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. 2020 Oct 1;38(5):550-557.
doi: 10.7518/hxkq.2020.05.014.

[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]

[Article in Chinese]
Xu Weng  1 , Jin-Song Li  2 , Song Fan  2
Affiliations

[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]

[Article in Chinese]
Xu Weng et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

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, OD492 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).

Conclusions: 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.

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TGFβ2/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OD492 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TGFβ2/Smad2信号通路，从而促进OSCC的进展。.

Keywords: long-chain noncoding RNA PCGEM1; oral squamous cell carcinomas; transforming growth factor β2/Smad2 signaling pathway.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. lncRNA PCGEM1、miR-148a在不同组织及细胞中的表达
Fig 1 Expression of lncRNA PCGEM1 and miR-148a in different tissues and different cell lines *与OMEC相比，P<0.05；#与其他OSCC细胞相比，P<0.05。
图 2
图 2. lncRNA PCGEM1在各组KB中的表达
Fig 2 Expression of lncRNA PCGEM1 in KB of each group
图 3
图 3. lncRNA PCGEM1对OSCC细胞株KB增殖能力的影响
Fig 3 Effect of lncRNA PCGEM1 on KB proliferation of OSCC cell line *与空白对照组相比，P<0.05；#与KB-NC组相比，P<0.05。
图 4
图 4. lncRNA PCGEM1对OSCC细胞株KB侵袭能力的影响 倒置显微镜 × 200
Fig 4 Effect of lncRNA PCGEM1 on KB invasion ability of OSCC cell line inverted microscope × 200 从左到右依次为空白对照组、KB-NC组和KB-siPCGEM1组。
图 5
图 5. lncRNA PCGEM1对OSCC细胞株KB迁移能力的影响 倒置显微镜 × 200
Fig 5 Effect of lncRNA PCGEM1 on KB migration of OSCC cell line inverted microscope × 200
图 6
图 6. 双荧光素酶报告基因检测
Fig 6 Double luciferase reporter gene detection 左：lncRNA PCGEM1与miR-148a双荧光素酶报告基因检测，A为对照组，B为lncRNA PCGEM1-wild组，C为lncRNA PCGEM1-mutant组；右：TGF β2与miR-148a双荧光素酶报告基因检测，A为对照组，B为TGF β2-wild组，C为TGF β2-mutant组。
图 7
图 7. lncRNA PCGEM1与TGF β2/Smad2的关系
Fig 7 Relationship between lncRNA PCGEM1 and TGF β2/Smad2 左：miR-148a在KB细胞中的相对表达；中：Western blotting检测TGF β2和p-Smad2在KB细胞中的表达；右：TGF β2和p-Smad2在KB细胞中的相对表达，*与空白对照组相比，P<0.05；#与KB-NC组相比，P<0.05。A、B、C分别为空白对照组、KB-NC组、KB-siPCGEM1组。
See this image and copyright information in PMC

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