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
|
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m RNA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。
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 CP15-5 a cells(mutation group,wild group,and vector group,respectively). The m RNA 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 migration activity was evaluated by wound-healing assay. Results:Lentiviral vectors containing mutant MTUS1 or wild MTUS1 or empty vectors were successfully co-infected into CP15-5 a cells,which was confirmed by fluorescence staining and real-time PCR. Immunofluorescence 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 m RNA 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 significantly 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 CP15-5 a cells and increasing cell migration activity by regulating the expression of RhoA.
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 CP15-5 a cells(mutation group,wild group,and vector group,respectively). The m RNA 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 migration activity was evaluated by wound-healing assay. Results:Lentiviral vectors containing mutant MTUS1 or wild MTUS1 or empty vectors were successfully co-infected into CP15-5 a cells,which was confirmed by fluorescence staining and real-time PCR. Immunofluorescence 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 m RNA 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 significantly 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 CP15-5 a cells and increasing cell migration activity by regulating the expression of RhoA.
引文
[1] Kubik M,Dabrowska-Kugacka A,Lewicka E,et al. Predictors of poor outcome in patients with left ventricular noncompaction:Review of the literature[J]. Adv Clin Exp Med,2018,27(3):415-422.
[2] Finsterer J,Stollberger C,Towbin JA. Left ventricular noncompaction cardiomyopathy:cardiac,neuromuscular,and genetic factors[J]. Nat Rev Cardiol,2017,14(4):224-237.
[3] Towbin JA,Jefferies JL. Cardiomyopathies due to left ventricular noncompaction,mitochondrial and storage diseases,and inborn errors of metabolism[J]. Circ Res,2017,121(7):838-854.
[4] Takasaki A,Hirono K,Hata Y,et al. Sarcomere gene variants act as a genetic trigger underlying the development of left ventricular noncompaction[J]. Pediatr Res,2018,84(5):733-742.
[5] Ajima R,Bisson JA,Helt JC,et al. DAAM1 and DAAM2 are corequired for myocardial maturation and sarcomere assembly[J]. Dev Biol,2015,408(1):126-139.
[6] Deng X,Fink G,TAM B,et al. Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability[J]. Proc Natl Acad Sci U S A,2017,114(29):E5950-5958.
[7] Bozgeyik I,Yumrutas O,Bozgeyik E. MTUS1,a gene encoding angiotensin-Ⅱtype 2(AT2)receptor-interacting proteins,in health and disease,with special emphasis on its role in carcinogenesis[J]. Gene,2017,626:54-63.
[8] Xu Y,Liu X,Li H. Improvement of the diagnosis of left ventricular noncompaction cardiomyopathy after analyzing both advantages and disadvantages of echocardiography and CMRI[J]. Prog Cardiovasc Dis,2018,61(5-6):491-493.
[9] Zhao T,Ding X,Chang B,et al. MTUS1/ATIP3a down-regulation is associated with enhanced migration,invasion and poor prognosis in salivary adenoid cystic carcinoma[J]. BMC Cancer,2015,15:203.
[10] Dong X,Fan P,Tian T,et al. Recent advancements in the molecular genetics of left ventricular noncompaction cardiomyopathy[J]. Clin Chim Acta,2017,465:40-44.
[11] Choquet C,THM N,Sicard P,et al. Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[J]. PLoS Genet,2018,14(7):e1007502
[12] Piccolo P,Attanasio S,Secco I,et al. MIB2 variants altering NOTCH signalling result in left ventricle hypertrabeculation/non-compaction and are associated with Ménétrier-like gastropathy[J]. Hum Mol Genet,2017,26(1):33-43.
[13] Miller EM,Hinton RB,Czosek R,et al. Genetic testing in pediatric left ventricular noncompaction[J]. Circ Cardiovasc Genet,2017,10(6):e001735.
[14] Parbin S,Pradhan N,Das L,et al. DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma[J]. Exp Cell Res,2019,374(2):323-332.
[15] Liu Y,Chen H,Shou W. Potential common pathogenic pathways for the left ventricular noncompaction cardiomyopathy(LVNC)[J]. Pediatr Cardiol,2018,39(6):1099-1106.
[16] Rodrigues-Ferreira S,Nehlig A,Monchecourt C,et al. Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis[J]. Breast Cancer Res Treat,2019,173(3):573-583.
[17] Ito S,Asakura M,Liao Y,et al. Identification of the Mtus1 splice variant as a novel inhibitory factor against cardiac hypertrophy[J]. J Am Heart Assoc,2016,5(7):e003521.
[18] van Dijk J,Bompard G,Cau J,et al. Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation[J]. BMC Biol,2018,16(1):116.
[19] Roostalu J,Rickman J,Thomas C,et al. Determinants of polar versus nematic organization in networks of dynamic microtubules and mitotic motors[J]. Cell,2018,175(3):796-808.e14.
[20] López-Doménech G,Covill-Cooke C,Ivankovic D,et al. Miro proteins coordinate microtubule-and actin-dependent mitochondrial transport and distribution[J]. EMBO J,2018,37(3):321-336.
