miR-5787通过靶向HSPG2基因对乳腺癌细胞侵袭和增殖的影响

Effect of miR-5787 on breast cancer cell invasion and proliferation by targeting HSPG2 gene

目的观察微小RNA(miR)-5787在乳腺癌组织及多种乳腺癌细胞系中的表达,分析过表达miR-5787对乳腺癌细胞侵袭和增殖的影响并探讨其可能的作用机制。方法实时荧光定量PCR(qRT-PCR)法检测miR-5787在47例乳腺癌组织及癌旁组织、4种乳腺癌细胞系及正常乳腺上皮细胞中的表达。选择miR-5787表达最低的乳腺癌细胞系,分别转染miR-5787模拟物(实验组)和对照NC模拟物(对照组)。qRT-PCR检测两组细胞中miR-5787的表达。Transwell侵袭实验和细胞计数试剂盒(CCK-8)分别检测过表达miR-5787对乳腺癌细胞侵袭和增殖的影响。生物信息学软件和双荧光素酶报告基因实验分别预测和验证miR-5787可配对结合的靶基因。qRT-PCR和Western blot检测靶基因mRNA及蛋白的表达。结果与癌旁组织(5.05±0.82)相比,乳腺癌组织miR-5787的表达(1.32±0.33)明显降低(P<0.01)。与正常乳腺上皮细胞相比,4种乳腺癌细胞系miR-5787的表达均降低(P<0.05),HCC1937细胞中的表达最低(P<0.01)。转染miR-5787模拟物后,实验组HCC1937细胞中miR-5787的表达明显高于对照组(P<0.01),表明转染成功。过表达miR-5787可抑制乳腺癌HCC1937细胞的侵袭(P<0.05)和增殖(P<0.05)。生物信息学软件预测miR-5787的靶基因可能是硫酸乙酰肝素糖蛋白2(HSPG2),miR-5787可配对结合HSPG2 mRNA(P<0.01)。qRT-PCR和Western blot结果显示,过表达miR-5787可明显抑制HSPG2蛋白和mRNA的表达(P<0.01),波形蛋白(Vimentin)、神经性钙黏附蛋白(N-cadherin)、Ki67、增殖细胞核抗原(PCNA)表达明显降低(P<0.05)。结论乳腺癌组织和细胞系中miR-5787均呈低表达,过表达miR-5787通过靶向干扰HSPG2基因的表达,抑制乳腺癌HCC1937细胞的侵袭和增殖。

Objective To observe the expression of microRNA(miRNA,miR)-5787 in breast cancer tissues and various cell lines,analyze the effect of overexpression of miR-5787 on breast cancer cell invasion and proliferation,and explore its possible molecular mechanism.Methods Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)method was used to detect the expression of miR-5787 in 47 breast cancer tissues and adjacent tissues,4 breast cancer cell lines and normal breast epithelial cells.Breast cancer cell lines with the lowest miR-5787 expression were selected and transfected with miR-5787 mimics(experimental group)and control NC mimics(control group),respectively.qRT-PCR was used to detect the expression of miR-5787 in cells of the two groups.Transwell invasion experiment and cell counting kit-8(CCK-8)were used to detect the effect of miR-5787 overexpression on breast cancer cell invasion and proliferation.Bioinformatics software and dual luciferase reporter gene experiments were used to predict and verify the target genes that miR-5787 could complementally bind.qRT-PCR and Western blot were used to detect the expression of target gene mRNA and protein.Results Compared with adjacent tissues(5.05±0.82),the expression of miR-5787 in breast cancer tissues(1.32±0.33)was significantly reduced(P<0.01).Compared with normal breast epithelial cells,the expression of miR-5787 in the four breast cancer cell lines was reduced(P<0.05),and the expression in HCC1937 cells was the lowest(P<0.01).After transfection of miR-5787 mimics,the expression of miR-5787 in HCC1937 cells in the experimental group was significantly higher than that in the control group(P<0.01).Overexpression of miR-5787 could inhibit the invasion(P<0.05)and proliferation(P<0.05)of breast cancer HCC1937 cells.Bioinformatics software showed that the target gene of miR-5787 might be heparan sulfate proteoglycan 2(HSPG2),and miR-5787 could complement HSPG2 mRNA(P<0.01).qRT-PCR and Western blot indicated that overexpression of miR-5787 could significantly inhibit the expression of HSPG2 gene(P<0.01),with the decreased expression of Vimentin,N-cadherin,Ki67 and PCNA.Conclusions miR-5787 expression was low in breast cancer tissues and cell lines.Overexpression of miR-5787 could inhibit the invasion and proliferation of breast cancer HCC1937 cells by interfering with the expression of HSPG2 gene.