长链非编码RNA DLX6-AS1调控微小RNA-15b和磷脂酶D1影响口腔鳞状细胞癌侵袭转移的分子机制研究

doi:10.3969/j.issn.1005-4979.2020.04.006

摘要/Abstract

摘要：

目的：探讨长链非编码RNA（long non-coding RNA，lncRNA）生长停滞特异性蛋白6-反义RNA1（growth arrest specific protein 6-antisense RNA1，DLX6-AS1）对口腔鳞状细胞癌（口腔鳞癌）细胞增殖、迁移和侵袭的影响及作用机制。方法：实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测口腔鳞癌组织及癌旁组织中DLX6-AS1、微小RNA-15b（microRNA-15b，miR-15b）和磷脂酶D1（phospholipase D1，PLD1）mRNA的表达水平。转染DLX6-AS1小干扰RNA至口腔鳞癌细胞HSC3，四甲基偶氮唑盐微量酶反应比色法（MTT法）、Transwell小室和Western印迹法（Western blot）检测抑制DLX6-AS1表达对HSC3细胞的增殖、迁移和侵袭能力，以及对p21、基质金属蛋白酶2（matrix metalloproteinase 2，MMP-2）和E-钙黏附素（E-cadherin）蛋白表达的影响。分别共转染miR-15b抑制剂或PLD1过表达载体与DLX6-AS1小干扰RNA至HSC3细胞，用上述相同方法观察抑制miR-15b或过表达PLD1对DLX6-AS1表达抑制的HSC3细胞增殖、迁移和侵袭能力，以及对p21、MMP-2和E-cadherin蛋白表达的影响。双荧光素酶报告基因实验验证DLX6-AS1与miR-15b及miR-15b与PLD1的调控关系。结果：与癌旁组织比较，口腔鳞癌组织中DLX6-AS1和PLD1 mRNA表达水平升高（P<0.001），miR-15b表达水平降低（P<0.001）。抑制DLX6-AS1表达可降低HSC3细胞存活率、迁移和侵袭，以及MMP-2的蛋白表达水平（P<0.001），提高p21和E-cadherin蛋白表达水平（P<0.001）。抑制miR-15b表达或过表达PLD1均可降低抑制DLX6-AS1表达对HSC3细胞的存活率、迁移和侵袭及P21、MMP-2和E-cadherin蛋白表达的影响。DLX6-AS1在HSC3细胞中靶向负调控miR-15b，miR-15b靶向负调控PLD1。结论：抑制DLX6-AS1表达可能通过靶向调控miR-15b和PLD1轴抑制口腔鳞癌细胞的增殖、迁移和侵袭。

关键词: 生长停滞特异性蛋白6-反义RNA1, 微小RNA-15b, 磷脂酶D1, 口腔鳞状细胞癌, 侵袭转移

Abstract:

Objective: To investigate the effects of long non-coding RNA(lncRNA) growth arrest specific protein 6-antisense RNA1(DLX6-AS1) on the proliferation, migration and invasion of oral squamous cell carcinoma cells and its mechanism. Methods: The expression levels of DLX6-AS1, microRNA-15b (miR-15b) and phospholipase D1(PLD1) mRNA in oral squamous cell carcinoma and adjacent tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR). After DLX6-AS1 small interfering RNA was transfected into oral squamous cell carcinoma cells HSC3, MTT assay, Transwell and Western blot were used to detect the effects of inhibition of DLX6-AS1 expression on the proliferation, migration and invasion of HSC3 cells and the expression of p21, matrix metalloproteinase 2(MMP-2) and E-cadherin protein. Co-transfection of miR-15b inhibitor or PLD1 overexpression vector and DLX6-AS1 small interfering RNA into HSC3 cells. The same methods as above were used to observe the proliferation, migration and invasion and the expression of p21, MMP-2 and E-cadherin protein of HSC3 cells with DLX6-AS1 expression inhibition. The double luciferase reporter gene experiment verified the regulatory relationship between DLX6-AS1 and miR-15b or miR-15b and PLD1. Results: Compared with adjacent tissues, the expression levels of DLX6-AS1 and PLD1 mRNA in oral squamous cell carcinoma tissues were increased (P<0.001), and the expression level of miR-15b was decreased (P<0.001). Inhibition of DLX6-AS1 expression could reduce the survival rate, migration and invasion number of HSC3 cells and the expression level of MMP-2 protein (P<0.001), and increase the expression levels of p21 and E-cadherin protein (P<0.001). Inhibition of miR-15b expression or over-expression of PLD1 could reduce the effects of inhibition of DLX6-AS1 expression on the survival rate, migration and invasion number of HSC3 cells and the expression of p21, MMP-2 and E-cadherin protein. DLX6-AS1 targets negative regulation of miR-15b in HSC3 cells, and miR-15b targets negative regulation of PLD1. Conclusion: Inhibition of DLX6-AS1 expression may inhibit the proliferation, migration and invasion of oral squamous cell carcinoma cells through targeted regulation of miR-15b/PLD1 axis.

