miR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

As a well-known cancer-related miRNA, miR-193b-3p is enriched in skeletal muscle and dysregulated in muscle disease. However, the mechanism underpinning this has not been addressed so far. Here, we probed the impact of miR-193b-3p on myogenesis by mainly using goat tissues and skeletal muscle satellite cells (MuSCs), compared with mouse C2C12 myoblasts. miR-193b-3p is highly expressed in goat skeletal muscles, and ectopic miR-193b-3p promotes MuSCs proliferation and differentiation. Moreover, insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) is the most activated insulin signaling gene when there is overexpression of miR-193b-3p; the miRNA recognition element (MRE) within the IGF1BP1 3′ untranslated region (UTR) is indispensable for its activation. Consistently, expression patterns and functions of IGF2BP1 were similar to those of miR-193b-3p in tissues and MuSCs. In comparison, ectopic miR-193b-3p failed to induce PAX7 expression and myoblast proliferation when there was IGF2BP1 knockdown. Furthermore, miR-193b-3p destabilized IGF2BP1 mRNA, but unexpectedly promoted levels of IGF2BP1 heteronuclear RNA (hnRNA), dramatically. Moreover, miR-193b-3p could induce its neighboring genes. However, miR-193b-3p inversely regulated IGF2BP1 and myoblast proliferation in the mouse C2C12 myoblast. These data unveil that goat miR-193b-3p promotes myoblast proliferation via activating IGF2BP1 by binding to its 3′ UTR. Our novel findings highlight the positive regulation between miRNA and its target genes in muscle development, which further extends the repertoire of miRNA functions.

