目的：观察miR-26b-3p在食管鳞状细胞癌（ESCC）组织中的表达水平及其对ESCC细胞增殖、侵袭和迁移能力的影响，并探讨其分子调控机制。方法: 选取河北医科大学第四医院2018年4月1日至2018年12月25日手术切除的ESCC组织及相应癌旁组织各60例，利用qPCR法检测ESCC组织、癌旁组织和ESCC细胞中miR-26b-3p的表达。选取miR-26b-3p表达水平较低的ESCC细胞TE1和KYSE150，转染miR-26b-3p mimic，并设立阴性对照。利用细胞增殖实验、划痕愈合实验和Transwell实验检测过表达miR-26b-3p对TE1和KYSE150细胞增殖、迁移和侵袭能力的影响。荧光素酶报告基因实验检测miR-26b-3p与STAT3的 3'UTR 靶点部位的结合情况。随后同时转染 miR-26b-3p mimic 和 pcDNA3.0-STAT3，利用细胞增殖实验、划痕愈合实验和Transwell实验检测STAT3是否可逆转过表达miR-26b-3p对细胞增殖、迁移和侵袭能力的影响。qPCR 和 WB法检测甲基化酶抑制剂5-Aza-DC对ESCC细胞甲基化的影响和miR-26b-3p与STAT3表达的影响。结果: miR-26b-3p在ESCC组织中的表达低于癌旁组织（P<0.01），其在 ESCC 细胞TE1、KYSE150和LYSE170中表达水平显著低于人正常食管上皮细胞HEEC（均P<0.01）。与miR-26b-3p NC组相比，miR-26b-3p mimic转染可明显上调TE1和KYSE150细胞中miR-26b-3p的表达（均P<0.01），但可明显抑制两种细胞的增殖、迁移和侵袭能力（P<0.05或P<0.01）。荧光素酶报告基因实验结果表明，在TE1和KYSE150细胞中，miR-26b-3p明显抑制了野生型STAT3载体的荧光素酶活性（P<0.05或P<0.01），而突变型的荧光素酶活性不受影响。同时转染miR-26b-3pmimic和pcDNA3.0-STAT3可部分逆转miR-26b-3p mimic对TE1 和 KYSE150 细胞增殖、迁移和侵袭能力的抑制作用（P<0.05或P<0.01）。5-Aza-DC 处理后 ，TE1 和 KYSE150 细胞中 miR-26b-3p 表达上调（均 P<0.01）、STAT3 mRNA 和蛋白水平降低（P<0.05），miR-26b-3p呈现去甲基化状态。结论: miR-26b-3p的启动子高甲基化导致其在ESCC组织和细胞中的表达下调，其作为抑癌因子可通过靶向STAT3而抑制ESCC细胞的增殖、侵袭和迁移能力。

Objective: To detect the expression level of miR-26b-3p in esophageal squamous cell carcinoma (ESCC) tissues, and to explore its effect on the proliferation, invasion and migration of ESCC cells and its molecular mechanism. Methods: 60 pairs of ESCC tissues and corresponding adjacent tissues that surgically resected from April 1, 2018 to December 25, 2018 were collected from the Fourth Hospital of Hebei Medical University. qPCR was used to detect the expression of miR-26b-3p in ESCC tissues, corresponding adjacent tissues and ESCC cells. ESCC cell lines TE1 and KYSE150 with low miR-26b-3p expression were selected and transfected with miR-26b-3p mimics. At the same time, the negative control was set up. The effects of miR-26b-3p on proliferation, migration and invasion of TE1 and KYSE150 cells were detected by cell proliferation assay, Wound healing assay and Transwell assay. Luciferase reporter gene analysis was used to detect the binding between miR-26b-3p and STAT3 3'UTR. Then, miR-26b-3p mimics and pcDNA3.0-STAT3 were co-transfected into the cells to verify whether STAT3 could reverse the effect of miR-26b-3p on cell proliferation, migration and invasion using cell proliferation assay, Wound healing assay and Transwell test. The effects of methylase inhibitor 5-Aza-DC on the methylation of ESCC cells as well as the expression of miR-26b-3p and STAT3 were detected by qPCR and WB. Results: The expression of miR-26b-3p in ESCC tissues was lower than that in the adjacent normal tissues (P<0.05), and the expression was also significantly lower in ESCC cell lines (TE1, KYSE150 and LYSE170) as compared with normal human esophageal epithelial HEEC cells (all P<0.01). Compared with miR-26b-3p NC group, transfection of miR-26b-3p mimics significantly up[1]regulated the expression of miR-26b-3p in TE1 and KYSE150 cells and obviously inhibited the proliferation, invasion and migration of the cells (P<0.05 or P<0.01). The luciferase reporter gene assay showed that miR-26b-3p significantly inhibited the luciferase activity of wild-type STAT3 vector in TE1 and KYSE150 cells (P<0.05 or P<0.01), while the luciferase activity of mutant-type was not affected. Co-transfection with miR-26b-3p mimics and pcDNA3.0-STAT3 partially reversed the inhibitory effect of miR-26b-3p on proliferation, migration and invasion of TE1 and KYSE150 cells (P<0.05 or P<0.01). After 5-aza-DC treatment, the expression of miR[1]26b-3p was up-regulated (P<0.01), mRNA and protein levels of STAT3 were decreased (P<0.05), and miR-26b-3p was demethylated in TE1 and KYSE150 cells. Conclusion: miR-26b-3p is down-regulated in ESCC tissues and cells due to the hypermethylation of its promoter. As a tumor suppressor, miR-26b-3p inhibits the proliferation, invasion and migration of ESCC cells via targeting STAT3.

国家自然科学基金资助项目（No. 81972722）；河北省卫健委青年科技课题基金资助项目（No. 20190720）