微小RNA-135a抑制骨肉瘤干细胞的迁移、侵袭和自我更新

MicroRNA-135a inhibits the migration,invasion,and self-renewal of osteosarcoma stem cells

目的探讨微小RNA(miR)-135a对骨肉瘤干细胞(OSCs)迁移、侵袭和自我更新的影响。方法微球体筛选移植瘤原代细胞的OSCs(P-OSCs)和Saos-2的OSCs(S-OSCs)。实时定量聚合酶链反应(RT-qPCR)检测分化群(CD)CD133、CD44、CD24和BMI1的转录水平,蛋白质印迹法(Western blot)检测CD133、CD44、CD24、BMI1和B细胞淋巴瘤2(bcl-2)的蛋白水平。构建miR-135a过表达的P-OSCs或S-OSCs为miR-135a组,相应的阴性病毒转染P-OSCs或S-OSCs为对照组(NC)。划痕、Transwell和球体形成实验分别检测细胞的迁移、侵袭和自我更新能力。采用GraphPad Prism 8.4.2进行分析。结果miR-135a组P-OSCs和S-OSCs的伤口愈合率[(46.33±11.25)%比(73.46±2.24)%和(31.25±2.61)%比(47.20±1.94)%,t=3.346、8.489,均P<0.05]、穿膜细胞数[(287.00±34.29)个比(401.33±14.66)个和(255.67±17.02)个比(574.67±31.58)个,t=4.336、12.570,均P<0.05]、成球率[(1.89±0.44)%比(3.72±0.21)%和(1.57±0.17)%比(2.65±0.05)%,t=5.360、6.789,均P<0.01]和球体直径均低于NC组[(97.21±8.89)μm比(184.91±15.32)μm和(90.68±7.02)μm比(163.75±14.14)μm,t=7.003、6.546,均P<0.01]。CD44和bcl-2蛋白在骨肉瘤组织中表达高于骨组织(分别为0.87±0.11比0.46±0.08和1.96±0.69比0.41±0.13,t=4.286、3.126,均P<0.05),细胞中的表达趋势与组织一致。miR-135a组P-OSCs和S-OSCs中CD44(0.68±0.02比1.02±0.06和0.29±0.01比1.03±0.01,t=9.311、90.630)和bcl-2(0.46±0.01比0.95±0.01和0.41±0.02比0.84±0.01,t=60.010、33.310)的蛋白表达量低于NC组,均P<0.01。结论miR-135能够通过抑制CD44和bcl-2抑制OSCs的迁移、侵袭和自我更新。

Objective To investigate the role of microRNA(miR)-135a in migration,invasion and self-renewal of osteosarcoma stem cells(OSCs).Methods The OSCs of transplanted tumor primary cells(P-OSCs)and Saos-2 OSCs(S-OSCS)were screened by microspheres.The transcriptional levels of cluster of differentiation(CD)133,CD44,CD24 and BMI1 were detected by real-time quantitative polymerase chain reaction(RT-qPCR),and the protein levels of CD133,CD44,CD24,BMI1 and B cell lymphoma 2(bcl-2)were detected by Western blotting.miR-135a overexpressed P-OSCs or S-OSCs were constructed as miR-135a group,and the corresponding negative virus transfected P-OSCs or S-OSCs were used as control group(NC).The effects of miR-135a on migration,invasion,and self-renewal of OSCs were detected by wound healing,transwell and sphere formation experiments,respectively.GraphPad Prism 8.4.2 was used for statistical analysis.Results The wound healing rate[(46.33±11.25)%vs.(73.46±2.24)%and(31.25±2.61)%vs.(47.20±1.94)%,t=3.346,8.489,respectively,all P<0.05],the number of transmembrane cells[(287.00±34.29)cells vs.(401.33±14.66)cells and(255.67±17.02)cells vs.(574.67±31.58)celss,t=4.336,12.570,all P<0.05],the sphere forming rate[(1.89±0.44)%vs.(3.72±0.21)%and(1.57±0.17)%vs.(2.65±0.05)%,t=5.360,6.789,all P<0.01]and sphere diameter[(97.21±8.89)μm vs.(184.91±15.32)μm and(90.68±7.02)μm vs.(163.75±14.14)μm,t=7.003,6.546,all P<0.01]in P-OSCs and S-OSCs of miR-135a group were lower than NC group.The expression of CD44 and bcl-2 proteins in osteosarcoma tissues was higher than bone tissues(0.87±0.11 vs.0.46±0.08 and 1.96±0.69 vs.0.41±0.13,t=4.289,3.126,respectively,all P<0.05),and their expression in cells was consistent with that in tissues.CD44(0.68±0.02 vs.1.02±0.06 and 0.29±0.01 vs.1.03±0.01,t=9.311,90.630)and bcl-2(0.46±0.01 vs.0.95±0.01 and 0.41±0.02 vs.0.84±0.01,t=60.010,33.310)in P-OSCs and S-OSCs of miR-135a group were lower than NC group,all P<0.01.Conclusion miR-135a can inhibit the migration,invasion,and self-renewal of OSCs by inhibiting CD44 and bcl-2.