[21] Cappello S. Small Rho-GTPases and cortical malformations:finetuning the cytoskeleton stability[J]. Small GTPases,2013,4(1):51-56.
[22] Wang X,Tang P,Guo F,et al. RhoA regulates activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling[J]. Biochim Biophys Acta Gen Subj,2017,1861(1 Pt A):3011-3018.
[23] Wang T,Hamilla S,Cam M,et al. Extracellular matrix stiffness and cell contractility control RNA localization to promote cell migration[J]. Nat Commun,2017,8(1):896.
[24] Kitakaze M. Dysfunction of microtubules induces cardiac dysfunction[J]. EBioMedicine,2018,37:3-4.
[2] Finsterer J,Stollberger C,Towbin JA. Left ventricular noncompaction cardiomyopathy:cardiac,neuromuscular,and genetic factors[J]. Nat Rev Cardiol,2017,14(4):224-237.
[3] Towbin JA,Jefferies JL. Cardiomyopathies due to left ventricular noncompaction,mitochondrial and storage diseases,and inborn errors of metabolism[J]. Circ Res,2017,121(7):838-854.
[4] Takasaki A,Hirono K,Hata Y,et al. Sarcomere gene variants act as a genetic trigger underlying the development of left ventricular noncompaction[J]. Pediatr Res,2018,84(5):733-742.
[5] Ajima R,Bisson JA,Helt JC,et al. DAAM1 and DAAM2 are corequired for myocardial maturation and sarcomere assembly[J]. Dev Biol,2015,408(1):126-139.
[6] Deng X,Fink G,TAM B,et al. Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability[J]. Proc Natl Acad Sci U S A,2017,114(29):E5950-5958.
[7] Bozgeyik I,Yumrutas O,Bozgeyik E. MTUS1,a gene encoding angiotensin-Ⅱtype 2(AT2)receptor-interacting proteins,in health and disease,with special emphasis on its role in carcinogenesis[J]. Gene,2017,626:54-63.
[8] Xu Y,Liu X,Li H. Improvement of the diagnosis of left ventricular noncompaction cardiomyopathy after analyzing both advantages and disadvantages of echocardiography and CMRI[J]. Prog Cardiovasc Dis,2018,61(5-6):491-493.
[9] Zhao T,Ding X,Chang B,et al. MTUS1/ATIP3a down-regulation is associated with enhanced migration,invasion and poor prognosis in salivary adenoid cystic carcinoma[J]. BMC Cancer,2015,15:203.
[10] Dong X,Fan P,Tian T,et al. Recent advancements in the molecular genetics of left ventricular noncompaction cardiomyopathy[J]. Clin Chim Acta,2017,465:40-44.
[11] Choquet C,THM N,Sicard P,et al. Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[J]. PLoS Genet,2018,14(7):e1007502
[12] Piccolo P,Attanasio S,Secco I,et al. MIB2 variants altering NOTCH signalling result in left ventricle hypertrabeculation/non-compaction and are associated with Ménétrier-like gastropathy[J]. Hum Mol Genet,2017,26(1):33-43.
[13] Miller EM,Hinton RB,Czosek R,et al. Genetic testing in pediatric left ventricular noncompaction[J]. Circ Cardiovasc Genet,2017,10(6):e001735.
[14] Parbin S,Pradhan N,Das L,et al. DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma[J]. Exp Cell Res,2019,374(2):323-332.
[15] Liu Y,Chen H,Shou W. Potential common pathogenic pathways for the left ventricular noncompaction cardiomyopathy(LVNC)[J]. Pediatr Cardiol,2018,39(6):1099-1106.
[16] Rodrigues-Ferreira S,Nehlig A,Monchecourt C,et al. Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis[J]. Breast Cancer Res Treat,2019,173(3):573-583.
[17] Ito S,Asakura M,Liao Y,et al. Identification of the Mtus1 splice variant as a novel inhibitory factor against cardiac hypertrophy[J]. J Am Heart Assoc,2016,5(7):e003521.
[18] van Dijk J,Bompard G,Cau J,et al. Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation[J]. BMC Biol,2018,16(1):116.
[19] Roostalu J,Rickman J,Thomas C,et al. Determinants of polar versus nematic organization in networks of dynamic microtubules and mitotic motors[J]. Cell,2018,175(3):796-808.e14.
[20] López-Doménech G,Covill-Cooke C,Ivankovic D,et al. Miro proteins coordinate microtubule-and actin-dependent mitochondrial transport and distribution[J]. EMBO J,2018,37(3):321-336.
[21] Cappello S. Small Rho-GTPases and cortical malformations:finetuning the cytoskeleton stability[J]. Small GTPases,2013,4(1):51-56.
[22] Wang X,Tang P,Guo F,et al. RhoA regulates activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling[J]. Biochim Biophys Acta Gen Subj,2017,1861(1 Pt A):3011-3018.
[23] Wang T,Hamilla S,Cam M,et al. Extracellular matrix stiffness and cell contractility control RNA localization to promote cell migration[J]. Nat Commun,2017,8(1):896.
[24] Kitakaze M. Dysfunction of microtubules induces cardiac dysfunction[J]. EBioMedicine,2018,37:3-4.