外泌体源性长链非编码RNA同源框基因A-反义转录本3通过吸附微小RNA-29b-3p调节前列腺癌去势抵抗的作用机制

The mechanism of cancer derived exosomal homeobox gene a-antisense transcript 3 regulating castration resistance of prostate cancer by sponging

目的探讨外泌体来源长链非编码RNA(lncRNA)同源框基因A-反义转录本3(HOXA-AS3)对前列腺癌(PCa)去势抵抗的作用机制。方法建立PC-3与LNCaP共培养体系,用双荧光素酶报告基因检测、荧光定量聚合酶链反应(PCR)和挽救实验等验证HOXA-AS3参与PCa去势抵抗和进展的机制。采用动物实验和人PCa组织切片,探究HOXA-AS3在由雄激素依赖性向非依赖性的转化过程中的作用。采用两样本t检验和单因素方差分析(One-WayANOVA)进行统计分析。结果ASO-NC转染组LNCaP细胞侵袭数量显著低于ASO-miR-29b-3p转染组[(116.00±5.10)个比(146.33±4.78)个,t=6.135,P<0.05],NC转染PC-3组显著高于miR-29b-3pmimic转染组[(394.33±11.95)个比(111.67±7.41)个,t=28.425,P<0.05]。LNCaP-HOXA-AS3-exo中HOXA-AS3显著高于LNCaP-exo(1.46±0.08比1.01±0.02,t=7.634,P<0.05),而PC-3-HOXA-AS3-ASO-exo中HOXA-AS3显著低于PC-3-exo(0.53±0.06比1.00±0.09,t=5.943,P<0.05)。转染miR-29b-3p-mimic组的野生型HOXA-AS3、野生型STAT3-3和野生型Mcl-1组的细胞荧光素酶活性低于对照组(1.00±0.04比0.51±0.02、1.00±0.02比0.56±0.04、1.00±0.02比0.57±0.02,t=18.058、15.857、26.081,P<0.01)。同时转染pcDNA3.1-STAT3和Mcl-1-promotor载体组免疫荧光强度高于对照组(1.72±0.05比1.00±0.03,t=18.717,P<0.05),并且cytochromec和Caspase-9的表达低于对照组(0.46±0.01比0.81±0.01、0.28±0.01比0.59±0.01,t=92.492、97.212,P<0.01)。结论HOXA-AS3通过吸附miR-29b-3p调节STAT3/Mcl-1/cytochromec/Caspase-9轴,调节PCa的去势抵抗。

Objective To investigate the role of exosomal long no-coding RNA(lncRNA)ho-meobox gene a-antisense transcript 3(HOXA-AS3)in castration resistance and progression of prostate cancer(PCa).Methods Construct a co-culture system between PC-3 and LNCaP.The dual luciferase re-porter gene detection,PCR(polymerase chain reaction)and Rescue were conducted to verify the role of ex-osomes in castration resistance and progression of PCa.Animal experiments and human prostate cancer tis-sue sections were conducted to investigate the role of HOXA-AS3 in the transition from androgen dependent prostate cancer(ADPC)to castration-resistant prostate cancer(CRPC).Two sample t-test or One-Way ANOVA was used for statistically analysis.Results The number of invasive cells in LNCaP containing ASO-NC group was significantly less than that in LNCaP containing miR-29b-3p-ASO group(116.00±5.10 vs.146.33±4.78,t=6.135,P<0.05).The number of invasive cells in PC-3 containing mimic-NC group was significantly higher than that in PC-3 containing miR-29b-3p group(394.33±11.95 vs.111.67±7.41,t=28.425,P<0.05).The content of HOXA-AS3 in LNCaP-HOXA-AS3-exo group is significantly higher than that in LNCaP-exo group(1.46±0.08 vs.1.01±0.02,t=7.634,P<0.05).The content of HOXA-AS3 in PC-3-HOXA-AS3-ASO-exo group is significantly higher than that in PC-3-exo group(0.53±0.06 vs.1.00±0.09,t=5.943,P<0.05).The overexpressed miR-29b-3p significantly reduced the luciferase intensity of HOXA-AS3-3'UTR wide type,STAT3-3'UTR wide type and Mcl-1-3'UTR wide type(1.00±0.04 vs.0.51±0.02,1.00±0.02 vs.0.56±0.04,1.00±0.02 vs.0.57±0.02,t=18.058,15.857,26.081,P<0.01).The relative luciferase intensity of the group containing with pcDNA3.1-STAT3 and Mcl-1-promotor was significantly higher than that of the controlled group(1.72±0.05 vs.1.00±0.03,t=18.717,P<0.05),and the expression of cytochrome c and caspase-9 was significantly less than that of the controlled group(0.81±0.01 vs.0.46±0.01,0.59±0.01 vs.0.28±0.01,t=92.492,97.212,P<0.01).Conclusion HOXA-AS3 contributes to the castration resistance of PCa via sponging miR-29b-3p and regulating STAT3/Mcl-1/cytochrome c/caspase-9 axis.