Experimental study of miR-613 targeting PTBP1 to inhibit the invasion and migration of endometrial cancer cells
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摘要:
目的 探讨微RNA-613(miR-613)靶向多聚嘧啶区结合蛋白1(PTBP1)对子宫内膜癌细胞(Ishikawa)增殖、迁移及侵袭的影响。 方法 采用RT-qPCR法检测子宫内膜癌组织miR-613及PTBP1 mRNA表达；体外培养Ishikawa细胞并对Ishikawa细胞进行干预，过表达miR-613、抑制PTBP1表达或共同过表达miR-613与PTBP1，分别检测Ishikawa细胞增殖、迁移与侵袭及PTBP1、基质金属蛋白酶2(MMP-2)、MMP-9蛋白表达情况；双荧光素酶报告实验验证miR-613与PTBP1的靶向关系。 结果 子宫内膜癌组织中miR-613表达水平为0.48±0.09，显著低于癌旁组织的1.03±0.11(P＜0.05)，PTBP1 mRNA表达水平为2.78±0.23，显著高于癌旁组织的1.01±0.12(P＜0.05)，miR-613与PTBP1 mRNA呈负相关关系(r=-0.523，P＜0.05)；双荧光素酶报告基因检测结果显示，miR-613靶向负调控PTBP1表达；过表达miR-613与抑制PTBP1表达，Ishikawa细胞存活率、细胞迁移与侵袭数、PTBP1、MMP-2及MMP-9表达水平显著降低(均P＜0.05)；过表达PTBP1逆转过表达miR-613对Ishikawa细胞增殖、迁移与侵袭的抑制作用。 结论 miR-613在子宫内膜癌组织中低表达，过表达miR-613可通过靶向抑制PTBP1表达，抑制人子宫内膜癌细胞的增殖、迁移及侵袭。 Abstract:Objective To investigate the effects of miR-613 targeting polypyrimidine tract-binding protein 1 (PTBP1) on the proliferation, migration and invasion of endometrial cancer cells (Ishikawa). Methods Real-time fluorescent quantitative PCR (RT-qPCR) was conducted to detect the expression of miR-613 and PTBP1 mRNA in endometrial cancer tissues. Ishikawa cells were cultured in vitro, and intervened to overexpress miR-613, inhibit PTBP1 expression, or co-overexpress miR-613 and PTBP1. The proliferation, migration and invasion of Ishikawa cells and the protein expression levels of PTBP1, matrix metalloproteinase-2(MMP-2) and MMP-9 were detected respectively. Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-613 and PTBP1. Results The expression level of miR-613 in endometrial cancer tissue was 0.48±0.09, which was significantly lower than that in paracancerous tissue (1.03±0.11, P < 0.05). The expression level of PTBP1 mRNA was 2.78±0.23, which was significantly higher than that in paracancerous tissue (1.01±0.12, P < 0.05). MiR-613 was negatively correlated with PTBP1 mRNA (r=-0.523, P < 0.05). The results of dual-luciferase reporter gene assay showed that miR-613 targeted and negatively regulated the expression of PTBP1. Overexpression of miR-613 and inhibition of PTBP1 expression significantly decreased the survival rate of Ishikawa cells, the number of cell migration and invasion, and the expression levels of PTBP1, MMP-2 and MMP-9 (all P < 0.05). Overexpression of PTBP1 reversed the inhibitory effects of overexpression of miR-613 on Ishikawa cell proliferation, migration and invasion. Conclusion The expression of miR-613 is low in endometrial cancer tissues. Overexpression of miR-613 can inhibit the proliferation, migration and invasion of human endometrial cancer cells by targeting and inhibiting the expression of PTBP1. -
Key words:
- MicroRNA-613 /
- Polypyrimidine tract-binding protein 1 /
- Endometrial cancer cells /
- Proliferation /
- Migration /
- Invasion
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图 1 各组Ishikawa细胞PTBP1、MMP-2、MMP-9蛋白表达电泳图
Figure 1. Electrophoretic graph of the protein expression of PTBP1, MMP-2 and MMP-9 in Ishikawa cells
