Screening Of Cardiac Development - Related IncRNAs And Study On The Function And Mechanism Of Uc.167 In Embryonic Heart Development

Congenital heart disease is a group of congenital malformations due to developmental defects or partial paused development of fetal hearts during their mothers’ gestation. It is the most common birth defects, with the incidence of 0.8-1%, and also has been the leading cause of infant death. Annual increase of CHD infants were about 140-150 thousands, and CHD can not only lead to fetal growth retardation, but also lead to miscarriage, stillbirth, etc. If CHD did not receive timely treatment, 30 percent of babies would die in infancy, even the survival are often secondary to the heart, brain, lungs and other vital organ damage, seriously affecting the quality of life and physical and mental health of children, bringing heavy economic pressure and mental burden to their families and the community. Therefore, to explore the mechanism of malformations of the embryonic heart development, to control and reduce the incidence of congenital heart disease fundamentally has become a focus of research in prevention medicine field.The etiology of congenital heart disease(CHD) has not yet been fully elucidated, although most scholars believe that CHD is developmental abnormalities caused by joint action of environmental and / or genetic factors during the process of the embryonic heart developing from the primitive heart tube to the mature heart, but studies found that CHD simply caused by environmental factors only accounted for 2-5% and more CHD are associated with genes and genetic defects. The heart develop process is extremely complex, involving related genes expressed in temporal sequence differentially and precise control of a number of signaling pathways. Any disorder in this procedure is likely to affect heart development, leading to the occurrence of CHD. It has been found that Nkx2-5, GATA4, MEF2 A, MEF2 C, TBX5 and other genes and Wnt, Notch signaling pathway are closely related cardiac development, which provides a molecular basis and clues to understand the development of congenital heart disease. However, the molecular regulatory networks of embryonic heart dysplasia are not yet entirely clear, exploring new regulator is still extremely important.In recent years, the life science research has entered the post-genomic era, the ENCODE program in September 2003 reveals that the majority of non-coding RNA, previously considered "transcriptional noise", also have biological functions, which attract more and more scientists’ attention. Discovery of non-coding RNA greatly expanded people’s understanding of mechanism in disease development. Researches regarding to mi RNA(length approximately 22 nt, a kind of non-coding RNA regulating encoding genes negatively at the post-transcriptional level) are the most active field, there have been many report that mi RNA play an important role in mechanism of heart development, cardiovascular disease; because long non-coding RNA(Long non-coding RNA, lnc RNA, transcript length is greater than the non-coding RNA 200nt) ①are far richer than mi RNA in type, quantity, function, and mechanism; ② have many important biological functions, such as genetic modification, chromosomal rearrangements, m RNA stability, epigenetics, gene transcription and post-transcriptional regulation and protein synthesis metabolism regulation and other function; ③ are closely related to the occurrence of many embryonic development diseases; ④may be a regulatory hub of the RNA world, has begun to attract the attention of researchers. Although studies about lnc RNA in the cardiovascular field are rarely reported, but there is no doubt that exploring the function and mechanisms of lnc RNA in heart development will further deepen our understanding of the mechanisms of heart development.Part 1 Screening and Integrated Analysis of Lnc RNAs related to Heart DevelopmentObjective: 1) Detecting lnc RNAs expression profiles between embryonic heart with cardiac ventricular septal defect and normal cardiac tissue 2) Screening lnc RNAs associated with abnormal heart development, providing dataMethod: 1) We use embryonic heart tissue diagnosed by ultrasound with ventricular septal defect at 17 weeks gestational age as test sample, and embryonic heart tissue samples at the same gestational age from abortion embryo with non-disease factors as control. lnc RNAs chip technology from US Arraystar companies were used to detecte the expression of m RNAs and lnc RNAs between the two groups. Original data were preprocessed and statistically analysed after homogenization. For the criteria, more than two-fold change were considered statistically significant(P <0.05), in this way, differentially expressed Lnc RNAs and m RNAs were screened. 2) 10 lnc RNAs in differential expression were randomly selected from chip results and Real-Time PCR method was used to validate repeatability and reliability of microarray data. 3) For differentially expressed m RNAs, we applyed GO analysis and KEGG pathway analysis. 4) Based on sequence conservation, heart development correlation, tissue-specific and some other standards, we screened lnc RNAs which were closelyrelated to cardiac development 5) Screened lnc RNAs were bioinformatic analysed, forecasting their target genes and potential biological function.Results: 1) After the raw data of the chip were standardized and the two groups were compared, we found the number of differential expression lnc RNAs was 1508,among which 880 were increased, 628 were decreased; the number of differentially expressed m RNA was 1784, and among which 691 were increased, 1093 were decreased. 2) Validation results of RT-PCR and microarray data are basically the same. 3) GO analysis revealed that differentially expressed lnc RNAS mainly involved development process, cell proliferation, cell cycle, cell differentiation, DNA replication, DNA repair, sugar molecules binding, protein binding, as well as some glucose and lipid metabolism on cell surface and some other functions. Pathway analysis showed that differentially expressed lnc RNAS participate in the signal path includeing JAK-STAT signaling pathway, Wntsignaling pathway, Hedgelog signaling pathway, etc., and these signaling pathways are closely related to heart development. 