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目的探讨AS-miR-21对鼻咽癌转移潜能及其对Bcl-2调控作用机制。方法应用MTT检测鼻咽癌细胞增殖抑制率;Giemsa染色检测鼻咽癌细胞克隆形成;细胞划痕实验检测鼻咽癌细胞迁移能力;半定量逆转录-聚合酶链反应(RT-PCR)检测Bcl-2 mRNA表达;免疫组化方法和蛋白质印迹法检测Bcl-2蛋白表达水平。结果结果显示,AS-miR-21、miR-21和miR-NC组24hA值分别为0.12±0.01、0.23±0.02和0.22±0.02,F=106.47,P<0.000 1;48hA值分别为0.21±0.02、0.53±0.03和0.52±0.02,F=340.01,P<0.000 1;72hA值分别为0.43±0.03、0.75±0.03和0.66±0.02,F=209.45,P<0.000 1;96hA值分别为0.74±0.02、0.95±0.03和0.93±0.01,F=114.52,P<0.000 1。Giemsa染色结果显示,AS-miR-21组克隆形成率为(0.35±0.02)%,比对照组的(0.65±0.06)%减少46.2%,t=8.215 8,P=0.001 2;miR-21组克隆形成率为(0.82±0.08)%,比对照组的(0.65±0.06)%增加20.7%,t=2.944 5,P=0.042 2。划痕实验结果显示,12h后AS-miR-21组愈合率为(0.207±0.015)%,比对照组的(0.487±0.032)%减少57.49%,F=185.68,P<0.000 1;24h后AS-miR-21组愈合率为(0.471±0.002)%,比对照组的(0.810±0.036)%减少41.85%,F=264.96,P<0.000 1。RT-PCR结果显示,AS-miR-21组Bcl-2 mRNA表达为0.252±0.053,比对照组的1.000±0.095减少74.8%,t=11.909 6,P=0.000 3;miR-21组Bcl-2 mRNA表达为1.837±0.152,比对照组的1.000±0.095增加83.7%,t=8.087 9,P=0.001 3。免疫组化结果显示,AS-miR-21组Bcl-2蛋白表达灰度值为196.2±11.8,比对照组的126.9±7.3减少54.6%,t=15.793 7,P<0.000 1。蛋白质印迹结果显示,AS-miR-21组Bcl-2蛋白表达为0.125±0.073,比对照组的1.000±0.163减少87.5%,t=15.492 7,P<0.000 1;miR-21组Bcl-2蛋白表达为2.018±0.182,比对照组的1.000±0.095增加101.8%,t=13.176 1,P<0.000 1。结论AS-miR-21能抑制鼻咽癌迁移及增殖并促进其凋亡,此外,AS-miR-21通过负调控Bcl-2有可能成为鼻咽癌新靶点之一。
Objective To investigate the metastatic potential of AS-miR-21 to nasopharyngeal carcinoma and its mechanism of regulating Bcl-2 expression. Methods The inhibitory rate of proliferation of nasopharyngeal carcinoma cells was detected by MTT assay. Clone formation of nasopharyngeal carcinoma cells was detected by Giemsa staining. Migration assay of nasopharyngeal carcinoma cells was performed by cell scratch assay. Bcl-2 was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) -2 mRNA expression was detected by immunohistochemistry and Western blotting to detect the expression of Bcl-2 protein. The results showed that the values of 24hA in AS-miR-21, miR-21 and miR-21 were 0.12 ± 0.01, 0.23 ± 0.02 and 0.22 ± 0.02 respectively, F = 106.47, P <0.0001; , 0.53 ± 0.03 and 0.52 ± 0.02, F = 340.01, P <0.000 1; 72hA values were 0.43 ± 0.03, 0.75 ± 0.03 and 0.66 ± 0.02, respectively, F = 209.45 and P <0.0001; 96hA values were 0.74 ± 0.02 , 0.95 ± 0.03 and 0.93 ± 0.01, F = 114.52, P <0.0001. Giemsa staining showed that the rate of colony formation was (0.35 ± 0.02)% in AS-miR-21 group, 46.2% less than that in control group (0.65 ± 0.06)%, t = 8.215 8, P = 0.001 2; The rate of colony formation was (0.82 ± 0.08)%, increased by 20.7% (t = 2.944 5, P = 0.042 2) compared with the control group (0.65 ± 0.06)%. Scratch test results showed that the healing rate of AS-miR-21 group after 12h was (0.207 ± 0.015)%, which was 57.49% lower than that of control group (0.487 ± 0.032)%, F = 185.68, P < The healing rate of -miR-21 group was (0.471 ± 0.002)%, which was 41.85% lower than that of the control group (0.810 ± 0.036)%, F = 264.96, P <0.0001. RT-PCR results showed that the expression of Bcl-2 mRNA in AS-miR-21 group was 0.252 ± 0.053, which was 74.8% lower than that in control group (t = 11.909 6, P = 0.0003) The mRNA expression was 1.837 ± 0.152, which was 83.7% higher than 1.000 ± 0.095 in the control group, t = 8.087 9, P = 0.001 3. Immunohistochemistry showed that the gray value of Bcl-2 protein in AS-miR-21 group was 196.2 ± 11.8, which was 54.6% lower than 126.9 ± 7.3 in control group, t = 15.793 7, P <0.0001. Western blot results showed that the expression of Bcl-2 protein in AS-miR-21 group was 0.125 ± 0.073, which was 87.5% less than 1.000 ± 0.163 in control group, t = 15.492 7, P <0.0001; Expressed as 2.018 ± 0.182, an increase of 101.8% compared with 1.000 ± 0.095 in the control group, t = 13.176 1, P <0.0001. Conclusions AS-miR-21 can inhibit nasopharyngeal carcinoma migration and proliferation and promote its apoptosis. In addition, AS-miR-21 may be one of the new targets of nasopharyngeal carcinoma by negatively regulating Bcl-2.