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目的研究胶质瘤细胞系U251中上皮剪接调控蛋白1(ESRP1)对人髓细胞白血病基因1(MCL1)转录本剪接的调控作用。方法对胶质瘤患者数据库中MCL1的测序结果进行数据挖掘,寻找关键位点。构建pc DNA-ESRP1外源表达载体并在U251细胞中进行过表达,通过反转录PCR和Western blot法检测MCL1不同异构体的表达情况。结果 ESRP1在U251细胞中的蛋白表达水平较正常胶质细胞低,MCL1异构体1在U251细胞中的表达高于正常胶质细胞,而异构体2和异构体3的表达则低于正常胶质细胞;在U251细胞中过表达ESRP1以后,发现异构体1表达较未转染组下降,而异构体3表达则显著升高,异构体2表达无明显变化。此外,第801G、802A缺失的MCL1无法被ESRP1正确剪切,异构体1和异构体3的表达与ESRP1转染前后无显著差异。结论抑制ESRP1表达或RNA识别位点突变可以引起肿瘤中癌基因转录本剪接异常。
Objective To study the regulation of the transcription of human myeloid leukemia gene 1 (MCL1) on the glioma cell line U251 epithelial splicing regulatory protein 1 (ESRP1). Methods Data mining of the sequencing results of MCL1 in the database of glioma patients, looking for key sites. The pcDNA-ESRP1 expression vector was constructed and overexpressed in U251 cells. The expression of different MCL1 isoforms was detected by reverse transcription PCR and Western blot. Results The protein expression level of ESRP1 in U251 cells was lower than that in normal glial cells. The expression of MCL1 isoform 1 in U251 cells was higher than that in normal glial cells, while the expressions of isoform 2 and isoform 3 were lower than Normal glial cells. After overexpression of ESRP1 in U251 cells, the expression of isoform 1 was lower than that of the untransfected cells, while the expression of isoform 3 was significantly increased. There was no significant change in isoform 2 expression. In addition, the 801G, 802A deletion of MCL1 can not be correctly cut ESRP1, isoform 1 and isoform 3 expression and ESRP1 transfected no significant difference. Conclusions Inhibition of ESRP1 expression or RNA recognition site mutations can cause abnormal oncogene transcript splicing in tumors.