Preliminary Study On HDAC11 Gene Regulating The Proliferation And Differentiation Of Muscle Stem Cells Of Guangxi Longlin Cattle

Longlin cattle,as an excellent local breed in Guangxi,has tender meat and rich nutrition.However,due to its small size and low meat production,improving meat production performance is an important direction of genetic improvement.In this study,RNA sequencing（RNA-Seq）was used to screen differentially expressed genes by sequencing the longissimus dorsi muscle tissue of Longlin cattle at weaning（6 months old）and juvenile（18 months old）period.Meanwhile,the function and mechanism of the significantly differentially expressed histone deacetylase 11（HDAC11）gene regulating Muscle Stem Cells（BMuSCs）were studied.This study is expected to provide a theoretical basis for the improvement of meat use direction of Longlin cattle in Guangxi.1.Transcriptomics analysis of longissimus dorsi muscle tissue of Guangxi Longlin cattle aged at 6 months and 18 months.High-quality Clean Reads were obtained,and more than 90%of them were comparable to the latest bovine reference genome.A total of 3,800 differentially expressed genes were selected by using|log₂Ratio|>1 and P<0.05 as screening conditions.Compared with the 6-month-old group,2784 genes were up-regulated and 1016 genes were down-regulated in the 18-month-old group.The reliability of the sequencing results was verified by q RT-PCR,and the results showed that the q RT-PCR results were consistent with the sequencing results.2.The effect of HDAC11 gene on the function of BMuSCs was explored.Functional genes were screened by q RT-PCR from the candidate histone deacetylase（HDACs）gene family.The results showed that the expression of HDAC11 in the 18-month-old group was significantly higher than that in other members of the gene family（P<0.01）.The effects of overexpression of HDAC11（pc DNA3.1-e GFP,p-HDAC11）in BMuSCs on cell proliferation,differentiation and apoptosis were examined.CCK-8 assay showed that HDAC11 promoted the proliferation of BMuSCs,and EDU assay also showed that HDAC11 increased the proportion of EDU-positive cells;the results of flow cytometry showed that HDAC11 promoted BMuSCs to enter S phase, and overexpression of HDAC11 increased the expression of proliferation-related gene Cyclin D1（P<0.05）.Myogenic differentiation was induced in BMuSCs overexpressing HDAC11.The results showed that the control group formed myotubes on the third day of induction of differentiation,while overexpressing HDAC11 inhibited myotube formation and decreased the m RNA and protein expression levels of myogenic marker gene MYOD1（P<0.05）.The results showed that overexpression of HDAC11 significantly reduced the apoptotic rate of BMuSCs,increased the expression level of BCL-2 and decreased the expression levels of BAX and Caspase3.Interference with the expression levels of HDAC11（LV201,Sh-HDAC11-A）in BMuSCs was performed to detect its effects on cell proliferation,differentiation and apoptosis,and the results were contrary to those of overexpression.3.The study preliminarily explore the effect and mechanism of HDAC11 gene on the proliferation and differentiation of BMuSCs.The results showed that the expression levels of Notch signaling pathway-related genes were significantly different before and after BMuSCs differentiation（P<0.05）,and overexpression of HDAC11 significantly upregulated the expression of Notch signaling receptors and downstream target genes（P<0.05）.The addition of 2μg/m L of the Notch signaling pathway inhibitor DAPT could effectively inhibit the expression of Notch1 and Hes1 m RNA and protein（P<0.05）,inhibit the proliferation of BMuSCs,reduce the proportion of S-phase cells,and promote the myogenic differentiation of BMuSCs and the expression of myogenic marker genes MYOD1 and MYOG（P<0.05）.Inhibiting Notch signaling pathway while overexpressing HDAC11 could alleviate the effect of DAPT on BMuSCs in inhibiting proliferation and promoting differentiation.The above results showed that the important functional genes regulating muscle development were screened out from the differential expression profiles of the muscle tissues of Longlin cattle at different months of age.It was found that HDAC11 gene could promote the proliferation,inhibit the differentiation and apoptosis of BMuSCs,and its regulatory role was closely related to Notch signaling pathway.