Key words: growth arrest specific protein 6-antisense RNA1(DLX6-AS1), microRNA-15b(miR-15b), phospholipase D1(PLD1), oral squamous cell carcinoma, invasion and metastasis

中图分类号:

R739.8

杨利杰, 田欣欣, 管臻洁. 长链非编码RNA DLX6-AS1调控微小RNA-15b和磷脂酶D1影响口腔鳞状细胞癌侵袭转移的分子机制研究[J]. 《口腔颌面外科杂志》, 2020, 30(4): 222-229.

YANG Lijie, TIAN Xinxin, GUAN Zhenjie. Molecular Mechanism of LncRNA DLX6-AS1 Regulating MiR-15b/PLD1 to Affect Invasion and Metastasis of Oral Squamous Cell Carcinoma[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(4): 222-229.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I4/222

图/表 7

表1 DLX6-AS1、miR-15b与临床分期及转移之间的关系

Table 1 Relationship between DLX6-AS1, miR-15b and clinical stage and metastasis

组别	例数	DLX6-AS1	P值	miR-15b	P值
TNM分期
Ⅰ~Ⅱ	20	0.73±0.028	<0.001	0.82±0.032	<0.001
Ⅲ~Ⅳ	18	1.64±0.062		0.18±0.011
有无转移
无	22	0.61±0.017	<0.001	0.68±0.027	<0.001
有	16	1.29±0.053		0.24±0.018

图1 口腔鳞癌组织中DLX6-AS1、miR-15b和PLD1 mRNA的表达水平注：A. DLX6-AS1的表达；B. miR-15b的表达；C. PLD1的表达。*表示与癌旁组织比较， P<0.001。

Figure 1 Expression level of DLX6-AS1, miR-15b and PLD1 mRNA in oral squamous cell carcinoma

图2 抑制DLX6-AS1对HSC3细胞的细胞活性、迁移和侵袭及p21、E-cadherin和MMP-2蛋白表达的影响注：A. DLX6-AS1的表达；B. 抑制DLX6-AS1对HSC3细胞活性的影响；C. 抑制DLX6-AS1对HSC3细胞迁移和侵袭的影响；D. 抑制DLX6-AS1对HSC3细胞中p21、E-cadherin和MMP-2蛋白表达的影响；E. Western Blot检测抑制DLX6-AS1后HSC3细胞中p21、E-cadherin和MMP-2蛋白的表达。*表示与si-NC组比较， P<0.001。

Figure 2 Inhibition of DLX6-AS1 on the cell activity, migration and invasion of HSC3 cells, and the expression of p21, E-cadherin and MMP-2

图3 DLX6-AS1靶向调控miR-15b 注：A. DLX6-AS1与miR-15b核苷酸序列的结合位点，红色字体为突变后的位点；B.共转染miR-15b模拟物与WT-DLX6-AS1或MUT-DLX6-AS1后的细胞荧光素酶活性；C.miR-15b的表达。*表示与miR-NC组比较， P<0.001；#表示与pcDNA3.1组比较，P<0.001；&表示与si-NC组比较， P<0.001。