Typically, miRNAs negatively regulate gene expression through miRNA-mRNA interaction [9], e.g., miRNAs bind complementary target RNAs and block translation (11-16%) t [10] or trigger the destruction of targeted mRNA (a majority, >84%) [11]. Nevertheless, accumulating evidence support that cytoplasmic miRNAs can also activate gene expression unconventionally through enhancing the translation by seed-matching binding on the 5 [12] or 3 untranslated region (UTR) [13], or even non-seed-matching sites in the 5 UTR  miR-193b-3p is different from that of other animals (low). (B) Expression profiles of human MIR193BHG, miR-193b-5p, and miR-193b-3p in multiple tissues from GTEx. Arrows mark the skeletal muscle. (C) Levels of miR-193b-3p in tissues from embryonic 75 days (E75) and 3 d postnatal (B3) goat kids, or longissimus dorsi (LD) muscle from E45 to B3, as well as in cultured skeletal muscle satellite cells (MuSCs). RNA levels are quantified by employing RT-qPCR and calculated by the 2 −∆∆Ct methods, with normalization to β-actin (ACTB) and values of the first tissue or timepoint set to 1. Data are shown as mean ± MSE.
Using RNAs extracted from goat tissues and cultured MuSCs, we also found out that compared with other internal organs, extinguished miR-193b-3p levels were presented in goat muscles from 75 d embryos (E75) and newborn goats ( Figure 1C). In addition, miR-193b-3p abundance continually increased in LD muscles with goat development ( Figure 1C). Moreover, miR-193b-3p was dramatically elevated when shifting MuSC from GM to DM ( Figure 1C). These suggest that miR-193b-3p most likely plays a critical role in mammal skeletal muscle development.
These results imply that miR-193b-3p closely regulates the myogenesis of MuSC, which may be mainly attributed to the activation of proliferation ability.
These results imply that miR-193b-3p closely regulates the myogenesis of MuSC, which may be mainly attributed to the activation of proliferation ability. Ectopic miR-193b-3p elevates the mRNA abundance of myogenic genes. The cultured goat MuSCs were transfected with miR-193b-3p mimic or control (mi-193b-3p or mi-Ctrl, 50 nM). The RNA levels are quantified via RT-qPCR and calculated using the 2 −ΔΔCt , with β-actin (ACTB) as an internal control and values of the mi-Ctrl set to 1. (B) miR-193b-3p upregulates the proteins of PAX7 and MyoG. Western blotting (WB) assay was typically performed to detect MyoG and PAX7 protein in MuSCs treated with miR-193b-3p or mi-Ctrl. GAPDH works as a loading control. (C) miR-193b-3p promotes cell proliferation measured using Cell-Counting Kit-8 (CCK-8). (D) and (E) miR-193b-3p increases EdU + cells and myotube formation. Left panel, Representative immunofluorescence images of EdU and MyHC staining cells after transfected mi-193b-3p or mi-Ctrl (50 nM). Cells are cultured in GM consisting of 10 μM EdU, stained with anti-DAPI (blue) and anti- Figure 2. miR-193b-3p promotes the proliferation and differentiation of goat skeletal muscle satellite cells (MuSCs). (A) Ectopic miR-193b-3p elevates the mRNA abundance of myogenic genes. The cultured goat MuSCs were transfected with miR-193b-3p mimic or control (mi-193b-3p or mi-Ctrl, 50 nM). The RNA levels are quantified via RT-qPCR and calculated using the 2 −∆∆Ct , with β-actin (ACTB) as an internal control and values of the mi-Ctrl set to 1. (B) miR-193b-3p upregulates the proteins of PAX7 and MyoG. Western blotting (WB) assay was typically performed to detect MyoG and PAX7 protein in MuSCs treated with miR-193b-3p or mi-Ctrl. GAPDH works as a loading control. (C) miR-193b-3p promotes cell proliferation measured using Cell-Counting Kit-8 (CCK-8). (D,E) miR-193b-3p increases EdU + cells and myotube formation. Left panel, Representative immunofluorescence images of EdU and MyHC staining cells after transfected mi-193b-3p or mi-Ctrl (50 nM). Cells are cultured in GM consisting of 10 µM EdU, stained with anti-DAPI (blue) and anti-MyHC (red). Scale bar = 100 µm. Right panel, EdU-stained cells (ratio of EdU + myoblasts to all) are evaluated using randomly selected fields and normalized to control. Each treatment is at least tripled. (F) Deficiency of miR-193b-3p suppresses the mRNA abundance of myogenic genes. The RNA levels are quantified via RT-qPCR and calculated using the 2 −∆∆Ct , with β-actin as an internal control and values of the mi-Ctrl set to 1. (G) Proteins of MyoG and PAX7 downregulated by deficiency of miR-193b-3p. Western blotting (WB) assay is typically performed to detect protein levels of MyoG and PAX7 in MuSCs treated within-193b-3p or in-Ctrl. GAPDH works as a loading control. (H) Deficiency of miR-193b-3p delays cell proliferation. (I) Representative images of EdU and MyHC staining cells transfected with in-193b-3p or in-Ctrl (50 nM). Cells are cultured in GM consisting of 10 µM EdU, stained with anti-DAPI (blue) and anti-MyHC (red). Scale bar = 100 µm. (J) The EdU-stained cells are evaluated using randomly selected fields and normalized to control. Each treatment is at least tripled. An unpaired two-tailed t-test is used to evaluate the means difference. Data are shown as mean ± MSE. *** p < 0.001, ** p < 0.01, * p < 0.05, and § p < 0.1.

miR-193b-3p Targets and Induces IGF2BP1 Expression
To systematically anchor the overall effect of miR-193b-3p on global gene expression in the myoblast, we performed transcriptome profiling in MuSCs treated with miR-193b-3p mimic and control (n = 4). Total RNA from mycoplasma-free cells was extracted, qualified (Supplementary Figure S2B,C), and then sequenced to analyze protein-coding. Over 8.29 Gb clean bases were identified for each sample, and the total mapped reads and unique mapped reads were as high as 97% and 94%, respectively (Supplementary Figure S2D). PCA analysis suggests that these samples were expectedly distinguished into two groups (Supplementary Figure S2E).
Among the detected 20,268 transcripts, 471 were differentially expressed (DE, Padj < 0.05, Supplementary Figure S3A). The functional analysis suggested that the upregulated DE genes (n = 264) were significantly enriched in cell proliferation and muscle development (Supplementary Tables S2 and S3). Among these, insulin-like growth factor binding was noticeable (GO:0005520, Padj = 0.015, Supplementary Figure S3B), since insulin-like growth factors (IGFs) play critical roles in muscle hypertrophy and regeneration through interplaying with myogenic regulators such as MyoD and MyoG [35]. We analyzed all 17 insulin family genes detected and found out that IGF2BP1 was the most dramatically affected one out of 14 genes induced by miR-193b-3p (Supplementary Figure S3C).

