miR-29b通过靶向STAT3调控免疫因子IL-6/10参与多发性骨髓瘤细胞增殖和侵袭的机制研究

miR-29b participates in the proliferation and invasion of multiple myeloma cells by regulating the immune factors IL-6/10 through targeting STAT3

目的分析微小RNA(microRNA,miR)-29b通过靶向转录激活因子3(signal transducer and activator of transcription3,STAT3)调控免疫因子白介素(interleukin,IL)-6和IL-10参与多发性骨髓瘤(multiple myeloma,MM)细胞增殖和侵袭的机制。方法双荧光素酶报告实验验证miR-29b和STAT3的靶向关系。将人MM细胞系U266细胞分为对照(normal control,NC)组、miR-29b模拟物(mimic)组、STAT3过表达(over expression STAT3,OE-STAT3)组和miR-29b mimic+OE-STAT3组,根据组别转染miR-29b mimic或者STAT3过表达质粒。通过CCK-8法、细胞划痕和Transwell实验检测细胞增殖活力、迁移和侵袭能力。通过ELISA检测培养基中IL-6和IL-10水平。结果通过双荧光素酶报告在U266细胞中验证miR-29b靶向STAT3。与NC组比较,miR-29b mimic组细胞的STAT3 mRNA和蛋白的表达水平、OD值、划痕前缘迁移距离百分比(26.79%±1.56%)、侵袭细胞数目(34.59±2.93)、培养基中IL-6(9.37 pg/ml±1.12 pg/ml)和IL-10(3.86 pg/ml±0.48 pg/ml)水平显著降低,而OE-STAT3组的上述指标均升高(P<0.05)。miR-29b mimic+OE-STAT3组的STAT3 mRNA和蛋白的表达水平、划痕前缘迁移距离百分比(49.06%±3.27%)、侵袭细胞数目(90.31±4.07)、培养基中IL-6(16.23 pg/ml±1.75 pg/ml)和IL-10(7.55 pg/ml±0.82 pg/ml)水平显著低于OESTAT3组且显著高于miR-29b mimic组(P<0.05)。结论miR-29b可能通过靶向STAT3的表达来抑制MM细胞的增殖、迁移、侵袭,并抑制IL-6和IL-10的表达解除免疫抑制。

This study was designed to explore the roles of microRNA(miR)-29b in the proliferation and invasion of multiple myeloma(multiple myeloma,MM)cells by regulating the immune factors interleukin(IL)-6 and IL-10 through targeting signal transducer and activator of transcription 3(STAT3).The dual luciferase report experiment was applied to verify the targeting relationship between miR-29b and STAT3.MM cell line U266 were divided into normal control(NC)group,miR-29b mimic group,over expression STAT3(OE-STAT3)group and miR-29b mimic+OE-STAT3 group.CCK-8,cell scratch and Transwell assay were used to detect cell proliferation,migration and invasion ability.The levels of IL-6 and IL-10 in the culture medium were detected by ELISA.Dualluciferase report assay indicated that miR-29b wastargeted STAT3 in U266 cells.Compared with the NC group,the miR-29b mimic group demonstrated lower levels of STAT3 mRNA and protein,the OD value,themigration distance percentage of the leading edge of the scratch(26.79%±1.56%),the number of invaded cells(34.59±2.93),IL-6(9.37 pg/ml±1.12 pg/ml)and IL-10(3.86 pg/ml±0.48 pg/ml)(P<0.05),while the OE-STAT3 group showed higher levels of the above indicators(P<0.05).Furthermore,the levels of these indicators mentionedabove in the miR-29b mimic+OE-STAT3 group were significantly lower than those in the OE-STAT3 group andsignificantly higher than those in the miR-29b mimic group(P<0.05).Taken together,miR-29b can inhibit theproliferation,migration and invasion of MM cells by targeting the expression of STAT3,and inhibit the expression ofIL-6 and IL-10 to relieve immunosuppression.