Screening Of Long - Chain Non - Coding RNA (Lnc RNA) Associated With Heart Failure In PDK1 Knockout Mice

Previous studies have indicated that long non-coding RNAs (lncRNA) are related to the occurrence and development of many human diseases, such as cancer and the HELLP and the brachydactyly syndromes. However, studies of LncRNA in heart failure (HF) have not yet been reported. Here, we investigated cardiac lncRNA expression profiles in the myocardial-specific knockout (KO) pdkl gene mouse model of heart failure. Methods:Cardiac samples were obtained from KO and wild type (WT) mice on postnatal (P) day8(P8) and day40(P40), and lncRNA expression profiles were analyzed by sequencing and screening using the Arraystar mouse lncRNA microarray. Quantitative real-time PCR analysis of these lncRNAs confirmed the identity of some genes. Results:Comparisons of the KO and control groups showed fold changes of>1.5in the expression levels of2,024lncRNAs at P8, while fold changes of>2in the expression levels of4,095lncRNAs were detected at P40. Nineteen lncRNAs were validated by RT-PCR. Bioinformatic and pathway analyses indicated that mkk7, a sense overlap lncRNA, may be involved in the pathological processes of heart failure through the MAPK signaling pathway. Conclusion:These data reveal differentially expressed lncRNA in mice with a myocardial-specific deletion of the pdkl gene, which may provide new insights into the mechanism of heart failure in PDK1knockout mice.Part I Screening LncRNA from cardiac tissue of PDK1knockout mice with heart failureObjective:To observe LncRNA expression levels whether change in PDK1knockout mice with heart failureMethods:First, scissors mouse tail lysis, extraction of DNA, PCR Identification of myocardial specific knockout (knockout, KO) mice, Western blot method validation knockout PDK1protein levels. Second, sent three smples both from PDK1KO and WT mice at40days after birth to the company to do LncRNA chip, Last, through bioinformatic analysis, screening may be associated with heart failure LncRNA, verified by RT-PCR screening results.Results:1) A total of4,095lncRNAs were differentially (fold change>2, P<0.05) expressed in the KO group compared to the control group;2) Among these,2,094lncRNAs were upregulated (>2-fold) in experimental group compared to the control group, while1,965lncRNA were downregulated;3) We randomly selected six lncRNAs for qPCR validation. The qPCR and microarray data were in accordance.Conclusion:1) LncRNAs are aberrantly expressed in the heart failure group compared to the matched normal groupPart â…¡ Screening LncRNA from PDK1gene stable knockout mice without heart failureObjective:To observe LncRNA expression levels whether change in PDK1knockout mice without heart failureMethods:PDK1knockout mice identified with the former, take myocardial tissue-specific knockout (KO) mice and wild type of the same age (wild type, WT) mice for the study, select mice from birth sixth and the eighth day to ninth day to observe PDK1protein levels. When PDK1protein levels stable knockout, weigh heart weight (HW) and body weight (BW), calculate the heart weight index (HW/BW); observe the overall appearance of the heart and cardiac morphology histological change use light microscope, assessment cardiac function in mice use echocardiography. Take PDK1KO and WT mice of three cardiac tissue samples sent to the company to do LncRNA chip, through bioinformatic analysis, screening may be associated with heart failure LncRNA, verified by RT-PCRResults:1) At P8, PDK1gene stable knockout in myocardial tissue;2) KO mice compared with WT mice heart weight index was significant difference;3) The appearance of the heart was no significantly difference;4) KO and WT mice in the heart slice shape without significant differences;5) echocardiography KO mice detected no significant changes in heart function;6) A total of2,024lncRNAs were differentially (fold change>1.5, P<0.05) expressed in the KO group compared to the control group;7) Among these,1,224lncRNAs were upregulated (>1.5-fold) in experimental group compared to the control group, while800lncRNA were downregulated;8) We randomly selected five lncRNAs for qPCR validation. The qPCR and microarray data were in accordanceConclusion:1) PDK1gene stable knockout mice compared to wild-type mice, the overall appearance of cardiac morphology and function were not significantly different;2) the heart weight index was significant difference;3) Although stable PDK1gene knockout mice that did not develop heart failure state, but there were2024LncRNAs expression levels change greater than1.5times in the heart tissue, and in part were verified by RT-PCR.Part â…¢ Compared analysis of lncRNAs microarray at two time points and reveal LncRNA possible mechanismObjective:To identify the specific lncRNAs that maybe participated in the development of heart failure and investigate LncRNA involved in the development and progression of heart failure possible mechanism.Methods:Based on the first and second data, we compared the microarray data of the two experimental time-points (P8and P40) and validate the results by RT-PCR. At the same time, by the target mRNA through LncRNA the GO and Pathway bioinformatics analysis to explore the possible mechanismResults:The results by compared screening as followed:1)93lncRNAs were consistently upregulated at the two experimental time-points;2)52were consistently downregulated at the two experimental time-points;3)136lncRNAs were downregulated at P40after the upregulation at P8;4)51lncRNAs were downregulated at P8before upregulation at P40;5) random verification of eight LncRNAs were statistically significant (P<0.05). The results by bioinformatics analysis as followed:1) P8day,9signaling pathways may be involved in heart function, including MAPKsignaling, Cell adhesion molecules and Calcium signaling pathway (P<0.05);2) P40day,10pathways may be involved in heart failure, including the MAPK and Cell adhesion molecules Pathway (P<0.05);3) screening found MKK7(a sense overlap lncRNA) possibly through MAPK signaling pathways involved in the pathogenesis of heart failure.Conclusions:1) At the two time points, the expression level of LncRNA was change. And LncRNA may be involved in the development of heart failure;2) LncRNA may be involved in the development of heart failure;3) LncRNA possibly through MAPK signaling pathways involved in the development of heart failure.