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[长链非编码RNA LINC-PINT通过靶向miR-524-5p调控骨肉瘤细胞的增殖和凋亡]

[LncRNA LINC-PINT regulating proliferation and apoptosis of osteosarcoma cells by targeting miR-524-5p].

作者信息

Yang H, Cao C, Wang L J

机构信息

Department of Orthopedics, Zhumadian Central Hospital, Zhumadian 463000, China.

Department of Orthopedics, Henan People's Hospital, Zhengzhou 450000, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2020 Apr 23;42(4):325-330. doi: 10.3760/cma.j.cn112152-20190726-00471.

Abstract

To study the effect of long non-coding RNA LINC-PINT on proliferation and apoptosis of osteosarcoma cells. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of LINC-PINT and miR-524-5p in normal osteoblast hFOB and human osteosarcoma cell lines HOS, MG63 and SAOS2 cells. The pcDNA plasmid, pcDNA-LINC-PINT plasmid, negative control siRNA (si-NC), si-LINC-PINT, negative control mimics (miR-NC), miR-524-5p mimics (miR-524-5p), pcDNA-LINC-PINT combined with miR-NC, pcDNA-LINC-PINT combined with miR-524-5p were transfected into HOS cells with liposome, respectively. The protein expressions of PCNA and cleaved-caspase-3 in the cells were detected by western blot. Cell proliferation ability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The apoptosis was detected by flow cytometry. The transcriptional activity was detected by double luciferase reporter assay. Compared with normal osteoblast hFOB cell (1.00±0.08 vs 1.00±0.06), the expressions of LINC-PINT were down-regulated (0.18±0.01; 0.33±0.01; 0.42±0.01), while the expressions of miR-524-5p were up-regulated (2.65±0.23; 1.68±0.14; 1.51±0.13) in human osteosarcoma cell lines HOS, MG63 and SAOS2 cells, respectively. Overexpression of LINC-PINT significantly inhibited the proliferation (0.41±0.05 vs. 0.62±0.05 for 48 h; 0.57±0.05 vs. 1.06±0.09 for 72 h, both <0.05) while promoted the apoptosis (25.28±2.15 vs. 9.01±0.17, <0.01) of HOS cells. Knockdown of LINC-PINT or overexpression of miR-524-5p can significantly promote the proliferation and inhibit apoptosis of HOS cells. Moreover, miR-524-5p inhibited the fluorescence activity of wild-type LINC-PINT (0.31±0.03) in HOS cells when comparred with miR-NC (1.00±0.03) and was negatively regulated by LINC-PINT. Overexpression of miR-524-5p reversed the proliferation inhibition and apoptosis-promotion effects of LINC-PINT in HOS cells. Long non-coding RNA LINC-PINT can inhibit the proliferation and promote apoptosis of osteosarcoma cells through targeting miR-524-5p, which will provide a new target for the treatment of osteosarcoma.

摘要

研究长链非编码RNA LINC-PINT对骨肉瘤细胞增殖和凋亡的影响。采用实时定量逆转录聚合酶链反应(qRT-PCR)检测正常成骨细胞hFOB及人骨肉瘤细胞系HOS、MG63和SAOS2细胞中LINC-PINT和miR-524-5p的表达。分别用脂质体将pcDNA质粒、pcDNA-LINC-PINT质粒、阴性对照小干扰RNA(si-NC)、si-LINC-PINT、阴性对照模拟物(miR-NC)、miR-524-5p模拟物(miR-524-5p)、pcDNA-LINC-PINT与miR-NC组合、pcDNA-LINC-PINT与miR-524-5p组合转染至HOS细胞。通过蛋白质免疫印迹法检测细胞中增殖细胞核抗原(PCNA)和裂解的半胱天冬酶-3的蛋白表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H四氮唑溴盐(MTT)法检测细胞增殖能力。通过流式细胞术检测细胞凋亡情况。采用双荧光素酶报告基因检测法检测转录活性。与正常成骨细胞hFOB细胞相比(1.00±0.08 vs 1.00±0.06),人骨肉瘤细胞系HOS、MG63和SAOS2细胞中LINC-PINT的表达下调(0.18±0.01;0.33±0.01;0.42±0.01),而miR-524-5p的表达上调(2.65±0.23;1.68±0.14;1.51±0.13)。LINC-PINT的过表达显著抑制HOS细胞的增殖(48小时时为0.41±0.05 vs. 0.62±0.05;72小时时为0.57±0.05 vs. 1.06±0.09,均<0.05),同时促进其凋亡(25.28±2.15 vs. 9.01±0.17,<0.01)。敲低LINC-PINT或过表达miR-524-5p均可显著促进HOS细胞的增殖并抑制其凋亡。此外,与miR-NC(1.00±0.03)相比,miR-524-5p抑制了HOS细胞中野生型LINC-PINT的荧光活性(0.31±0.03),且受LINC-PINT负调控。miR-524-5p的过表达逆转了LINC-PINT对HOS细胞的增殖抑制和凋亡促进作用。长链非编码RNA LINC-PINT可通过靶向miR-524-5p抑制骨肉瘤细胞的增殖并促进其凋亡,这将为骨肉瘤的治疗提供新的靶点。

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