miR-515-5p靶向RING1增强NSCLC A549/DDP细胞顺铂敏感性分析

doi:10.3877/cma.j.issn.1674-6902.2021.05.003

目的

探讨微小RNA-515-5p(miR-515-5p)对非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞顺铂敏感性的影响及机制。

方法

实时荧光定量PCR(RT-qPCR)检测BEAS-2B、A549及A549/DDP细胞中miR-515-5p的表达情况。A549/DDP细胞分为NC组、miR-515-5p组、si-RING1组及miR-515-5p inh+si-RING1组，CCK-8法检测A549/DDP细胞增殖活性，用WB检测RING1和凋亡相关蛋白的表达情况。用双荧光素酶报告基因系统验证miR-515-5p与RING1的靶向关系。

结果

miR-515-5p在BEAS-2B、A549及A549/DDP中的表达水平分别为(1.00±0.03)、(0.61±0.03)、(0.31±0.04)；A549/DDP细胞转染miR-515-5p mimics后，NC组和miR-515-5p组细胞在24、48、72和96 h时的OD₄₅₀值分别为(0.61±0.02)、(0.52±0.02)，(0.84±0.02)、(0.68±0.04)，(1.03±0.03)、(0.79±0.04)，(1.08±0.03)、(0.86±0.04)；以上指标组间差异均有统计学意义(P<0.05)。miR-515-5p组cleaved caspase-3、cleaved PARP和Bax表达水平显著高于NC组，Bcl-2的表达水平显著低于NC组。A549/DDP细胞转染si-RING1、miR-515-5p inhibitor后，NC组，si-RING1组和miR-515-5p inh+si-RING1组细胞在24、48、72和96 h时的OD₄₅₀值分别为(0.63±0.02)、(0.49±0.04)、(0.58±0.03)、(0.83±0.03)、(0.62±0.05)、(0.80±0.02)、(1.05±0.04)、(0.76±0.03)、(1.02±0.04)、(1.15±0.05)、(0.86±0.03)、(1.12±0.06)；以上指标组间差异均有统计学意义(P<0.05)。si-RING1组cleaved caspase-3、cleaved PARP和Bax表达水平显著高于NC组和miR-515-5p inh+si-RING1组，Bcl-2的表达水平显著低于NC组和miR-515-5p inh+si-RING1组。

结论

miR-515-5p通过靶向下调RING1的表达增强A549/DDP细胞顺铂敏感性。

关键词: 微小RNA-515-5p, 无名指蛋白1, 非小细胞肺癌, 顺铂敏感性

Objective

To investigate the effect and mechanism of microRNA-515-5p (miR-515-5p) on the cisplatin sensitivity of non-small cell lung cancer (NSCLC).

Methods

The expression of miR-515-5p in BEAS-2B, A549 and A549/DDP cells was detected by real-time fluorescent quantitative PCR (RT-qPCR). A549/DDP cells were divided into NC group, miR-515-5p group, si-RING1 group and miR-515-5p inh+ si-RING1 group. CCK-8 assay was used to assess the proliferation of A549/DDP cells. WB was performed to measure the expression of RING1 and apoptosis related proteins. Dual luciferase reporter gene system was used to verify the targeting relationship between miR-515-5p and RING1.

Results

The expression of miR-515-5p in BEAS-2B, A549 and A549/DDP cells were (1.00±0.03), (0.61±0.03), (0.31±0.04), respectively. A549/DDP cells were transfected with miR-515-5p mimics, the OD₄₅₀ values of cells in NC group and miR-515-5p group at 24, 48, 72 and 96 h were (0.61±0.02), (0.52±0.02), (0.84±0.02), (0.68±0.04), (1.03±0.03), (0.79±0.04), (1.08±0.03), (0.86±0.04), respectively. The differences of those items were all statistically significant (all P<0.05). The expression of cleaved caspase-3, cleaved PARP and Bax in miR-515-5p group were significantly higher than those in NC group, and the expression of Bcl-2 were significantly lower than those in NC group. A549/DDP cells were transfected with si-RING1 and miR-515-5p inhibitor, the OD₄₅₀ values of cells in NC group, si-RING1 group and miR-515-5p inh+ si-RING1 group at 24, 48, 72 and 96 h were (0.63±0.02), (0.49±0.04), (0.58±0.03), (0.83±0.03), (0.62±0.05), (0.80±0.02), (1.05±0.04), (0.76±0.03), (1.02±0.04), (1.15±0.05), (0.86±0.03), (1.12±0.06), respectively. The differences of those items were all statistically significant (all P<0.05). The expression of cleaved caspase-3, cleaved PARP and Bax in si-RING1 group were significantly higher than those in NC group and miR-515-5p inh+ siRING1 group, and the expression of Bcl-2 were significantly lower than those in NC group and miR-515-5p inh+ siRING1 group.

Conclusion

miR-515-5p enhanced the cisplatin sensitivity of A549/DDP cells through targeting downregulate the expression of RING1.

Key words: MicroRNA-515-5p, Ring finger protein 1, Non-small cell lung cancer, Cisplatin sensitivity

图1 WB检测A549/DDP细胞中cleaved caspase-3、cleaved PARP、Bax及Bcl-2的表达

表1 过表达miR-515-5p对A549/DDP细胞增殖的影响(OD₄₅₀，±s)

分　组	24 h	48 h	72 h	96 h
NC组	0.61±0.02	0.84±0.02	1.03±0.03	1.08±0.03
miR-515-5p组	0.52±0.02^a	0.68±0.04^a	0.79±0.04^a	0.86±0.04^a

表1 过表达miR-515-5p对A549/DDP细胞增殖的影响(OD₄₅₀，±s)

分　组	24 h	48 h	72 h	96 h
NC组	0.61±0.02	0.84±0.02	1.03±0.03	1.08±0.03
miR-515-5p组	0.52±0.02^a	0.68±0.04^a	0.79±0.04^a	0.86±0.04^a

图2 WB检测A549/DDP细胞中RING1的表达

图3 WB检测A549/DDP细胞中RING1、cleaved caspase-3、cleaved PARP、Bax及Bcl-2的表达

表2 miR-515-5p通过RING1对A549/DDP细胞的影响(OD₄₅₀，±s)

分　组	24 h	48 h	72 h	96 h
NC组	0.63±0.02	0.83±0.03	1.05±0.04	1.15±0.05
si-SING1组	0.49±0.04^a	0.62±0.05^a	0.76±0.03^a	0.86±0.03^a
miR-515-5p inh　+si-RING1组	0.58±0.03^b	0.80±0.02^b	1.02±0.04^b	1.12±0.06^b

表2 miR-515-5p通过RING1对A549/DDP细胞的影响(OD₄₅₀，±s)

分　组	24 h	48 h	72 h	96 h
NC组	0.63±0.02	0.83±0.03	1.05±0.04	1.15±0.05
si-SING1组	0.49±0.04^a	0.62±0.05^a	0.76±0.03^a	0.86±0.03^a
miR-515-5p inh　+si-RING1组	0.58±0.03^b	0.80±0.02^b	1.02±0.04^b	1.12±0.06^b

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Study on the mechanism of miR-515-5p enhanced the cisplatin sensitivity of NSCLC A549/DDP cells via targeting RING1

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Study on the mechanism of miR-515-5p enhanced the cisplatin sensitivity of NSCLC A549/DDP cells via targeting RING1