Expression Analysis of Qinchuan Cattle (Bos taurus) LRRN1 Gene and Its Effect on Proliferation and Differentiation of Myoblasts
SONG Gui-Bing1, JIA Hong-Ru1, JIANG Lei1, ZAN Lin-Sen1,2, WANG Hong-Bao1,2*
1 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; 2 National Beef Cattle Improvement Center, Yangling 712100, China
Abstract：Leucine rich repeat neuronal protein-1 gene (LRRN1) encodes a type Ⅰ transmembrane protein that plays an important role in neural development and regeneration. The proliferation and differentiation of bovine myoblasts directly affect the growth and development of muscle and thus affect the yield of beef. In the present study, to investigate the expression pattern of LRRN1 gene in different tissues of bovine myoblasts and its effect on the proliferation and differentiation of bovine myoblasts, to determine its major role in myogenic differentiation. qRT-PCR was used to detect the expression pattern of LRRN1 in different tissues of newborn (3 d old) and adult (24 months old) Qinchuan cattle (Bos taurus), and to detect the expression characteristics of bovine skeletal muscle myoblasts at different differentiation stages in vitro. Adenovirus mediated shRNA and pcDNA3.1(+ ) were used to construct LRRN1 gene interference and overexpression vectors, which were transfected into Qinchuan bovine myoblasts. Then EdU method was used to detect cell proliferation, and qRT- PCR and Western blot were used to detect the expression changes of proliferation marker molecules. Myogenic differentiation inducing medium was used, then the phenotype of myotube formation was observed and the expression changes of differentiation marker molecules was detected. The results showed that LRRN1 was widely expressed in various tissues of Qinchuan cattle, and the expressions of LRRN1 in kidney and longissimus dorsi muscle of newborn cattle (3 d old) and adult cattle (24 months old) were significantly higher than that in other tissues. The expression trend of LRRN1 gene was consistent with the in vitro differentiation rate of myoblasts, which increased first and then decreased, peaked at the 4th day of differentiation, and gradually decreased thereafter. The results of interfering with LRRN1 gene expression in myoblasts showed that the proportion of proliferating cells with EdU positive staining was significantly reduced (P<0.05). Proliferation marker genes CCNA2 (P<0.01), CCNB1 (P<0.05), PCNA (P<0.01), CCND2 (P<0.05) and CDK1 (P<0.05) were down-regulated, and the protein expressions of CCND2, CDK1, CCNB1 and PCNA were also decreased. Overexpression of LRRN1 significantly inhibited the proliferation of myoblasts (P<0.05). The expressions of CCNA2 (P<0.05), CCNB1 (P<0.01), PCNA (P<0.01) and CCNE2 (P<0.05) were inhibited, and the protein expressions of CCNB1, PCNA and CDK1 were also decreased. By observing the cell differentiation phenotype, it was found that interfered LRRN1 inhibited myotube formation and the expression of differentiation marker genes MYOG (P<0.01), MYF5 (P<0.01), MYF6 (P<0.01), MYH3 (P<0.01) and CKM (P<0.05), and the protein expressions of MYOG, MYF5 and MYH3 were also decreased. Overexpression of LRRN1 promoted the formation of myotube, and the gene expressions of MYH3 (P<0.01), MYOD (P<0.05), MYF5 (P<0.05) and MYF6 (P<0.05) were significantly increased, as were the protein expressions of MYOD and MYH3. Above results suggest that LRRN1 might be a positive regulator of myoblast differentiation, but its effect on myoblast proliferation needs more comprehensive and in-depth studies. Therefore, LRRN1 has a potential regulatory effect on the growth and development of muscle tissue of Qinchuan cattle, and further studies on its function can be used for molecular breeding practice of Qinchuan cattles.
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