溶质载体家族成员SLC25A39基因促进乳腺癌细胞线粒体分裂

Solute carrier family member SLC25A39 gene promotes fission of mitochondria in breast cancer cells

目的 构建带Flag标签的人溶质载体家族成员蛋白SLC25A39基因的真核表达载体,在获得FlagSLC25A39融合蛋白表达后,研究SLC25A39表达对人乳腺癌细胞线粒体稳态的影响。方法 以人乳腺文库为模板采用PCR技术扩增出人SLC25A39的编码区序列,双酶切后将其插入到带Flag标签的pcDNA3.0载体上,转化大肠杆菌DH5α感受态细胞,菌液PCR鉴定阳性的克隆再进行质粒提取,测序正确后将重组质粒与空载体分别转染人乳腺癌细胞ZR75-1。Western印迹鉴定SLC25A39目的基因的表达;Mitotracker对乳腺癌细胞的线粒体进行染色,共聚焦显微镜对线粒体进行观察;Western印迹检测线粒体融合蛋白1(Mfn1)、线粒体分裂蛋白1(Fis1)的表达变化。结果双酶切鉴定表明,Flag-SLC25A39真核表达载体构建成功;Western印迹验证SLC25A39基因表达成功;激光共聚焦显微镜下观察发现SLC25A39过表达的乳腺癌ZR75-1细胞分裂的线粒体较多;Western印迹证实SLC25A39基因过表达后线粒体分裂蛋白Fis1表达增高,线粒体融合蛋白Mfn1表达减弱。结论 成功构建带Flag标签的SLC25A39基因真核表达载体,该基因可促进人乳腺癌细胞的线粒体分裂,有望成为靶向线粒体治疗乳腺癌的潜在新靶点。

Objective To construct the eukaryotic expression vector of Flag tagged human solute carrier family member SLC25A39 gene,and to study the effect of SLC25A39 expression on the homeostasis of human mitochondria in breast cancer cells after Flag-SLC25A39 fusion protein is obtained. Methods The coding sequence of SLC25A39 was amplified by PCR using the human mammary gland library as the template. After double enzyme digestion,SLC25A39gene was inserted into the pcDNA3.0 vector with Flag tag,and the plasmid was transformed into E.coli DH5α. PCR positive clones were selected,then the recombinant plasmid and empty vector were transfected into human breast cancer cell line ZR75-1 respectively. The expression of SLC25A39 gene was identified by Western blotting. The mitochondria of breast cancer cells were stained by Mitotracker and observed under a confocal microscope. The expressions of Mitofusins(Mfn1)and mitochondrial fission protein 1(Fis1)were detected by Western blotting. Results The eukaryotic expression vector pcDNA3.0-Flag-SLC25A39 was constructed. Western blotting showed that SLC25A39 gene was expressed. More fissioned mitochondria were observed in ZR75-1 cells after overexpressing SLC25A39 by confocal laser scanning microscopy.Western blotting showed that the expression of Fis1 was increased,while the expression of Mfn1 was decreased. Conclusion The eukaryotic expression vector of pcDNA3.0-Flag-SLC25A39 has been constructed,which can promote the mitochondrial fission of human breast cancer cells. SLC25A39 gene can be a potential new target for mitochondrial therapy of breast cancer.