4) By making strict screening criteria, we selected 10 lnc RNAs which is closely related to heart development, they have highly conservative and tissue specificity, respectively: ENST00000513542 、 RP11-473L15.2 、 uc.167 、 HIT000242541 、BX648912、BC040935、AY927503、 LOC440839、G65566、AK127225 5) Bioinformatics analysis revealed chromosomal regions of two lnc RNAs ENST00000513542, and RP11-473L15.2, have rich regulatory information of epigenetic regulation, including a variety of histone modification methods, DNA methylation, and can be regulated by binding multiple transcription factor related todevelopment.Conclusions: 1) Compared with normal control, lnc RNAs expression in embryonic cardiac tissue with ventricular septal defect significant changed, indicating that lnc NRAs were involved in the regulation of heart development process. 2) By validation through Real-time PCR, we concluded that lnc RNAs microarray results were reliable and can be used for subsequent analysis.Part 2 Bioinformatics analysis and Biological Characteristic of Lnc RNA-uc.167Objective: 1) To explore importance of uc.167 potential function by the use of bioinformatics methods 2) To analyze the basic biological characteristics of uc.167, including uc.167 spatial distribution and expression pattern over time in the heart development process of mice, and uc.167 expression pattern over time during P19 cells differentiating into cardiomyocytesMethod: 1) Using the UCSC genome browser of ENCODE for preliminary bioinformatics analysis of our screened lnc RNAs; predicting TFBs components combinedwith lnc RNAs by the use of a relational database. Using Cat RAPIDalgorithm to predict interactions between RNA and protein2) Take the different stages(P8.5, P11.5, P14.5, P18.5) embryonic mouse heart, using RT-PCR method to detect the expression of uc.167 and neighboring genes in myocardial tissue, and detecting uc.167 in myocardial tissue distribution by the use of in situ hybridization 3) Using DMSO method to induce P19 cell differenting into cardiomyocytes, using RT-PCR method to detect expression changes uc.167 and neighboring genes during differentiationResults: 1) Full-length of human uc.167 is 201 bp, and human uc.167 is located in the genome 5q14.3(chr5: 88179623-88179824, GRCh37 / hg19), according to lnc RNA classification principles, it is Intronic antisense lnc RNA. 2) uc.167 related chromosomal regions are affected by a number of key transcription factors binding regulation,such as CEBPB, GATA2, SRF, etc.There is a wealth of epigenetic regulation information in these chromosomal regions, for example, a variety of histone modifications, DNA methylation and so on. 3) uc.167 mainly expressed in the ventricular muscle tissue; uc.167 expression gradually decreased in mouse embryonic heart development process, and its neighboring Mef2 c gene expression gradually increased, showing a opposite trend; 4) The expression of uc.167 during P19 cell differentiating into cardiomyocytes, showed a "first increased and then decreased" pattern. The expression reached the highest level in embryonic-like body formation(d4), then gradually reduced. The expression of neighboring gene Mef2 c was in the opposite trend.Conclusions: 1) uc.167 may have important potential biological functions; 2) uc.167 possibly play a role through negative regulation of its neighboring genes Mef2 c.Part 3 Function and Mechanism Research of uc.167 in the Process of Heart DevelopmentObjection: 1) To explore the influence of uc.167 overexpression has in P19 on proliferation, cell cycle, apoptosis and differentiation and the related mechanisms; 2) To explore the effects and possible mechanisms of uc.167 heart during heart development in miceMethod: 1) Design and construct lnc RNA-uc.167 overexpression vector, along with the blank control vector, both of which transfected P19 cells, and screened with puromycin for 14 d. Using RT-PCR method to verify and obtain stable expression cell lines. CCK-8 assay was used to predict cell proliferation, flow cytometry was used to to detect the impact of uc.167 overexpression on P19 cell cycle, RT-PCR method was used to detect its impact on the P19 cell differentiation, and phosphatidylcholine with valgus law, Capase3 activity detection and Hoechst staining and other methods were used to analyze its effects on apoptosis. 2) We designed mef2 c overexpression vector and co-transfected P19 cells with uc.167, then detected P19 cell proliferation, cell cycle, apoptosis and differentiation 3) We built uc.167 cardiac tissue specific transgenic mice by embryonic microinjection, and preliminary analyze its phenotype by using echocardiography, HE, Masson and other experimental techniques.Results: 1) Over-expression vector was successfully constructed uc.167 and uc.167 stably overexpressed cell lines was obtained. Over-expression of uc.167 significantlyinhibited cell proliferation, which was blocked in S / G2 phase. And over-expression of uc.167 promotes apoptosis. Over-expression of uc.167 significantly inhibited P19 cell differentiating into cardiomyocytes. 2) Overexpression of mef2 c together with uc.167 can save the adverse effects on P19 cells when uc.167 was over-expressed alone. 3) uc.167 transgenic mice did not show significant cardiac malformation phenotype. Compared with normal control, cardiac structure, heart function parameters of transgenic mice did not significant change. Myocardial tissue morphology, degree of fibrosis did not change significantly either.Conclusions: 1) Overexpression of uc.167 can produce significant cell phenotype, significantly affecting P19 the cell cycle, apoptosis and differentiation. 2) uc.167 possibly play its biological function through negative regulation of its neighboring gene Mef2c; 3) uc.167 have a regulatory role in heart development process, but it may not play an unique and key role.