长链非编码RNA ANRIL对口腔鳞状细胞癌增殖和凋亡的影响及其机制
| 作者: |
1杨艳飞,
1邢育珍
1 华中科技大学同济医学院附属协和医院口腔科，武汉 430000 |
| 通讯: |
邢育珍
Email: xinyuzhen898@126.com |
| DOI: | 10.3978/j.issn.2095-6959.2018.01.002 |
| 基金: | 湖北省自然科学基金(2016CFB670)。 |
摘要
目的：研究长链非编码RNA(long non-coding RNA，lncRNA)ANRIL对口腔鳞状细胞癌细胞增殖及凋亡的影响，并探讨其机制。方法：qRT-PCR检测LncRNA ANRIL在SCC-4，Cal-27，TSCCA及Tca8113口腔鳞癌细胞系和正常口腔上皮细胞系HOK中的表达，将TSCCA细胞系分成ANRIL基因沉默组和对照组，采用LipofetamineTM 2000分别转染lncRNA-ANRIL-shRNA及随机对照序列lncRNA-ANRIL-NC，采用CCK-8和克隆形成实验测定细胞增殖，流式细胞术测定细胞凋亡，Western印迹检测Cleaved Caspase-3，KLF2和P21的蛋白表达。结果：LncRNA ANRIL在口腔鳞癌细胞系SCC-4，Cal-27，TSCCA及Tca8113中的相对表达量显著高于正常口腔上皮细胞系HOK(P<0.05)。转染0，24及48 h后，对照组与ANRIL基因沉默组OD450 nm值比较差异均无统计学意义(P>0.05)；转染72及96 h后，对照组与ANRIL基因沉默组OD450 nm值比较差异均有统计学意义(P<0.05)。ANRIL基因沉默组克隆形成率为21.54%±2.03%，显著低于对照组的53.72%±5.93%(P<0.01)。ANRIL基因沉默组凋亡率(14.7%±0.9%)高于对照组(4.9%±0.7%，P<0.01)。ANRIL基因沉默组裂解型Caspase-3蛋白相对表达量(4.1±0.44)显著高于对照组的1.0±0.03(P<0.01)。ANRIL基因沉默组KLF2蛋白相对表达量(0.53±0.06)低于对照组(1.0±0.03，P<0.05)。ANRIL基因沉默组P21蛋白相对表达量(0.46±0.05)低于对照组(1.0±0.03，P<0.05)。结论：LncRNA ANRIL高表达于口腔鳞癌细胞系，lncRNA ANRIL沉默抑制细胞增殖、诱导凋亡，其机制可能与上调裂解型Caspase-3，下调KLF2和P21蛋白表达有关。
关键词：
长链非编码RNA；口腔鳞状细胞癌；增殖；凋亡
Effect of long non-coding RNA ANRIL on proliferation and apoptosis in oral squamous cell carcinoma and its mechanism
CorrespondingAuthor: XING Yuzhen Email: xinyuzhen898@126.com
DOI: 10.3978/j.issn.2095-6959.2018.01.002
Foundation: This work was supported by Natural Science Foundation of Hubei Province, China
Abstract
Objective: To investigate the influence of long non-coding RNA (lncRNA) ANRIL on proliferation and apoptosis in oral squamous cell carcinoma cell line and its mechanism. Methods: The real time fluorescence quantitative PCR was used to detect and compare lncRNA ANRIL level in normal squamous cell line, HOK and four kinds of human oral squamous cell carcinoma cell line (SCC-4, Cal-27, TSCCA and Tca8113). The TSCCA cell line was divided into two groups, ANRIL gene silencing group (transfected with lncRNA-ANRIL-shRNA by LipofetamineTM 2000) and control group (transfected with negative randomized LncRNA-ANRIL-NC sequence by LipofetamineTM 2000). The proliferation capability and colony forming efficiency was detected by CCK-8 kit and colony formation assay respectively. The cell apoptosis rate was detected and compared upon FACS method between two groups. Finally, the protein levels of Cleaved Caspase-3, KLF2 and P21 was measured and compared between two groups by western blot. Results: The expression level of LncRNA ANRIL among four kinds of human oral squamous cell carcinoma cell line (SCC-4, Cal-27, TSCCA and Tca8113) was significantly higher than that in normal squamous cell line, HOK (P<0.05). After transfection for 0, 24, 48, 72 and 96 hours, the OD 450 nm value of control group vs ANRIL gene silencing group was 0.38±0.04 vs 0.37±0.04 (P>0.05), 0.58±0.04 vs 0.51±0.05 (P>0.05), 0.89±0.1 vs 0.67±0.06 (P>0.05), 1.20±0.09 vs 0.83±0.07 (P<0.05) and 1.87±0.13 vs 1.16±0.10 (P<0.01), respectively. The colony formation rate was 21.54%±2.03% and 53.72%±5.93% in ANRIL gene silencing group and control group, respectively (P<0.01). The result of FACS method demonstrated that the cell apoptosis rate was 14.7%±0.9% in the ANRIL gene silencing group and 4.9%±0.7% in the control group, respectively (P<0.01). Western blot indicated that the expression level of cleaved caspase-3 protein was 4.1±0.44 in ANRIL gene silencing group, which was significantly higher than that in control group (P<0.01). The expression level of KLF2 protein was 0.53±0.06 in ANRIL gene silencing group, which was significantly lower than 1.0±0.03 in the control group (P<0.05). While the expression level of P21 was 0.46±0.05 in the ANRIL gene silencing group, which was significantly lower than 1.0±0.03 in the control group (P<0.05). Conclusion: LncRNA ANTIL was over-expression in human oral squamous cell carcinoma cell line. Silencing lncRNA ANTIL could inhibit proliferation and induce apoptosis in human oral squamous cell carcinoma, which mechanism may be associated with up-regulation of Caspase-3, down-regulation of KLF2 and P21 protein.
Keywords：
lncRNA ANRIL; oral squamous cell carcinoma; proliferation; apoptosis