfected to insert the target fragment miR-4319 plasmid overexpression miR-4319,which was,miR-4319 overexpression;(3)MDA-MB-231/miR-4319+Con group,transiently transfected into miR-4319 overexpression plasmid and USP2 overexpression control plasmid at the same time,namely miR-4319 overexpression and USP2 overexpression control;(4)MDA-MB-231/miR-4319+USP2 group,transiently transfected into miR-4319 overexpression plasmid and USP2 overexpression plasmid at the same time,namely miR-4319 overexpression and USP2 overexpression;(5)MDA-MB-231/miR-4319 inhibitor group,transiently transfected with the antisense sequence of miR-4319 to inhibit the expression of miR-4319,which inhibited the expression of miR-4319;(6)MDA-MB-231/miR-4319 inhibitor NC group,transiently transfected to insert a scramble sequence(scramble 2),as a control group for inhibiting miR-4319 group.qRT-PCR detects the transfection efficiency of miR-4319 in MDA-MB-231 cells.Luciferase assay detected whether there was a binding site between miR-4319 and USP2 mRNA.qRT-PCR was used to detect the expression level of USP2 mRNA after miR-4319 was overexpressed in breast cancer cells.Western blotting was used to detect the expression level of USP2 protein after overexpression of miR-4319 or inhibition of miR-4319 expression.Transwell invasion assay detected the invasion ability of MDA-MB-231 cells after overexpression of miR-4319 or USP2.The dual-luciferase assay was used to detect the effect of miR-4319 on the activity of the NF-κB signaling pathway and the effect of miR-4319 on the activity of the NF-κB signaling pathway after over-expression of USP2.Results qRT-PCR results showed that the relative expression of miR-4319 RNA in breast cancer cells MDA-MB-231 and MCF7 were 0.330±0.075 and 0.57±0.082,which were lower than normal breast cancer epithelial cells MCF10 A(1.012±0.051),t=14.860,P<0.001;t=12.770,P<0.001.qRT-PCR results showed that the relative expression of miR-4319 was 3.980±0.083 in the MDA-MB-231/miR-4319 group,which was higher than the MDA-MB-231/NC group(1.009±0.058).The difference was statistically significant,t=102.900,P<0.001.Luciferase experiment results showed that the luciferase activity of pGL3-USP23′-UTR-WT reporter vector co-transfected with miR-4319 plasmid decreased significantly(t=15.740,P<0.001).qRT-PCR results showed that the relative expression of USP2 mRNA in the MDA-MB-231/NC group(1.013±0.058)and MDA-MB-231/miR-4319 group(0.988±0.062)were no significant change,the difference was not statistically significant,t=1.201,P=0.296.Western blot results showed that the relative expression of USP2 protein in the miR-4319 group(0.371±0.083)was lower than that in the miR-4319/NC group(1.003±0.064);miR-4319/inhibitor group(1.982±0.093)was higher than inhibitor/NC group(1.104±0.072).The relative expression of USP2 protein showed statistically significant difference between groups,F=212.4,P<0.001.Transwell invasion experiment showed that the number of breast cancer cells that passed through matrigel in the MDA-MB-231/miR-4319 group was 58.337±5.972,which was significantly lower than the MDA-MB-231/NC group(192.371±5.476),the difference was statistically significant,t=29.720,P<0.001.The number of breast cancer cells that passed through matrigel in the MDA-MB-231/miR-4319+USP2 group was 167.197±8.292,which was higher than the MDA-MB-231/miR-4319+Con group(63.283±10.397),the difference was statistically significant,t=14.150,P=0.001.The results of the dual-luciferase experiment showed that the luciferase activity of the miR-4319/NF-κB-luc group was 0.324±0.06,which was lower than the NC/pRL-TK group(1.024±0.080)and the NC/NF-κB-luc group(2.023±0.11),miR-4319/pRL-TK group(1.109±0.050),the difference among the groups is statistically significant,F=237.1,P<0.001.The luciferase activity of the miR-4319+USP2/NF-κB-luc group was 2.032±0.120,which was higher than the miR-4319+USP2/pRL-TK group(1.094±0.100),and the miR-4319+Con/pRL-TK group(1.063±0.080),miR-4319+Con/NF-κB-luc group(0.334±0.050),the difference among groups was statistically significant,F=164.2,P<0.001.Conclusions MiR-4319 is low-expression in breast cancer cells and can target USP2.Over-expression of USP2 reverses the inhibitory effect of miR-4319 on the invasion ability of breast cancer cells.Over-expression of USP2 reverses miR-4319 on the NF-κB inhibition of transcriptional activity.miR-4319 inhibits the invasion of breast cancer cells by targeting the USP2 to inhibit the NF-κB signaling pathway.