c-Myc靶向LncRNA SNHG3对乳腺癌细胞生物学作用的影响

Effects of biological role of LncRNA SNHG3 targeted by c-Myc on breast cancer cells

目的:探讨c-Myc靶向LncRNA SNHG3对乳腺癌细胞的生物学作用。方法:通过实时定量PCR(RT-qPCR)法分析c-Myc和LncRNA SNHG3在乳腺癌细胞中的表达情况;生物信息学分析c-Myc与LncRNA SNHG3的调控关系;采用染色质免疫沉淀实验(ChIP)和双荧光素酶报告基因实验验证它们的相互作用,并通过对c-Myc进行RNAi和过表达后,RT-qPCR和Western blot法检测SNHG3的表达变化;CCK8、划痕愈合和Transwell^(TM)实验分别检测细胞的增殖能力、横向迁移能力和纵向迁移及侵袭能力;采用流式细胞术检测细胞的周期分布;RT-qPCR和Western blot法检测上皮型钙黏蛋白(E-cadherin)、基质金属蛋白酶2(MMP2)、波形蛋白(Vimentin)、锌指转录因子(Snail)、细胞周期蛋白D1(cyclinD1)的mRNA和蛋白表达水平。结果:c-Myc和LncRNA SNHG3在乳腺癌细胞中异常高表达(P<0.001);生物信息学发现LncRNA SNHG3是c-Myc转录调控的主要靶标,且c-Myc在LncRNA SNHG3启动子区存在多个结合位点;LncRNA SNHG3与Myc信号通路成正相关(P<0.001)。实验结果证实c-Myc结合LncRNA SNHG3启动子,促进LncRNA SNHG3转录和表达(P<0.001);此外,功能挽救实验结果显示c-Myc靶向LncRNA SNHG3增加MDA-MB-231细胞的增殖数目,迁移与侵袭的穿膜数量(P<0.001)。RT-qPCR和Western blot结果显示过表达LncRNA SNHG3部分可抵消敲低c-Myc表达对细胞的EMT进程;并下调E-cadherin的表达(P<0.01),上调MMP2(P<0.01)、Vimentin(P<0.05)和Snail(P<0.05)的表达。干扰LncRNA SNHG3表达能将细胞周期阻滞在G_(0)/G_(1)期(P<0.001),cyclinD1表达减少(P<0.001)。结论:由c-Myc激活转录的LncRNA SNHG3一方面通过激活上皮间充质转化(EMT)促进乳腺癌细胞迁移、侵袭;另一方面通过加速G_(1)/S细胞周期进程促进乳腺癌细胞增殖。

Objective:To investigate the biological role of c-Myc-targeted LncRNA SNHG3 on breast cancer cells.Methods:The expression of c-Myc and LncRNA SNHG3 in breast cancer cell lines was analyzed by real-time quantitative PCR(RT-qPCR)method.Bioinformatics was used to analyze the regulatory relationship between c-Myc and LncRNA SNHG3.Chromatin immunoprecipitation assay(ChIP)and dual-luciferase reporter gene assay were used to further confirm their direct effects,and expression changes of SNHG3 were detected by RT-qPCR and Western blot after RNAi and overexpression of c-Myc.CCK8,wound healing experiment and Transwell^(TM) experiment were used to observe the proliferation,lateral migration,longitudinal migration and invasion of cells,respectively.The cell cycle distribution was measured by flow cytometry.The mRNA and protein expression levels of E-cadherin,MMP2,Vimentin,Snail and cyclinD1 were detected by RT-qPCR and Western blot.Results:The expression levels of c-Myc and LncRNA SNHG3 were significantly high in breast cancer cell lines(P<0.001).Bioinformatics found that LncRNA SNHG3 was the main target for c-Myc transcriptional regulation,and that c-Myc had multiple binding sites in the LncRNA SNHG3 promoter region.Moreover,LncRNA SNHG3 was positively associated with the Myc signaling pathway(P<0.001).It was proved that c-Myc bound to the promoter of LncRNA SNHG3 and promoted the transcription and expression of LncRNA SNHG3 by ChIP,dual-luciferase reporter gene assay RT-qPCR and Western blot(P<0.001).The results of functional rescue experiments clarified that c-Myc-LncRNA SNHG3 promoted the proliferation ability of MDA-MB-231 cells and increased the number of cells that migrated and invaded(P<0.001).RT-qPCR and Western blot results showed that overexpression of LncRNA SNHG3 partially counteracted the EMT progression of knocking down c-Myc expression on cells,inhibiting the expression of E-cadherin (P<0.01),upregulating the expression of MMP2(P<0.01),Vimentin(P<0.05)and Snail(P<0.05).In addition,LncRNA SNHG3 silencing blocked the cell cycle in the G_(0)/G_(1) phase(P<0.001),and the expression of cyclinD1 was reduced(P<0.001).Conclusion:LncRNA SNHG3,which is transcribed by c-Myc,promoted migration and invasion of breast cancer cells by activating epithelial-mesenchymal transition(EMT)pathway,while promoting the proliferation of breast cancer cells throught accelerating the G_(1)/S cell cycle process.