长链非编码RNA PCGEM1通过转化生长因子β2/Smad2信号通路调控口腔鳞状细胞癌侵袭和转移的机制研究

doi:10.7518/hxkq.2020.05.014

摘要/Abstract

摘要： 目的探讨长链非编码RNA（lncRNA）PCGEM1通过转化生长因子β2（TGF β2）/Smad2信号通路调控口腔鳞状细胞癌（OSCC）侵袭和转移的机制。方法将60例OSCC患者癌组织及距离癌组织超过2 cm处的正常组织纳入研究,实时定量聚合酶链反应（qRT-PCR）检测miR-148a、lncRNA PCGEM1在OSCC组织及正常组织、人正常口腔黏膜上皮细胞（OMEC）及人源OSCC细胞株KB、BcaCD885、SCC-4、CAL27、SCC-15中的表达情况;分析lncRNA PCGEM1和miR-148a表达与患者临床病理信息之间的关系。构建lncRNA PCGEM1沉默细胞系KB-siPCGEM1及阴性对照（KB-NC）,并以KB作为空白对照组,采用MTT、Transwell和划痕实验检测lncRNA PCGEM1对KB细胞增殖、侵袭和迁移能力的影响;使用生物信息学网站starBase预测lncRNA PCGEM1可以互补结合的微小RNA（miRNA）,再根据www.microRNA.org网站预测相应miRNA可靶向结合的基因;免疫印迹（Western blotting）检测TGF β2/Smad2信号通路蛋白表达情况。结果 qRT-PCR结果显示,OSCC组织中lncRNA PCGEM1、miR-148a的表达水平高于正常组织（P<0.05）;lncRNA PCGEM1和miR-148a在不同TNM分期、淋巴结转移和组织分化程度的患者癌组织中表达差异均有统计学意义（P<0.05）;与空白对照组和KB-NC组相比,KB-siPCGEM1组细胞OD_{492 nm}值下降,细胞侵袭数量及迁移率降低,差异均具有统计学意义（P<0.05）;生物信息学预测结果显示,lncRNA PCGEM1可与miR-148a互补结合,miR-148a与TGFβ2存在靶向结合位点;qRT-PCR和Western blotting检测结果显示,KB-siPCGEM1组中miR148a、TGFβ2及p-Smad 2的表达明显低于空白对照组与KB-NC组（P<0.05）,空白对照组和KB-NC组的差异无统计学意义（P>0.05）。结论 lncRNA PCGEM1在OSCC中高表达,高表达lncRNA PCGEM1可能通过上调miR-148a水平,强化TGF β2/Smad2信号通路,从而促进OSCC的进展。

关键词: 长链非编码RNA PCGEM1, 口腔鳞状细胞癌, 转化生长因子β2/Smad2信号通路;

Abstract:

Objective To investigate the mechanism underlying the regulation of the invasion and metastasis of oral squamous cell carcinoma (OSCC) by long-chain noncoding RNA (lncRNA) PCGEM1 through the transforming growth factor (TGF) β2/Smad2 signaling pathways. Methods A total of 60 OSCC cases were collected. Cancer tissues and normal tissues more than 2 cm away from cancer tissues were also collected. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-148a and lncRNA PCGEM1 in OSCC, adjacent normal tissues, oral mucosa epithelial cells, KB, BcaCD885, SCC-4, CAL27, and SCC-15. The relationship between the expression of lncRNA PCGEM1 and miR-148a and the clinicopathological information of patients was analyzed. The lncRNA PCGEM1-silenced cell line KB-siPCGEM1 and negative control (KB-NC) group were constructed, and KB was used as the blank control group. The effects of lncRNA PCGEM1 on the proliferation, invasion, and migration of KB cells were determined via MTT, Transwell, and scratch assays. The bioinformatics website starBase was used to predict the complementary binding microRNA (miRNA) of lncRNA PCGEM1. Furthermore, the genes that the miRNA could target and bind were predicted in accordance with the website www.microRNA.org. Western blotting analysis was used to detect the expression of TGF β2/Smad2 signaling pathway proteins. Results qRT-PCR results showed that the expression level of lncRNA PCGEM1 and miR-148a in OSCC tissues was higher than that in normal tissues (P<0.05). The expression of lncRNA PCGEM1 and miR-148a in the cancer tissues of patients with different TNM grades, lymph node metastasis, and tissue differentiation was statistically significant (P<0.05). Compared with those in the blank control group and the KB-NC group, OD_{492 nm} value was significantly decreased and cell mobility was significantly reduced in the KB-siPCGEM1 group (P<0.05). Bioinformatics predictions showed that lncRNA PCGEM1 could bind to miR-148a in a complementary manner and that miR-148a had a targeted binding site with TGF β2. qRT-PCR and Western blotting analysis results showed that the expression levels of miR-148a, TGF β2, and p-Smad2 in the KB-siPCGEM1 group were significantly lower than those in the blank control and KB-NC groups (P<0.05), and no statistically significant difference between the blank control group and the KB-NC group was observed (P>0.05). Conclusion LncRNA PCGEM1 is highly expressed in OSCC. The high expression of lncRNA PCGEM1 may enhance the TGF β2/Smad2 signaling pathway by upregulating miR-148a, thus promoting the development of OSCC.

