Role of G protein-coupled receptor kinase 3 in the proliferation, migration and invasion of oral squamous carcinoma cells and its possible mechanism
目的： 探讨沉默G蛋白偶联受体激酶3（G protein-coupled receptor kinase 3，GRK3）对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞增殖、迁移和侵袭的影响及其可能的机制。 方法： 利用Oncomine数据库分析GRK3在正常口腔组织及OSCC组织中的表达水平。用RNA干扰技术敲降GRK3在OSCC细胞WSU-HN6和CAL27中的表达，用qPCR法验证干扰效率后，采用CCK-8法和流式细胞术分别检测敲降GRK3对OSCC细胞增殖和凋亡的影响，Transwell小室法检测对OSCC细胞迁移、侵袭能力的影响，qPCR法检测对OSCC细胞周期、上皮间质转化（epithelial to mesenchymal transition，EMT）和基质金属蛋白酶（matrix metallopeptidase，MMP）相关分子mRNA水平表达的影响，WB法检测EMT及MMP相关分子的蛋白表达水平变化。 结果： OSCC组织中GRK3的表达水平显著高于正常口腔组织（P<0.01）。转染si-GRK3后，OSCC细胞中GRK3 mRNA表达水平均下调70%以上。敲降GRK3可显著抑制OSCC细胞的增殖、迁移和侵袭能力（均P<0.01），对细胞凋亡无显著影响（P>0.05）。敲降GRK3表达后，OSCC细胞的G0/G1期比例显著增高（P<0.01），细胞周期蛋白D1（Cyclin D1）、Cyclin D3、细胞周期蛋白依赖性激酶2（cyclin-dependent kinases 2，CDK2）和CDK4基因的mRNA表达降低（均P<0.05）；EMT相关分子波形蛋白（Vimentin）、Zeb1和Slug表达降低，E-钙黏蛋白（E-cadherin）表达升高（均P<0.05）；MMP3和MMP9表达降低（均P<0.05），MMP2和MMP7表达无明显变化（均P>0.05）。 结论： GRK3可通过调节细胞周期促进OSCC细胞的增殖能力，并通过调控EMT和MMP增强细胞的迁移和侵袭能力。Abstract: Objective: To investigate the effects of silencing G protein-coupled receptor kinase 3 (GRK3) on the proliferation, migration and invasion of oral squamous cell carcinoma (OSCC) cells and the possible underlying mechanisms. Methods: GRK3 expression levels in normal oral tissues and OSCC tissues were analyzed using Oncomine database. RNA interference technology was used to down-regulate GRK3 expression in OSCC cell lines WSU-HN6 and CAL27. The interference efficiency was verified by qPCR. The effects of knockdown of GRK3 on proliferation and apoptosis of OSCC cells were detected by CCK-8 assay and Flow cytometry, respectively; the effects on migration and invasion abilities of OSCC cells were determined by Transwell assay; and the effects on mRNA expression levels of molecules associated with cell cycle, epithelial-mesenchymal transition (EMT), and matrix metallopeptidase (MMP) were determined by qPCR. The changes in protein levels of molecules associated with EMT and MMP were detected using WB assay. Results: GRK3 expression level in OSCC tissues was significantly higher than that in normal oral tissues ( P<0.01). After being transfected with si-GRK3, the mRNA expression of GRK3 in OSCC cells was down-regulated by more than 70%. Silencing GRK3 significantly inhibited the proliferation, migration and invasion of OSCC cells (all P<0.01), but had no significant effect on apoptosis (P>0.05). After down-regulation of GRK3, the percentage of OSCC cells in G0/G1 phase was significantly increased (P<0.01); the mRNA expression levels of Cyclin D1, Cyclin D3, CDK2 and CDK4 were decreased (all P<0.05); expression levels of EMT-related proteins (Vimentin, Zeb1 and Slug) were decreased, while E-cadherin was increased (all P<0.05); MMP3 and MMP9 were decreased (all P<0.05), while MMP2 and MMP7 showed no significant changes (all P>0.05). Conclusion: GRK3 promotes the proliferation of OSCC cells by regulating cell cycle-related molecules and enhances the migration and invasion through regulating EMT and MMPs.
图 1 GRK3在OSCC组织中的表达及敲降GRK3对OSCC细胞增殖的影响
Figure 1. The expression level of GRK3 in OSCC tissues and the effect of GRK3 silence on the proliferation of OSCC cells
**P<0.01 vs si-NC group A: The mRNA level of GRK3 in normal oral tissues and OSCC tissues; B: Interference efficiency of GRK3 mRNA expression in OSCC cells was determined using qPCR; C and D: CCK-8 assay was performed to detect the proliferation of OSCC cells
图 3 敲降GRK3对OSCC细胞凋亡和细胞周期的影响
Figure 3. The effects of knocking down GRK3 on the apoptosis and cell cycle of OSCC cells
*P<0.05, **P<0.01 vs si-NC group A: The apoptosis rate of WSU-HN6 cells; B: The percentage of WSU-HN6 cells at different phases of the cell cycle; C: The mRNA levels of molecules associated with cell cycle in WSU-HN6 cells
图 4 敲降GRK3对OSCC细胞EMT及MMP相关分子表达的影响
Figure 4. The effects of knocking down GRK3 on the expression levels of molecules associated with EMT and MMPs in OSCC cells
*P<0.05, **P<0.01 vs si-NC group A: The mRNA levels of EMT-associated molecules in WSU-HN6 cells; B: The mRNA levels of MMPs in WSU-HN6 cells; C: The protein levels of molecules associated with EMT and MMPs in WSU-HN6 cells
表 1 引物序列
Table 1. Primer sequences
Gene Forward primer (5’-3’) Reverse primer (5’-3’) Cyclin D1 TCTACACCGACAACTCCATCCG TCTGGCATTTTGGAGAGGAAGTG Cyclin D3 AGATCAAGCCGCACATGCGGAA ACGCAAGACAGGTAGCGATCCA CDK2 ATGGATGCCTCTGCTCTCACTG CCCGATGAGAATGGCAGAAAGC CDK4 CCATCAGCACAGTTCGTGAGGT TCAGTTCGGGATGTGGCACAGA Vimentin AGGCAAAGCAGGAGTCCACTGA ATCTGGCGTTCCAGGGACTCAT Zeb1 GGCATACACCTACTCAACTACGG TGGGCGGTGTAGAATCAGAGTC E-Cadherin GCCTCCTGAAAAGAGAGTGGAAG TGGCAGTGTCTCTCCAAATCCG Slug ATCTGCGGCAAGGCGTTTTCCA GAGCCCTCAGATTTGACCTGTC MMP2 AGCGAGTGGATGCCGCCTTTAA CATTCCAGGCATCTGCGATGAG MMP3 CACTCACAGACCTGACTCGGTT AAGCAGGATCACAGTTGGCTGG MMP7 TCGGAGGAGATGCTCACTTCGA GGATCAGAGGAATGTCCCATACC MMP9 GCCACTACTGTGCCTTTGAGTC GGATCAGAGGAATGTCCCATACC GAPDH GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAA
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