细胞周期蛋白依赖性激酶抑制剂2B反义RNA1靶向微小RNA-339-5p对乳腺癌细胞增殖、迁移、侵袭的影响

Effects of CDKN2B-AS1 on breast cancer cell proliferation,migration,and invasion by targeting miR-339-5p

目的 探讨长链非编码RNA细胞周期蛋白依赖性激酶抑制剂2B反义RNA1(lncRNA CDKN2B-AS1)对乳腺癌细胞增殖、迁移、侵袭的影响及分子机制。方法 收集南阳市中心医院2017年1月2019年1月收治的37例乳腺癌患者的癌组织及癌旁组织。将MDA-MB-453细胞分为CDKN2B-AS1阴性对照(si-NC组)、CDKN2B-AS1小分子干扰RNA(si-CDKN2B-AS1组)、CDKN2B-AS1小分子干扰RNA+miR-339-5p阴性对照(si-CDKN2B-AS1+anti-miR-NC组)以及CDKN2B-AS1小分子干扰RNA+miR-339-5p特异性寡核苷酸抑制剂(si-CDKN2B-AS1+anti-miR-339-5p组)。采用RT-qPCR法对CDKN2B-AS1及微小RNA-339-5p(miR-339-5p)表达水平进行检测;细胞周期及增殖活性检测分别采用流式细胞术及MTT实验;采用Transwell小室技术对细胞迁移和侵袭进行检测;采用Western blotting法对增殖标记蛋白细胞增殖核抗原-67(Ki67)、细胞周期依赖性蛋白激酶抑制因子1A(P21)、上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)蛋白表达进行检测;荧光素酶报告实验检测CDKN2BAS1对miR-339-5p的靶向调控。结果 在乳腺癌组织中CDKN2B-AS1表达水平上调[(2.23±0.08)比(1.00±0.06)](P<0.05)。抑制CDKN2B-AS1可增加G0期细胞比例[(43.29±3.76)%比(30.25±3.01)%]、P21表达水平升高,S期细胞比例[(21.91±3.10)%比(34.19±3.32)%]、细胞存活率[(53.02±5.38)%比(100.00±7.12)%]、迁移、侵袭数以及Ki67、E-cadherin、N-cadherin蛋白表达水平降低(P<0.05)。CDKN2B-AS1靶向调控miR-339-5p,抑制miR-339-5p逆转抑制CDKN2B-AS1对MDA-MB-453细胞增殖、迁移、侵袭的作用。结论 抑制CDKN2B-AS可抑制乳腺癌MDA-MB-453细胞增殖、迁移、侵袭,且靶向调控miR-339-5p表达。

Objective To investigate the effect of Long non-coding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1(lncRNA CDKN2B-AS1) on the proliferation,migration and invasion of breast cancer cells and its molecular mechanism.Methods The cancer tissues and paracancerous tissues of 37 breast cancer patients who were admitted to Nanyang Central Hospital from January2017 to January 2019 were collected.MDA-MB-453 cells were assigned into CDKN2B-AS1 negative control(si-NC group),CDKN2BAS1 small interfering RNA(si-CDKN2B-AS1 group),CDKN2B-AS1 small interfering RNA+miR-339-5p Negative control(si-CDKN2BAS1+anti-miR-NC group) and CDKN2B-AS1 small interfering RNA+miR-339-5p specific oligonucleotide inhibitor(si-CDKN2B-AS1+anti-miR-339-5p group).The expression levels of CDKN2B-AS1 and Mir-339-5p were detected by RT-qPCR.The cell cycle and proliferation activity were detected by flow cytometry and MTT assay.Cell migration and invasion were detected by Transwell chamber technique.The expressions of the proliferation marker protein proliferating nuclear antigen-67(Ki67),cyclin-dependent protein kinase inhibitor 1A(P21),epithelial cadherin(E-cadherin) and N-cadherin proteins were detected by Western blot.The targeted regulation of Mir-339-5p by CDKN2B-AS1 was detected by luciferase reporting assay.Results The expression level of CDKN2B-AS1 was up-regulated in breast cancer tissues [(2.23±0.08) vs.(1.00±0.06)](P<0.05).Inhibition of CDKN2B-AS1 could increase the proportion of cells in G0 phase [(43.29±3.76)% vs.(30.25±3.01)% ],increase the expression level of P21,decrease the proportion of cells in S phase[(21.91±3.10)% vs.(34.19±3.32)%],survival rate [(53.02±5.38)% vs.(100.00±7.12)%],migration and invasion number,and decrease the expression levels of Ki67,E-cadherin and N-cadherin(P<0.05).CDKN2B-AS1 could target and regulate miR-339-5p,and inhibiting miR-339-5p could reverse the effect of inhibiting CDKN2B-AS1 on the proliferation,migration and invasion of MDA-MB-453 cells.Conclusions and target the expression of miR-339-5p.