LncRNA LOC730101通过Wnt/β-catenin信号通路对骨肉瘤U2OS细胞生物学特性的研究

Effect of lncRNA loc730101 on the biological characteristics of osteosarcoma U2OS cells through Wnt/β-catenin signal pathway

目的 探讨长链非编码RNA (lncRNA)LOC730101对骨肉瘤U2OS细胞增殖、侵袭和迁移的影响及其机制。方法 2019年2月至2019年10月,将体外培养的U2OS细胞分为对照组(正常培养)、阴性组(转染阴性对照)和干扰组(转染靶向LOC730101的干扰序列),采用RT-PCR检测细胞中LOC730101表达,噻唑蓝(MTT)法检测细胞增殖活力,平板克隆形成实验检测细胞克隆形成率,流式细胞仪检测细胞周期分布情况, Transwell小室检测细胞侵袭与迁移,Western blotting法检测细胞中上皮间质转化相关蛋白波形蛋白(Vi- mentin)、N-钙黏附素(N-cadherin)、E-钙黏附蛋白(E-cadherin)和Wnt/β-连环蛋白(β-catenin)信号通路相关蛋白β-catenin、c-Myc、细胞周期蛋白D1 (Cyclin D1)和基质金属蛋白酶-7 (MMP-7)蛋白的表达水平。结果进行统计学分析,P<0.05为差异有统计学意义。结果 与对照组比较,干扰组细胞中LOC730101表达水平(干扰组、对照组分别为0.25±0.03、1.00±0.06,下同)、细胞成活率[(57.65±3.26)%、(100.00±7.39)%]、克隆形成率[(13.03 ± 2.12)%、(25.35±3.58)%]、侵袭细胞数(51.36±3.48、92.85±6.62)、迁移细胞数(77.15 ± 5.05、136.92 ± 15.35)和细胞在S期[(20.54±2.15)%、(28.15±2.38)%]、G2/M期所占百分比[(16.87±2.12)%、(23.36±3.12)%]以及细胞中Vimentin (0.52 ± 0.04、1.17 ± 0.13)、N-cadherin (0.31 ± 0.03、0.65 ± 0.04)、β-catenin (0.42 ± 0.03、0.73 ± 0.04)、c-Myc (0.29±0.03、0.65±0.03)、Cyclin D1 (0.26±0.02、0.58±0.04)、MMP-7蛋白表达水平(分别为0.55± 0.03、0.86±0.06)均明显降低,而细胞在G0/G1期所占百分比[分别为(62.62±5.15)%、(48.46±3.65)%]以及细胞中E-cadherin蛋白的表达水平(分别为0.82±0.06、0.38±0.03)均明显升高,差异均有统计学意义(P<0.05);但阴性组的各指标与对照组之间差异均无统计学意义(P>0.05)。结论 下调LOC730101可抑制骨肉瘤U2OS细胞增殖、侵袭和迁移,其作用机制可能与抑制Wnt/β-catenin信号通路有关。

Objective To investigate the effect of long-chain non coding RNA(lncrna)loc730101 in the proliferation,invasion and migration of U2OS cells,and its mechanism.Methods From February,2019 to October,2019,U2OS cells cultured in vitro were divided into control group(normal culture),negative group(transfection nega-tive control),and interference group(transfection of interference sequences targeting LOC730101).The expression of LOC730101 in cells was detected by reverse transcription-polymerase chain reaction(RT-PCR).Cell proliferation ac tivity was tested by methylthiazolyldiphenyl-tetrazolium bromide(MTT)method.Cell clone formation rate was mearused by plate clone formation test.Cell cycle distribution was tested by flow cytometry.Cell invasion and migra-tion were examed by Transwell chamber.The expression levels of epithelial-mesenchymal transition-related proteins Vimentin,N-cadherin,E-cadherin,and Wnt/β-catenin signaling pathway related proteins in cellsβ-catenin,c-Myc,cyclin D1(CyclinD1)and matrix metalloproteinase-7(MMP-7)proteins were detected by Western blotting method.The date was statistical analysed and considered as statistically significant when P<0.05.Results Compared with the control group,the expression level of LOC730101(0.25±0.03 and 1.00±0.06)in interference group and control group,respectively.The same below),cell survival rate[(57.65±3.26)%and(100.00±7.39)%],clone formation rate[(13.03±2.12)%and(25.35±3.58)%],number of invasive cells(51.36±3.48 and 92.85±6.62),number of migrating cells(77.15±5.05 and 136.92±15.35),the percentage of cells in S phase[(20.54±2.15)%and(28.15±2.38)%]and G2/M phase[(16.87±2.12)%and(23.36±3.12)%],as well as the expression level of Vimentin(0.52±0.04 and 1.17±0.13),N-cadherin(0.31±0.03 and 0.65±0.04),β-catenin(0.42±0.03 and 0.73±0.04),c-Myc(0.29±0.03 and 0.65±0.03),CyclinD1(0.26±0.02 and 0.58±0.04),MMP-7 protein(0.55±0.03 and 0.86±0.06)was decreased significantly(P<0.05),while the per-centage of cells in G0/G1 phase[(62.62±5.15)%and(48.46±3.65)%]and the expression level of E-cadherin protein(0.82±0.06 and 0.38±0.03)were increased significantly in the interference group(P<0.05).But there was no significant differ-ence in each index in the negative group(P>0.05).Conclusion Reduce the regulation of LOC730101 can inhibit the proliferation,invasion and migration of U2OS cells,and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.