环状RNA GRIN2B通过调控微小RNA-135b对食管癌细胞增殖和侵袭的影响实验研究

Effects of circular RNA GRIN2B on proliferation and invasion of esophageal cancer cells through regulation of microRNA-135b

目的:探讨环状RNA GRIN2B(circ-GRIN2B)对食管癌细胞增殖和侵袭的影响及其分子机制。方法:采用RT-qPCR检测circ-GRIN2B在食管癌细胞株Eca109、TE-13、KYSE30、OE33、EC9706和正常食管上皮细胞HET-1A中的表达,筛选circ-GRIN2B表达最低的细胞株进行后续实验。在食管癌细胞中转染circ-GRIN2B过表达质粒(circ-GRIN2B组)和空载对照质粒(NC组)。采用平板克隆实验和Transwell实验检测circ-GRIN2B对食管癌细胞增殖和侵袭的影响。采用circRNADb软件预测circ-GRIN2B与微小RNA-135b(miR-135b)的结合位点并用双荧光素酶报告基因验证两者的靶向关系。采用RT-qPCR检测两组miR-135b表达。采用Western blot检测细胞增殖相关蛋白[周期蛋白依赖性激酶8(CDK8)、周期素C(Cyclin C)]和侵袭相关蛋白[纤维结合蛋白(FN)、转录因子8(ZLF8)、叉头盒蛋白C2(FOXC2)]表达。结果:与HET-1A细胞比较,circ-GRIN2B在细胞株Eca109、TE-13、KYSE30、OE33、EC9706表达降低,且在EC9706细胞中表达量最低(均P<0.05)。circ-GRIN2B组circ-GR IN2B表达量高于NC组(P<0.01)。circ-GRIN2B组EC9706细胞集落数量少于NC组(P<0.01)。circ-GRIN2B组EC9706细胞侵袭数目小于NC组(P<0.01)。miR-135b是circ-GRIN2B的靶基因。circ-GRIN2B组miR-135b表达量低于NC组(P<0.01)。circ-GRIN2B组EC9706细胞增殖相关蛋白和侵袭相关蛋白表达水平较NC组降低(均P<0.05)。结论:circ-GRIN2B在食管癌细胞中低表达,其可能通过下调miR-135b表达抑制食管癌细胞的增殖和侵袭。

Objective:To investigate the effects of circular RNA GRIN2B(circ-GRIN2B)on the proliferation and invasion of esophageal carcinoma cells and its molecular mechanism.Methods:The expression of circ-GRIN2B in esophageal cancer cell lines Eca109,TE-13,KYSE30,OE33,EC9706 and normal esophageal epithelial cells HET-1A was detected by RT-qPCR,and the lowest expression cell of circ-GRIN2B was screened for follow-up experiments.Circ-GRIN2B overexpression plasmid(circ-GRIN2B group)and no-load control plasmid(NC group)were transfected into esophageal cancer cells.The effects of circ-GRIN2B on the proliferation and invasion of esophageal carcinoma cells were examined by plate cloning assay and Transwell assay.CircRNADb software was used to predict the binding sites of circ-GRIN2B and miR-135b,and dual luciferase reporter gene was used to verify the targeting relationship between the two.The expression of miR-135b was detected by RT-qPCR.The expressions of proliferation-related proteins(Cyclin dependent kinase 8[CDK8],Cyclin C)and invasion-related proteins(Fibronection[FN],transcription factor 8[ZLF8],forkhead box protein C2[FOXC2])were detected by Western blot.Results:Compared with HET-1A cells,the expressions of circ-GRIN2B were decreased in Eca109,TE-13,KYSE30,OE33 and EC9706 cells,and the expression level was lowest in EC9706 cells(all P<0.05).The expression of circ-GRIN2B in circ-GRIN2B group was higher than that in NC group(P<0.01).The number of EC9706 cell colonies in circ-GRIN2B group was lower than that in NC group(P<0.01).The number of EC9706 cells invaded in circ-GRIN2B group was less than that in NC group(P<0.01).MiR-135b was a target gene for circ-GRIN2B.The expression level of miR-135b in circ-GRIN2B group was lower than that in NC group(P<0.01).The expression levels of proliferation-related proteins and invasion-related proteins in EC9706 cells in circ-GRIN2B group were lower than those in NC group(all P<0.05).Conclusion:Circ-GRIN2B is underexpressed in esophageal cancer cells,and it may inhibit the proliferation and invasion of esophageal cancer cells by down-regulating the expression of miR-135b.