miR-4497靶向TMEM88通过PI3K/AKT信号通路调控下咽癌FaDu细胞增殖和凋亡的研究

Study of miR-4497 targeting TMEM88 regulates the proliferation and apoptosis of hypopharyngeal carcinoma FaDu cells through thePI3K/AKT signaling pathway

目的探讨microRNA-4497(miR-4497)在下咽癌患者化疗前后血清外泌体中的表达及对人咽鳞状细胞癌FaDu细胞株(简称FaDu细胞)增殖和凋亡的作用机制。方法收集2021年6月~2022年12月于连云港市第一人民医院确诊的7例局部晚期下咽癌患者,给予TPF方案辅助化疗2个周期,分别抽取患者化疗前后的外周血,采用测序技术检测化疗前后两组血清外泌体miRNA,并对其中差异表达的miRNA进行生物信息学分析。通过转染技术下调miR-4497的表达,提取FaDu细胞外泌体。分别利用CCK-8、EdU、流式细胞术和Western blot实验检测FaDu细胞外泌体来源miR-4497对FaDu细胞的增殖和凋亡情况。数据库miRDB、miRWalk、miR2Target和TargetScan联合预测miR-4497潜在靶点,分析下调TMEM88的表达并同时抑制miR-4497的表达对FaDu细胞增殖和凋亡的影响。根据测序预测分析KEGG通路图,Western blot实验验证FaDu细胞内p-PI3K、PI3K、p-AKT和AKT的表达。结果与化疗前相比,化疗后下咽癌患者外周血血清外泌体中miR-4497的表达明显降低。FaDu细胞中miR-4497的表达明显高于人鼻咽上皮细胞系NP69及人鼻咽癌细胞系6-10B和HONE-1(F=109.3,P<0.01)。与转染miR-4497 inhibitor的FaDu细胞外泌体共培育抑制FaDu细胞增殖,促进细胞凋亡,Bax蛋白表达升高(t=84.69,P<0.01),Bcl-2蛋白表达降低(t=31.11,P<0.01)。数据库预测抑癌基因TMEM88可能是miR-4497的靶向。荧光素酶报告基因检测证实TMEM88与miR-4497存在结合位点。使用外泌体来源的miR-4497 inhibitor与TMEM88低表达的FaDu细胞共培育,观察到细胞增殖能力回升,p-PI3K和p-AKT表达降低(t=91.54,P<0.01;t=58.30,P<0.01)。结论FaDu细胞外泌体来源miR-4497可通过下游靶点TMEM88激活PI3K/AKT信号通路,调控FaDu细胞的增殖和凋亡。

OBJECTIVE To explore the expression of microRNA-4497(miR-4497)in serum exosomes before and after chemotherapy in hypopharyngeal carcinoma patients and the mechanism of proliferation and apoptosis of FaDu cells.METHODS Seven patients with local advanced hypopharyngeal cancer treated in Lianyungang first people's hospital from June 2021 to December 2022 were included in this study.Two cycles of TPF regimen neoadjuvant chemotherapy were conducted.The peripheral blood of the patients before and after chemotherapy was taken,and the serum exosomes miRNA was detected by sequencing technique,and the differentially expressed miRNA was analyzed by bioinformatics.FaDu cell exosomes were extracted by downregulating miR-4497 expression through transfection technique.The miR-4497 in serum exosomes of FaDu cells was detected by CCK-8,EdU,flow cytometry and Western blot assay,and the proliferation and apoptosis of FaDu cells were observed.The database miRDB,miRWalk,miR2Target and TargetScan jointly predicted the potential targets of miR-4497 and analyzed the effect of downregulation of TMEM88 expression and simultaneous inhibition of miR-4497 expression on proliferation and apoptosis of FaDu cells.Based on the sequencing prediction analysis of KEGG pathway maps,the expression of p-PI3-K,PI3K,p-AKT and AKT in FaDu cells was verified by Western blot experiments.RESULTS The expression of miR-4497 was significantly decreased in peripheral serum exosomes of hypopharyngeal carcinoma patients after chemotherapy compared with that before chemotherapy.The expression of miR-4497 was significantly higher in FaDu cells than in the human nasopharyngeal epithelial cell line NP69 and other NPC cell lines(F-109.3,P<0.01).Co-cultivation with exosomes of FaDu cells transfected with miR-4497 inhibitor inhibited FaDu cell proliferation and promoted apoptosis,increased Bax expression(f-84.69,P<0.01)and decreased expression of Bcl-2(t-31.11,P<0.01).The database predicted that the tumor suppressor gene TMEM88 may be targeted by miR-4497.Luciferase reporter assay confirmed the presence of a binding site for TMEM88 with miR-4497.Co-cultured using exosome-derived miR-4497 inhibitor with FaDu cells with low TMEM88 expression,increased proliferative capacity and decreased p-PI3K and p-AKT expression were observed(t-91.54,P<0.01;t-58.30,P<0.01).CONCLUSION FaDu cell exosomes-derived miR-4497 can activate the PI3K/AKT signaling pathway through the downstream target TMEM88 and regulate the proliferation and apoptosis of FaDu cells.