IGF2BP1 Intermediates Function of MiR-193b-3p on Myoblast Proliferation
It was previously reported that IGF2BPs (also named IMPs) participate in the development of many tissues through cell proliferation, especially HMGA2-IGF2BP2 axis is critical in regulating the activation of satellite cells toward myogenesis [36]. To address the function of IGF2BP1 in myogenesis, we constructed overexpressing vectors using the CDS of IGF2BP1 (pIGF2BP1, 3 μg/mL) and designed small interfering RNAs against IGF2BP1 (siIGF2BP1, 100 nM), and transfected them into MuSC separately. As shown in Figure 4A, pIGF2BP1 dramatically promoted its mRNA (p < 0.001) and protein levels, while siIGF2BP1 resulted in ~20% downregulation of IGF2BP1 mRNA (p < 0.01) but no dominant protein density was altered, which likely resulted from the low endogenous IGF2BP1 in cells. Additionally, pIGF2BP1-treated cells contained higher transcripts of PAX7, MyoD, and MyoG. Conversely, deficiency of IGF2BP1 (siIGF2BP1) was accompanied by downregulation of all myogenic genes (p > 0.10) ( Figure 4B). Correspondingly, the protein levels of PAX7 and MyoG elevated in pIGF2BP1-treated MuSCs coincided with (E) The addition of miR-193b-3p activates luciferase activity of wild-type (R-Luc-IGF2BP1-wt) in MuSCs. (F) miR-193b-3p activates luciferase activity of R-Luc-IGF2BP1-wt in HeLa and mouse C2C12 myoblast. Data are shown as mean ± MSE. An unpaired two-tailed t-test is used to evaluate the means difference. *** p < 0.001, ** p < 0.01, * p < 0.05, and § p < 0.1.

IGF2BP1 Intermediates Function of MiR-193b-3p on Myoblast Proliferation
It was previously reported that IGF2BPs (also named IMPs) participate in the development of many tissues through cell proliferation, especially HMGA2-IGF2BP2 axis is critical in regulating the activation of satellite cells toward myogenesis [36]. To address the function of IGF2BP1 in myogenesis, we constructed overexpressing vectors using the CDS of IGF2BP1 (pIGF2BP1, 3 µg/mL) and designed small interfering RNAs against IGF2BP1 (siIGF2BP1, 100 nM), and transfected them into MuSC separately. As shown in Figure 4A, pIGF2BP1 dramatically promoted its mRNA (p < 0.001) and protein levels, while siIGF2BP1 resulted in~20% downregulation of IGF2BP1 mRNA (p < 0.01) but no dominant protein density was altered, which likely resulted from the low endogenous IGF2BP1 in cells. Additionally, pIGF2BP1-treated cells contained higher transcripts of PAX7, MyoD, and MyoG. Conversely, deficiency of IGF2BP1 (siIGF2BP1) was accompanied by downregulation of all myogenic genes (p > 0.10) ( Figure 4B). Correspondingly, the protein levels of PAX7 and MyoG elevated in pIGF2BP1-treated MuSCs coincided with their mRNA abundance ( Figure 4C), and EdU + cells were increased in pIGF2BP1 and decreased in siIGF2BP1-treated cells, compared to the control group ( Figure 4D). Moreover, we simply discriminated sub-cycling cells using flow cytometric analysis. The pIGF2BP1 treatment upregulated S-phase cells but downregulated G1/G0 cells ( Figure 4D). These results phenocopy overexpression or deficiency of the miR-193b phenotype, respectively. their mRNA abundance ( Figure 4C), and EdU + cells were increased in pIGF2BP1 and decreased in siIGF2BP1-treated cells, compared to the control group ( Figure 4D). Moreover, we simply discriminated sub-cycling cells using flow cytometric analysis. The pIGF2BP1 treatment upregulated S-phase cells but downregulated G1/G0 cells ( Figure 4D). These results phenocopy overexpression or deficiency of the miR-193b phenotype, respectively.  The fold change of EdU + cells is evaluated using randomly selected fields and normalized to control. Data are shown as mean ± MSE. An unpaired two-tailed t-test is used to evaluate the means difference. *** p < 0.001, ** p < 0.01, and * p < 0.05. Furthermore, to validate whether function of miR-193b-3p is mediated by IGF2BP1, we transfected miR-193b-3p mimic (mi-193b-3p) and simultaneously interfering IGF2BP1 (siIGF2BP1) in MuSC. As shown in Figure 4E, IGF2BP1 deficiency neutralized the abundance of miR-193b-3p-elevated proliferation genes, including PAX7 and PCNA (p > 0.10), but not differentiation genes including MyoD, MyoG, and Myomaker (p < 0.05). Furthermore, comparing with the control, the fold change of EdU + cells at 24 h, 48 h, and 72 h were insignificantly altered in cotransfected cells ( Figure 4E), suggesting that instead of myogenic differentiation, the IGF2BP1 is more likely mediating function of miR-193b-3p in myoblast proliferation.

