Role Of A Novel Protein LOC339524 On Regulating The Division And Proliferation Of Cardiomyocytes

With leaving the cell cycle, the division and proliferation of adult cardiomyocytes is inhibited. So it is hard to repair damages through self-division and proliferation after suffering ischemia, hypoxia and so on. These result in reducing the number of myocardial cells, heart dysfunction and even causing heart failure.Recent studies have revealed that the proliferation and update of adult mammalian ventricular cardiomyocytes are sporadic but feasible [1] [2]. Some signal pathways and methods which can induce division and proliferation of adult cardiomyocytes have been found [3][4][5]. However, the molecular mechanisms of impeding adult cardiomyocytes to return the cell cycle remains unclear. Once the proliferation of adult cardiomyocytes can be regulated, it will have had significant therapeutic value for myocardial infarction, heart failure and so on, but also have had vital implications for patients with cardiovascular disease to reduce anesthesia and surgical risk.Objective:This study aims to analyze the expression pattern of LOC339524 protein in the development of rat heart, and to clarify the role of LOC339524 protein on regulating the division and proliferation of H9C2 cardiomyocytes, which will provide theoretical basis for inventing gruds to induce myocardial regeneration.Methods:1. The embryonic hearts from13 to 21 days(E13 ~ E21), the postnatal hearts from 2 hours to seven days(P0 ~ P7), adult(two months) and old(18 months) hearts of SD rats were obtained after anaesthesia. The expression and location of LOC339524 gene were detected by RT-qPCR,Western Blot and immunofluorescence.2. Constructing a recombinant adenovirus vector containing LOC339524, overexpression of LOC339524 gene and protein in H9C2 cells was acquired. Its amount and location were valued by Western-Blot, RT-PCR and immunofluorescence.3. Three RNAi(si-h-LOC339524) segments for different target sequences were synthesized and transfected into H9C2 cells by lipofectamineTM 2000 as the carrier. The most effective si-h-LOC339524 sequence, the best transfection concentration and optimal duration were confirmed through RT-PCR and Western Blot.4. After overexpression and silence of LOC339524 gene in H9C2 cardiomyocytes, the cell proliferation was calculated by cell counting kit-8(CCK-8) method, DNA synthesis phase was marked by EdU; expression levels of p21 and cyclin D1 protein were detected by Western Blot.Results:1. The expression of LOC339524 gene was increased in E15 and E19 that the atrial septation and the modification of atrioventricular valve leaflets and coronary arteries have been completed respectively. With the gradual loss of myocardial proliferation capacity, the expression of LOC339524 gene was gradually increased in postnatal cardiomyocytes, and was mainly in the cytoplasm, little in the nucleus. With low proliferation capacity of adult and old rat cardiomyocytes, the expression of LOC339524 was low and concentrated in the nucleus.2. The recombinant adenovirus vector containing LOC339524(pAd-LOC339524) was constructed successfully and LOC339524 was overexpressed in H9C2 cells by transfecting this recombinant adenovirus vector.3. Expression of LOC339524 gene in H9C2 cells was most significantly kockdowned by transfection with si-h-LOC339524-002 sequence with targeting sequence GCAAGTCCATCTCATCGCT in 200nmol/L and maintaining 24 hours.4. After overexpressed LOC339524 gene, the proliferation of H9C2 cardiac cells was inhibited significantly, and the number of cells in DNA synthesis was obviously reduced, the expression of cyclin-dependent kinase inhibitor p21 was upregulated. At the same time, cyclin D1 was downregulated. However, these results were reversed when LOC339524 gene was silenced,Conclusions:1. LOC339524 may be involved in the regulation of division and proliferation of cardiomyocytes during development of rat hearts, and the expression of LOC339524 in the nucleus may inhibit the proliferation of cardiac cells in adult rats.2. LOC339524 can inhibit the proliferation of H9C2 cardiomyocytes through upregulating the expression of P21 gene and downregulating the expression of cyclin D1.3. LOC339524 protein might play a key role in preventing cardiomyocytes re-entrying cell cycle and it may be a new target to develop drug for inducing self-repairation in cardiac cells.