Effect of LncRNA LOC106505926 on Myogenesis and Lipogenesis of Porcine Primary cells

Effect of LncRNA LOC106505926 on Myogenesis and Lipogenesis of Porcine Primary cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of LncRNA LOC106505926 on Myogenesis and Lipogenesis of Porcine Primary cells Mingyue Shi, Shuai Yang, Xiaolei Zhao, Di Sun, Yifei Li, Jingxian Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3977166/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Skeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving carcass quality and meat quality. In the present study, RNA sequencing was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age. Results The results showed that a total of 245 differentially expressed miRNAs were screened, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Bioinformatics predicted that long non-coding RNA (lncRNA) LOC106505926 and CXXC 5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC 5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC 5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN. Conclusions Collectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition. pig LOC106505926 skeletal muscle satellite cells preadipocytes myogenesis lipogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pork is the main source of meat for the Chinese people. Skeletal muscle accounts for about 40% of the body weight and 50%-75% of all body proteins [ 1 ]. The characteristics of skeletal muscle also directly affect pork quality. Muscle fiber numbers are already established in the embryonic period [ 2 ]. Muscle development after birth mainly depends on the proliferation and differentiation of skeletal muscle satellite cells [ 3 , 4 ], which are muscle tissue stem cells located between the muscle fiber substrate and the plasma coat [ 5 ]. Skeletal muscle satellite cells are activated in response to muscle injury and undergo cell proliferation, differentiation, and fusion to form new myotubes [ 6 ]. Skeletal muscle development is a complex process, current studies have found that many transcription factors are involved in the regulation of myogenesis, such as myogenic regulatory factors (MRFs) including Myf 5, MyoD , MyoG , Myf 6, myocyte enhancer factor-2 (MEF-2) family [ 7 – 9 ] and Pax family members [ 10 , 11 ]. Recent studies have found that, in addition to transcription factors, non-coding RNAs are also involved in the regulation of muscle development, including miRNAs and lncRNAs. Fat deposition is also an important biological process that affects the quality and growth efficiency of pork. Adipocytes originate from the embryonic mesoderm and are formed by the differentiation of preadipocytes. The transformation of preadipocytes to mature adipocytes determines the process of fat deposition [ 12 ]. Peroxisome proliferator-activated receptor γ (PPARγ) [ 13 ], fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4) [ 14 ] and many key regulators of adipogenic differentiation play an important role in adipocyte differentiation and deposition. Many lncRNAs have been found to play a role in adipocyte thermogenesis [ 15 , 16 ], adipocyte metabolism [ 17 ] and adipocyte differentiation [ 18 ]. Long non-coding RNA (lncRNA) is a kind of linear RNA with a length of more than 200 nt, which has no protein coding ability and has a wide subcellular distribution in cells [ 19 ]. This wide subcellular distribution determines the diversity of its functional mechanism. The mechanism of lncRNAs acting as ceRNAs has attracted much attention [ 20 ], but lncRNAs also have other mechanisms that play an important role in a variety of biological processes. H19 is one of the most known lncRNA, and it plays a key role in the differentiation of skeletal muscle [ 21 , 22 ]. By competitively binding to miR-140-5p, H19 inhibits the differentiation of skeletal muscle satellite cells in porcine [ 23 ]. In vivo, overexpression of H19 improved insulin sensitivity and mitochondrial biogenesis, silencing H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown adipocytes [ 24 ]. Previous studies have found an obesity-related lncRNA lnc-ORA, which had a seven-fold higher expression in ob/ob mice than in WT mice. Lnc-ORA knockdown inhibits adipocyte differentiation by regulating the PI3K/AKT/mTOR signaling pathway [ 25 ]. In addition, lnc-ORA was found to play a role in myogenesis. It can inhibit skeletal muscle myogenesis and reduce the stability of myogenic genes by acting as a sponge for miR-532-3p or interacting with insulin-like growth factor 2 mRNA binding protein 2 [ 26 ]. These indicate that lncRNA plays a role in a variety of biological processes through different mechanisms and lncRNA is indispensable in the development of various tissues. In the present research, whole transcriptome sequencing was performed on longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90 and 180 days of age to screen candidate miRNAs that can regulate porcine myogenesis. The LOC106505926/miR-22-5p/ CXXC 5 regulatory network was constructed to regulate the proliferation and differentiation of PSCs. In addition, LOC106505926 was testified to inhibit the differentiation of preadipocytes by directly binding to FASN. This study may provide a molecular basis for understanding the development process of porcine skeletal muscle and adipose tissue. Results Candidate miRNAs regulating muscle development were screened by RNA-seq Whole transcriptome sequencing was performed on LDM of Jinfen White pigs at 1, 90 and 180 days of age. On average, 14.45 million, 17.68 million and 15.07 million raw reads and 14.12 million, 17.44 million, 14.87 million clean reads were obtained from LDM of Jinfen White pigs at 1, 90 and 180 days of age respectively. In addition, the error rate of all sequencing data sets was less than 0.01%, the value of Q20 was above 98%, Q30 value was above 94% and the average GC content was 49%, Specific data are presented in the supplementary Table S3 . A total of 245 differentially expressed miRNAs (DE miRNAs) were screened (Fig. 1 A). Specifically, 100 miRNAs were up-regulated and 89 miRNAs were down-regulated in 90-day-old muscle tissues compared with 1-day-old (Fig. 1 B). There were 106 up-regulated and 106 down-regulated miRNAs in 180-day-old muscle tissues compared with 1-day-old (Fig. 1 C). Compared with the 90-day-old group, there were 37 up-regulated miRNAs and 44 down-regulated miRNAs in the 180-day-old group (Fig. 1 D). The heat map showed the DE miRNAs expression patterns at different days of age (Fig. 1 E). GO and KEGG results showed that the target genes of DE miRNAs were mainly enriched in MAPK, AMPK, JAK-STAT, VEGF and other signaling pathways, which were related to muscle development (Fig. 1 F and 1 G). Thus, DE miRNAs may be involved in the regulation of porcine myogenesis. Among DE miRNAs, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group, with the log 2 (fold change) approaching 3. Therefore, miR-22-5p attracted our attention and we decided to further investigate its function. MiR-22-5p inhibits the proliferation and differentiation of PSCs The expression of miR-22-5p was extremely significant increased after transfected with miR-22-5p mimic (Fig. 2 A, P < 0.01). The expression of proliferation marker genes was significantly decreased after transfected with miR-22-5p mimic (Fig. 2 B, P < 0.05). the number of EdU positive cells was extremely significant decreased (Fig. 2 C, P < 0.01). After transfected with miR-22-5p inhibitor, the expression of miR-22-5p was extremely significant decreased (Fig. 2 D, P < 0.01) and the expression of proliferation marker genes was extremely significant increased (Fig. 2 E, P < 0.01) and the number of EdU positive cells was extremely significant increased (Fig. 2 F, P < 0.01). These results indicated that miR-22-5p inhibited the proliferation of PSCs. To explore the effect of miR-22-5p on the myogenic differentiation of PSCs, the expression changes of key myogenic factors were detected. The results showed that, after transfected with miR-22-5p mimic, the expression of MyoG , MyoD and MHC were extremely significant decreased (Fig. 2 G, P < 0.01). Western blot showed the same results (Fig. 2 H). The immunofluorescence staining results showed that the number of myotubes was extremely significant decreased in miR-22-5p mimic group (Fig. 2 I, P < 0.01). After transfected with miR-22-5p inhibitor, the opposite results were shown (Fig. 2 J-L). The above results proved that miR-22-5p inhibited the differentiation of PSCs. MiR-22-5p binds to the 3′UTR of CXXC5 and LOC106505926 Based on sequencing results, there were multiple mRNAs and lncRNAs associated with miR-22-5p. Among them, CXXC5 and LOC106505926 had a stronger negative correlation with miR-22-5p (Fig. 3 A), with Pearson coefficient of -0.896 and − 0.852 (Fig. 3 B). Expression patterns analysis found that the expression of miR-22-5p showed an upward trend with the increase of age, while CXXC5 expression showed a downward trend (Fig. 3 C). Expression profiling revealed that CXXC5 had the highest expression in the liver and LDM (Fig. 3 D, P < 0.01). The expression of CXXC5 and LOC106505926 was significantly decreased after transfection of miR-22-5p mimics (Fig. 3 E, F, P < 0.05), while transfection of miR-22-5p inhibitor showed the opposite results (Fig. 3 G, H). RNAhybird prediction showed that the 3′UTR of CXXC5 and LOC106505926 could bind to the seed sequence of miR-22-5p (Fig. 3 I, J). The wild-type and mutant vectors of 3′UTR of CXXC5 and LOC106505926 were constructed respectively. The luciferase activity of miR-22-5p mimics and CXXC5-wt-psiCHECK2 group was extremely significant lower than other groups (Fig. 3 K, P < 0.01), The luciferase activity of miR-22-5p mimics and LOC106505926-wt-psiCHECK2 group was also extremely significant lower than other groups (Fig. 3 L, P < 0.01), indicated that miR-22-5p can bind to CXXC5 and LOC106505926, then regulate their expression. Biological characteristics of LOC106505926 LOC106505926 is located on chromosome 14 of pig (Fig. 4 A). To verify the existence of LOC106505926, LOC106505926 was amplified by using the cDNA of the longissimus dorsi muscle of Jinfen white pig as a template, and 731 bp LOC106505926 partial sequence was obtained, which confirmed the existence of LOC106505926 (Fig. 4 B). The subcellular localization pattern of LOC106505926 was predicted by online software lncLocator, the results showed that LOC106505926 was mostly located in the cytoplasm, subsequently, RNA was extracted from the cytoplasm and nucleus of skeletal muscle satellite cells, the results also showed that LOC106505926 was mostly localized in the cytoplasm and also expressed in the nucleus (Fig. 4 C). We detected the expression pattern of LOC106505926, its relative expression level showed a downward trend with the increase of age in Jinfen white pigs (Fig. 4 D). In addition, LOC106505926 has a high expression level in the lung, subcutaneous fat and longissimus dorsi muscle (Fig. 4 E). LOC106505926 promotes the proliferation and differentiation of PSCs The overexpression vector of LOC106505926 was constructed and transfected into PSCs. The expression of LOC106505926 in OE-LOC106505926 group was extremely significant increased (Fig. 5 A, P < 0.01). The expression of PCNA , cyclin D , CDK 1 and CDK 4 was extremely significant increased ( P < 0.01), the expression of Ki 67 was significantly increased (Fig. 5 B, P < 0.05) and the number of EdU-positive cells was significantly