Figure 3 DLX6-AS1 targeting regulation miR-15b

图4 抑制miR-15b表达降低抑制DLX6-AS1对HSC3细胞的细胞活性、迁移、侵袭及p21、E-cadherin和MMP-2蛋白表达的影响注：A. miR-15b的表达；B. 抑制miR-15b表达降低抑制DLX6-AS1对HSC3细胞迁移、侵袭的影响；C. 抑制miR-15b表达降低抑制DLX6-AS1对HSC3细胞存活率的影响；D. 抑制miR-15b表达降低抑制DLX6-AS1对HSC3细胞中p21、E-cadherin和MMP-2蛋白表达的影响；E. Western Blot检测同时抑制miR-15b和DLX6-AS1表达后，HSC3细胞中p21、E-cadherin和MMP-2蛋白的表达。*表示与si-DLX6-AS1+anti-miR-NC组比较，P<0.001。

Figure 4 Inhibition of miR-15b reduces the effect of inhibition of DLX6-AS1 on cell activity, migration, invasion and expression of p21, E-cadherin and MMP-2 in HSC3 cells

图5 过表达PLD1能减弱抑制DLX6-AS1对HSC3细胞的细胞活性、迁移、侵袭及p21、E-cadherin和MMP-2蛋白表达的影响注：A. 过表达PLD1能减弱抑制DLX6-AS1对HSC3细胞的细胞活性的影响；B. 过表达PLD1能减弱抑制DLX6-AS1对HSC3细胞迁移、侵袭的影响；C. 过表达PLD1能减弱抑制DLX6-AS1对HSC3细胞中p21、E-cadherin和MMP-2蛋白表达的影响；D. Western Blot检测过表达PLD1且抑制DLX6-AS1后HSC3细胞中p21、E-cadherin和MMP-2蛋白表达。*表示与si-DLX6-AS1+pcDNA3.1组比较，P<0.001。

Figure 5 Overexpression of PLD1 attenuates the inhibition of DLX6-AS1 on cell activity, migration, invasion and expression of p21, E-cadherin and MMP-2 in HSC3 cells

图6 MiR-15b靶向调控PLD1 注：A. PLD1的3′UTR含有miR-15b的互补序列，红色字体为突变后的位点；B. 共转染miR-15b模拟物与WT-PLD1或MUT-PLD1后的双荧光素酶报告基因实验；C. miR-15b对PLD1的mRNA和蛋白表达的影响；D. Western Blot检测过表达或抑制miR-15b后PLD1蛋白表达。与miR-NC组比较，* P<0.001; 与anti-miR-NC组比较，# P<0.001。

