Mechanisms Of Missense Mutation Microtubule Associated Tumor Suppressor 1 (MTUS1)in The Development Of Compaction Of Ventricular Myocardium Through Microtubule-Associated Pathway

Objective: To investigate molecular mechanisms of missense mutation Microtubule-associated tumor suppressor 1(MTUS1)in compaction of ventricular myocardium,and to preliminarily explain the genetic mechanism of noncompaction of ventricular myocardium.Methods: In the first place,Lentiviral vectors containing MTUS1 wild type and containing the mutation MTUS1 were constructed and co-infected into CP15-5a cells(mutantion LV-M-MTUS1 group,wild type LV-W-MTUS1 group and vector CON group,respectively).The mRNA expression of MTUS1 and ras homolog family member A(RhoA)were evaluated by RT-PCR.Expression level of RhoA protein was measured by Western blot.The proteins ?-tubulin was detected by immunofluorescence assays.Cell migration of each group was determined by Wound-Healing Assay.In the second place,Lentiviral vectors containing MTUS1 wild type and containing the mutation MTUS1 were constructed and co-infected into HEK-293 cells.The mRNA expression of MTUS1 was evaluated by RT-PCR.The fluorescence intensity and the location in cells of ?-tubulin,?/?-tubulin,polarity protein(PAR6)and were measured by Immunofluorescence assays.The phosphorylation level of Rac1/Cdc42 was determined by Western blot.Meanwhile,the distance between spindle and membrane and the size of daughter cells after division of HEK293 cells were measured according to the Immunofluorescence result of ?-tubulin.Results: The lentiviral vectors of mutantion LV-M-MTUS1 group,wild type LV-W-MTUS1 group and vector CON group in CP15-5a cells and HEK-293 cells were constructed successfully,verified by fluorescence staining and RT-PCR.In CP15-5a cell models,Immunofluorescence assays revealed the fluorescence intensity of ?-tubulin decreased in the cells expressing the mutated MTUS1(P<0.01).The ?-tubulin in mutated MTUS1 intracellular is sparse and disorganized.RT-PCR and Western blot showed that expression of RhoA was significantly increased in mutation than that in wild type group(P<0.01).In Wound-Healing Assay,after the scratch 6 h and 12 h,the percent wound closure of the mutation group was significantly increased than that in wild type group(P<0.01).In HEK-293 cell models,the RNA and protein expression levels of MTUS1 in the mutant and wild type groups were significantly higher than in the blank and vector groups(P<0.05).Immunofluorescence assays revealed the fluorescence intensity of ?-tubulin to be decreased and ?/?–tubulin to be increased in the cells expressing the mutated MTUS1(P<0.01).Meanwhile,the fluorescence intensity of PAR6 was higher and location of PAR6 abnormal(P<0.01).According to the Immunofluorescence of ?-tubulin,the area in the daughter cells staining positive for ?-tubulin in the mutation group was significantly larger than that in wild type groups(P<0.01).And the distance between the spindle and cytoplasmic membrane of the daughter cells in the mutation group were significantly larger than that in the wild type group(P<0.01).Phosphorylation of Rac1/Cdc42 in the mutation group was significantly lower than that in the wild type group(P<0.01).Conclusion: In CP15-5a cell models,the mutant MTUS1 decreased the stability of microtubules and increased cell migration by regulating the mRNA and protein expression of RhoA.In HEK-293 cell models,the mutant MTUS1 affected the expression of ?-tubulin and decreased the stability of microtubules,thus affected cell polarity division.The possible mechanism is that the phosphorylation level of Rac1/Cdc42 protein was further reduced after the stability of microtubules decreased.The expression of ?/?–tubulin protein,which regulated transportation was increased,and PAR6 protein was transported from the cytoplasm to the cell membrane,thereby mediating the trend of cell polarity division increased.Above all,the missense mutation MTUS1 is a protective mutation,which may be promote densification of the myocardium and reducing the occurrence of myocardial densification arrest.