increased (Fig. 5 C, P < 0.05). Three siRNA sequences of LOC106505926 were synthesized and detected the interference efficiency after transfection of PSCs (Fig. 5 D). The siLOC106505926-1 was selected to transfect PSCs to detect the expression of proliferation marker genes. Compared with the si-NC group, the expression of proliferation marker genes was extremely significant reduced (Fig. 5 E, P < 0.01), the EdU results showed that the number of positive cells was extremely significant reduced (Fig. 5 F, P < 0.01). Then, the effect of LOC106505926 on PSC differentiation was explored. The results showed that after transfected with OE-LOC106505926, the expression of MyoD was extremely significant increased ( P < 0.01), the expression of MyoG was significantly increased ( P < 0.05), there was no significant difference in MHC expression (Fig. 5 G). Western blot results showed that the expression of MYOD, MYOG and MHC were significantly increased (Fig. 5 H, P < 0.01). Immunofluorescence staining results showed that the number of myotubes was extremely significant increased in OE-LOC106505926 group (Fig. 5 I, P < 0.01). While after transfection with si-LOC106505926, the opposite results were shown (Fig. 5 J-L). CXXC5 promotes the proliferation and differentiation of PSCs The overexpression vector of CXXC5 was constructed and the immunofluorescence results showed a positive result after transfected of PSCs (Fig. 6 A). Compared with OE-NC group, the expression of CXXC5 protein in OE-CXXC5 group was extremely significant increased (Fig. 6 B, P < 0.01). The effect of CXXC5 on the cell cycle was detected by flow cytometry, the results showed that the proportion of S phase cells was extremely significant increased (Fig. 6 C, P < 0.01), accompanied with the significant upregulation of PCNA , Cyclin D , CDK 1 and CDK 4 (Fig. 6 D, P < 0.05). EdU results showed that the number of positive cells increased extremely significant after transfection of OE-CXXC5 (Fig. 6 E, P < 0.01). Three siRNA sequences of CXXC5 were designed to transfect PSCs and the interference efficiency of three siRNAs was detected. The results showed that siCXXC5-3 had the highest interference efficiency (Fig. 6 F). Transfection of siCXXC5-3 in PSCs showed the opposite results to transfection of OE-CXXC5 (Fig. 6 G, H). Transfected PSCs were induced to differentiate to detect the effect of CXXC5 on PSCs differentiation. The expression of MyoD , MyoG and MHC were significantly increased after transfection of OE-CXXC5 (Fig. 6 I, P < 0.05). Western blot results showed the same results (Fig. 6 J, P < 0.05). The immunofluorescence assay showed that the number of myotubes in OE-CXXC5 group was extremely significant increased compared with OE-NC group (Fig. 6 K, P < 0.01). In addition, the effect of siCXXC5-3 on the differentiation of PSCs was detected. The results were contrary to the transfection of OE-CXXC5 (Fig. 6 L-N), indicating that CXXC5 promotes the differentiation of PSCs. The rescue assay certified LOC106505926/miR-22-5p/CXXC5 regulates PSCs proliferation and differentiation To further explore the relationship between miR-22-5p and CXXC5, rescue experiments were performed. Overexpression of CXXC5 alleviates the inhibitory effect of miR-22-5p on the expression of PCNA and MYOD (Fig. 7 A). In addition, transfected with OE-LOC106505926 also alleviates the inhibitory effect of miR-22-5p on the proliferation and myogenic differentiation of PSCs (Fig. 7 B). Thus, LOC106505926 may reduce the inhibitory effect of miR-22-5p on CXXC5 by competitively binding miR-22-5p. LOC106505926 inhibits preadipocyte differentiation and directly interacts with FASN After si-LOC106505926 was transfected in porcine precursor adipocytes, Oil Red O staining showed that LOC106505926 could inhibit lipid-droplet formation (Fig. 8 A), the mRNA and protein levels of lipogenic factors were dramatically improved in the si-LOC106505926 group compared with the si-NC group (Fig. 8 B, C). RNA pull-down assay was performed with biotinylated LOC106505926 to identify the LOC106505926 interacting proteins. After SDS-PAGE, the gel was stained with silver nitrate (Fig. 8 D) and analyzed by MS (Fig. 8 E). The proteins that specifically bind to LOC106505926 (including FASN) were identified. The association of LOC106505926 with FASN was validated by Western blot, and the results show that the target protein FASN was detected in LOC106505926 pull-down protein samples but not in the samples associated with antisense LOC106505926 (Fig. 8 F). Discussion In recent years, there were many examples that miRNAs affect skeletal muscle development by participating in various processes. MiR-142a-5p was able to function as an important regulator of denervation-induced skeletal muscle atrophy via targeting MFN1 [ 27 ]. MiR-135a-5p regulated skeletal muscle fibrosis by Tgfbr2/Smad4 signaling pathway [ 28 ]. MiR-491 can specifically bind to the 3′UTR of myomaker and regulates myocyte differentiation and muscle regeneration [ 29 ]. Previous studies demonstrated that the expression of miR-22-5p in mice was significantly up-regulated with the increase of age [ 30 ]. MiR-22-5p has also been testified to play important roles in a variety of diseases, such as acute myocardial infarction, myocardial damage and hepatocellular carcinoma [ 31 – 33 ]. MiR-22-5p regulated the proliferation of human osteoblasts via MALAT1 and VEGF [ 34 ]. Many miRNAs have been proven to be involved in the regulation of myoblast proliferation and differentiation [ 35 – 38 ]. At present, the regulation of myogenesis by miR-22-5p remains unclear. In this study, we demonstrated that miR-22-5p was differentially expressed in the LDM of Jinfen White pigs at three developmental stages, suggesting its involvement in the regulation of porcine muscle development. CXXC5 is a member of the CXXC-type zinc finger protein family, which has a CXXC-type zinc finger (ZF-CxxC) protein structural domain. The CXXC structural domain normally binds to unmethylated CpG islands in gene promoters and plays a key role in epigenetic regulation [ 39 , 40 ]. CXXC5 does not have any catalytic domain, but it has been shown to regulate transcription by directly binding to DNA [ 41 ]. CXXC5 was expressed in a variety of tissues [ 42 ], and was involved in the development of nephron and heart, as well as the differentiation of osteoblasts, oligodendrocytes and endothelial cells [ 43 – 45 ]. Recent studies showed that CXXC5 affected cell cycle regulation and was required for myeloid cell maturation and differentiation [ 46 ]. In addition, CXXC5 regulates multiple physiological processes via several important cellular signaling pathways. It was demonstrated that CXXC5 mediated the BMP signaling pathway and induced endothelial cell differentiation and vessel formation [ 44 ]. CXXC5 also participated in the Wnt signaling pathway to regulate the differentiation of neural stem cells [ 47 ] and osteoblast [ 43 ]. Moreover, the ZF-CXXC domain of CXXC5 interacted with the SMAD2/3/4 and activated TGF-β signaling and BMP signaling to promote normal heart development [ 48 ] and mediate TNF-α induced apoptosis [ 49 ]. Besides, CXXC5 was involved in cell cycle arrest and DNA repair by activating ATM-p53 signaling axis [ 50 ]. Therefore, CXXC5 may be an important medium for various signal inputs. CXXC5 was necessary for the myogenic differentiation of C2C12 cells [ 51 ]. Previous studies showed that CXXC5 regulated the regeneration of skeletal muscle through TGF-β, Wnt, Vitamin D signaling pathways [ 49 , 52 – 54 ]. Wu demonstrated that CXXC5 may be associated with skeletal muscle development and Ca 2+ release from muscle via whole-genome sequencing in Qingyu pigs [ 23 ]. In the present study, CXXC5 was proved to promote the proliferation and differentiation of PSCs, which is consistent with previous research. This study demonstrated that miR-22-5p inhibited the proliferation and differentiation of PSCs via binding to the 3′UTR of CXXC5. With the discovery of a large number of lncRNAs, people have gradually realized that lncRNA has a very important regulatory function and can participate in various biological processes and pathway regulation. In recent years, lncRNA has played an important role in participating in transcription as a transcriptional regulator [ 55 ], regulating chromatin stability [ 56 ], regulating subcellular organs [ 57 ] and regulating miRNA expression [ 58 ]. Studies have found that lncRNA-TBP promotes myoblast differentiation, reduces fat deposition, activates slow muscle phenotype and induces muscle hypertrophy. It is worth noting that lncRNA-TBP, as a regulatory RNA, directly interacts with TBP protein to regulate TBP-target genes [ 59 ]. In a study of lipogenic transdifferentiation of myoblasts, 114 core lncRNAs were identified, lncRNA-GM43652 gene was a potential regulator of adipogenesis in muscle cells [ 60 ]. In our study, LOC106505926 regulates the differentiation of satellite cells through the ceRNA mechanism, while in adipocytes, it inhibits the differentiation of preadipocytes by directly binding to FASN. Our study provides a basis for a variety of biological functions and specific mechanisms of lncRNAs. Conclusion The present study demonstrated that miR-22-5p inhibited the proliferation and differentiation of PSCs via binding to the 3′UTR of CXXC5. LOC106505926 was found to play a role in the proliferation and differentiation of PSCs as a molecular sponge of miR-22-5p. In addition, LOC106505926 inhibited the differentiation of preadipocytes by directly binding to FASN. These results provide insights into the molecular regulation mechanism in porcine skeletal muscle development and fat deposition. Materials and methods Tissues collection All experimental protocols were approved by the Ethics Committee of Shanxi Agricultural University (Shanxi, China, Approval No. SXAU-EAW-2021Sus.AB.0926001). LDM from Jinfen White pigs was collected at 1, 90 and 180 days of age. Seven tissues including heart, liver, spleen, lung, kidney, longissimus dorsi, and subcutaneous fat from 3-month-old Jinfen White pigs were collected strictly according to the anatomical structure of the pigs. All samples were stored at -80 ℃ for later use. Construction of library and identification of differentially expressed miRNAs The libraries were sequenced on an Illumina HiseqTM 2500 platform. HISAT2 (version 2.0.2) was used to get the clean reads mapped to the pig reference genome (Sus scrofa 11.1). The sequencing data was publicly available in the NCBI SRA database ( https://www.ncbi.nlm.nih.gov/sra/ ), with accession number: PRJNA867525. The expression level of miRNAs was calculated using the HTseq software (0.6.1). Transcript per million (TPM) values were used to determine miRNA counts and levels of expression [ 61 ]. Subsequently, differentially expressed miRNAs were identified using the R packages DEG seq2 [ 62 ]. PSCs isolation, culture and differentiation PSCs were isolated from 3-day-old Jinfen White male pigs. The skin of pigs was disinfected with 75% alcohol after slaughter. The LDM were collected and kept in PBS supplement with 1% penicillin-streptomycin. The tissue was digested with digestive solution (DMEM containing 15% collagenase II, 15% dispase II, 1% penicillin-streptomycin) at 37 ℃ for 1 h, and the digestion was terminated with DMEM (including 10% FBS). The mixture was filtered through a 100 µm cell strainer and centrifuged at 1500 r/min for 10 min. Then, the supernatant was removed