长链非编码RNA CRNDE对胰腺癌细胞增殖、迁移及侵袭的影响
| 作者: |
1刘向梅,
1许达峰,
1王春玲,
1符于正,
1王丹,
1武金才
1 海南省人民医院，海南医学院附属海南医院肝胆胰外科，海口 570000 |
| 通讯: |
武金才
Email: jincaiwu73@sina.com |
| DOI: | 10.3978/j.issn.2095-6959.2021.10.003 |
| 基金: | 海南省自然科学基金(819QN356, Hnky2019-53)。 |
摘要
目的：探讨胰腺癌细胞系中长链非编码RNA CRNDE(LncRNA CRNDE)的表达及对细胞增殖、迁移和侵袭的影响。方法：通过real-time RT-PCR检测LncRNA CRNDE在胰腺癌细胞系和正常胰腺导管上皮细胞系中的表达。将胰腺癌细胞系Panc-1分成3组，LncRNA CRNDE沉默表达组(si-CRNDE组)﹑阴性对照组(si-NC组)及空白对照组(Blank组)，对si-CRNDE组及si-NC组采用LipofectamineTM 3000分别转染LncRNA CRNDE沉默序列(si-CRNDE)及阴性对照序列(si-NC)，Blank组仅给予缓冲盐溶液对照。采用real-time RT-PCR验证沉默效率，CCK-8实验测定细胞增殖能力，细胞划痕实验测定细胞迁移能力，Transwell实验测定细胞侵袭能力，蛋白质印迹实验测定c-Myc和磷酸化细胞外信号调节激酶(p-ERK)蛋白的表达。结果：胰腺癌细胞系Panc-1(P<0.01)、AsPC-1(P<0.01)、HPAC(P<0.05)、BxPC-3(P<0.05)中LncRNA CRNDE相对表达量高于HPDE6-C7细胞系。转染siRNA 48 h后，si-CRNDE组LncRNA CRNDE相对表达量低于si-NC组(P<0.01)。CCK-8实验示细胞铺板后在0、24、48 h，si-CRNDE组与si-NC组的OD450 nm值差异无统计学意义(P>0.05)，在72和96 h，二者差异有统计学意义(分别P<0.05和P<0.01)；Blank组与si-NC组差异无统计学意义(P>0.05)。细胞划痕实验结果示si-CRNDE组细胞划痕愈合率低于si-NC组[(26.3±2.8)% vs (52.1±3.7)%，P<0.05]。Transwell实验示si-CRNDE组侵袭细胞数少于si-NC组(108±15 vs 319±21，P<0.05)。蛋白质印迹结果示si-CRNDE组c-Myc蛋白质表达量低于si-NC组(0.47±0.04 vs 1.02±0.03，P<0.05)。si-CRNDE组p-ERK蛋白质表达量低于si-NC组(0.27±0.04 vs 1.04±0.05，P<0.01)。结论：LncRNA CRNDE在胰腺癌细胞系中高表达，沉默LncRNA CRNDE表达可抑制胰腺癌细胞的增殖﹑迁移和侵袭，机制可能与c-Myc﹑p-ERK蛋白下调表达有关。
关键词：
长链非编码RNA CRNDE；胰腺癌；增殖；迁移；侵袭
Effects of long-chain non-coding RNA CRNDE on proliferation, migration and invasion of pancreatic cancer cells
CorrespondingAuthor: WU Jincai Email: jincaiwu73@sina.com
DOI: 10.3978/j.issn.2095-6959.2021.10.003
Foundation: This work was supported by the Natural Science Foundation of Hainan Province, China (819QN356, Hnky2019-53).
Abstract
Objective: To investigate the expression of long-chain non-coding RNA CRNDE (LncRNA CRNDE) in pancreatic cancer cell lines and its effect on cell proliferation, migration, and invasion. Methods: The expression of LncRNA CRNDE in pancreatic cancer cell lines and normal pancreatic duct epithelial cell lines was detected by real-time RT-PCR. PANC-1 cell line was divided into a LncRNA CRNDE silence expression group (Si-CRNDE group), a negative control group (Si-NC group) and a Blank control group (Blank group). In the Si-CRNDE group and Si-NC group, lipofectamineTM 3000 was used to transfect LncRNA CRNDE silence sequence (Si-CRNDE) and negative control sequence (Si-NC). The Blank group was only given buffer salt solution. Real-time RT-PCR was used to verify the silencing efficiency. CCK-8 was used to measure cell proliferation. Cell scratch assay was used to measure cell migration ability. Transwell assay was used to measure cell invasion ability. Western blotting was used to measure the expression of c-myc and p-ERK proteins expression. Results: The relative expression of LncRNA CRNDE in PANC-1 (P<0.01), AsPC-1 (P<0.01), HPAC (P<0.05), BXPC-3 (P<0.05) was higher than that in HPDE6-C7. After 48 hours of siRNA transfection, it was found that the relative expression of LncRNA CRNDE in Si-CRNDE group was lower than that in Si-NC group (P<0.01). The results of CCK-8 showed that at 0, 24, 48, 72 and 96 hours after the cell laying, the OD450 nm values of the Si-CRNDE group vs the Si-NC group were 0.33±0.04 vs 0.32±0.04 (P>0.05), 0.49±0.05 vs 0.52±0.06 (P>0.05), 0.72±0.07 vs 0.74±0.08 (P>0.05), 0.97±0.09 vs 1.28±0.10 (P<0.05), and 1.25±0.10 vs 1.97±0.14(P<0.01), respectively. There was no significant difference between the Blank group and the Si-NC group (P>0.05). The results of cell scratch test showed that the cell scratch healing rate of the Si-CRNDE group was lower than that of Si-NC group (26.3%±2.8% vs 52.1%±3.7%, P<0.05). The transwell experiment showed that the invasive cell number of si-CRNDE group was significantly less than that in the si-NC group (108±15 vs 319±21, P<0.05). Western blot showed that the expression of c-Myc protein in the Si-CRNDE group was lower than that in the Si-NC group (0.47±0.04 vs 1.02±0.03, P<0.05). The expression of p-ERK protein in Si-CRNDE group was lower than that in the Si-NC group (0.27±0.04 vs 1.04±0.05, P<0.01). Conclusion: LncRNA CRNDE is highly expressed in pancreatic cancer cell lines. Silencing the expression of LncRNA CRNDE can inhibit the proliferation, migration, and invasion of pancreatic cancer cells. The mechanism may be related to the down-regulation of c-Myc and p-ERK protein.
Keywords：
LncRNA CRNDE; pancreatic cancer; proliferation; migration; invasion