LncRNA-BLACAT1调节CyclinD1/CDKN2B轴对非小细胞肺癌细胞增殖的影响及其机制

doi:10.13481/j.1671-587x.20190403

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (04): 759-765.doi: 10.13481/j.1671-587x.20190403

• 基础研究 • 上一篇

LncRNA-BLACAT1调节CyclinD1/CDKN2B轴对非小细胞肺癌细胞增殖的影响及其机制

霍小蕾¹, 裴振², 杨浩³, 张毅强⁴, 贾建桃⁵, 韩玲娜²

1. 长治医学院组织胚胎学教研室, 山西长治 046000;
2. 长治医学院生理学教研室, 山西长治 046000;
3. 长治医学院附属和平医院肿瘤科, 山西长治 046000;
4. 长治医学院生物化学教研室, 山西长治 046000;
5. 长治医学院病理生理学教研室, 山西长治 046000

收稿日期:2018-10-26 发布日期:2019-08-02
通讯作者: 韩玲娜,副教授(Tel:0355-3012487,E-mail:hanlingna124@163.com) E-mail:hanlingna124@163.com
作者简介:霍小蕾(1981-),女,山西省阳泉市人,副教授,医学硕士,主要从事组织胚胎学及肿瘤学相关方面的研究。
基金资助:
国家自然科学基金资助课题（81600951）；长治医学院科研启动基金资助课题（普及QDZ201601）

Effect of LncRNA-BLACAT1 on cell proliferation of non-small cell lung cancer through regulation of CyclinD1/CDKN2B axis and its mechanism

HUO Xiaolei¹, PEI Zhen², YANG Hao³, ZHANG Yiqiang⁴, JIA Jiantao⁵, HAN Lingna²

1. Department of Histology and Embryology, Changzhi Medical College, Changzhi 046000, China;
2. Department of Physiology, Changzhi Medical College, Changzhi 046000, China;
3. Department of Oncology, Affiliated Heping Hospital, Changzhi Medical College, Changzhi 046000, China;
4. Department of Biochemistry, Changzhi Medical College, Changzhi 046000, China;
5. Department of Pathophysiology, Changzhi Medical College, Changzhi 046000, China

Received:2018-10-26 Published:2019-08-02

摘要/Abstract

摘要： 目的：探讨非小细胞肺癌（NSCLC）患者癌组织和癌细胞中长链非编码RNA（LncRNAs）BLACAT1的表达特征及其调控机制，阐明BLACAT1在NSCLC发生发展中的作用和临床意义。方法：高通量基因表达数据库（GEO数据库）分析BLACAT1在NSCLC组织的表达特征，Kmplot网站分析BLACAT1表达与NSCLC患者生存预后的联系。qRT-PCR法检测BLACAT1在25例NSCLC患者癌组织和癌旁组织及NSCLC细胞系中的表达情况。将si-NC（阴性对照）和si-BLACAT1（抑制物）转染入NSCLC A549细胞作为si-NC组和si-BLACAT1组，qRT-PCR法分别检测各组A549细胞中BLACAT1 mRNA表达水平，流式细胞术检测各组细胞不同细胞周期细胞百分率，细胞集落生成实验检测各组细胞克隆形成能力，CCK8实验检测各组细胞增殖活性，qRT-PCR和Western blotting法检测各组A549细胞中细胞周期蛋白D1（CyclinD1）和细胞周期蛋白依赖性激酶抑制剂2B（CDKN2B）mRNA和蛋白表达水平。结果：GEO数据库的NSCLC数据集GSE18842和GSE19804、qRT-PCR法检测以及Kmplot分析，与癌旁组织比较，NSCLC患者癌组织中BLACAT1表达水平明显升高（P< 0.01）；NSCLC患者癌组织中BLACAT1低表达患者的存活时间明显高于BLACAT1高表达患者（P=0.011）。与si-NC组比较，si-BLACAT1组A549细胞中BLACAT1 mRNA表达水平明显降低（P< 0.05）；与si-NC组比较，si-BLACAT1组A549细胞周期G₁期细胞百分率增加（P< 0.05），S期细胞百分率降低（P< 0.05），细胞克隆形成能力和细胞增殖活性均降低（P< 0.05或P<0.01）；与si-NC组比较，si-BLACAT1组A549细胞中CyclinD1 mRNA和蛋白表达水平均明显降低（P< 0.05），而CDKN2B mRNA和蛋白表达水平均明显升高（P< 0.05或P<0.01）。结论：LncRNA-BLACAT1在NSCLC患者癌组织和癌细胞中表达升高，下调BLACAT1表达可通过调节CyclinD1/CDKN2B轴从而抑制NSCLC A549细胞增殖，BLACAT1可作为NSCLC患者的潜在治疗靶点。