and the precipitate was resuspended with PBS. Next, 70 and 40 µm cell strainers were used to filter the suspension. The supernatant was removed and resuspended with erythrocyte lysate, an equal volume of PBS was added, centrifuged at 1000 r/min for 5 min. Last, the cells were suspended with 20% FBS (Gibco, Life Technologies, United States) and 1% penicillin-streptomycin. Cells were cultured in thermostatic incubator (Thermo Fisher, United States) for 1 h, the cell suspension was transferred to the new culture dishes, which were coated with Matrigel (Corning, United States). Isolated PSCs were cultured on Matrigel coated 10 cm plates (Corning, United States) with the medium (containing 20% FBS, 1% penicillin-streptomycin, 2.5 ng/mL human recombinant basic fibroblast growth factor) (Gibco, United States). When the cells density was up to 70%, the differentiation medium (containing 5% horse serum) (HyClone, United States) was used to induce cell myogenic differentiation, the medium was changed every 2 days. Porcine preadipocytes isolation, culture and differentiation The subcutaneous adipose tissue of piglets was collected and placed in a sterile petri dish to remove fascia and connective tissue. Then, the tissue was cut into pieces and digested with 2 mg/mL type I collagenase (Gibco, USA) for 1 h, the digestion was terminated with DMEM (including 10% FBS). After filtration, the cells were centrifuged at 1000 r/min for 10 minutes, resuspended and inoculated into a petri dish for culture. Preadipocytes were cultured with DMEM (including 10% FBS, 1% penicillin-streptomycin). When the cell density reached 80% -90%, the preadipocytes were digested with 0.25% trypsin. Replace the medium every two days. When the cell density reached 95%, lipogenic induction was performed. The complete induction medium was supplemented with dexamethasone (1 µmol/L), indomethacin (100 µmol/L), IBMX (0.5 mmol/L) and insulin (10 µg/mL). After 4 days of differentiation, the maintenance medium was replaced. The maintenance medium was a complete medium supplemented with insulin (10 µg/mL). After that, the maintenance medium was replaced every 2 days, and the cells were observed under a microscope. Nuclear and cytoplasmic RNA fractionation PSCs were collected and washed twice with cold PBS and centrifuged at 500 g for 3 min. The precipitate was resuspended with 0.3 mL Cell Fractionation Buffer, placed on ice for 10 min, and centrifuged at 500 g at 4°C for 3 min. The supernatant was transferred to a new 1.5 mL micro-centrifugal tube without RNase, and 0.3 mL 2×Lysis/Binding Buffer was added and mixed, which was cytoplasmic RNA. The precipitate was washed with 0.3 mL Cell Disruption Buffer, and 0.3 mL 2×Lysis/Binding Buffer was added after shaking and mixing to obtain nuclear RNA. Then, 0.3 mL absolute ethanol was added to the cytoplasm and nucleus RNA, poured into the filter extraction, centrifuged at 1,400 r/min for 1 min, discarded the liquid, added Wash Solution centrifuged at 1,400 r/min for 1 min, repeated several times, 0.3 mL Elution Solution was poured into the filter, replaced the new centrifuge tube, centrifuged at 10,000 r/min for 30 s to obtain RNA and stored at -80°C. Cell transfection Firstly, the overexpression vector of CXXC5: pHBLV-CXXC5-puro (OE-CXXC5) and control vector of CXXC5: pHBLV-CMVIE-puro control (NC-CXXC5) were constructed and synthesized. When the density of HEK-293T cells reached to 70% confluence, the Lipo3000 (Invitrigen, USA) was used to transfect the plasmid and two package plasmids psPAX2 and pMD2G (Public Protein/Plasmid Library, Jiangsu, China) at the ratio of 1:1:1 for 6 h following the introduction. The supernatants were collected and stored at -80 ℃ for infection. After 48 h of transfection, the fluorescence rate was observed. The overexpression vector of LOC106505926 was synthesized by Tsingke Biotechnology according to the sequence on NCBI. The RNA oligo against of porcine CXXC5, miR-22-5p and LOC106505926 were purchased from GenePharma (Shanghai, China). The 50 nM RNA oligo specific were transfected to cells at a density about 60%. The siRNA sequences were shown in the supplementary Table S1 . Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from samples according to the instruction of TaKaRa RNAiso Plus (Takara, Japan). The cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). The relative expression level of genes was normalized by 18sRNA . Primers′ information was shown in Table S2 . The qRT-PCR reaction system: cDNA 2 µL, 2×SYBR Premix Ex Taq II 10 µL, forward and reverse primers 0.5 µL, RNAase Free ddH 2 O supplemented to 20 µL. Reaction procedure: 95°C 30 s; 95°C 5 s, 58°C 30 s, 35 cycles; the melting curve program is 95°C 15 s, 60°C 35 s, 95°C. Each sample was repeated three times. For miRNAs, U6 snRNA was selected as the internal control. The primers of miRNAs were designed using the stem-loop approach and the information of primers was shown in Supplementary Table S2 . The cDNA was synthesized according to the protocol of miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China). Each 20 µL qRT-PCR reaction mixture contained 2×miRNA Universal SYBR qPCR Master Mix 10 µL (Vazyme, China, MQ101), cDNA 1 µL, nuclease-free H 2 O 7.8 µL, 0.5 µL specific primer and mQ primer R. The following parameters were used for qRT-PCR: pre-denaturation for 5 min at 95 ℃, then 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s, and 72 ℃ for 8 s, the melting curve program is 95°C 15 s, 60°C 60 s, 95°C 15 s. Relative expression levels of genes and miRNAs were calculated by the 2 −ΔΔCt method. Flow cytometry After 48 h of transfection, the cells were collected and resuspended with 75% ethanol when the cell density reached 90%. The cell suspension was placed at -20°C for 10 h. After centrifugation, RNase A was added and incubated at 37°C for 30 min. PI staining solution was added and incubated at 4°C for 30 min. The proportion of cells in different cell cycles was analyzed by flow cytometry. EdU assay PSCs were incubated with EdU-containing medium for 2 h, then, fixed with 4% paraformaldehyde for 30 min, washed with PBS, incubated with 2 mg/mL glycine for 5 min, washed with PBS for 5 min, incubated with 0.5% TritonX-100 for 10 min, then incubated with Apollo staining reaction solution (Ribobio, China) for 30 min, washed with PBS and stained with DAPI reaction solution for 10 min. Observed with a fluorescence microscope. Dual-luciferase reporter assay The wild-type of 3′UTR of CXXC5 (CXXC5-wt-psiCHECK2) and mutant-type of 3′UTR of CXXC5 (CXXC5-wut-psiCHECK2) were constructed and inserted into psi-CHECK2 vectors. HEK-293T cells were seeded and cultured on 24-well plates. Then, cells were co-transfected with the miRNAs (mimic NC, miR-22-5p mimic, inhibitor NC or miR-22-5p inhibitor) and the CXXC5-wt-psiCHECK2 or CXXC5-wut-psiCHECK2 vectors. The cells were collected after 24 h of cell transfection. A standard plate reader (BioTek, Vermont, United States) was used to measure the luciferase activity. Western blot In PSCs, the expression levels of MYOG, MYOD, MHC and CXXC5 protein were detected. The protein expression levels of FABP4, PPARγ, SREBP1 and FASN were detected in preadipocytes. Transfected cells were lysed in RIPA buffer with 1% PMSF. 5×Buffer was added to the sample and denatured at 100°C for 10 min. SDS-PAGE gel electrophoresis was performed at 80 V 30 min, 120 V 90 min. Then transferred them onto a PVDF membrane and non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Then, they were incubated with 1:1000 diluted polyclonal rabbit MYOG (Abclonal, China), MYOD (Proteintech, China), CXXC5 (Bioss, China), 1:500 diluted polyclonal mouse MHC (DSHB, America), 1:1000 diluted polyclonal rabbit FABP4 (Proteintech, China), PPARγ (Proteintech, China), SREBP1 (Proteintech, China) and FASN (Proteintech, China) at 4 ℃ overnight. The blots were subsequently incubated with secondary antibody (1:10000) for 1 h. Secondary antibody include goat anti-mouse IgG (Servicebio, China) and goat anti-rabbit IgG (Servicebio, China). GAPDH (Servicebio, China) was used as an endogenous protein for normalization. Image J software was used to conduct quantitative analysis of western blot results according to the gray value of the strip. Immunofluorescent analysis Cells were fixed with precooled 4% paraformaldehyde for 30 min. Then permeabilized in 0.5% Triton X-100 for 10 min. After that, cells were blocked with 3% bovine serum albumin (BSA), and incubated with 1:1000 diluted polyclonal mouse MHC (DSHB, America) antibodies overnight at 4 ℃. Cells were washed with PBS for 1–3 times and incubated with goat anti-mouse IgG antibodies (Bioss, China) for 1 h at room temperature. Then, the cells were stained with Hoechst 33342 (Sanofi-Aventis, Germany) for 10 min. Images were acquired by a Leica SP8 confocal microscope. Immunofluorescence results were quantified by Image J software. Oil red O staining After lipogenic differentiation of porcine preadipocytes, oil red O staining was performed, 4% paraformaldehyde was added, fixed at room temperature for 30 min, and washed with PBS. Oil red O dye solution (Solarbio, China) was added and incubated at room temperature for 30 min. After washing with PBS, the cells were observed under an inverted microscope. Isopropanol was added to extract triglyceride, and the OD value was detected by enzyme-labeled instrument. Silver nitrate staining Take out the gel after SDS-PAGE, put it in the staining box, wash the gel twice with distilled water, then pour out the distilled water, add 25% ethanol to denature for 3 minutes, recover the ethanol, wash the gel with distilled water, add 0.1% silver nitrate solution, incubate for 30 minutes on the shaker, and fix the gel on the glass plate to observe the staining. RNA Pull-Down Assay The biotinylated probe of LOC106505926 was synthesized by RiboBio. Preparation of cell lysates with standard lysis buffers, wash the beads with 20mM Tris (pH 7.5), add an equal volume of 1×RNA Capture Buffer, resuspend beads by pipetting or vortexing, then, add 50 pmol of labeled RNA to the beads, mix gently by pipetting, incubate for 15–30 minutes at room temperature with agitation. The RNAs were targeted with streptavidin beads, add 100 µL of Master Mix (including Protein-RNA Binding Buffer, glycerol, salts, lysate, nuclease-free water) to the RNA-bound beads, incubate 30–60 minutes at 4°C with agitation or rotation. The protein complexes were obtained after several elutions. Finally, the protein complexes associated with the beads were analyzed by mass spectrometry (MS) and western blot. Statistical analysis All results were presented as mean ± standard error of mean (SEM). Multigroup comparisons of the means were carried out by a one-way analysis of variance test. The two-tailed t-test was performed for differences analysis between the two groups. * represents P < 0.05, ** represents P < 0.01. Abbreviations LDM Longissimus dorsi muscle PSCs Porcine skeletal muscle satellite cells LncRNA Long non-coding RNA GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes CXXC5 CXXC finger protein 5 MRFs Myogenic regulatory factors MYOD Myogenic differentiation MYOG Myogenin MHC Myosin heavy chain MEF2 Myocyte enhancer factor 2 Myf5 Myogenic factor 5 Myf6 Myogenic factor 6 GAPDH Glyceraldehyde-3-phosphate dehydrogenase PPARγ Peroxisome proliferator-activated receptorγ FABP4 Fatty acid binding protein 4 SREBP1 Sterol regulatory element binding transcription factor 1 FASN fatty acid synthase. Declarations Authors′ contributions All authors contributed to the study's conception and design. Study conception and design: MS, SY, GC Conducted RNA-seq and bioinformatics clustering: XZ, SD, YL, JY, performed experimental study: MS, SY, SW, WL. Article drafting and revising: ML, CC, XG, BL, GC. Funding acquisition: CL, GC. Funding This work was supported by the National Natural Science Foundation of China (31872336); the Key Research and Development Project of Shanxi Privince (202102140601005); Special Funds for Scholars Support Program of Shanxi Province (2017); the University Science and Technology Innovation Project of Shanxi Province (2021L158); Science and Technology Innovation Foundation of Shanxi Agricultural University (2020BQ56). Conflict of interest The authors state that they have no competing economic interests or personal relationships. Data Availability The original data for the RNA-seq data were submitted to the SRA Database (BioProject ID: PRJNA867525). 