miR-149-5p靶向FGF5调节人口腔鳞状细胞癌细胞凋亡和上皮-间质转换及微管形成

miR-149-5p targeting FGF5 regulation of apoptosis, epithelial-mesenchymal transition and microtubule formation in human oral squamous cell carcinoma

摘要:

目的： 探究miR-149-5p对人口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞凋亡、上皮-间质转换（epithelial-mesenchymal transition，EMT）及微管形成的影响及作用机制。方法： miR-149-5p mimic（mimic）和pcDNA-FGF5（FGF5）单转或共转CAL-27细胞系，RT-PCR、Western blot检测FGF5表达；双荧光素酶报告实验验证miR-149-5p和FGF5靶向关系；流式细胞术检测细胞凋亡率，Western blot检测Bax/Bcl-2蛋白表达；观察EMT细胞形态转化，Western blot检测CAL-27细胞上皮标记物E-钙黏蛋白（E-cadherin，E-cad）及间质标记物N-钙黏蛋白（N-cadherin，N-cad）表达；成管实验检测CAL-27细胞微管样形成能力，Western blot检测血管内皮生长因子（vascular endothelial growth factor，VEGF）表达。结果： miR-149-5p mimic能明显抑制CAL-27细胞中FGF5的表达（P=0.000）；miR-149-5p mimic能明显减弱FGF5野生质粒的荧光素酶活性（P=0.000）；miR-149-5p mimic可明显上调CAL-27细胞凋亡率及Bax/Bcl-2比值（P=0.000），并逆转FGF5过表达导致的细胞凋亡率（P=0.014）及Bax/Bcl-2比值上调（P=0.000）。miR-149-5p mimic抑制EMT细胞形态由圆形到纺锤形转变，并逆向转换FGF5过表达导致的间质样细胞形态。miR-149-5p mimic可上调CAL-27细胞中E-cad的表达（P=0.000）、下调N-cad的表达（P=0.000），逆转FGF5过表达引起的E-cad表达下调（P=0.020）和N-cad表达上调（P=0.000）。miR-149-5p过表达能明显抑制CAL-27细胞微管结节形成（P=0.002），降低VEGF蛋白表达水平（P=0.000），逆转过表达FGF5引起的VEGF表达上调（P=0.000）。结论： miR-149-5p能抑制OSCC细胞的EMT和微管形成能力并促进细胞凋亡，作用机制与靶向抑制FGF5表达有关。

Abstract:

Objective: To explore the effect and mechanism of miR-149-5p in apoptosis, epithelial-mesenchymal transition (EMT) and microtubule formation in human oral squamous cell carcinoma (OSCC). Methods: miR-149-5p mimic (mimic) and pcDNA-FGF5 (FGF5) were singly or co-transfected with CAL-27 cell line, and RT-PCR and Western blot were used to detected the expression FGF5; dual luciferase report test was used to verify the targeting relationship between miR-149-5p and FGF5. The apoptosis rate was detected by flow cytometry, and the expression of Bax/Bcl-2 protein was detected by Western blot. The morphological transformation of EMT cells was observed, and the expression of epithelial and interstitial markers E-cadherin (E-cad) and N-cadherin (N-cad) in CAL-27 cells was detected by Western blot. The microtubule-like formation ability of CAL-27 cells was detected by tube-forming test, and the expression of vascular endothelial growth factor (VEGF) was detected by Western blot. Results: miR-149-5p mimic could significantly inhibit the expression of FGF5 in CAL-27 cells (P=0.000); miR-149-5p mimic could significantly reduce the luciferase activity of FGF5 wild plasmid (P=0.000); miR-149-5p mimic increased the apoptosis rate of CAL-27 cells and Bax/Bcl-2 ratio significantly (P=0.000), and reversed the increase of apoptosis rate (P=0.014) and Bax/Bcl-2 ratio caused by FGF5 overexpression (P=0.000). miR-149-5p mimic inhibited the EMT transformation on cell morphology from round to spindle shape, and reversely transformed the mesenchymal-like cell morphology caused by FGF5 overexpression. miR-149-5p mimic up-regulated the expression of E-cad in CAL-27 cells (P=0.000), down-regulated the expression of N-cad (P=0.0009), reversed the down-regulated expression of E-cad (P=0.020) and the up-regulated expression of N-cad (P=0.000) caused by FGF5 overexpression. miR-149-5p overexpression significantly inhibited the formation of microtubule nodules in CAL-27 cells (P=0.002), reduced the expression level of VEGF protein (P=0.000), and reversed the upregulation of VEGF expression caused by overexpression of FGF5 (P=0.000). Conclusion: miR-149-5p can inhibit EMT and microtubules formation and apoptosis promotion of OSCC cells. The mechanism of action is related to the targeted inhibition of FGF5 expression.