Key words: long-chain noncoding RNA PCGEM1, oral squamous cell carcinomas, transforming growth factor β2/Smad2 signaling pathway

中图分类号:

R739.8

翁旭, 李劲松, 范松. 长链非编码RNA PCGEM1通过转化生长因子β2/Smad2信号通路调控口腔鳞状细胞癌侵袭和转移的机制研究[J]. 华西口腔医学杂志, 2020, 38(5): 550-557.

Weng Xu, Li Jinsong, Fan Song. Mechanism of long-chain noncoding RNA PCGEM1 in the regulation of the invasion and metastasis of oral squamous carcinoma cells via transforming growth factor β2/Smad2 signaling pathway[J]. West China Journal of Stomatology, 2020, 38(5): 550-557.

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参考文献 17

[1]	粟芃芃, 叶健斌, 邱晓媚 , 等. 人DLL1真核表达质粒的构建及过表达DLL1抑制人口腔鳞癌细胞SCC15的增殖[J]. 暨南大学学报(自然科学与医学版), 2017,38(3):185-191.
	Su PP, Ye JB, Qiu XM , et al. Construction of eukaryotic expression vector of human Delta-like 1 and the overexpression of DLL1 inhibites proliferation of human oral squamous cell carcinoma SCC15 cells[J]. J Jinan Univ (Nat Sci Med Edit), 2017,38(3):185-191.
[2]	Yamashita K, Iwatake M, Okamoto K , et al. Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas in vitro[J]. Oral Dis, 2017,23(4):518-525. doi: 10.1111/odi.12643 URL pmid: 28117540
[3]	Fox MD, Xiao L, Zhang M , et al. Plasmacytoid urothelial carcinoma of the urinary bladder: a clinicopathologic and immunohistochemical analysis of 49 cases[J]. Am J Clin Pathol, 2017,147(5):500-506. doi: 10.1093/ajcp/aqx029 URL pmid: 28371875
[4]	张小路, 杜梅红 . lncRNA MALAT1调控miR-204表达影响胰腺癌细胞的生物学行为[J]. 中国肿瘤生物治疗杂志, 2018,25(1):79-84.
	Zhang XL, Du MH . Long chain non-coding MALAT1 affects biological behavior of pancreatic cancer cells by regulating miR-204 expression[J]. Chin J Cancer Biother, 2018,25(1):79-84.
[5]	Xue Y, Wang M, Kang M , et al. Association between lncrna PCGEM1 polymorphisms and prostate cancer risk[J]. Prostate Cancer Prostatic Dis, 2013,16(2):139-144. doi: 10.1038/pcan.2013.6 URL pmid: 23459097
[6]	施文斌, 甄超, 王旭 , 等. 口腔鳞癌中PTEN、Bcl-2和CyclinD1蛋白表达及其相关性研究[J]. 现代口腔医学杂志, 2017,31(4):210-213.
	Shi WB, Zhen C, Wang X , et al. Correlation among expression of PTEN, Bcl-2, CyclinD1 protein of oral squamous cell cancer[J]. J Modern Stomatol, 2017,31(4):210-213.
[7]	Wang W, Dong J, Wang MX , et al. MiR-148a-3p suppresses epithelial ovarian cancer progression primarily by targeting c-Met[J]. Oncol Lett, 2018,15(5):6131-6136. doi: 10.3892/ol.2018.8110 URL pmid: 29616095
[8]	Kim CR, Kim YM, Lee MK , et al. Pyropia yezoensis peptide promotes collagen synjournal by activating the TGF-β/Smad signaling pathway in the human dermal fibroblast cell line Hs27[J]. Int J Mol Med, 2017,39(1):31-38. doi: 10.3892/ijmm.2016.2807 URL pmid: 27878236
[9]	Zhang J, Jin HY, Wu Y , et al. Hypoxia-induced LncRNA PCGEM1 promotes invasion and metastasis of gastric cancer through regulating SNAI1[J]. Clin Transl Oncol, 2019,21(9):1142-1151. doi: 10.1007/s12094-019-02035-9 URL pmid: 30690667
[10]	Chen S, Wang LL, Sun KX , et al. LncRNA PCGEM1 induces ovarian carcinoma tumorigenesis and progression through RhoA pathway[J]. Cell Physiol Biochem, 2018,47(4):1578-1588. doi: 10.1159/000490931 URL pmid: 29949791
[11]	车明文, 龚翔, 张小金 , 等. LncRNA-ATB在高糖诱导的人腹膜间皮细胞表型转换及增殖中的作用[J]. 解放军医学杂志, 2017,42(11):985-991.
	Che MW, Gong X, Zhang XJ , et al. Effects of LncRNA-ATB on human peritoneal mesothelial cells phenotypic transition and proliferation induced by high glucose[J]. Med J Chin PLA, 2017,42(11):985-991.
[12]	Kou B, Liu W, Tang XS , et al. HMGA2 facilitates epithelial-mesenchymal transition in renal cell carcinoma by regulating the TGF-β/Smad2 signaling pathway[J]. Oncol Rep, 2018,39(1):101-108. doi: 10.3892/or.2017.6091 URL pmid: 29138866
[13]	Li BW, Wang WZ, Li Z , et al. MicroRNA-148a-3p enhances cisplatin cytotoxicity in gastric cancer through mitochondrial fission induction and cyto-protective autophagy suppression[J]. Cancer Lett, 2017,410:212-227. doi: 10.1016/j.canlet.2017.09.035 URL pmid: 28965855
[14]	Jiang F, Mu J, Wang XX , et al. The repressive effect of miR-148a on TGF beta-SMADs signal pathway is involved in the glabridin-induced inhibition of the cancer stem cells-like properties in hepatocellular carcinoma cells[J]. PLoS One, 2014,9(5):e96698. doi: 10.1371/journal.pone.0096698 URL pmid: 24806207
[15]	Deng GQ, Chen L, Zhang YQ , et al. Fucosyltransferase 2 induced epithelial-mesenchymal transition via TGF-β/Smad signaling pathway in lung adenocarcinaoma[J]. Exp Cell Res, 2018,370(2):613-622. doi: 10.1016/j.yexcr.2018.07.026 URL pmid: 30031128
[16]	孙雪, 梅峰 . TGF-β/Smad信号通路诱导肾上皮细胞间充质转化机制的研究进展[J]. 山东医药, 2017,57(30):108-110.
	Sun X, Mei F . Research progress of TGF-β/Smad signaling pathway in inducing mesenchymal transformation in renal epithelial cells[J]. Shandong Med J, 2017,57(30):108-110.
[17]	Yu CP, Liu ZW, Chen QH , et al. Nkx2.8 inhibits epithelial-mesenchymal transition in bladder urothelial carcinoma via transcriptional repression of Twist1[J]. Cancer Res, 2018,78(5):1241-1252. doi: 10.1158/0008-5472.CAN-17-1545 URL pmid: 29311157