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Bioinformatics. 2010;26(1):136–8. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Additional file 1: Table S1. The siRNA sequences TableS2.docx Additional file 2: Table S2. Primers of qRT-PCR TableS3.docx Additional file 3: Table S3. Quality assessment of sRNA sequencing data Rawimages.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Mar, 2024 Reviews received at journal 15 Mar, 2024 Reviewers agreed at journal 06 Mar, 2024 Reviewers invited by journal 29 Feb, 2024 Editor assigned by journal 29 Feb, 2024 Editor invited by journal 29 Feb, 2024 Submission checks completed at journal 29 Feb, 2024 First submitted to journal 21 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3977166","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276005470,"identity":"a3747639-45a5-4000-a0ff-1852d2e8e9d3","order_by":0,"name":"Mingyue Shi","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Shi","suffix":""},{"id":276005471,"identity":"f400d806-3c03-4b45-b8be-b4ec99e1b20e","order_by":1,"name":"Shuai Yang","email":"","orcid":"","institution":"Shanxi Animal Husbandry Technology 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00:50:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3977166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3977166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51948279,"identity":"50d2eb02-d75b-4921-84c6-73a61a8cb199","added_by":"auto","created_at":"2024-03-04 11:57:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":892097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCandidate miRNAs regulating muscle development were screened.\u003c/strong\u003e (A) Venn diagram of DE miRNAs. (B-D) Volcano plot of DE miRNAs. (E) Heat map of DE miRNAs. (F) GO enrichment results of DE miRNAs. (G) The results of KEGG analysis of DE miRNAs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/513cc445617059bebd8731ac.png"},{"id":51948283,"identity":"e80ec81a-eb96-4bbe-af21-b8f9cfc21c0e","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1348783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of miR-22-5p on the proliferation and differentiation of PSCs. \u003c/strong\u003e(A) Cell transfection efficiency of miR-22-5p mimic. (B) The expression changes of proliferation marker genes after transfected with miR-22-5p mimic. (C) The EdU results after transfected with miR-22-5p mimic. (D) Cell transfection efficiency of miR-22-5p inhibitor. (E) The expression changes of proliferation marker genes after transfected with miR-22-5p inhibitor. (F) The EdU results after transfected with miR-22-5p inhibitor. (G) The expression changes of differentiation marker genes after transfected with miR-22-5p mimic at mRNA level. (H) The expression changes of differentiation marker genes after transfected with miR-22-5p mimic at protein level. (I) The results of immunofluorescence after transfected with miR-22-5p mimic. (J) The expression changes of differentiation marker genes after transfected with miR-22-5p inhibitor at mRNA level. (K) The expression changes of differentiation marker genes after transfected with miR-22-5p inhibitor at protein level. (L) The results of immunofluorescence after transfected with miR-22-5p inhibitor.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/9aa732b16db8727de1bd2dce.png"},{"id":51948460,"identity":"9ad51869-d839-4d47-8fa9-8bb19df83080","added_by":"auto","created_at":"2024-03-04 12:05:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":616827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-22-5p binds to the 3′UTR of CXXC5 and LOC106505926. \u003c/strong\u003e(A) The correlation between miR-22-5p expression and its target genes. Blue from light to deep indicates that the pearson coefficient changes from 0 to -1. (B) The pearson coefficient between miR-22-5p and CXXC5, miR-22-5p and LOC106505926. (C) The expression levels of miR-22-5p and CXXC5 in LDM at three developmental stages. (D) Expression profiling of CXXC5. (E) Effect of miR-22-5p mimics transfection on relative expression of CXXC5. (F) Effect of miR-22-5p inhibitor transfection on relative expression of CXXC5. (G) Effect of miR-22-5p mimics transfection on relative expression of LOC106505926. (H) Effect of miR-22-5p inhibitor transfection on relative expression of LOC106505926. (I) RNAhybird predicts the binding of miR-22-5p to CXXC5-3′UTR. (J) RNAhybird predicts the binding of miR-22-5p to LOC106505926. (K) Dual-luciferase reporter assay verifies the binding of miR-22-5p to CXXC5-3′UTR. (L) Dual-luciferase reporter assay verifies the binding of miR-22-5p to LOC106505926. Different uppercase indicates extremely significant differences.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/4fe95c233e1b2dd478554905.png"},{"id":51948461,"identity":"b923e510-357c-44ad-9846-8c3bc636c928","added_by":"auto","created_at":"2024-03-04 12:05:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiological characteristics of LOC106505926.\u003c/strong\u003e (A) The location of LOC106505926 on the chromosome. (B) Agarose gel electrophoresis of LOC106505926. (C) The results of nuclear cytoplasmic localization of LOC106505926. (D) Temporal expression characteristics of LOC106505926. (E) The tissue expression characteristics of LOC106505926. Different uppercase indicates extremely significant differences.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/788d0dde050b1458b6550b24.png"},{"id":51948462,"identity":"a0e05c45-fd8d-4633-841e-d061b3997cfd","added_by":"auto","created_at":"2024-03-04 12:05:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1387761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of LOC106505926 on the proliferation and differentiation of PSCs.\u003c/strong\u003e(A) Cell transfection efficiency of OE-LOC106505926. (B) The expression changes of proliferation marker genes after transfected with OE-LOC106505926. (C) The EdU results after transfected with OE-LOC106505926. (D) Cell transfection efficiency of siRNA of LOC106505926. (E) The expression changes of proliferation marker genes after transfected with si-LOC106505926. (F) The EdU results after transfected with si-LOC106505926. (G) The expression changes of differentiation marker genes after transfected with OE-LOC106505926 at mRNA level. (H) The expression changes of differentiation marker genes after transfected with OE-LOC106505926 at protein level. (I) The results of immunofluorescence after transfected with OE-LOC106505926. (J) The expression changes of differentiation marker genes after transfected with si-LOC106505926 at mRNA level. (K) The expression changes of differentiation marker genes after transfected with si-LOC106505926 at protein level. (L) The results of immunofluorescence after transfected with si-LOC106505926.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/e46aaded6b4752f102e9d246.png"},{"id":51948285,"identity":"012e9801-2350-487a-81c4-4014bd970878","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1542676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of CXXC5 on the proliferation and differentiation of PSCs.\u003c/strong\u003e(A) Construction of OE-CXXC5 overexpression vector and detection of transfection efficiency. (B) Transfection efficiency of OE-CXXC5 was detected by western blot. (C) Effect of OE-CXXC5 on cell cycle by flow cytometry. (D) The expression changes of proliferation marker genes after transfected with OE-CXXC5. (E) The results of EdU after transfected with OE-CXXC5. (F) Transfection efficiencies of siCXXC5-1, 2, 3 were detected by qRT-PCR. (G) The expression changes of proliferation marker genes after transfected with siCXXC5-3. (H) The results of EdU after transfected with siCXXC5-3. (I) The expression changes of differentiation marker genes after transfected with OE-CXXC5 at mRNA level. (J) The expression changes of differentiation marker genes after transfected with OE-CXXC5 at protein level. (K) The results of immunofluorescence after transfected with OE-CXXC5. (L) The expression changes of differentiation marker genes after transfected with siCXXC5 at mRNA level. (M) The expression changes of differentiation marker genes after transfected with siCXXC5 at protein level. (N) The results of immunofluorescence after transfected with siCXXC5.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/7944e4ac5138907e1e2d56e1.png"},{"id":51948287,"identity":"20c28a4a-3a61-42af-9e30-06eca3e4456e","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":355604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of proliferation and differentiation marker genes in rescue experiment.\u003c/strong\u003e (A) Detection of PCNA and MYOD expression after transfection of OE-CXXC5 and miR-22-5p at protein level. (B) Detection of PCNA and MYOD expression after transfection of OE-LOC106505926 and miR-22-5p at protein level.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/522b59ea41c8e0031040c49a.png"},{"id":51948289,"identity":"49b89729-6a0e-4fc9-bf6d-896e9b54a3c9","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":846269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of LOC106505926 on the differentiation of preadipocytes.\u003c/strong\u003e (A) Cell oil red O staining. (B) The expression changes of differentiation marker genes after transfected with si-LOC106505926 at mRNA level. (C) The expression changes of differentiation marker genes after transfected with si-LOC106505926 at protein level. (D) Protein silver staining gel map. (E) Venn diagram of mass spectrometry detection of the pull-down protein. (F) LOC106505926 interacts with FASN.