长链非编码RNA LINC-PINT通过靶向miR-524-5p调控骨肉瘤细胞增殖凋亡的分子机制  

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

作　　者：杨豪 曹臣[2] 王力军 Yang Hao;Cao Chen;Wang Lijun(Department of Orthopedics,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Orthopedics,Henan People′s Hospital,Zhengzhou 450000,China)

机构地区：[1]驻马店市中心医院骨二科,463000 [2]河南省人民医院骨科,郑州450000

出　　处：《中华肿瘤杂志》2020年第4期325-330,共6页Chinese Journal of Oncology

基　　金：河南省自然科学基金(1611040376)。

摘　　要：目的:探讨长链非编码RNA LINC-PINT对骨肉瘤细胞增殖和凋亡的影响机制。方法:应用实时荧光定量聚合酶链反应检测正常成骨细胞hFOB和人骨肉瘤细胞HOS、MG63和SAOS2中LINC-PINT和miR-524-5p的表达。应用脂质体法转染至pcDNA组(转染pcDNA)、pcDNA-LINC-PINT组(转染pcDNA-LINC-PINT)、si-NC组(转染si-NC)、si-LINC-PINT组(转染si-LINC-PINT)、miR-NC组(转染miR-NC)、miR-524-5p组(转染miR-524-5p mimics)、pcDNA-LINC-PINT+miR-NC组(共转染pcDNA-LINC-PINT和miR-NC)和pcDNA-LINC-PINT+miR-524-5p组(共转染pcDNA-LINC-PINT和miR-524-5p)HOS细胞,Western blot检测细胞中PCNA和cleaved-caspase-3蛋白的表达,四甲基偶氮唑蓝(MTT)法检测细胞的增殖情况,流式细胞术检测细胞的凋亡情况,双荧光素酶报告基因检测实验检测细胞的荧光活性。结果:人骨肉瘤细胞HOS、MG63和SAOS2中LINC-PINT的表达水平分别为0.18±0.01、0.33±0.01和0.42±0.01,miR-524-5p的表达水平分别为2.65±0.23、1.68±0.14和1.51±0.13,与正常成骨细胞hFOB(分别为1.00±0.08和1.00±0.06)比较,差异均有统计学意义(均 P<0.05)。培养48、72 h时,pcDNA-LINC-PINT组HOS细胞的吸光度( A)值分别为0.41±0.05和0.57±0.05,pcDNA组细胞的 A值分别为0.62±0.05和1.06±0.09,差异均有统计学意义(均 P<0.01)。pcDNA-LINC-PINT组和pcDNA组细胞的凋亡率分别为(25.28±2.15)%和(9.01±0.17)%,差异有统计学意义( P<0.01)。与miR-NC组(1.00±0.03)比较,miR-524-5p组WT-LINC-PINT细胞的荧光活性(0.31±0.03)降低,差异有统计学意义(均 P<0.01)。过表达miR-524-5p可逆转LINC-PINT对HOS细胞的增殖抑制和凋亡促进作用。 结论:长链非编码RNA LINC-PINT可抑制骨肉瘤细胞的增殖,促进凋亡,其作用机制与靶向miR-524-5p有关,可为骨肉瘤的治疗提供新的作用靶点。Objective To study the effect of long non-coding RNA LINC-PINT on proliferation and apoptosis of osteosarcoma cells.Methods 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.Results 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 P<0.05)while promoted the apoptosis(25.28±2.15 vs.9.01±0.17,P<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.Conclusions Long non-coding RNA LINC-PINT can inhibit the proliferation and promote apoptos

关 键 词：Lnc RNA LINC-PINT miR-524-5p 骨肉瘤 增殖 凋亡 

分 类 号：R73[医药卫生—肿瘤]