MicroRNA-196a对食管癌预后的评估价值及其生物学行为的调控机制

Prognostic value of miR-196a in esophageal carcinoma and its regulatory mechanism of biological behavior

摘要:

探讨microRNA-196a（miR-196a）对食管癌预后的评估价值及其生物学行为的调控机制。方法收集120 例行食管癌根治性手术切除的食管癌组织及癌旁组织，逆转录聚合酶链反应（RT-PCR）检测组织miR-196a 表达水平，分析miR-196a 表达与食管癌患者临床资的关系。对食管癌患者进行随访，记录患者总生存期（OS）和无病生存期（DFS）；以OS 和DFS 作为评价指标，采用单变量和多变量Cox 比例风险模型评价患者预后的影响因素。分别采用miR-196a mimic、NC-mimic、miR-196a inhibitor、NC-inhibitor 转染 食管癌TE1 细胞，MTT实验检测细胞增殖能力，Transwell实验检测细胞侵袭能力，细胞划痕实验检测细胞迁移能力，Western blot检测细胞ANXA1、NTN4、HMGA2、HOXB8 蛋白表达。结果miR-196a在食管癌组织中表达水平高于癌旁组织（p <0.05）；在TE1 细胞中，miR-196a mimic 组miR-196a表达水平高于NC-mimic组，miR-196a inhibitor组miR-196a 表达水平低于NC-inhibitor 组（P<0.05），证实细胞转染实验成功。将食管癌患者按照miR-196a 表达分为miR-196a高表达组51 例和低表达组69 例，miR-196a 表达与年龄、性别、分化程度、N 分期、肿瘤位置等无关（p >0.05），随着T 分期、TNM 分期和肿瘤直径增加，miR-196a 高表达率升高（p <0.05）。生存分析显示，miR-196a 高表达患者总生存期（ p=0.015）和无病生存期（ p=0.017）低于miR-196a低表达者；单因素和多因素分析显示，T 分期、miR-196a表达是影响患者总生存期和无病生存期的的独立危险因素（p<0.05）。MTT 实验结果显示，第48～120 h，miR-196a mimic 组吸光度值高于NC-mimic 组，miR-196a inhibitor 组吸光度值低于NC-inhibitor 组（ p<0.05）；Transwell 小室实验显示，miR-196a mimic 组穿膜细胞数量高于NC-mimic 组，miR-196a inhibitor 组穿膜细胞数量低于NC-inhibitor组（p < 0.05）；细胞划痕实验显示，miR-196a mimic 组细胞迁移距离高于NC-mimic 组，miR-196a inhibitor组细胞迁移距离低于NC-inhibitor 组（p <0.05）。Western blot实验结果显示，miR-196a mimic 组、NC-mimic组、miR-196a inhibitor 组、NC-inhibitor 组HMGA2、HOXB8 蛋白表达比较差异均无统计学意义（p >0.05），miR-196a mimic 组ANXA1、NTN4 蛋白表达低于NC-mimic 组，miR-196a inhibitor 组ANXA1、NTN4 蛋白表达高于NC-inhibitor 组（p <0.05）。结论miR-196a 能够作为食管癌预后的预测指标之一；miR-196a可能通过抑制ANXA1、NTN4 基因的表达参与调控食管癌细胞的增殖、侵袭和迁移等生物学行为。

Abstract:

To explore the prognostic value of miR-196a in esophageal carcinoma and its regulatory mechanism of biological behavior. Methods Esophageal carcinoma tissues and paracancerous tissues were collected from 120 patients. miR-196a expression level was detected by RT-PCR. The relationships between miR-196a expression and clinical data were analyzed. The esophageal carcinoma patients were followed up. The overallsurvival (OS) and disease-free survival (DFS) were recorded. Taking OS and DFS as evaluation indexes, the prognostic factors were evaluated by univariate and multivariate Cox proportional hazards models. The TE1 cells were transfected with miR-196a mimic, NC-mimic, miR-196a inhibitor and NC-inhibitor. Cell proliferative ability was detected by MTT assay, invasive ability was detected by Transwell assay, migratory ability was detected by cell scratch assay. ANXA1, NTN4, HMGA2 and HOXB8 protein expressions in the cells were detected by Western blot. Results The expression of miR-196a in the esophageal carcinoma tissues was significantly higher than that in the paracancerous tissues (p < 0.05). In the TE1 cells, the expression of miR-196a in the miR-196a mimic group was significantly higher than that in the NC-mimic group, the expression of miR-196a in the miR-196a inhibitor group was significantly lower than that in the NC -inhibitor group ( p< 0.05), which confirmed the cell transfection experiment was successful. The esophageal carcinoma patients were divided into miR-196a high-expression group (51 cases) and miR-196a low-expression group (69 cases) according to the miR-196a expression. miR-196a expression was not related to age, sex, differentiation, N stage or tumor location (p > 0.05). With the increase of T staging, TNM stage and tumor diameter, the high-expression rate of miR-196a was significantly increased (p < 0.05). Survival analysis showed that OS and DFS in the miR-196a high-expression group were significantly lower than those in the miR-196a low-expression group ( p< 0.05). Univariate and multivariate analyses showed that T stage and miR-196a expression were the independent risk factors for OS and DFS ( p< 0.05). MTT experimental showed that in the 48th-120th h, the absorbance value of the miR-196a mimic group was significantly higher than that of the NC-mimic group, while the absorbance value of the miR-196a inhibitor group was significantly lower than that of the NC-inhibitor group ( p< 0.05). Transwell chamber experiment showed that the number of transmembrane cells in the miR -196a mimic group was significantly larger than that in the NC -mimic group, and the number of transmembrane cells in the miR-196a inhibitor group was significantly smaller than that in the NC-inhibitor group ( p< 0.05). Cell scratch test showed that the cell migration distance in the miR-196a mimic group was significantly longer than that in the NC-mimic group, and the cell migration distance in the miR-196a inhibitor group was significantly shorter than the NC-inhibitor group (p< 0.05). Western blot showed that there was no significant difference in HMGA2 or HOXB8 protein expression among the 4 TE1 cell groups (P > 0.05); the expressions of ANXA1 and NTN4 proteins in the miR-196a mimic group were significantly lower than those in the NC-mimic group, but the expressions of ANXA1 and NTN4 proteins in the miR-196a inhibitor group were significantly higher than those in the NC-inhibitor group (p < 0.05). Conclusions miR-196a can be used as one of the indexes predicting the prognosis of esophageal carcinoma. It may inhibit the proliferation, invasion and migration of esophageal cancer cells by inhibiting the expressions of and genes.