Goat miR-193b-3p Activates IGF2BP1 Transcription
To address the spatial localization of miR-193b-3p and IGF2BP1 mRNA in cells, we designed fluorescence-labeled oligonucleotide probes and performed RNA-RNA duallabeled fluorescence in situ hybridization (FISH). Transcripts of miR-193b-3p and IGF2BP1 were dominantly overlapped in the cytoplasm of MuSC culturing, in growth (GM 48h) and differentiation media (DM 7d, Figure 5A). Typically, over 84% of miRNAs bind complementary target RNAs and trigger their destruction [11]. To monitor the effect of miR-193b-3p on turnover of endogenous IGF2BP1 mRNA, we treated culturing MuSCs with actinomycin D (ActD, 5 µg/mL) to compromise transcription effectively [37]. In addition, we then quantified the remaining levels of IGF2BP1 transcripts at three sequential time points. Expectedly, miR-193b-3p mimic dramatically degraded IGF2BP1 transcripts, in particular at 120 min (p < 0.01). In contrast, interfering miR-193b-3p exhibited no effect on the stability of IGF2BP1 mRNAs ( Figure 5B). These suggest that miR-193b-3p destabilizing IGF2BP1 transcripts in the cytoplasm is similar to the typical function [38], and otherwise the mechanism implicates promoting IGF2BP1.
Furthermore, using data from UCSC, we found that the hsa-miR-193b region in human skeletal muscle myoblasts (HSMM) is enriched with the H3K4Me3 and H3K27Ac histone mark ( Figure 5E upper panel, and Supplementary Figure S5A), a signal used for a promoter and active enhancer discovery, respectively [41]. Whereas mouse miR-193b barely overlaps with eRNA markers (Supplementary Figure S5B). On the other hand, both human and mouse IGF2BP1 3 UTR region lack histone modification (Supplementary Figure S6A,B), suggesting that the activation of miR-193b-3p on IGF2BP1 is most likely attributed to the enhancer-related characteristics of miR-193b-3p.
A mature miRNA and its passenger strand present synergistic function and the same phenotype [42]. Therefore, to validate whether the goat miR-193b gene is similar to enhancer-related hsa-miR-193b-3p, we amplified~60 bp 5p and 3p region, inserted them into a PGL3-Promoter vector separately (named pre-193b-5p and pre-193b-3p, Figure 5E lower panel), and transfected these vectors into MuSC and HeLa. As shown in Figure 5F, comparing to the basic (pBasic) and control (pCtrl), pre-193b-3p efficiently promoted F-Luc/R-Luc levels in GM and DM myoblasts, as well as in HeLa cells (p < 0.01 or 0.001). Meanwhile, pre-193b-5p enhanced F-Luc/R-Luc values significantly in DM cells (p < 0.001). These imply that the sequence of goat miR-193b could activate gene expression. Sci. 2022, 23, x FOR PEER REVIEW 9 of 21  Another characteristic of activation-related miRNAs is their cis-activation on neighboring genes [15]. According to the goat genome, the adjacent genes for miR-193b include MKL2 (MKL/Myocardin Like 2), PARN (Poly(A)-Specific Ribonuclease), and BFAR (Bifunctional Apoptosis Regulator) ( Figure 5G). We therefore quantified transcripts of these genes in goat MuSCs after perturbing miR-193b-3p, and found that in GM myoblasts, their expression was markedly waved along with the miR-193b-3p levels; while in DM cells, miR-193b-3p changed these three genes modestly ( Figure 5H). Furthermore, we constructed vectors overexpressing the pri-miR-193b region (~400 bp) and unveiled that IGF2BP1 and PARN mRNA were significantly increased by ectopic pri-miR-193b in GM cells (p < 0.01, Figure 5I). Additionally, disturbing miR-193b-3p failed to alter miR-365-3p in goat MuSCs ( Figure 5J, left panel), inconsistent with the results reported in mouse C2C12 [25], which may be ascribed to the differed location of miR-365 and miR-193b among species ( Figure 5J, right panel, and Supplementary Figure S7).
These imply that goat miR-193b-3p is most likely similar to has-miR-193b, working as an enhancer-related miRNA that activates the target gene transcriptionally. These imply that goat miR-193b-3p is most likely similar to has-miR-193b, working as an enhancer-related miRNA that activates the target gene transcriptionally.

