连翘苷调控miR-34c-5p表达对鼻咽癌细胞CNE-1、CNE-2凋亡的影响及作用机制分析

The effect and mechanism of pillyrin on apoptosis of nasopharyngeal carcinoma CNE-1 and CNE-2 cells by regulating miR-34c-5p

摘要:

目的 探究连翘苷通过调控微小RNA-34c-5p(miR-34c-5p)表达对鼻咽癌(NPC)细胞系CNE-1、CNE-2增殖抑制及凋亡诱导的影响及其作用机制。方法 体外培养NPC细胞CNE-1、CNE-2，采用MTT法筛选连翘苷对NPC细胞增殖的作用浓度。将CNE-1、CNE-2细胞分别分为对照组、连翘苷组、阴性对照模拟物(miR-NC)组、miR-34c-5p模拟物(miR-34c-5p)组、连翘苷+阴性对照抑制剂(in-miR-NC)组和连翘苷+miR-34c-5p抑制剂(in-miR-34c-5p)组。采用集落形成实验、EdU法、TUNEL法及流式细胞术检测各组细胞的增殖与凋亡情况，利用Western blot检测各组NPC细胞增殖与凋亡相关蛋白的表达。结果 CNE-1和CNE-2细胞增殖活力随着连翘苷浓度的升高呈剂量依赖性降低(P<0.05)，8 μmol/L连翘苷对CNE-1和CNE-2细胞增殖活力的抑制率接近50%。与对照组比较，连翘苷组CNE-1和CNE-2细胞中miR-34c-5p表达、TUNEL阳性细胞率、细胞凋亡率、p53水平及FasL水平均显著升高，集落形成、EdU阳性细胞比例、c-Myc、PCNA、Survivin均降低(P<0.05)；与miR-NC组比较，miR-34c-5p组CNE-1和CNE-2细胞中miR-34c-5p表达、TUNEL阳性细胞率、细胞凋亡率、p53水平、FasL水平均升高，集落形成、EdU阳性细胞比例、c-Myc、PCNA、Survivin均降低(P<0.05)；与连翘苷+in-miR-NC组比较，连翘苷+in-miR-34c-5p组CNE-1和CNE-2细胞中miR-34c-5p表达、TUNEL阳性细胞率、细胞凋亡率、p53水平、FasL水平均降低，集落形成、EdU阳性细胞比例、c-Myc、PCNA、Survivin均升高(P<0.05)。结论 连翘苷可通过上调miR-34c-5p表达，有效抑制NPC细胞CNE-1和CNE-2增殖并促进细胞凋亡。

Abstract:

Objective To explore the effect and mechanism of pillyrin on the proliferation inhibition and apoptosis induction of nasopharyngeal carcinoma (NPC) cell lines CNE-1 and CNE-2 by regulating the expression of microRNA-34c-5p (miR-34c-5p). Methods CNE-1 and CNE-2 cells were cultured, and the effect of pillyrin on the proliferation activity of NPC cells was screened by methyl thiazolyl tetrazolium (MTT) assay. The CNE-1 and CNE-2 cells were respectively assigned into control group, pillyrin group, negative control mimic (miR-NC) group, miR-34c-5p mimic (miR-34c-5p) group, pillyrin+negative control inhibitor (in-miR-NC) group, and pillyrin+miR-34c-5p inhibitor (in-miR-34c-5p) group. The colony formation assay, EdU method, TUNEL method and flow cytometry were used to detect the proliferation and apoptosis of cells in each group. Western blot was adopted to analyze the expression of proliferation and apoptosis-related proteins in NPC cells of each group. Results The proliferation activities of CNE-1 and CNE-2 cells decreased with the increase of pillyrin concentration (P<0.05). At 8 μmol/L of pillyrin, the inhibitory ability of the proliferation activities of CNE-1 and CNE-2 was close to 50%. Compared with the control group, the expression of miR-34c-5p, the rate of TUNEL positive cells, the apoptosis rate, the level of p53, and the level of FasL in CNE-1 and CNE-2 cells of the pillyrin group were all increased, while the colony formation, the proportion of EdU positive cells, c-Myc, PCNA, and Survivin were all decreased (P<0.05). Compared with the miR-NC group, the expression of miR-34c-5p, the rate of TUNEL positive cells, the apoptosis rate, the level of p53, and the level of FasL in CNE-1 and CNE-2 cells of the miR-34c-5p group were all increased, while the colony formation, the proportion of EdU positive cells, c-Myc, PCNA, and Survivin were all decreased (P<0.05). Compared with the pillyrin + in-miR-NC group, the expression of miR-34c-5p, the rate of TUNEL positive cells, the apoptosis rate, the level of p53, and the level of FasL in CNE-1 and CNE-2 cells of the pillyrin+in-miR-34c-5p group were all decreased, while the colony formation, the proportion of EdU positive cells, c-Myc, PCNA, and Survivin were all increased (P<0.05). Conclusions Pillyrin can effectively promote the proliferation of NPC cells CNE-1 and CNE-2 and promote their apoptosis by up-regulating the expression of miR-34c-5p.