关键词: 癌,非小细胞肺, 长链非编码RNA, 细胞增殖, 预后

Abstract: Objective:To explore the expression charateristics of long non-coding RNA bladder cancer associated transcript-1(BLACAT1) in the cancer tissue and cancer cells in the patients with non-small cell lung cancer(NSCLC) and the regulation mechanism,and to elucidate the effect and clinical significance of BLACAT1 in the occurrence and development of NSCLC. Methods:Gene Expression Omnibus (GEO) database was used to analyze the expression characteristics of BLACAT1 in the NSCLC tissue. Kmplot website was used to analyze the correlations between the expression of BLACAT1 and the survival and prognosis of the NSCLC patients. Real-time quantitative PCR(qRT-PCR) method was applied to detect the expressions of BLACAT1 in cancer tissue and corresponding adjacent normal tissue and NSCLC cell lines of the NSCLC patients.The specific small interfering RNA for BLACAT1(si-BLACAT1 group) or negative control sequence(si-NC group) were transfected into the A549 cells. The mRNA expression level of BLACAT1 in A549 cells in various groups were detected by qRT-PCR method; the percentages of A549 cells in different cell cycles were detected by flow cytometry; the clone formation abilities of A549 cells in various groups were detected by cell clone formation experiment. The proliferation activities of cells in various groups were detected by CCK8 assay. qRT-PCR and Western blotting methods were used to detect the expression level of CyclinD1 and CDKN2B mRNA and protein in the A549 cells in various groups. Results:The results of GSE18842 and GSE19804 in GEO datatase,qRT-PCR and Kmplot analysis showed that the expression level of BLACAT1 in NSCLC tissue was significantly increased compared with adjacent normal lung tissue (P< 0.05); the survival time in the patients with low expression of BLACAT1 in cancer tissue was significantly longer than that in the patients with high expression of BLACAT1 (P=0.011). Compared with si-NC group,the BLACAT1 expression level in the A549 cells in si-BLACAT1 group was significantly decreased(P<0.05).Compared with si-NC group, the percentage of A459 cells in G₁ phase in si-BLACAT1 group was increased (P<0.05), and the percentage of A549 cells in S phase was decreased(P<0.05);the cell clone formation ability and the cell proliferation activity were decreased(P<0.05 or P<0.01). Compared with si-NC group, the expression levels of CyclinD1 mRNA and protein in the A549 cells in si-BLACAT1 group were significantly decreased(P<0.05), and the expression levels of CDKN2B mRNA and protein were significantly increased(P<0.05). Conclusion:LncRNA-BLACAT1 is up-regulated in cancer tissue and cancer cells of the NSCLC patients, and down-regulation of BLACAT1 expression can inhibit the proliferation of A549 cellsvia modulating the CyclinD1/CDKN2B axis, which may serve as a potential therapeutic target for the NSCLC patients.

Key words: long non-coding RNA, cancer, non-small cell lung, cell proliferation, prognosis

中图分类号:

R734.2

霍小蕾, 裴振, 杨浩, 张毅强, 贾建桃, 韩玲娜. LncRNA-BLACAT1调节CyclinD1/CDKN2B轴对非小细胞肺癌细胞增殖的影响及其机制[J]. 吉林大学学报(医学版), 2019, 45(04): 759-765.

HUO Xiaolei, PEI Zhen, YANG Hao, ZHANG Yiqiang, JIA Jiantao, HAN Lingna. Effect of LncRNA-BLACAT1 on cell proliferation of non-small cell lung cancer through regulation of CyclinD1/CDKN2B axis and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(04): 759-765.

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LncRNA-BLACAT1调节CyclinD1/CDKN2B轴对非小细胞肺癌细胞增殖的影响及其机制

Effect of LncRNA-BLACAT1 on cell proliferation of non-small cell lung cancer through regulation of CyclinD1/CDKN2B axis and its mechanism

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