MYSM1全长及截短质粒构建及其对乳腺癌细胞增殖的影响

Construction of MYSM1 full-length and truncated plasmids and its effect on proliferation in breast cancer cells

目的构建去泛素化酶Myb样SWIRM和MPN结构域1(MYSM1)全长及各结构域缺失的截短表达质粒并验证其在乳腺癌细胞MCF7中的表达与定位,探讨MYSM1对MCF7细胞增殖能力的影响并初步评估相关机制。方法以人基因组DNA为模板扩增MYSM1基因片段,构建其全长及截短质粒。将测序结果正确的上述MYSM1质粒分别转染于MCF7细胞中,进行免疫荧光共聚焦实验探究外源MYSM1蛋白在乳腺癌细胞中的定位。MTS和平板克隆实验检测对照或MYSM1过表达后,MCF7细胞的增殖能力。STRING数据库预测及免疫共沉淀实验验证MYSM1与E2F1蛋白结合;Western blot检测转染MYSM1后的E2F1蛋白表达及H2A泛素化水平。结果构建出了FLAG-MYSM1全长及截短质粒;MYSM1全长及截短重组质粒均能在乳腺癌细胞中表达并定位于细胞核;过表达MYSM1后,细胞生长加快,且与对照组相比差异有统计学意义(t=6.389,P<0.01);MYSM1能够结合并上调E2F1表达;过表达MYSM1后,H2A泛素化水平显著降低。结论MYSM1蛋白的168-371 aa及471-576 aa在其核定位中发挥重要作用。MYSM1结合并增加E2F1表达,发挥促进MCF7细胞增殖的作用。

Objective To construct full-length and truncated expression plasmids of deubiquitinase Myb like,SWIRM and MPN Domains 1(MYSM1),and verify the expression and localization of exogenous MYSM1 in breast cancer MCF7 cells.Methods Human genomic DNA was used as the template to amplify the MYSM1 gene fragment and construct its full-length and truncated plasmids.The MYSM1 plasmids with correct sequencing verification results were transfected into MCF7 cells respectively and immunofluorescence staining was performed to explore the localization of exogenous MYSM1 proteins in breast cancer cells.Proliferation ability was detected in control or MYSM1 over-expression MCF7 cells terms of MTS and plate colony assay.Besides,the binding of MYSM1 to E2F1 protein was predicted and verified by STRING database and immunoprecipitation assay.Western Blot was used to detect the expression of E2F1 protein and histone H2A ubiquitination after MYSM1 plasmid transfection.Results The full-length and truncated FLAG-MYSM1 plasmids were constructed and these recombinant plasmids could be expressed in cells with the protein bands in line with the expected protein molecular weight.The Confocal experiment showed the nucleus distribution of these FLAG-MYSM1 plasmids in breast cancer cells.Cell growth was significantly accelerated after MYSM1 overexpression(t=6.389,P<0.01).MYSM1 could bind and upregulate E2F1 expression.After MYSM1 plasmid transfection,H2A ubiquitination level was significantly reduced.Conclusions The 168-371 aa and 471-576 aa of MYSM1 protein might play an essential role in its nuclear localization.MYSM1 could bind to and increase the expression of E2F1,promoting the proliferation of MCF7 cells.