临床病理参数	n	lncRNA PCGEM1		χ²值	P值	miR-148a		χ²值	P值
临床病理参数	n	低表达	高表达	χ²值	P值	低表达	高表达	χ²值	P值
性别				1.482	0.224			0.234	0.628
男	32	14（63.64%）	18（47.37%）			18（56.25%）	14（50.00%）
女	28	8（36.36%）	20（52.63%）			14（43.75%）	14（50.00%）
年龄/岁				0.861	0.353			0.357	0.550
<45	45	15（68.18%）	30（78.95%）			25（78.13%）	20（71.43%）
≥45	15	7（31.82%）	8（21.05%）			7（21.88%）	8（28.57%）
TNM分期				8.086	0.004			14.464	0.000
Ⅰ、Ⅱ	24	14（63.64%）	10（26.32%）			20（62.50%）	4（14.29%）
Ⅲ、Ⅳ	36	8（36.36%）	28（73.68%）			12（37.50%）	24（85.71%）
组织分化				10.757	0.005			24.501	0.000
高	12	8（36.36%）	4（10.53%）			10（31.25%）	2（7.14%）
中	25	11（50.00%）	14（36.84%）			19（59.38%）	6（21.43%）
低	23	3（13.64%）	20（52.63%）			3（9.38%）	20（71.43%）
淋巴结转移				4.781	0.029			5.249	0.022
是	38	10（45.45%）	28（73.68%）			16（50.00%）	22（78.57%）
否	22	12（54.55%）	10（26.32%）			16（50.00%）	6（21.43%）
肿瘤直径/cm				0.064	0.801			0.005	0.944
≥2	34	12（54.55%）	22（57.89%）			18（56.25%）	16（57.14%）
<2	26	10（45.45%）	16（42.11%）			14（43.75%）	12（42.86%）