lncRNA H19对乳腺癌细胞增殖、侵袭、迁移能力的影响及其分子机制

Effects of lncRNA H19 on proliferation,invasion,and migration of breast cancer cells and the mechanism

目的观察长链非编码RNA H19(lncRNA H19)对乳腺癌细胞增殖、侵袭、迁移能力的影响,并探讨其可能的机制。方法采用实时荧光定量PCR法检测乳腺癌细胞系(SKBR3、MDA-MB-453、MCF-7)和正常乳腺上皮细胞系(MCF-10A)中lncRNA H19表达。将SKBR3细胞分为三组,pcDNA-H19组和si-H19组分别转染pcDNA-H19、H19 siRNA,空白对照组不进行转染,转染48 h后收集细胞。采用CCK-8法检测三组培养0、24、48、72、96 h的增殖能力(光密度值),Transwell小室侵袭实验检测细胞侵袭能力(穿膜细胞数),细胞划痕实验检测细胞迁移能力(划痕愈合率)。双荧光素酶实验检测miR-194-5p与lncRNA H19的靶向结合作用,实时荧光定量PCR法检测三组miR-194-5p表达。结果乳腺癌细胞系SKBR3、MDA-MB-453、MCF-7中lncRNA H19相对表达量分别为8.37±1.21、5.62±0.85、3.43±0.96,均高于正常乳腺上皮细胞系MCF-10A中的1.08±0.24(P均<0.01)。培养24、48、72、96 h,si-H19组、空白对照组、pcDNA-H19组相同时间点光密度值均依次升高(P均<0.01);三组穿膜细胞数及划痕愈合率均依次升高,miR-194-5p相对表达量均依次降低(P均<0.01)。共转染miR-194-5p mimic+H19-wt或NC mimic+H19-wt的SKBR3细胞荧光素酶活性分别为0.33±0.02、0.97±0.06(P<0.01),共转染miR-194-5p mimic+H19-mut或NC mimic+H19-mut的SKBR3细胞荧光素酶活性分别为1.02±0.04、1.06±0.05(P>0.05)。结论乳腺癌细胞lncRNA H19表达升高,lncRNA H19可能通过负靶向调控miR-194-5p而促进乳腺癌细胞的增殖、侵袭和迁移。

Objective To observe the effects of long non-coding RNA H19(lncRNA H19)on the proliferation,invasion,and migration of breast cancer cells,and to explore the possible mechanism.Methods Quantitative real-time PCR(qRT-PCR)was used to detect the expression of lncRNA H19 in the breast cancer cell lines(SKBR3,MDA-MB-453 and MCF-7)and normal breast epithelial cell line(MCF-10A).SKBR3 cells were divided into three groups.The cells in the pcDNA-H19 group and H19 siRNA group were transfected with pcDNA-H19 and H19 siRNA,respectively.The blank control group was not transfected.Then the cells were collected at 48 h after transfection.Cell counting kit-8(CCK-8)was used to detect the proliferation ability(OD value)of the three groups at 0,24,48,72,96 h.Transwell invasion assay was used to detect the cell invasion(number of cells in the lower layer of the chamber).The wound healing assay was used to detect the cell migration(wound healing rate).Dual-luciferase reporter assay was used to check the targeted binding effect between lncRNA H19 and miR-194-5p.The expression of miR-194-5p in the three groups was detected by qRT-PCR.Results The relative expression of lncRNA H19 in SKBR3,MDA-MB-453,and MCF-7 cells were 8.37±1.21,5.62±0.85 and 3.43±0.96,respectively,which were higher than that(1.08±0.24)in MCF-10A cells(all P<0.01).After culturing for 24,48,72,96 h,the OD values of the si-H19 group,blank control group,and pcDNA-H19 group increased in turn at the same time point(P<0.01).In the three groups,the number of cells in the lower layer of the chamber and wound healing rate increased in turn,while the relative expression of miR-194-5p decreased in turn(all P<0.01).The luciferase activities of SKBR3 cells co-transfected with miR-194-5p mimic+H19-wt or NC mimic+H19-wt were 0.33±0.02 and 0.97±0.06,respectively(P<0.01).The luciferase activities of SKBR3 cells co-transfected with miR-194-5p mimic+H19-mut or NC mimic+H19-mut were 1.02±0.04 and 1.06±0.05,respectively(both P>0.05).Conclusion LncRNA H19 is highly expressed in the breast cancer cells,and it may promote the proliferation,invasion,and migration of breast cancer cells by negatively targeting miR-194-5p.