图 2 各组Ishikawa细胞PTBP1、MMP-2、MMP-9蛋白表达电泳图
Figure 2. Electrophoretic graph of the protein expression of PTBP1, MMP-2 and MMP-9 in Ishikawa cells
图 3 miR-613与PTBP1 mRNA结合位点预测结合
Figure 3. miR -613 is predicted to bind to the PTBP1 mRNA binding site
图 4 各组Ishikawa细胞PTBP1、MMP-2、MMP-9蛋白表达电泳图
Figure 4. Electrophoretic graph of the protein expression of PTBP1, MMP-2 and MMP-9 in Ishikawa cells
表 1 miR-613及PTBP1 mRNA在子宫内膜癌中表达情况(x±s)
Table 1. The expression of miR-613 and PTBP1 mRNA in endometrial carcinoma(x±s)
组别 例数 miR-613 PTBP1 mRNA 癌旁组织 39 1.03±0.11 1.01±0.12 子宫内膜癌组织 39 0.48±0.09 2.78±0.23 t值 24.167 42.609 P值 < 0.001 < 0.001 
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表 2 转染miR-613 mimics对Ishikawa细胞增殖、迁移与侵袭的影响(x±s)
Table 2. Effects of transfection with miR-613 mimics on proliferation, migration and invasion of Ishikawa cells(x±s)
组别 n 细胞存活率(%) 细胞迁移数(个) 细胞侵袭数(个) PTBP1/ β-actin MMP-2/ β-actin MMP-9/ β-actin miR-NC组 6 96.54±1.24 122.16±5.20 103.54±5.13 1.02±0.10 0.98±0.10 1.01±0.12 miR-613 mimics组 6 57.26±2.41 69.03±4.19 42.75±4.69 0.63±0.07 0.57±0.08 0.45±0.06 t值 35.500 19.488 21.423 7.826 7.842 10.224 P值 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
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表 3 抑制PTBP1对Ishikawa细胞增殖、迁移与侵袭的影响(x±s)
Table 3. Inhibition of PTBP1 on proliferation, migration and invasion of Ishikawa cells(x±s)
组别 n 细胞存活率(%) 细胞迁移数(个) 细胞侵袭数(个) PTBP1/ β-actin MMP-2/ β-actin MMP-9/ β-actin si-NC 6 97.28±1.13 131.32±4.95 98.47±3.17 1.01±0.12 1.05±0.13 0.97±0.11 Si-PTBP1组 6 49.53±2.89 57.24±3.78 45.58±2.67 0.43±0.05 0.54±0.06 0.58±0.07 t值 37.693 29.095 31.258 10.928 8.725 7.327 P值 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
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表 4 各组Ishikawa细胞双荧光素酶报告基因检测结果(x±s)
Table 4. Results of double luciferase reporter gene detection in Ishikawa cells(x±s)
组别 n 荧光素酶相对光活性 PTBP1-3'UTR-WT+miR-NC组 6 0.87±0.09 PTBP1-3'UTR-WT+miR-613 mimics组 6 0.29±0.05a PTBP1-3'UTR-MUT+miR-613 mimics组 6 0.82±0.07 PTBP1-3'UTR-MUT+miR-NC组 6 0.79±0.05 F值 97.455 P值 < 0.001 注：与PTBP1-3 ' UTR-WT+miR-NC组比较，aP＜0.05。
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表 5 过表达PTBP1逆转过表达miR-613对Ishikawa细胞增殖、迁移与侵袭的影响(x±s)
Table 5. Overexpression of PTBP1 reversed the effects of overexpression of miR-613 on proliferation, migration and invasion of Ishikawa cells(x±s)
组别 n 细胞存活率(%) 细胞迁移数(个) 细胞侵袭数(个) PTBP1/ β-actin MMP-2/ β-actin MMP-9/ β-actin miR-613 mimics组 6 57.26±2.41 69.03±4.19 42.75±4.69 0.63±0.07 0.57±0.08 0.45±0.06 miR-613 mimics+pcDNA组 6 56.37±1.49 72.32±4.67 44.63±4.52 0.59±0.09 0.58±0.07 0.47±0.08 PTBP1组 6 112.38±3.34 153.56±5.33 126.28±6.54 1.13±0.12 1.31±0.13 1.24±0.15 miR-613 mimics+PTBP1组 6 97.05±1.43ab 129.03±4.78abc 108.51±5.76abc 0.92±0.08abc 1.12±0.11abc 0.97±0.12abc F值 909.075 467.298 377.921 46.172 84.903 77.215 P值 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 注：与miR-613 mimics组比较，aP＜0.05；与miR-613 mimics+pcDNA组比较，bP＜0.05；与PTBP1组比较，cP＜0.05。
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