LncRNA-TUG1通过调节miR-31-5p/YWHAE轴影响22Rv1细胞的增殖和迁移
LncRNA-TUG1 Affects 22Rv1 Cell Proliferation and Migration by Modulating miR-31-5p/YWHAE Axis
赵佳福 1李永 1周明帅 1温晓艳 1陆情梅 1刘彬1
作者信息
- 1. 贵州大学高原山地动物遗传育种与繁殖教育部重点实验室/动物科学学院,贵阳 550025
- 折叠
摘要
酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白ε(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein ε,YWHAE)在前列腺癌(prostate cancer,PCa)中高表达,miR-31-5p可靶向调控YWHAE的表达影响前列腺癌细胞的增殖.但是,参与miR-31-5p对YWHAE靶向调控的长链非编码RNA(long non-coding RNA,LncRNA)目前尚不明确.本研究根据前人研究结果及生物信息学分析软件筛选出LncRNA-牛磺酸上调基因(LncRNA-tauronic upregulation,LncRNA-TUG1)、LINC02604和PDCD6IP-DT 3条LncRNAs能靶向调控miR-31-5p的表达.通过RT-qPCR检测3条LncRNAs在前列腺癌细胞系LNCaP、PC3、22Rv1及正常前列腺基质永生化细胞(WPMY-1)中的表达,筛选调控miR-31-5p/YWHAE轴的最佳LncRNA;RT-qPCR检测干扰LncRNA-TUG1及过表达miR-31-5p对22Rv1细胞中LncRNA-TUG1、miR-31-5p和YWHAE表达的影响;同时,通过CCK-8、细胞划痕实验检测共转染sh-TUG1及miR-31-5p mimics对22Rv1细胞增殖和迁移的影响;Western blot检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、B细胞淋巴瘤-2蛋白(B-cell lymphoma-2,BCL-2)、Bcl-2相关X蛋白(BCL2-associated X,BAX)和半胱氨酸蛋白酶-3(cysteine proteinase-3,Caspase-3)表达的影响.结果表明,LncRNA-TUG1、LINC02604和PDCD6IP-DT为调控miR-31-5p/YWHAE轴的3条LncRNAs,且与正常细胞相比,LncRNA-TUG1在PCa各细胞系中高表达,尤其在22Rv1细胞中的表达量最高.以LncRNA-TUG1为研究对象,分别转染sh-TUG1及miR-31-5p mimics到22Rv1细胞后,qPCR结果显示,与NC mimics组相比,转染miR-31-5p mimics组中miR-31-5p的表达量极显著上调(P＜0.01),LncRNA-TUG1和YWHAE基因的表达显著下调(P＜0.05);与sh-NC组相比,转染sh-TUG1组中miR-31-5p的表达无显著变化,LncRNA-TUG1的表达显著下调(P＜0.05),YWHAE基因的表达极显著下调(P＜0.01);CCK-8及细胞划痕实验表明,共转染sh-TUG1及miR-31-5p mimics后,能够显著抑制细胞增殖及迁移;Western blot结果表明,共转染sh-TUG1及miR-31-5p mimics能够显著抑制PCNA、BCL-2/BAX蛋白的表达,促进caspase-3蛋白的表达.以上结果说明,LncRNA-TUG1可通过调节miR-31-5p/YWHAE来调控PCNA、BCL-2、BAX和caspase-3蛋白的表达,进而影响22Rv1细胞的增殖.本研究为前列腺癌症靶向药物的开发提供理论参考和实验思路.
Abstract
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein ε(YWHAE)was highly expressed in prostate cancer,and miR-31-5p could target and regulate the expression of YWHAE to affect the proliferation of prostate cancer cells.However,it was not clear which lncRNAs were involved in the targeted regulation of miR-31-5p against YWHAE.In this study,3 LncRNAs with long non-coding RNA tauronic upregulation gene(LncRNA-TUG1),LINC02604 and PDCD6IP-DT were screened to target and regulate the expression of miR-31-5p based on the results of previous studies and bioanalysis software.The expression of 3 LncRNAs in prostate cancer LNCaP,PC3,22Rv1 and normal prostate stromal immortem cells(WPMY-1)was detected by RT-qPCR,and the optimal LncRNAs regulating the miR-31-5p/YWHAE axis were screened.The effects of interfering LncRNA-TUG1 and overexpression of miR-31-5p on the expression of LncRNA-TUG1,miR-31-5p and YWHAE in 22Rv1 cells were detected by RT-qPCR.Meanwhile,the effects of co-transfection of sh-TUG1 and miR-31-5p mimics on the proliferation and migration of 22Rv1 cells were detected by CCK-8 and cell scratch assay.Western blot was used to detect the expression of proliferating cell nuclear antigen(PCNA),B cell lymphoma-2(BCL-2),Bcl-2 associated X protein(BAX)and cysteine proteinase-3(Caspase-3).The results showed that LncRNA-TUG1,LINC02604 and PDCD6IP-DT were the 3 LncRNAs regulating the miR-31-5p/YWHAE axis,and compared with normal cells,LncRNA-TUG1 was highly expressed in all PCa cell lines,especially in 22Rv1 cells.Taking LncRNA-TUG1 as the research object,after transfecting sh-TUG1 and miR-31-5p mimics into 22Rv1 cells,respectively,qPCR results showed that the expression of miR-31-5p in the miR-31-5p mimics group was significantly up-regulated compared with the NC mimics group(P＜0.01).The expression of LncRNA-TUG1 and YWHAE genes was significantly down-regulated(P＜0.05).Compared with the sh-NC group,there was no significant change in the expression of miR-31-5p in the sh-TUG1 transfection group,and the expression of LncRNA-TUG1 and YWHAE were significantly down-regulated(P＜0.05).CCK-8 and cell scratch experiments showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit cell proliferation and migration.In addition,Western blot results showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit the expression of PCNA and BCL-2/BAX protein,and promoted the expression of caspase-3 protein.These results indicated that LncRNA-TUG1 could regulate the expression of PCNA,BCL-2,BAX and caspase-3 proteins by regulating miR-31-5p/YWHAE,and further affected the proliferation of 22Rv1 cells.This study provides theoretical basis and experimental ideas for the study of targeted drugs for prostate cancer.
关键词
LncRNA-牛磺酸上调基因(LncRNA-TUG1)/miR-31-5p/前列腺癌/22Rv1/细胞增殖/细胞迁移Key words
LncRNA-tauronic upregulation(LncRNA-TUG1)/miR-31-5p/Prostate cancer/22Rv1/Cell proliferation/Cell migration引用本文复制引用
基金项目
贵州省科技计划(黔科合基础[2020]1Y085)
出版年
2024
国家科技期刊平台
NSTL
万方数据