微管相关新发突变基因MTUS1在心肌致密化过程中的作用机制

Mechanism of de novo mutation in the MTUS1 gene involved in compaction of ventricular myocardium

摘要:

目的：探讨新发突变基因微管相关肿瘤抑制因子1（microtubule-associated tumor suppressor 1，MTUS1）在心肌致密化过程中的作用机制。方法：构建过表达慢病毒突变组MTUS1、野生组MTUS1及空载组（分别记为突变组、野生组及空载组）CP15-5a细胞模型，Real-time PCR检测各组MTUS1及小GTPase RhoA（ras homolog family member A，RhoA）的mRNA表达水平，Western blot检测各组RhoA蛋白表达水平，细胞免疫荧光检测各组α-tubulin荧光表达强度，并对各组细胞进行细胞划痕实验，检测各组细胞迁移情况。结果：成功构建过表达慢病毒突变组、野生组及空载组细胞模型。细胞免疫荧光结果显示，与野生组相比，突变组细胞α-tubulin荧光强度表达下降（P=0.006）。Real-time PCR及Western blot结果显示，与野生组相比，突变组RhoA的mRNA及蛋白表达水平明显增高（P=0.005，P=0.01）。划痕实验结果显示，与野生组相比，突变组细胞在划痕后6 h、12 h的迁移速率增快（均P=0.000）。结论：MTUS1新发突变是保护性突变，通过降低CP15-5a细胞微管稳定性，并调节RhoA表达增强细胞迁移，从而减低心肌致密化不全的发生率。

Abstract:

Objective：To investigate the mechanism of de novo mutation in the microtubule-associated tumor suppressor 1（MTUS1） gene in the compaction of ventricular myocardium. Methods：Lentiviral vectors containing mutant MTUS1 gene or wild MTUS1 gene or empty vectors were co-infected into CP15-5a cells（mutation group，wild group，and vector group，respectively）. The mRNA expression of MTUS1 and small GTPase-ras homolog family member A（RhoA） was measured by real-time PCR. The protein expression of RhoA was measured by Western blot. The fluorescence intensity of α-tubulin was determined by immunofluorescence assay. Cell mi－gration activity was evaluated by wound-healing assay. Results：Lentiviral vectors containing mutant MTUS1 or wild MTUS1 or empty vectors were successfully co-infected into CP15-5a cells，which was confirmed by fluorescence staining and real-time PCR. Immuno-fluorescence assay results showed that the mutation group had a lower fluorescence intensity of α-tubulin than the wild group（P=0.006，P<0.01）. Real-time PCR and Western blot results showed that the mutation group had significantly higher mRNA and protein expression of RhoA compared with the wild group（P=0.005，P=0.01）. Wound-healing assay showed that the mutation group had sig－nificantly higher migration rates at 6 and 12 hours after scratch compared with the wild group（P=0.000，P=0.000）. Conclusion：A de novo mutation in the MTUS1 gene is a protective mutation for decreasing the incidence of noncompaction of ventricular myocardium via reducing the stability of microtubules in CP15-5a cells and increasing cell migration activity by regulating the expression of RhoA.