miR-19b-3p靶向IGF-1参与绒毛膜癌发生发展的机制研究

Study on mechanism of miR-19b-3p targeting IGF-1 in the development of choriocarcinoma

目的探究miR-19b-3p靶向胰岛素样生长因子-1(IGF-1)参与绒毛膜癌增殖、侵袭和转移的机制。方法以人绒毛膜癌细胞系JEG-3为研究对象,A组:miR-19b-3p mimics组,B组:miR-19b-3p mimics NC组,C组:miR-19b-3p inhibitor组,D组:miR-19b-3p inhibitor NC组,E组:空白对照组。A组、B组、C组、D组按照分组情况进行相应转染,E组不做任何处理。分别用Real-time PCR和Western blot检测五组miR-19b-3p RNA和IGF-1蛋白表达水平,采用CCK-8法测定细胞增殖,采用划痕实验和Transwell小室侵袭实验测定细胞迁移和侵袭能力,并进行比较;采用双荧光素酶(Dualluciferase)报告实验验证miR-19b-3p对IGF-1的靶向关系。结果与E组的(0.98±0.13)相比,A组miR-19b-3p mRNA表达水平(2.23±0.16)明显上调,C组miR-19b-3p mRNA表达水平(0.73±0.08)明显下调(P<0.05)。与E组的(1.03±0.03)nmol/L相比,A组IGF-1蛋白表达水平(1.48±0.12)nmol/L明显上调,C组IGF-1蛋白表达水平(0.76±0.05)nmol/L明显下调(P<0.05)。72 h时,与E组的(5.03±0.14)%相比,A组细胞增殖活力(10.34±0.87)%明显增加,C组细胞增殖活力(2.42±0.13)%明显下调(P<0.05);与E组的(1.09±0.07)mm相比,A组细胞划痕迁移距离(1.45±0.08)mm明显增加,C组细胞划痕迁移距离(0.73±0.07)mm明显减少(P<0.05)。与E组的(103.00±7.00)个相比,A组侵袭细胞数(173.00±4.00)个明显增加,C组侵袭细胞数(82.00±1.00)个明显减少(P<0.05)。双荧光素酶报告实验结果显示:miR-19b-3p转染WT-IGF-1的细胞荧光素酶活性(0.57±0.08)Kat低于miR-NC的(0.95±0.06)Kat(P<0.05);miR-19b-3p转染MUT-IGF-1的细胞荧光素酶活性(0.98±0.05)Kat与miR-NC的(0.96±0.06)Kat比较无明显差异(P>0.05)。结论miR-19b-3p靶向上调IGF-1促进绒毛膜癌JEG-3细胞增殖、侵袭、迁移。

Objective To explore the mechanism of miR-19b-3p targeting insulin like growth factor-1(IGF-1)in the development of choriocarcinoma.Methods Human chorionic cancer cell line JEG-3 was selected as the research object and divided into five groups:group A:miR-19b-3p mimics group;group B:miR-19b-3p mimics NC group;group C:miR-19b-3p inhibitor group;group D:Mir-19b-3p inhibitor group;group E:blank control group.Groups A,B,C,and D were transfected accordingly to the grouping,and group E did not receive any treatment.The expression levels of miR-19b-3p RNA and IGF-1 protein in five groups were detected by Real-time PCR and Western blot,respectively,and cell proliferation was measured by CCK-8 method.Scratch assay and Transwell cell invasion assay were used to determine cell migration and invasion ability in each group,and then compared.Dual-luciferase reporter assay was used to verify the targeting relationship between miR-19b-3p and IGF-1.Results Compared with(0.98±0.13)in group E,miR-19b-3p mRNA expression level of(2.23±0.16)was significantly up-regulated in group A,and miR-19b-3p mRNA expression level of(0.73±0.08)was significantly down-regulated in group C(P<0.05).Compared with(1.03±0.03)nmol/L in group E,IGF-1 protein expression level of(1.48±0.12)nmol/L in group A was significantly up-regulated,and that of(0.76±0.05)nmol/L in group C was significantly down-regulated(P<0.05).At 72 h,compared with(5.03±0.14)%in group E,the cell proliferation viability of(10.34±0.87)%in group A was significantly increased,and the cell proliferation viability of(2.42±0.13)%in group C was significantly down-regulated(P<0.05).Compared with(1.09±0.07)mm in group E,the cell scratch migration distance was significantly increased in group A[(1.45±0.08)mm]and significantly decreased in group C[(0.73±0.07)mm](P<0.05).The number of invaded cells was significantly increased in group A[(173.00±4.00)]and significantly decreased in group C[(82.00±1.00)]compared to(103.00±7.00)in group E(P<0.05).The results of dual luciferase assay showed that the cellular luciferase activity of miR-19b-3p transfected with WT-IGF-1was(0.57±0.08)Kat,which was lower than(0.95±0.06)Kat of miR-NC(P<0.05).The cellular luciferase activity of miR-19b-3p transfected MUT-IGF-1 was(0.98±0.05)Kat,which was not significantly different from(0.96±0.06)Kat of miR-NC(P>0.05).Conclusion Upregulation of IGF-1 by miR-19b-3p targeting is involved in promoting proliferation,invasion and migration of JEG-3 cells in choriocarcinoma.