miR-146b-5p调控CD82介导食管鳞状细胞癌恶性表型

miR-146b-5p mediates malignant phenotype of esophageal squamous cell carcinoma(ESCC)by targeting CD82

目的探究miR-146b-5p促进食管鳞状细胞癌恶性表型的分子机制。方法选取2020年10月-2021年12月在四川省西南医科大学附属医院行食管癌根治术治疗的38名食管鳞状细胞癌患者的癌组织和癌旁正常组织(NAT)样本,核酸原位杂交法检测miR-146b-5p在食管鳞癌及癌旁正常组织中的表达水平;通过siRNA敲低miR-146b-5p的表达水平,细胞增殖实验、划痕实验检测敲低miR-146b-5p后细胞行为学表型变化;生物信息学分析预测miR-146b-5p的下游靶基因为CD82;免疫印迹法(Western-blot)检测靶基因CD82的表达水平,双荧光素酶报告基因实验确定miR-146b-5p与CD82之间的调控关系。结果核酸原位杂交法检测结果显示,与NAT组织病理评分比较,miR-146b-5p在ESCC组织中的病理评分较高(P<0.05);EdU细胞增殖实验结果显示,与NC组比较,KD组EdU阳性细胞比例显著降低(P<0.05)。细胞划痕实验结果显示,与NC组比较,KD组划痕愈合比例显著下降(P<0.05)。ENCORI数据库预测CD82是miR-146b-5p的下游靶基因;Western-blot实验结果显示,相比NC组,KD组CD82在蛋白水平显著性上调;双荧光素酶报告基因实验结果显示,wt+mimic组荧光强度较mut+mimic组显著性下降(P<0.05)。结论miR-146b-5p通过靶向调控CD82促进食管鳞状细胞癌细胞的迁移,为食管鳞状细胞癌的治疗提供新的靶点。

Objective To explore the molecular mechanism by which miR-146b-5p promoted the malignant phenotype of esophageal squamous cell carcinoma(ESCC).Methods The cancer tissue and adjacent normal tissue(NAT)samples from 38 patients with ESCC who underwent radical esophageal cancer treatment at the hospital from October 2020 to December 2021 were selected,and the expression of miR-146b-5p was detected using an in situ hybridization kit.Knockdown the expression of miR-146b-5p was achieved through siRNA transfection,and ensuing cellular changes of cellular behavior phenotype were evaluated through EdU and wound healing assay;Bioinformatics analysis predicted that CD82 was one of downstream targets of miR-146b-5p;expression variation of CD82 was examined by Western blot;Dual luciferase reporter assays was adopted to confirm the regulation between miR-146b-5p and target genes.Results The results of in situ hybridization assay showed that miR-146b-5p was highly expressed in ESCC tissues compared to the pathological score of NAT group(P<0.05);The EdU assay results showed that the KD group had a significant decrease in the proportion of EdU positive cells compared with the NC group(P<0.05).The results of the wound healing assay showed that KD group had a significant decrease in the proportion of wound healing compared with the NC group(P<0.05).The ENCORI database predicted that CD82 was a downstream target gene of miR-146b-5p.The Western-blot results showed that CD82 was significantly upregulated at protein level in KD group compared to NC group.The results of the dual luciferase reporter assay showed that the fluorescence intensity of wt+mimic group was significantly reduced compared to mut+mimic group(P<0.05).Conclusion miR-146b-5p promotes the malignant phenotype of esophageal squamous cell carcinoma cells via CD82,and it will provide a new target for treatment of esophageal squamous cell carcinoma.