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/878bcc24796a3c5c0d2def13.png"},{"id":51948697,"identity":"2314c67d-b62e-475f-ad06-d830d7bf7d89","added_by":"auto","created_at":"2024-03-04 12:13:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4344942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/1dbcf83f-39c3-4b45-9f76-78f17071c16a.pdf"},{"id":51948280,"identity":"11d5b0b3-0825-48a7-ae95-214451baee60","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: \u003c/strong\u003eTable S1. The siRNA sequences\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/e65eae4c349dffbd8f1e97f8.docx"},{"id":51948281,"identity":"2260626d-830a-49b4-a723-deade1ccf517","added_by":"auto","created_at":"2024-03-04 11:57:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: \u003c/strong\u003eTable S2. Primers of qRT-PCR\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/97d4fff43861958880c1fbb7.docx"},{"id":51948291,"identity":"913b5728-c0ba-4b27-9c0a-4d316e4db78f","added_by":"auto","created_at":"2024-03-04 11:57:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: \u003c/strong\u003eTable S3. Quality assessment of sRNA sequencing data\u003c/p\u003e","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/b80a63b78340361703b446ac.docx"},{"id":51948290,"identity":"ccbda2af-8833-44ec-9f7e-0ee1ae58edc2","added_by":"auto","created_at":"2024-03-04 11:57:03","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":922171,"visible":true,"origin":"","legend":"","description":"","filename":"Rawimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3977166/v1/420a243687601502f2a510e9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of LncRNA LOC106505926 on Myogenesis and Lipogenesis of Porcine Primary cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePork is the main source of meat for the Chinese people. Skeletal muscle accounts for about 40% of the body weight and 50%-75% of all body proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The characteristics of skeletal muscle also directly affect pork quality. Muscle fiber numbers are already established in the embryonic period [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Muscle development after birth mainly depends on the proliferation and differentiation of skeletal muscle satellite cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which are muscle tissue stem cells located between the muscle fiber substrate and the plasma coat [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Skeletal muscle satellite cells are activated in response to muscle injury and undergo cell proliferation, differentiation, and fusion to form new myotubes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Skeletal muscle development is a complex process, current studies have found that many transcription factors are involved in the regulation of myogenesis, such as myogenic regulatory factors (MRFs) including \u003cem\u003eMyf\u003c/em\u003e5, \u003cem\u003eMyoD\u003c/em\u003e, \u003cem\u003eMyoG\u003c/em\u003e, \u003cem\u003eMyf\u003c/em\u003e6, myocyte enhancer factor-2 (MEF-2) family [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and Pax family members [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent studies have found that, in addition to transcription factors, non-coding RNAs are also involved in the regulation of muscle development, including miRNAs and lncRNAs.\u003c/p\u003e \u003cp\u003eFat deposition is also an important biological process that affects the quality and growth efficiency of pork. Adipocytes originate from the embryonic mesoderm and are formed by the differentiation of preadipocytes. The transformation of preadipocytes to mature adipocytes determines the process of fat deposition [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Peroxisome proliferator-activated receptor γ (PPARγ) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and many key regulators of adipogenic differentiation play an important role in adipocyte differentiation and deposition. Many lncRNAs have been found to play a role in adipocyte thermogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], adipocyte metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and adipocyte differentiation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLong non-coding RNA (lncRNA) is a kind of linear RNA with a length of more than 200 nt, which has no protein coding ability and has a wide subcellular distribution in cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This wide subcellular distribution determines the diversity of its functional mechanism. The mechanism of lncRNAs acting as ceRNAs has attracted much attention [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], but lncRNAs also have other mechanisms that play an important role in a variety of biological processes. H19 is one of the most known lncRNA, and it plays a key role in the differentiation of skeletal muscle [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. By competitively binding to miR-140-5p, H19 inhibits the differentiation of skeletal muscle satellite cells in porcine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In vivo, overexpression of H19 improved insulin sensitivity and mitochondrial biogenesis, silencing H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown adipocytes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Previous studies have found an obesity-related lncRNA lnc-ORA, which had a seven-fold higher expression in ob/ob mice than in WT mice. Lnc-ORA knockdown inhibits adipocyte differentiation by regulating the PI3K/AKT/mTOR signaling pathway [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, lnc-ORA was found to play a role in myogenesis. It can inhibit skeletal muscle myogenesis and reduce the stability of myogenic genes by acting as a sponge for miR-532-3p or interacting with insulin-like growth factor 2 mRNA binding protein 2 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These indicate that lncRNA plays a role in a variety of biological processes through different mechanisms and lncRNA is indispensable in the development of various tissues.\u003c/p\u003e \u003cp\u003eIn the present research, whole transcriptome sequencing was performed on longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90 and 180 days of age to screen candidate miRNAs that can regulate porcine myogenesis. The LOC106505926/miR-22-5p/\u003cem\u003eCXXC\u003c/em\u003e5 regulatory network was constructed to regulate the proliferation and differentiation of PSCs. In addition, LOC106505926 was testified to inhibit the differentiation of preadipocytes by directly binding to FASN. This study may provide a molecular basis for understanding the development process of porcine skeletal muscle and adipose tissue.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCandidate miRNAs regulating muscle development were screened by RNA-seq\u003c/h2\u003e \u003cp\u003eWhole transcriptome sequencing was performed on LDM of Jinfen White pigs at 1, 90 and 180 days of age. On average, 14.45\u0026nbsp;million, 17.68\u0026nbsp;million and 15.07\u0026nbsp;million raw reads and 14.12\u0026nbsp;million, 17.44\u0026nbsp;million, 14.87\u0026nbsp;million clean reads were obtained from LDM of Jinfen White pigs at 1, 90 and 180 days of age respectively. In addition, the error rate of all sequencing data sets was less than 0.01%, the value of Q20 was above 98%, Q30 value was above 94% and the average GC content was 49%, Specific data are presented in the supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eA total of 245 differentially expressed miRNAs (DE miRNAs) were screened (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Specifically, 100 miRNAs were up-regulated and 89 miRNAs were down-regulated in 90-day-old muscle tissues compared with 1-day-old (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). There were 106 up-regulated and 106 down-regulated miRNAs in 180-day-old muscle tissues compared with 1-day-old (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Compared with the 90-day-old group, there were 37 up-regulated miRNAs and 44 down-regulated miRNAs in the 180-day-old group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The heat map showed the DE miRNAs expression patterns at different days of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). GO and KEGG results showed that the target genes of DE miRNAs were mainly enriched in MAPK, AMPK, JAK-STAT, VEGF and other signaling pathways, which were related to muscle development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Thus, DE miRNAs may be involved in the regulation of porcine myogenesis. Among DE miRNAs, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group, with the log\u003csub\u003e2\u003c/sub\u003e(fold change) approaching 3. Therefore, miR-22-5p attracted our attention and we decided to further investigate its function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMiR-22-5p inhibits the proliferation and differentiation of PSCs\u003c/h2\u003e \u003cp\u003eThe expression of miR-22-5p was extremely significant increased after transfected with miR-22-5p mimic (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The expression of proliferation marker genes was significantly decreased after transfected with miR-22-5p mimic (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). the number of EdU positive cells was extremely significant decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After transfected with miR-22-5p inhibitor, the expression of miR-22-5p was extremely significant decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the expression of proliferation marker genes was extremely significant increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the number of EdU positive cells was extremely significant increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicated that miR-22-5p inhibited the proliferation of PSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the effect of miR-22-5p on the myogenic differentiation of PSCs, the expression changes of key myogenic factors were detected. The results showed that, after transfected with miR-22-5p mimic, the expression of \u003cem\u003eMyoG\u003c/em\u003e, \u003cem\u003eMyoD\u003c/em\u003e and \u003cem\u003eMHC\u003c/em\u003e were extremely significant decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Western blot showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The immunofluorescence staining results showed that the number of myotubes was extremely significant decreased in miR-22-5p mimic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After transfected with miR-22-5p inhibitor, the opposite results were shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-L). The above results proved that miR-22-5p inhibited the differentiation of PSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMiR-22-5p binds to the 3\u0026prime;UTR of CXXC5 and LOC106505926\u003c/h2\u003e \u003cp\u003eBased on sequencing results, there were multiple mRNAs and lncRNAs associated with miR-22-5p. Among them, CXXC5 and LOC106505926 had a stronger negative correlation with miR-22-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with Pearson coefficient of -0.896 and \u0026minus;\u0026thinsp;0.852 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Expression patterns analysis found that the expression of miR-22-5p showed an upward trend with the increase of age, while CXXC5 expression showed a downward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Expression profiling revealed that CXXC5 had the highest expression in the liver and LDM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The expression of CXXC5 and LOC106505926 was significantly decreased after transfection of miR-22-5p mimics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while transfection of miR-22-5p inhibitor showed the opposite results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). RNAhybird prediction showed that the 3\u0026prime;UTR of CXXC5 and LOC106505926 could bind to the seed sequence of miR-22-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). The wild-type and mutant vectors of 3\u0026prime;UTR of CXXC5 and LOC106505926 were constructed respectively. The luciferase activity of miR-22-5p mimics and CXXC5-wt-psiCHECK2 group was extremely significant lower than other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), The