Effect Of LRRN1 Gene On The Proliferation And Differentiation Of Qinchuan Bovine Myoblasts

The gradual increase of beef consumption has put forward higher requirements for beef production and quality,so the mining and utilization of excellent germplasm resources of local beef cattle has become an issue that animal husbandry pays attention to.In the development stage of bovine skeletal muscle,muscle fibers form by the proliferation and differentiation of myoblasts,become the basis of adult muscle remodeling to affect the yield and quality of beef.Therefore,it is of great significance to explore the mechanism of gene regulation on myoblast proliferation and differentiation for the selection of molecular breeding targets.Leucine rich repeat neuronal protein 1(LRRN1)gene is a candidate gene selected by our group through transcripomics in the co-culture of bovine myoblasts and preadipocytes.At present,the effects of LRRN1 gene on muscle development and myoblast have not been reported.So this study to explore the target gene LRRN1 expression characteristics in important tissues of Qinchuan cattle at different months of age and at different stages of differentiation of bovine myoblasts.adenovirus and plasmid mediated sh RNA interference and overexpression methods were used to explore the influence of LRRN1 gene on proliferation and differentiation of bovine myoblasts,further reveal the mechanism of LRRN1 gene regulation the proliferation and differentiation of bovine myoblasts and muscle development.The experimental results and conclusions as follows:1.The expression level of LRRN1 gene in kidney and longissimus dorsi muscle of newborn cattle extremely significantly higher than other tissues(P<0.01).Longissimus dorsi muscle and kidney extremely significantly higher than other tissues at adult cattle(P<0.01).The expression level in muscle tissue of adult cattle extremely significantly higher than that of newborn cattle(P<0.01).The expression trend of LRRN1 gene in myoblast differentiation is firstly increase and then decrease.2.Interference and overexpression of LRRN1 gene inhibit bovine myoblasts proliferation,significantly inhibit proliferation marker genes CCNA2,PCNA,CCNB1 and CCND2(P<0.05).Interference of LRRN1 no effect on the number of living cells,overexpression of LRRN1 decrease the number of living cells.Interference with LRRN1 gene significantly inhibit the apoptosis of myoblasts(P<0.05),significantly decrease the expression of apoptosis factors BAX and BAD(P<0.05),overexpression of LRRN1 gene significantly promote the apoptosis of myoblasts(P<0.05),significantly increase the BAD(P<0.05).3.Interference with LRRN1 gene inhibit the formation of myotubes,the differentiation index significantly decrease(P<0.05);The expression level of myogenic differentiation marker genes in m RNA and protein level: MYOD extremely significantly decrease in D6differentiation(P<0.01),MYOD protein significantly decrease in D4 and D6(P<0.05).MYOG extremely significantly decrease in D4 and D6(P<0.01),MYOG protein significantly decrease in D2 and D4(P<0.05).MYF6 extremely significantly decrease in D2 and D4(P<0.01),significantly decrease in D6(P<0.05),MYF6 protein extremely significantly decrease in D4(P<0.01),significantly decrease in D2 and D6(P<0.05).MYF5,CKM and MEF2 A significantly decrease in D4 and D6(P<0.05),MYF5 and CKM protein significantly decrease in D4(P<0.05).MYH3 extremely significantly decrease in D0,D2 and D4(P<0.01)and significantly decrease in D6(P<0.05),MYH3 protein significantly decrease in D4 and D6(P<0.05).Overexpression LRRN1 gene promote the formation and maintenance of myotubes,significantly increase the differentiation index(P<0.05).MYOD extremely significantly increase in D4 and D6(P<0.01),significantly increase in D0 and D2(P<0.05),MYOD protein extremely significantly increase in D2(P<0.01)and significantly increase in D4(P<0.05).MYOG significantly increase in D2(P<0.05),MYOG protein significantly increase in D4 and D6(P<0.05).MYF5 extremely significantly increase in D4(P<0.01)and significantly increase in D6(P<0.05),MYF5 protein significantly increase in D6(P<0.05).MYF6 extremely significantly increase in D2,D4 and D6(P<0.01),significantly increase in D0(P<0.05),MYF6 protein extremely significantly increase in D4 and D6(P<0.01).CKM extremely significantly increase in D2 and D4(P<0.01),CKM protein extremely significantly increase in D2(P<0.01),significantly increase in D6(P<0.05);MYH3 significantly increase in D4(P<0.05),MYH3 protein extremely significantly increase in D6(P<0.01),significantly increase in D0 and D4(P<0.05).MEF2 A extremely significantly increase in D2(P<0.01).In conclusion,this study find that the expression level of LRRN1 gene is the higher in the kidney and longissimus dorsi muscle of newborn and adult cattle,the muscle tissue of adult cattle is significantly higher than that of newborn cattle.Interference and overexpression of LRRN1 gene inhibit the proliferation of bovine myoblasts.LRRN1 gene promote the differentiation of bovine myoblasts,It is speculate that LRRN1 gene is an important candidate gene for muscle development of beef cattle.