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目的用miR-140模拟物转染三阴型乳腺癌BT549细胞,检测Pin1在乳腺癌细胞系BT549中的表达情况,研究在BT549细胞中miR-140如何通过Pin1发挥对上皮细胞-间充质转化(EMT)的抑制作用。方法用miR-140模拟物(miR-140模拟物组)、miR-140阴性对照(阴性对照组)转染BT549细胞,同时设置未处理组为空白对照组。利用实时PCR法检测不同组BT549细胞中的Pin1 mRNA表达水平;利用Western Blot检测E-钙黏蛋白及Pin1蛋白的表达水平;利用划痕试验研究转染miR-140对BT549侵袭能力的影响。结果划痕后24 h,miR-140模拟物组、阴性对照组和空白对照组划痕宽度相对0 h变化的比例分别为23.4%、41.4%和53.1%。与阴性对照组和空白对照组相比,miR-140模拟物组BT549细胞迁移能力明显降低(χ~2=5.550,P<0.05;χ~2=6.995,P<0.05)。miR-140模拟物组、阴性对照组和空白对照组Pin1 mRNA表达水平分别为(0.55±0.02)、(0.89±0.06)和(1.05±0.06)。miR-140模拟物组Pin1 mRNA表达水平显著低于阴性对照组和空白对照组(t=8.823,P<0.05;t=11.480,P<0.05)。miR-140模拟物组、阴性对照组和空白对照组E-钙黏蛋白表达水平分别为(0.98±0.11)、(0.68±0.07)和(0.38±0.04)。miR-140模拟物组E-钙黏蛋白表达水平显著高于阴性对照组和空白对照组(t=7.990,P<0.05;t=21.348,P<0.05)。miR-140模拟物组、阴性对照组和空白对照组Pin1蛋白表达水平分别为(0.57±0.12)、(0.78±0.08)和(1.08±0.21)。miR-140模拟物组Pin1蛋白表达水平显著低于阴性对照组和空白对照组(t=8.921,P<0.05;t=18.657,P<0.05)。结论 miR-140可以抑制Pin1 mRNA转录,进而降低Pin1蛋白的表达水平,抑制EMT的发生。
Objective To detect the expression of Pin1 in BT549 breast cancer cell line BT549 by miR-140 mimics and to investigate the role of miR-140 in epithelial-mesenchymal transition through Pin1 in BT549 cells (EMT) inhibition. Methods BT549 cells were transfected with miR-140 mimics (miR-140 mimics) and miR-140 negative controls (negative controls). Meanwhile, the untreated group was set as the blank control group. The expression of Pin1 mRNA in different groups of BT549 cells was detected by real-time PCR. The expression of E-cadherin and Pin1 protein was detected by Western Blot. The scratch assay was used to study the effect of transfected miR-140 on the invasion ability of BT549 cells. Results The scratch width of miR-140 mock group, negative control group and blank control group were 23.4%, 41.4% and 53.1% respectively at 24 h after scratching. Compared with the negative control group and blank control group, the migration ability of BT549 cells in miR-140 mock group was significantly decreased (χ ~ 2 = 5.550, P <0.05; χ ~ 2 = 6.995, P <0.05). The expression levels of Pin1 mRNA in miR-140 mimics group, negative control group and blank control group were (0.55 ± 0.02), (0.89 ± 0.06) and (1.05 ± 0.06), respectively. The level of Pin1 mRNA in miR-140 mimics group was significantly lower than that in negative control group and blank control group (t = 8.823, P <0.05; t = 11.480, P <0.05). The expression levels of E-cadherin in miR-140 mock group, negative control group and blank control group were (0.98 ± 0.11), (0.68 ± 0.07) and (0.38 ± 0.04), respectively. The expression of E-cadherin in miR-140 mimics was significantly higher than that in the negative control group and the blank control group (t = 7.990, P <0.05; t = 21.348, P <0.05). The expression levels of Pin1 in miR-140 mock group, negative control group and blank control group were (0.57 ± 0.12), (0.78 ± 0.08) and (1.08 ± 0.21), respectively. The level of Pin1 protein in miR-140 mimics group was significantly lower than that in negative control group and blank control group (t = 8.921, P <0.05; t = 18.657, P <0.05). Conclusion miR-140 can inhibit Pin1 mRNA transcription, thereby reducing Pin1 protein expression and inhibiting the occurrence of EMT.