Effect Of Meis1,Apbb1 And RBL1 On Proliferation Of Cardiomyocytes

Background:Heart failure is a global epidemic,which was caused by the loss of a large number of cardiomyocytes in a pathological condition.Due to the limited proliferation capacity of remaining cardiomyocytes,the lost cardiomyocytes can not be completely replaced by the lost cardiomyocytes.Therefore,how to induce the cardiomyocyte proliferation and promote heart regeneration is an urgent problem to be solved.Bone marrow viral integration site 1(Meis1)is a homeodomain transcription factor in the triamino acid ring extension(TALE)family,which has been shown that deletion of Meis1 can significantly promote primary cardiomyocyte proliferation and cardiac regeneration in adult mice.However,the mechanism of Meis1 in cardiac regeneration and myocardial cell proliferation is not completely clear.It is noteworthy that amyloid precursor protein(Apbb1)and retinoblastoma transcription-inhibitory factor(RBL1)m RNA are significantly down-regulated in the hearts of Meis1 knockout mice,but the role of Apbb1 and RBL1 in cardiomyocyte proliferation have not been clarified.Therefore,we intend to further explore the molecular mechanism of Meis1 regulating cardiomyocyte proliferation,as well as the effect of Apbb1 and RBL1 on cardiomyocyte proliferation and its molecular mechanism,in an attempt to provide a new theoretical basis for cardiac regeneration.This study focused on exploring the effects of Meis1,Apbb1 and RBL1 on proliferation of H9c2 cardiomyocytes.Objective:To determine the effects of Meis1,Apbb1 and RBL1 on H9c2 cardiomyocyte proliferation.Methods:1.Si RNA technique was used to knock down the expressions of Meis1,Apbb1 and RBL1 in H9c2 cardiomyocytes respectively;2.H9c2 cardiomyocytes were induced to overexpress Meis1,Apbb1 and RBL1 by transfection of pc DNA3.1(+)Meis1,pc DNA3.1(+)Apbb1 and pc DNA3.1(+)RBL1 overexpression vectors respectively;3.The knockdown efficiency and overexpression efficiency of Meis1,Apbb1 and RBL1 were detected by q RT-PCR assay.Western Blot assay was used to detect the knockdown and overexpression efficiency of Meis1;4.The effects of Meis1,Apbb1 and RBL1 on H9c2 myocardial cell proliferation were determined by CCK-8 experiment respectively;5.The effect of Meis1 on proliferation of H9c2 cardiomyocytes was determined by cell clonogenesis assay;6.The effects of Meis1,Apbb1 and RBL1 on H9c2 cardiomyocyte proliferation were determined by q RT-PCR respectively;7.The effects of Meis1,Apbb1 and RBL1 on H9c2 cardiomyocyte proliferation were determined by immunofluorescence assay respectively.Results:1.The levels of m RNA expression of Meis1,Apbb1 and RBL1 were significantly changed under si RNA knockdown and plasmid overexpression,and the protein level of Meis1 was also significantly changed;2.Compared with the control group,CCK8 results showed that knockdown Meis1 and knockdown Apbb1 both increased the proliferation rate of H9c2 cardiomyocytes,while overexpression of Meis1 and overexpression of Apbb1 both reduced the proliferation rate of H9c2 cardiomyocytes.Knockdown and overexpression of RBL1 both reduced the proliferation rate of H9c2cardiomyocytes;3.Cell cloning and formation experiments showed that knockdown of Meis1 increased the clone number of H9c2 cardiomyocytes,while overexpression of Meis1 reduced the clone number of H9c2 cardiomyocytes;4.q RT-PCR results showed that knockdown of Meis1 and Apbb1 both significantly up-regulated Ki67,Ccnb1 and other proliferation-related markers.Overexpression is the opposite.Both knockdown and overexpression of RBL1 induced downregulation of Ki67,Ccnb1 and other proliferation-related markers;5.Immunofluorescence assay also showed that knockdown of Meis1 and Apbb1 both significantly increased Ph3 positive and Aurora B positive H9c2 cells.Overexpression is the opposite.Both knockdown and overexpression of RBL1 significantly reduced Ph3 positive and Aurora B positive H9c2 cardiomyocyte.Conclusion:Both of Meis1 and Apbb1 play a role in inhibiting cardiomyocyte proliferation,while RBL1 may at the optimal level of regulating cardiomyocyte proliferation.Knockdown and overexpression of RBL both inhibits the proliferation of cardiomyocytes.Meis1,Apbb1 and RBL1 may be potential intervention targets for cardiac regeneration.