luciferase activity of miR-22-5p mimics and LOC106505926-wt-psiCHECK2 group was also extremely significant lower than other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicated that miR-22-5p can bind to CXXC5 and LOC106505926, then regulate their expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiological characteristics of LOC106505926\u003c/h2\u003e \u003cp\u003eLOC106505926 is located on chromosome 14 of pig (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To verify the existence of LOC106505926, LOC106505926 was amplified by using the cDNA of the longissimus dorsi muscle of Jinfen white pig as a template, and 731 bp LOC106505926 partial sequence was obtained, which confirmed the existence of LOC106505926 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The subcellular localization pattern of LOC106505926 was predicted by online software lncLocator, the results showed that LOC106505926 was mostly located in the cytoplasm, subsequently, RNA was extracted from the cytoplasm and nucleus of skeletal muscle satellite cells, the results also showed that LOC106505926 was mostly localized in the cytoplasm and also expressed in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We detected the expression pattern of LOC106505926, its relative expression level showed a downward trend with the increase of age in Jinfen white pigs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In addition, LOC106505926 has a high expression level in the lung, subcutaneous fat and longissimus dorsi muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLOC106505926 promotes the proliferation and differentiation of PSCs\u003c/h2\u003e \u003cp\u003eThe overexpression vector of LOC106505926 was constructed and transfected into PSCs. The expression of LOC106505926 in OE-LOC106505926 group was extremely significant increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The expression of \u003cem\u003ePCNA\u003c/em\u003e, \u003cem\u003ecyclin D\u003c/em\u003e, \u003cem\u003eCDK\u003c/em\u003e1 and \u003cem\u003eCDK\u003c/em\u003e4 was extremely significant increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the expression of \u003cem\u003eKi\u003c/em\u003e67 was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the number of EdU-positive cells was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Three siRNA sequences of LOC106505926 were synthesized and detected the interference efficiency after transfection of PSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The siLOC106505926-1 was selected to transfect PSCs to detect the expression of proliferation marker genes. Compared with the si-NC group, the expression of proliferation marker genes was extremely significant reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the EdU results showed that the number of positive cells was extremely significant reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, the effect of LOC106505926 on PSC differentiation was explored. The results showed that after transfected with OE-LOC106505926, the expression of \u003cem\u003eMyoD\u003c/em\u003e was extremely significant increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the expression of \u003cem\u003eMyoG\u003c/em\u003e was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), there was no significant difference in MHC expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Western blot results showed that the expression of MYOD, MYOG and MHC were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Immunofluorescence staining results showed that the number of myotubes was extremely significant increased in OE-LOC106505926 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). While after transfection with si-LOC106505926, the opposite results were shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCXXC5 promotes the proliferation and differentiation of PSCs\u003c/h2\u003e \u003cp\u003eThe overexpression vector of CXXC5 was constructed and the immunofluorescence results showed a positive result after transfected of PSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Compared with OE-NC group, the expression of CXXC5 protein in OE-CXXC5 group was extremely significant increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The effect of CXXC5 on the cell cycle was detected by flow cytometry, the results showed that the proportion of S phase cells was extremely significant increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), accompanied with the significant upregulation of \u003cem\u003ePCNA\u003c/em\u003e, \u003cem\u003eCyclin D\u003c/em\u003e, \u003cem\u003eCDK\u003c/em\u003e1 and \u003cem\u003eCDK\u003c/em\u003e4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). EdU results showed that the number of positive cells increased extremely significant after transfection of OE-CXXC5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Three siRNA sequences of CXXC5 were designed to transfect PSCs and the interference efficiency of three siRNAs was detected. The results showed that siCXXC5-3 had the highest interference efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Transfection of siCXXC5-3 in PSCs showed the opposite results to transfection of OE-CXXC5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTransfected PSCs were induced to differentiate to detect the effect of CXXC5 on PSCs differentiation. The expression of \u003cem\u003eMyoD\u003c/em\u003e, \u003cem\u003eMyoG\u003c/em\u003e and \u003cem\u003eMHC\u003c/em\u003e were significantly increased after transfection of OE-CXXC5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Western blot results showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The immunofluorescence assay showed that the number of myotubes in OE-CXXC5 group was extremely significant increased compared with OE-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, the effect of siCXXC5-3 on the differentiation of PSCs was detected. The results were contrary to the transfection of OE-CXXC5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL-N), indicating that CXXC5 promotes the differentiation of PSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThe rescue assay certified LOC106505926/miR-22-5p/CXXC5 regulates PSCs proliferation and differentiation\u003c/h2\u003e \u003cp\u003eTo further explore the relationship between miR-22-5p and CXXC5, rescue experiments were performed. Overexpression of CXXC5 alleviates the inhibitory effect of miR-22-5p on the expression of PCNA and MYOD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In addition, transfected with OE-LOC106505926 also alleviates the inhibitory effect of miR-22-5p on the proliferation and myogenic differentiation of PSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Thus, LOC106505926 may reduce the inhibitory effect of miR-22-5p on CXXC5 by competitively binding miR-22-5p.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLOC106505926 inhibits preadipocyte differentiation and directly interacts with FASN\u003c/h2\u003e \u003cp\u003eAfter si-LOC106505926 was transfected in porcine precursor adipocytes, Oil Red O staining showed that LOC106505926 could inhibit lipid-droplet formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), the mRNA and protein levels of lipogenic factors were dramatically improved in the si-LOC106505926 group compared with the si-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C). RNA pull-down assay was performed with biotinylated LOC106505926 to identify the LOC106505926 interacting proteins. After SDS-PAGE, the gel was stained with silver nitrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) and analyzed by MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). The proteins that specifically bind to LOC106505926 (including FASN) were identified. The association of LOC106505926 with FASN was validated by Western blot, and the results show that the target protein FASN was detected in LOC106505926 pull-down protein samples but not in the samples associated with antisense LOC106505926 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, there were many examples that miRNAs affect skeletal muscle development by participating in various processes. MiR-142a-5p was able to function as an important regulator of denervation-induced skeletal muscle atrophy via targeting MFN1 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. MiR-135a-5p regulated skeletal muscle fibrosis by Tgfbr2/Smad4 signaling pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. MiR-491 can specifically bind to the 3\u0026prime;UTR of myomaker and regulates myocyte differentiation and muscle regeneration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies demonstrated that the expression of miR-22-5p in mice was significantly up-regulated with the increase of age [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. MiR-22-5p has also been testified to play important roles in a variety of diseases, such as acute myocardial infarction, myocardial damage and hepatocellular carcinoma [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. MiR-22-5p regulated the proliferation of human osteoblasts via MALAT1 and VEGF [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Many miRNAs have been proven to be involved in the regulation of myoblast proliferation and differentiation [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. At present, the regulation of myogenesis by miR-22-5p remains unclear. In this study, we demonstrated that miR-22-5p was differentially expressed in the LDM of Jinfen White pigs at three developmental stages, suggesting its involvement in the regulation of porcine muscle development.\u003c/p\u003e \u003cp\u003eCXXC5 is a member of the CXXC-type zinc finger protein family, which has a CXXC-type zinc finger (ZF-CxxC) protein structural domain. The CXXC structural domain normally binds to unmethylated CpG islands in gene promoters and plays a key role in epigenetic regulation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. CXXC5 does not have any catalytic domain, but it has been shown to regulate transcription by directly binding to DNA [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. CXXC5 was expressed in a variety of tissues [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and was involved in the development of nephron and heart, as well as the differentiation of osteoblasts, oligodendrocytes and endothelial cells [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recent studies showed that CXXC5 affected cell cycle regulation and was required for myeloid cell maturation and differentiation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In addition, CXXC5 regulates multiple physiological processes via several important cellular signaling pathways. It was demonstrated that CXXC5 mediated the BMP signaling pathway and induced endothelial cell differentiation and vessel formation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. CXXC5 also participated in the Wnt signaling pathway to regulate the differentiation of neural stem cells [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and osteoblast [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moreover, the ZF-CXXC domain of CXXC5 interacted with the SMAD2/3/4 and activated TGF-β signaling and BMP signaling to promote normal heart development [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and mediate TNF-α induced apoptosis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Besides, CXXC5 was involved in cell cycle arrest and DNA repair by activating ATM-p53 signaling axis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, CXXC5 may be an important medium for various signal inputs.