Figure 6 MiR-15b targeting PLD1

参考文献 17

[1]	王雪阳, 周昱川, 陈青立, 等. Hippo信号通路相关蛋白YAP, Mst 1, Lats 1/2在口腔鳞癌中的表达及临床意义[J]. 口腔医学研究, 2017, 33(4): 400-403. doi: 10.13701/j.cnki.kqyxyj.2017.04.013
[2]	王立山, 周福亭, 韩成冰, 等. 早期舌鳞状细胞癌颈淋巴结转移规律、手术方式选择及预后相关因素分析[J]. 中华口腔医学杂志, 2018, 53(2):73-78.
[3]	Li DY, Chen WJ, Luo L, et al. Prospective lncRNA-miRNA-mRNA regulatory network of long non-coding RNA LINC00968 in non-small cell lung cancer A549 cells: a miRNA microarray and bioinformatics investigation[J]. Int J Mol Med, 2017, 40(6): 1895-1906.
[4]	Zhang XL, Guo HH, Bao Y, et al. Exosomal long non-coding RNA DLX6-AS1 as a potential diagnostic biomarker for non-small cell lung cancer[J]. Oncol Lett, 2019, 18(5): 5197-5204. doi: 10.3892/ol.2019.10892 pmid: 31612030
[5]	Tian Y, Wang YR, Jia SH. Knockdown of long noncoding RNA DLX6-AS1 inhibits cell proliferation and invasion of cervical cancer cells by downregulating FUS[J]. Eur Rev Med Pharmacol Sci, 2019, 23(17): 7307-7313.
[6]	Zhou FR, Pan ZP, Shen F, et al. Long non-coding RNA DLX6-AS1 functions as a competing endogenous RNA for miR-577 to promote malignant development of colorectal cancer[J]. Eur Rev Med Pharmacol Sci, 2019, 23(9): 3742-3748.
[7]	郭艳, 尚超, 任美思, 等. MiR-15b对口腔鳞癌细胞Cal27增殖与凋亡能力影响研究[J]. 中国实用口腔科杂志, 2015, 8(8):458-460. doi: 10.7504/kq.2015.08.003
[8]	郑蕙, 张宏颖. 细胞膜磷脂与肿瘤侵袭、转移及靶向治疗的相关性研究进展[J]. 肿瘤防治研究, 2015, 42(6): 631-636.
[9]	饶倩雯, 王菲, 宋美怡, 等. 磷脂酶D1对胰腺癌细胞增殖的影响[J]. 同济大学学报(医学版), 2018, 39(3): 13-18.
[10]	Noble AR, Maitland NJ, Berney DM, et al. Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation[J]. Br J Cancer, 2018, 118(2): 189-199. doi: 10.1038/bjc.2017.391 URL
[11]	Kang DW, Lee SW, Hwang WC, et al. Phospholipase D1 acts through Akt/TopBP1 and RB1 to regulate the E2F1-dependent apoptotic program in cancer cells[J]. Cancer Res, 2017, 77(1): 142-152. doi: 10.1158/0008-5472.CAN-15-3032 pmid: 27793841
[12]	Fang C, Xu L, He W, et al. Long non-coding RNA DLX6-AS1 promotes cell growth and invasiveness in bladder cancer via modulating the miR-223-HSP90B1 axis[J]. Cell Cycle, 2019, 18(23): 3288-3299. doi: 10.1080/15384101.2019.1673633 URL
[13]	Liang Y, Zhang CD, Zhang C, et al. DLX6-AS1/miR-204-5p/OCT1 positive feedback loop promotes tumor progression and epithelial-mesenchymal transition in gastric cancer[J]. Gastric Cancer, 2020, 23(2): 212-227. doi: 10.1007/s10120-019-01002-1 pmid: 31463827
[14]	Yao RS, Han DY, Sun XY, et al. Histone deacetylase inhibitor NaBut suppresses cell proliferation and induces apoptosis by targeting p21 in multiple myeloma[J]. Am J Transl Res, 2017, 9(11): 4994-5002. pmid: 29218097
[15]	Ou YW, Wu QN, Wu CY, et al. Migfilin promotes migration and invasion in glioma by driving EGFR and MMP-2 signalings: a positive feedback loop regulation[J]. Yi Chuan Xue Bao, 2017, 44(12): 557-565.
[16]	Qiu Y, Liu Y, Li WH, et al. P2Y2 receptor promotes the migration and invasion of breast cancer cells via EMT-related genes Snail and E-cadherin[J]. Oncol Rep, 2018, 39(1): 138-150. doi: 10.3892/or.2017.6081 pmid: 29115551
[17]	Kang DW, Lee BH, Suh YA, et al. Phospholipase D1 inhibition linked to upregulation of ICAT blocks colorectal cancer growth hyperactivated by wnt/β-catenin and PI3K/Akt signaling[J]. Clin Cancer Res, 2017, 23(23): 7340-7350. doi: 10.1158/1078-0432.CCR-17-0749 pmid: 28939743

选择文件类型/文献管理软件名称

选择包含的内容