以hsa-miR-491-3p为靶标的反义寡核苷酸对NCI-H446细胞生物学特性的影响

摘要

背景与目的:miRNAs是一类小分子RNA,参与转录后调控.前期研究结果显示,hsa-miR-491-3p在肺痛和肾痛组织中高表达,目前鲜见其功能研究的报道.本研宄旨在探讨以hsa-miR-491-3p为靶标的反义寡核苷酸对人小细胞肺癌NCI-H446细胞生物学特性的影响.方法:合成以hsa-miR-491-3p为靶标的反义寡核苷酸和随机对照序列,转染NCI-H446细胞.实验分4组:空白对照组(组1)、空白转染组(组2) ⑺婊?对照组(组3)和反义寡核苷酸组(组4).用四甲基偶氮唑蓝(MTT)法筛选最佳作用浓度,Hoechst33258染色检测细胞核的形态变化,流式细胞术检测细胞凋亡和周期变化,划痕实验检测细胞的迁移能力.结果:转染反义寡核苷酸后细胞生长受到抑制,最佳作用浓度为0.5 μmol/L.Hoechst33258染色各组的凋亡率分别为(6.67±0.58)%、(7.00±1.00)%、(6.67±1.53)%和(26.33±1.53)%,组4的凋亡率明显高于前3组,差异有统计学意义(P<0.001).流式细胞仪检测各组的凋亡率分别为(2.44±0.60)%、(2.59±0.85)%、(2.62±1.11)%和(5.22±0.40)%,组4的凋亡率明显高于前3组,差异有统计学意义(P<0.01),且G1期细胞增多,S期细胞减少.划痕实验显示各组细胞24 h的迁移距离分别为(196.88±39.13)、(163.06±21.28)、(225.49±23.42)和(77.41±6.07)μ m,组4细胞的迁移距离小于前3组(P<0.05),迁移能力减弱.结论:以hsa-miR-491-3p为靶标的反义寡核苷酸能够抑制NCI-H446细胞增殖,诱导其凋亡,并降低其侵袭能力.%Background and purpose: MicroRNAs (miRNAs) are short RNAs that are involved in post-transcriptional regulation. The results of our previous research display that the expression of hsa-miR-491-3p was up-regulated in both pulmonary and renal cancer tissues. There are no reports yet about the function of hsa-miR-49 l-3p.The aim of this study was to explore the effects of antisense oligonucleotide targeted hsa-miR-491-3p on the biological properties of human small lung cancer (SCLC) NCI-H446 cells. Methods: NCI-H446 cells were distributed to into four groups. Group 1 was the blank control (cells without any treated factors); Group 2 was mock transfection cells group (cells which transfected with LipofectamineTM2000); Group 3 was the scramble control (cells which were transfected with scramble oligonucleotide); Group 4 was the antisense oligonucleotide cells group (cells transfected with antisense hsa-miR-491-3p oligonucleotide). The viability of the NCI-H446 cells was measured using the methyl thiazolyl tetrazolium (MTT) assay, and the optimal concentration for transfection was determined. The morphologic change of the nucleus was detected through Hoechst33258 staining. Flow cytometry was used to check apoptosis and the cell cycle. Cellular migrating ability was detected using a scratch test. Results: After being transfected through antisense oligonucleotide, cellular growth was inhibited, and the optimal concentration for transfection was 0.5 μmol/ L. The apoptosis rate in Group 4 [(26.33±1.53)％] was detected through Hoechst33258 staining and was higher than the first 3 groups; [(6.67±0.58)％, (7.00±1.00)％ and (6.67±1.53)％, respectively, P＜0.001]. The apoptosis rate in Group 4 [(5.22±0.40)％] was detected by flow cytometry and was higher than in the first 3 groups [(2.44±0.60)％, (2.59±0.85)％ and (2.62±1.11)％, respectively, P＜0.01 ]. Meanwhile, the cellular distribution in the G1 phase increased, but decreased in the S and G2/M phase. Cell scratch assay displayed that the cellular migration lengths during the 24 hours were (196.88±39.13), (163.06±21.28), (225.49±23.42) and (77.41±6.07) μm, respectively (P＜0.05). The cellular migration distance of Group 4 was shorter than that of Groups 1, 2 and 3 (P＜0.05). The migrating ability of the cells transfected through antiseuse oligonucleotide also decreased. Conclusion: Antisense oligonucleotide targeted hsa-miR-491-3p can inhibit cell proliferation, induce apoptosis and decrease cellular migration capacity in NCI-H446 cells.