\u003c/p\u003e \u003cp\u003eCXXC5 was necessary for the myogenic differentiation of C2C12 cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Previous studies showed that CXXC5 regulated the regeneration of skeletal muscle through TGF-β, Wnt, Vitamin D signaling pathways [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Wu demonstrated that CXXC5 may be associated with skeletal muscle development and Ca\u003csup\u003e2+\u003c/sup\u003e release from muscle via whole-genome sequencing in Qingyu pigs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the present study, CXXC5 was proved to promote the proliferation and differentiation of PSCs, which is consistent with previous research. This study demonstrated that miR-22-5p inhibited the proliferation and differentiation of PSCs via binding to the 3\u0026prime;UTR of CXXC5.\u003c/p\u003e \u003cp\u003eWith the discovery of a large number of lncRNAs, people have gradually realized that lncRNA has a very important regulatory function and can participate in various biological processes and pathway regulation. In recent years, lncRNA has played an important role in participating in transcription as a transcriptional regulator [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], regulating chromatin stability [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], regulating subcellular organs [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and regulating miRNA expression [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Studies have found that lncRNA-TBP promotes myoblast differentiation, reduces fat deposition, activates slow muscle phenotype and induces muscle hypertrophy. It is worth noting that lncRNA-TBP, as a regulatory RNA, directly interacts with TBP protein to regulate TBP-target genes [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In a study of lipogenic transdifferentiation of myoblasts, 114 core lncRNAs were identified, lncRNA-GM43652 gene was a potential regulator of adipogenesis in muscle cells [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In our study, LOC106505926 regulates the differentiation of satellite cells through the ceRNA mechanism, while in adipocytes, it inhibits the differentiation of preadipocytes by directly binding to FASN. Our study provides a basis for a variety of biological functions and specific mechanisms of lncRNAs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study demonstrated that miR-22-5p inhibited the proliferation and differentiation of PSCs via binding to the 3\u0026prime;UTR of CXXC5. LOC106505926 was found to play a role in the proliferation and differentiation of PSCs as a molecular sponge of miR-22-5p. In addition, LOC106505926 inhibited the differentiation of preadipocytes by directly binding to FASN. These results provide insights into the molecular regulation mechanism in porcine skeletal muscle development and fat deposition.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTissues collection\u003c/h2\u003e \u003cp\u003eAll experimental protocols were approved by the Ethics Committee of Shanxi Agricultural University (Shanxi, China, Approval No. SXAU-EAW-2021Sus.AB.0926001). LDM from Jinfen White pigs was collected at 1, 90 and 180 days of age. Seven tissues including heart, liver, spleen, lung, kidney, longissimus dorsi, and subcutaneous fat from 3-month-old Jinfen White pigs were collected strictly according to the anatomical structure of the pigs. All samples were stored at -80 ℃ for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of library and identification of differentially expressed miRNAs\u003c/h2\u003e \u003cp\u003eThe libraries were sequenced on an Illumina HiseqTM 2500 platform. HISAT2 (version 2.0.2) was used to get the clean reads mapped to the pig reference genome (Sus scrofa 11.1). The sequencing data was publicly available in the NCBI SRA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with accession number: PRJNA867525.\u003c/p\u003e \u003cp\u003eThe expression level of miRNAs was calculated using the HTseq software (0.6.1). Transcript per million (TPM) values were used to determine miRNA counts and levels of expression [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Subsequently, differentially expressed miRNAs were identified using the R packages DEG seq2 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePSCs isolation, culture and differentiation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePSCs were isolated from 3-day-old Jinfen White male pigs. The skin of pigs was disinfected with 75% alcohol after slaughter. The LDM were collected and kept in PBS supplement with 1% penicillin-streptomycin. The tissue was digested with digestive solution (DMEM containing 15% collagenase II, 15% dispase II, 1% penicillin-streptomycin) at 37 ℃ for 1 h, and the digestion was terminated with DMEM (including 10% FBS). The mixture was filtered through a 100 \u0026micro;m cell strainer and centrifuged at 1500 r/min for 10 min. Then, the supernatant was removed and the precipitate was resuspended with PBS. Next, 70 and 40 \u0026micro;m cell strainers were used to filter the suspension. The supernatant was removed and resuspended with erythrocyte lysate, an equal volume of PBS was added, centrifuged at 1000 r/min for 5 min. Last, the cells were suspended with 20% FBS (Gibco, Life Technologies, United States) and 1% penicillin-streptomycin. Cells were cultured in thermostatic incubator (Thermo Fisher, United States) for 1 h, the cell suspension was transferred to the new culture dishes, which were coated with Matrigel (Corning, United States).\u003c/p\u003e \u003cp\u003eIsolated PSCs were cultured on Matrigel coated 10 cm plates (Corning, United States) with the medium (containing 20% FBS, 1% penicillin-streptomycin, 2.5 ng/mL human recombinant basic fibroblast growth factor) (Gibco, United States). When the cells density was up to 70%, the differentiation medium (containing 5% horse serum) (HyClone, United States) was used to induce cell myogenic differentiation, the medium was changed every 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePorcine preadipocytes isolation, culture and differentiation\u003c/h2\u003e \u003cp\u003eThe subcutaneous adipose tissue of piglets was collected and placed in a sterile petri dish to remove fascia and connective tissue. Then, the tissue was cut into pieces and digested with 2 mg/mL type I collagenase (Gibco, USA) for 1 h, the digestion was terminated with DMEM (including 10% FBS). After filtration, the cells were centrifuged at 1000 r/min for 10 minutes, resuspended and inoculated into a petri dish for culture. Preadipocytes were cultured with DMEM (including 10% FBS, 1% penicillin-streptomycin). When the cell density reached 80% -90%, the preadipocytes were digested with 0.25% trypsin. Replace the medium every two days. When the cell density reached 95%, lipogenic induction was performed. The complete induction medium was supplemented with dexamethasone (1 \u0026micro;mol/L), indomethacin (100 \u0026micro;mol/L), IBMX (0.5 mmol/L) and insulin (10 \u0026micro;g/mL). After 4 days of differentiation, the maintenance medium was replaced. The maintenance medium was a complete medium supplemented with insulin (10 \u0026micro;g/mL). After that, the maintenance medium was replaced every 2 days, and the cells were observed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNuclear and cytoplasmic RNA fractionation\u003c/h2\u003e \u003cp\u003ePSCs were collected and washed twice with cold PBS and centrifuged at 500 g for 3 min. The precipitate was resuspended with 0.3 mL Cell Fractionation Buffer, placed on ice for 10 min, and centrifuged at 500 g at 4\u0026deg;C for 3 min. The supernatant was transferred to a new 1.5 mL micro-centrifugal tube without RNase, and 0.3 mL 2\u0026times;Lysis/Binding Buffer was added and mixed, which was cytoplasmic RNA. The precipitate was washed with 0.3 mL Cell Disruption Buffer, and 0.3 mL 2\u0026times;Lysis/Binding Buffer was added after shaking and mixing to obtain nuclear RNA. Then, 0.3 mL absolute ethanol was added to the cytoplasm and nucleus RNA, poured into the filter extraction, centrifuged at 1,400 r/min for 1 min, discarded the liquid, added Wash Solution centrifuged at 1,400 r/min for 1 min, repeated several times, 0.3 mL Elution Solution was poured into the filter, replaced the new centrifuge tube, centrifuged at 10,000 r/min for 30 s to obtain RNA and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eFirstly, the overexpression vector of CXXC5: pHBLV-CXXC5-puro (OE-CXXC5) and control vector of CXXC5: pHBLV-CMVIE-puro control (NC-CXXC5) were constructed and synthesized. When the density of HEK-293T cells reached to 70% confluence, the Lipo3000 (Invitrigen, USA) was used to transfect the plasmid and two package plasmids psPAX2 and pMD2G (Public Protein/Plasmid Library, Jiangsu, China) at the ratio of 1:1:1 for 6 h following the introduction. The supernatants were collected and stored at -80 ℃ for infection. After 48 h of transfection, the fluorescence rate was observed. The overexpression vector of LOC106505926 was synthesized by Tsingke Biotechnology according to the sequence on NCBI. The RNA oligo against of porcine CXXC5, miR-22-5p and LOC106505926 were purchased from GenePharma (Shanghai, China). The 50 nM RNA oligo specific were transfected to cells at a density about 60%. The siRNA sequences were shown in the supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from samples according to the instruction of TaKaRa RNAiso Plus (Takara, Japan). The cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). The relative expression level of genes was normalized by \u003cem\u003e18sRNA\u003c/em\u003e. Primers\u0026prime; information was shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The qRT-PCR reaction system: cDNA 2 \u0026micro;L, 2\u0026times;SYBR Premix Ex Taq II 10 \u0026micro;L, forward and reverse primers 0.5 \u0026micro;L, RNAase Free ddH\u003csub\u003e2\u003c/sub\u003eO supplemented to 20 \u0026micro;L. Reaction procedure: 95\u0026deg;C 30 s; 95\u0026deg;C 5 s, 58\u0026deg;C 30 s, 35 cycles; the melting curve program is 95\u0026deg;C 15 s, 60\u0026deg;C 35 s, 95\u0026deg;C. Each sample was repeated three times. For miRNAs, U6 snRNA was selected as the internal control. The primers of miRNAs were designed using the stem-loop approach and the information of primers was shown in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The cDNA was synthesized according to the protocol of miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China). Each 20 \u0026micro;L qRT-PCR reaction mixture contained 2\u0026times;miRNA Universal SYBR qPCR Master Mix 10 \u0026micro;L (Vazyme, China, MQ101), cDNA 1 \u0026micro;L, nuclease-free H\u003csub\u003e2\u003c/sub\u003eO 7.8 \u0026micro;L, 0.5 \u0026micro;L specific primer and mQ primer R. The following parameters were used for qRT-PCR: pre-denaturation for 5 min at 95 ℃, then 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s, and 72 ℃ for 8 s, the melting curve program is 95\u0026deg;C 15 s, 60\u0026deg;C 60 s, 95\u0026deg;C 15 s. Relative expression levels of genes and miRNAs were calculated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eAfter 48 h of transfection, the cells were collected and resuspended with 75% ethanol when the cell density reached 90%. The cell suspension was placed at -20\u0026deg;C for 10 h. After centrifugation, RNase A was added and incubated at 37\u0026deg;C for 30 min. PI staining solution was added and incubated at 4\u0026deg;C for 30 min. The proportion of cells in different cell cycles was analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEdU assay\u003c/h2\u003e \u003cp\u003ePSCs were incubated with EdU-containing medium for 2 h, then, fixed with 4% paraformaldehyde for 30 min, washed with PBS, incubated with 2 mg/mL glycine for 5 min, washed with PBS for 5 min, incubated with 0.5% TritonX-100 for 10 min, then incubated with Apollo staining reaction solution (Ribobio, China) for 30 min, washed with PBS and stained with DAPI reaction solution for 10 min. Observed with a fluorescence microscope.