Characteristics and Expression of circ_003628 and Its Promoted Effect on Proliferation and Differentiation of Skeletal Muscle Satellite Cells in Goats

Abstract

In our previous a study, circ_003628 was one of the most highly expressed circular RNAs (circRNAs) in the Longissimus dorsi muscle of goats found by RNA-seq, suggesting that the circRNA may be important for caprine muscle growth and development. However, there have been no reports describing the molecular mechanisms by which circ_003628 regulates the activities of goat skeletal muscle satellite cells (SMSCs). In this study, reverse transcriptase-PCR (RT-PCR) and DNA sequencing were used to validate the authenticity of circ_003628, and its characteristics, expression profile and effect on goat SMSCs were also studied using real-time quantitative-PCR (RT-qPCR), EdU, CCK-8 and immunofluorescence assays. Circ_003628 is partially originated from 13 exons, 12 introns and 3’-untranslated regions (UTR) of caprine Myosin Heavy Chain 1 (MYH1), and 25 exons and 5’ UTR of Myosin Heavy Chain 4 (MYH4), as well as intergenic sequences between the two genes. A total of 77.07% of circ_003628 were located in the nuclei of goat SMSCs, while 22.93% were expressed in the cytoplasm. The circRNAs were only expressed in triceps brachii, quadriceps femoris and longissimus dorsi muscle tissues in nine caprine tissues investigated, with the highest expression level in longissimus dorsi muscle. The expression level of circ_003628 gradually increased during differentiation periods of goat SMSCs and reached the maximum on day 6 after differentiation. The small interfering RNA of circ_003628 (named si-circ_003628) inhibited the viability and proliferation of goat SMSCs, and also decreased the expression of four cell proliferation marker genes: paired box 7 (Pax7), cyclin-dependent kinase 2 (CDK2), CDK4 and CyclinD1 in goat SMSCs. Transfection of si-circ_003628 significantly decreased the area of MyHC-labeled myotubes of goat SMSCs, as well as the expression levels of three differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C). These results suggest that circ_003628 promotes the viability, proliferation, and differentiation of goat SMSCs, and they also provide an improved understanding of the roles of circ_003628 in skeletal muscle growth and development in goats.

Unlocking the Transcriptional Control of NCAPG in Bovine Myoblasts: CREB1 and MYOD1 as Key Players

“…Following the method described by Zhen and Zhu for isolating and culturing muscle cells [ 27 , 28 ], we discovered that the majority of cells were digested by collagenase, adhered to the 25 cm 2 cell culture bottle, and exhibited a pebble-like morphology within 12–36 h ( Figure 1 A,B). After a duration of 72 h, the cells underwent deformation, resulting in an elongated fused morphology, and displayed an increased rate of proliferation ( Figure 1 C).…”

“…Subsequently, the cells should be placed in a 5% CO 2 incubator (CCL-170T-8, ESCO, Shanghai, China) set at 37 °C. Following the method described by Zhen [ 28 ], myoblast differentiation was induced, and morphological changes in the cells were observed.…”

“…After collecting the cell clumps, they should be washed three times with PBS, centrifuged at 1000 rpm/min, and then resuspended in the primary myoblast growth medium containing 80% DMEM/F12 (C11330500BT, Hyclone, New York, NY, USA) (stored at 4 • C), 20% fetal bovine serum (A6904, Invigentech, Carlsbad, CA, USA) (stored at −20 • C). Subsequently, the cells should be placed in a 5% CO 2 incubator (CCL-170T-8, ESCO, Shanghai, China) set at 37 • C. Following the method described by Zhen [28], myoblast differentiation was induced, and morphological changes in the cells were observed.…”