Identification Of Different Transcripts Of Bovine Lnc9141 And Their Regulation In Bovine Myoblast

Due to the slow growth of local yellow cattle,low meat production rate,lag in breeding progress,and weak conservation and utilization of germplasm resources,Chinese beef production has slowed down.The growth rate of beef production still can not balance the ever-increasing demand of beef by our country’s citizens.This contradiction not only greatly hindered the improvement of the meat consumption structure of urban and rural residents in China,but also seriously affected the international competitiveness of Chinese cattle industry.Muscle growth is one of the important factors affecting beef production.For this reason,excavating important functional genes and regulatory genes(including non-coding RNAs)that regulate cattle muscle development has practical significance for improving cattle breeding and beef production in China.At present,the molecular breeding of cattle has moved from classical genetic research to epigenetic modification.With the rapid development of high-throughput sequencing technologies,beef cattle breeding faces unprecedented development and challenges.Today,high-throughput sequencing technologies have been applied to beef muscle research.The transcriptome sequencing was performed on muscles of fetal bovine,calf and adult bovine and many differentially expressed long non-coding RNAs(lnc RNAs)lnc RNAs related to muscle development have been screened.Therefore,lnc9141 was selected as the research object.Flow cytometry,RACE,Western Blot,q PCR and other equipment and technical methods were used to explore its different transcript identification and function studies.It is as scientific data provided for molecular breeding and gene function of cattle.1.The RACE method was used to clone the full length of lnc9141,revealing that lnc9141 has two different transcripts,namely lnc9141-a and lnc9141-b,with full lengths of657 nt and 547 nt,respectively.Lnc9141-a and lnc9141-b were initially transcribed from the different start sites,and they both contained the longest ORF of 138 nt,encoding 45 amino acids.On-line software predictions showed that both lnc9141-a and lnc9141-b had weak coding capabilities;Prokaryotic expression experiments revealed that neither lnc9141-a nor lnc9141-b had the ability to encode large proteins.q PCR showed that the expression levels of lnc9141-a and lnc9141-b in fetal bovine muscle tissues were higher than in calf muscle tissues,which was consistent with the results of the previous transcriptome sequencing;Different tissue expression analysis showed that lnc9141-a and lnc9141-b were expressed highly in the heart and lungs.These results revealed the molecular characteristics of lnc9141-a and lnc9141-b,and provided the foundation for subsequent functional studies.2.In this study,bovine myoblasts were successfully isolated from muscle tissue of 3-m fetal bovine using collagenase digestion method,and bovine myoblasts were purified using differential adherent method.After 4 days induced differentiation of bovine myoblasts,microscopic observations revealed that a large number of myotubes appeared.Using My HC immunofluorescence to identify the myotubes,it was found that myotubes could be enriched by My HC.q PCR analysis showed a trend of first increasing and then decreasing in bovine myoblasts at day 0,day 1,day 3 and day 5 of differentiation,and the highest expression level at day 3 of differentiation.After nucleoplasm was separated,expression studies revealed that both lnc9141-a and lnc9141-b were highly expressed in the cytoplasm.3.To investigate the function of lnc9141-a and lnc9141-b in bovine myoblasts,overexpression vectors for lnc9141-a and lnc9141-b were constructed in this study.According to the sequence characteristics of lnc9141-a and lnc9141-b,the si RNAs(si RNA-a and si RNA-b)of lnc9141-a and lnc9141-b were designed.In detecting the over-expression and interference efficiency of overexpression vectors and si RNAs,overexpression of lnc9141-a was found to increase the expression level more than 10 fold.si RNA-a can significantly reduce the expression of lnc9141-a in bovine myoblast without affecting lnc9141-b expression level.Similarly,after overexpression of lnc9141-b,its expression increased more than 10 fold;si RNA-b could significantly reduce the expression of lnc9141-b without affecting the expression of lnc9141-a.4.After bovine myoblasts were overexpressed by lnc9141-a and lnc9141-b respectively,the proliferation of bovine myoblasts was detected by cell cycle,Ed U and MTT assay.The results showed that the proliferation of bovine myoblasts was inhibited.However,interference with lnc9141-a or lnc9141-b promotes the proliferation of bovine myoblasts.At the same time,the overexpression of lnc9141-a and lnc9141-b respectively,the expression of Cyclin D1 was down-regulated.After si RNAs were transfected,the expression of Cyclin D1 and Cyclin E increased.It was demonstrated that lnc9141-a and lnc9141-b inhibit the proliferation of bovine myoblasts.In bovine myoblasts,after overexpressing or interfering with lnc9141-a and lnc9141-b,Annexin V-FITC/PI double staining and detecting the expression of apoptosis marker genes(Bax,Bcl2,Caspase3)were performed.It was found that lnc9141-b can regulate the expression of Bax gene,but lnc9141-b does not affect the number of apoptotic cells.Similarly,the effects of lnc9141-a and lnc9141-b on the differentiation of bovine myoblasts were examined.The results showed that the differentiation of bovine myoblasts was not regulated by lnc9141-a,but overexpression of lnc9141-b reduced the expression of My HC.Lnc9141-b can potentially affect the differentiation of bovine myoblasts.5.Based on the functions performed by lnc9141,this study next explored the active promoter region of lnc9141 for providing a basis for in-depth functional studies.According to the position of lnc9141 in the genome,IRX5 gene was found at 2.0 kb upstream of lnc9141,and the transcription direction was opposite to that of lnc9141.The on-line software predicts the position of the Cp G island in the promoter region shared by lnc9141 and IRX5,and the lnc9141 and IRX5 promoter regions are divided into five truncated body regions respectively.The dual luciferase reporter system was used to detect the fluorescence activity of each truncated body.It was found that the-1449 to-885 region of the lnc9141 promoter was the active region,basic on that the first base of the lnc9141-b transcript was +1 position of lnc9141.There was no strong activity in the IRX5 promoter,except that some element sequences from-2042 to-1703 may weakly regulate IRX5 transcription.