Effect Of LYRM1 Knockdown On Proliferation,Apoptosis, Differentiation And Mitochondrial Function In The P19 Cell Model Of Cardiac Differentiation In Vitro

Heart development is an extremely complex process, which involves precisely temporal and spatial expression of many genes associated with embryonic development. Research shows cardiac structure and function are quite sensitive to genetic perturbation during cardiogenesis in human and that mutations in components of the cardiac gene network can lead to adverse consequences in the form of congenital heart disease (CHD). Thus, exploring genetic etiology is still the key to the prevention and treatment of CHDs.LYRM1 (LYR motif containing 1), a novel human gene, was found and characterizedby the research groupof the professor Guo Xirong. Surprisingly, LYRM1 is both abundantly expressed in human heart and adipocytes tissue by multi-tissue expression analysis. In addition, studies have shown that LYRM1 was a member of the Complexl LYR superfamily, and LYR proteins were predominantly mitochondrial proteins and influenced mitochondrial homeostasis. In our earlier studies, the group membersof the professor Guo Xirong found that LYRM1 had an effect on differentiation, proliferation and apoptosis in 3T3-L1 pre-adipocytes and also influenced mitochondrial function in mature 3T3-L1 adipocytes. In view of the fact that heart and adipose both derived from the mesoderm, the same germ layer organization have the similar structure and function, suggesting that LYRM1 may also play a role in the physiological processesof heart development. Further studies by our group revealed that overexpression of LYRM1 did not affect the differentiation of P19 cells into cardiomyocytes, but significantly promoted proliferation and inhibited apoptosis in embryonic myocardial cells, which indicated that the LYRM1 gene may play a vital role in the development of the human heart.In order to further explore the role of LYRM1 gene in heart development, we used P19 cells as the research object, induced their differentiation into cardiomyocytes and silenced the expression of LYRM1 genecombined with RNA interference technology to explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Through the analysis of the effect of LYRM1 gene on the apoptosis of myocardial cells and the relationship between mitochondrial function and apoptosis, we further demonstrate the important role of LYRM1 gene in embryonic heart development and thepossible teratogenic mechanism, which may provide a new direction for the role of LYRM1 physiopathologic in embryonic heart development.PartⅠ Effect of LYRM1 Silencing on Proliferation, Apoptosis and Differentiation of P19 CellsObjective:To explorethe effects of LYRM1 gene silencing on proliferation, apoptosis and differentiation of P19 cells in vitro.Methods:We designed short hairpin RNA (shRNA) construct contained a 19-nt double-stranded LYRM1 sequence, constructed the LYRM1 silencing slow virus vector. The recombinant vector was named pgLV-U6-puro-LYRM1-shRNA. The negative control vector was named pgLV-U6-puro-NC-shRNA.HEK-293 T cells were transfected with the pgLV-U6-puro-LYRM1-shRNA or pgLV-U6-puro-NC-shRNA. The medium containing lentiviruses was collectedand applied directly to P19 cells. Stably transduced P19 cells were selected in medium containing puromycin. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8 (CCK-8) proliferation assays and cell cycle analysis were used to detect cell proliferation. Flow cytometry and measurement of their caspase-3 activities were used to inspect cell apoptosis. Cell morphological changes during differentiation were observated by an inverted microscope and expression levels of specific differentiation marker genes (GATA4 and Nkx2-5) were detectedby quantitative real-time PCR and western blotting.Results:1) The CCK-8 assay showed that P19 cells silencing LYRM1 grew slower than control cells, and cell cycle analysis indicated a remarkable decrease in the percentage of cells entering the S-phase and distinctly increased Gl phase fraction in LYRM1-silenced P19 cells.2) Flow cytometry and measurement of caspase-3 activity revealed that apoptosis of P19 cells with silencing LYRM1 was significantly upregulated.3) Compared with the control group, cell morphology during differentiation was not neat and smooth in the LYRM1 knockdown group. GATA4 and Nkx2-5 expression was significantly down-regulated during P19 cell differentiation into cardiomyocytes on days 6 and 10 in the LYRMl knockdown group compared with the control group at the same time points. Although the expression of cardiomyogenesis-specific markers detected by western blots had no marked difference during P19 cell differentiation into cardiomyocytes on days 6, there was significantly down-regulated on days 10 in the LYRM1 knockdown group compared with the control group.Conclusion:LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.Part Ⅱ Effect of LYRM1 Silencing on Mitochondrial Function in the P19 Cell Model of Cardiac DifferentiationObjective:To investigatethe effects of LYRM1 knockdown on the mitochondrial function in the P19 cell model of cardiac differentiation.Methods:Stably transduced P19 cells with silencing LYRM1were selectedand differentiated intomyocardial cellswith 1% DMSO. Then, on the 10th day of differentiation, real-time PCR was used to determine the relative amounts of mitochondrial DNA (mtDNA). Using a Luciferase-Based Luminescence Assay Kit, we measured the cellular adenosine triphosphate (ATP) on the 10th day of differentiation. Finally, we measured the intracellular ROS levels by a 2’, 7’-dichlorodihydrofiuorescein diacetate acetyl ester (H2-DCFDA) probeand evaluated the mitochondria membrane potential (MMP) by a JC-1 fluorescent probe from the Mitochondrial Membrane Potential Detection Kit on the 10th day of differentiation. The fluorescence was analyzed with a FACScan flow cytometer.Results:1) There was no marked difference in mtDNA copy number between the LYRM1 knockdown group and control group.2) The cellular ATP production decreased dramatically in the LYRM1 silenced cells.3) LYRM1 knockdown significantly increased the ROS level.4) LYRM1 knockdown distinctly decreased the mitochondrial membrane potentialin myocardial cells.Conclusion:The suppression of LYRM1 expression caused mitochondria dysfunction, which revealed that LYRM1 might play an important role in maintain normal function of mitochondrion.