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe wild-type of 3\u0026prime;UTR of CXXC5 (CXXC5-wt-psiCHECK2) and mutant-type of 3\u0026prime;UTR of CXXC5 (CXXC5-wut-psiCHECK2) were constructed and inserted into psi-CHECK2 vectors. HEK-293T cells were seeded and cultured on 24-well plates. Then, cells were co-transfected with the miRNAs (mimic NC, miR-22-5p mimic, inhibitor NC or miR-22-5p inhibitor) and the CXXC5-wt-psiCHECK2 or CXXC5-wut-psiCHECK2 vectors. The cells were collected after 24 h of cell transfection. A standard plate reader (BioTek, Vermont, United States) was used to measure the luciferase activity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eIn PSCs, the expression levels of MYOG, MYOD, MHC and CXXC5 protein were detected. The protein expression levels of FABP4, PPARγ, SREBP1 and FASN were detected in preadipocytes. Transfected cells were lysed in RIPA buffer with 1% PMSF. 5\u0026times;Buffer was added to the sample and denatured at 100\u0026deg;C for 10 min. SDS-PAGE gel electrophoresis was performed at 80 V 30 min, 120 V 90 min. Then transferred them onto a PVDF membrane and non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Then, they were incubated with 1:1000 diluted polyclonal rabbit MYOG (Abclonal, China), MYOD (Proteintech, China), CXXC5 (Bioss, China), 1:500 diluted polyclonal mouse MHC (DSHB, America), 1:1000 diluted polyclonal rabbit FABP4 (Proteintech, China), PPARγ (Proteintech, China), SREBP1 (Proteintech, China) and FASN (Proteintech, China) at 4 ℃ overnight. The blots were subsequently incubated with secondary antibody (1:10000) for 1 h. Secondary antibody include goat anti-mouse IgG (Servicebio, China) and goat anti-rabbit IgG (Servicebio, China). GAPDH (Servicebio, China) was used as an endogenous protein for normalization. Image J software was used to conduct quantitative analysis of western blot results according to the gray value of the strip.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescent analysis\u003c/h2\u003e \u003cp\u003eCells were fixed with precooled 4% paraformaldehyde for 30 min. Then permeabilized in 0.5% Triton X-100 for 10 min. After that, cells were blocked with 3% bovine serum albumin (BSA), and incubated with 1:1000 diluted polyclonal mouse MHC (DSHB, America) antibodies overnight at 4 ℃. Cells were washed with PBS for 1\u0026ndash;3 times and incubated with goat anti-mouse IgG antibodies (Bioss, China) for 1 h at room temperature. Then, the cells were stained with Hoechst 33342 (Sanofi-Aventis, Germany) for 10 min. Images were acquired by a Leica SP8 confocal microscope. Immunofluorescence results were quantified by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eOil red O staining\u003c/h2\u003e \u003cp\u003eAfter lipogenic differentiation of porcine preadipocytes, oil red O staining was performed, 4% paraformaldehyde was added, fixed at room temperature for 30 min, and washed with PBS. Oil red O dye solution (Solarbio, China) was added and incubated at room temperature for 30 min. After washing with PBS, the cells were observed under an inverted microscope. Isopropanol was added to extract triglyceride, and the OD value was detected by enzyme-labeled instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSilver nitrate staining\u003c/h2\u003e \u003cp\u003eTake out the gel after SDS-PAGE, put it in the staining box, wash the gel twice with distilled water, then pour out the distilled water, add 25% ethanol to denature for 3 minutes, recover the ethanol, wash the gel with distilled water, add 0.1% silver nitrate solution, incubate for 30 minutes on the shaker, and fix the gel on the glass plate to observe the staining.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRNA Pull-Down Assay\u003c/h2\u003e \u003cp\u003eThe biotinylated probe of LOC106505926 was synthesized by RiboBio. Preparation of cell lysates with standard lysis buffers, wash the beads with 20mM Tris (pH 7.5), add an equal volume of 1\u0026times;RNA Capture Buffer, resuspend beads by pipetting or vortexing, then, add 50 pmol of labeled RNA to the beads, mix gently by pipetting, incubate for 15\u0026ndash;30 minutes at room temperature with agitation. The RNAs were targeted with streptavidin beads, add 100 \u0026micro;L of Master Mix (including Protein-RNA Binding Buffer, glycerol, salts, lysate, nuclease-free water) to the RNA-bound beads, incubate 30\u0026ndash;60 minutes at 4\u0026deg;C with agitation or rotation. The protein complexes were obtained after several elutions. Finally, the protein complexes associated with the beads were analyzed by mass spectrometry (MS) and western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). Multigroup comparisons of the means were carried out by a one-way analysis of variance test. The two-tailed t-test was performed for differences analysis between the two groups. * represents \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** represents \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLongissimus dorsi muscle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine skeletal muscle satellite cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLncRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLong non-coding RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCXXC5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCXXC finger protein 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyogenic regulatory factors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMYOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyogenic differentiation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMYOG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyogenin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyosin heavy chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEF2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyocyte enhancer factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMyf5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyogenic factor 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMyf6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyogenic factor 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPARγ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxisome proliferator-activated receptorγ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFABP4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFatty acid binding protein 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSREBP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSterol regulatory element binding transcription factor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFASN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid synthase.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026prime; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eAll authors contributed to the study\u0026apos;s conception and design. Study conception and design: MS, SY, GC Conducted RNA-seq and bioinformatics clustering: XZ, SD, YL, JY, performed experimental study: MS, SY, SW, WL. Article drafting and revising: ML, CC, XG, BL, GC. Funding acquisition: CL, GC. \u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThis work was supported by the National Natural Science Foundation of China (31872336); the Key Research and Development Project of Shanxi Privince (202102140601005); Special Funds for Scholars Support Program of Shanxi Province (2017); the University Science and Technology Innovation Project of Shanxi Province (2021L158); Science and Technology Innovation Foundation of Shanxi Agricultural University (2020BQ56).\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe authors state that they have no competing economic interests or personal relationships.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe original data for the RNA-seq data were submitted to the SRA Database (BioProject ID: PRJNA867525).\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe authors thank the Pig Breeding Engineering Center of Shanxi Agricultural University for its support in laboratory animals and facilities.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eAll experimental procedures involving animals were approved by the Animal Welfare and Ethics Committee of Shanxi Agricultural University with the approval No. SXAU-EAW-2021Sus.AB.0926001.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFrontera WR, Ochala J. 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Bioinformatics. 2010;26(1):136\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pig, LOC106505926, skeletal muscle satellite cells, preadipocytes, myogenesis, lipogenesis","lastPublishedDoi":"10.21203/rs.3.rs-3977166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3977166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSkeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving carcass quality and meat quality. In the present study, RNA sequencing was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that a total of 245 differentially expressed miRNAs were screened, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Bioinformatics predicted that long non-coding RNA (lncRNA) LOC106505926 and \u003cem\u003eCXXC\u003c/em\u003e5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and \u003cem\u003eCXXC\u003c/em\u003e5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of \u003cem\u003eCXXC\u003c/em\u003e5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.\u003c/p\u003e","manuscriptTitle":"Effect of LncRNA LOC106505926 on Myogenesis and Lipogenesis of Porcine Primary cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-04 11:56:57","doi":"10.21203/rs.3.rs-3977166/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-21T00:01:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-15T11:44:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38b355ca-35a4-40b2-8dc6-ca7840e31517","date":"2024-03-06T06:46:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-01T02:26:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-01T00:38:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-29T15:40:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-29T15:38:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-02-22T00:46:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d04ec320-8c7f-4e60-92f2-a24a94bd870c","owner":[],"postedDate":"March 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-16T09:09:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-04 11:56:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3977166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3977166","identity":"rs-3977166","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}