Discussion
Muscle formation is precisely mastered by a handful of hierarchical gene cascades covering protein-coding genes such as MRFs, as well as non-coding genes such as miR-NAs. It is well-accepted that miRNAs play critical roles in muscle development through inversely fine-tune gene enrichment, canonically [5,6]. Nevertheless, a few miRNAs have myoblasts to all) are evaluated using randomly selected fields and normalized to control. Each treatment is repeated ten times. (D) Cell cycle is affected by miR-193b-3p mimic. Flow cytometric assay is performed for cells treated with mi-193b-3p or mi-Ctrl (n = 3). Data are shown as mean ± MSE. ** p < 0.01, * p < 0.05, and § p < 0.1.

Discussion
Muscle formation is precisely mastered by a handful of hierarchical gene cascades covering protein-coding genes such as MRFs, as well as non-coding genes such as miRNAs. It is well-accepted that miRNAs play critical roles in muscle development through inversely fine-tune gene enrichment, canonically [5,6]. Nevertheless, a few miRNAs have gained much attention for newly appreciated activation roles indirectly targeting genes [14,15]. Here, we reported that the anti-tumor gene miR-193b-3p induces proliferation and differentiation of goat MuSCs, which phenocopies IGF2BP1 overexpression. Meanwhile, IGF2BP1 knockdown impairs myogenic proliferation, but not differentiation, of MuSCs induced by miR-193b-3p supplementation. Furthermore, miR-193b-3p located in the nucleus, blooms IGF2BP1 abundance transcriptionally and its neighboring genes. In addition, miR-193b-3p destructs IGF2BP1 mRNA in the cytoplasm, which is in line with the regulation of miR-193b-3p on IGF2BP1 transcripts in mice C2C12 myoblasts, reported previously [25]. These results suggest that miR-193b-3p/IGF2BP1 axis activates goat myogenesis, mainly attributed to its nucleus function.

Anti-Proliferation vs. Pro-Proliferation Function of miR-193b in Muscle
In cancer tissues, miR-193b-3p functions oppositely as an anti-tumor [19,43] or tumorinducer [44] under differing disease conditions. As for the normal development, miR-193b signaling induces adipogenesis for adipose-derived stem cells [28,29] or is even shifted from C2C12 cells [25]. On the contrary, deficiency of miR-193b induces cells from mouse BAT to the muscle lineage with upregulated levels of muscle-specific genes, including Cmk, Myf5, Myf6, MyoD, and MyoG [25]. Although muscle diseases such as Duchenne muscular dystrophy 1 (DMD1) and DMD2 share similar symptoms, including gradually worsening muscle loss and weakness, miR-193b is accumulating in DMD1 patients [22] but is downregulated in DMD2 [23], suggesting their miR-193b-related intrinsic mechanism differs. Using C2C12 myoblasts, we validated the suppressing effect of miR-193b-3p on myogenic proliferation reported previously [25]. On the contrary, the gene expression, CCK-8, EdU, and cell cycle analysis implied that miR-193b-3p induced goat MuSCs proliferation toward the myotube. These suggest that the myogenic function of miR-193b-3p is closely associated with the microenvironment and species.

Activation of miR-193b on IGF2BP1 Transcription
For a long time, miRNAs have been tacitly assumed as cytoplasm located and posttranscriptional negative regulators of its target gene expression [10,11]. With high-throughput assessment, numerous mature miRNAs are currently identified in the nucleus [39]. Correspondingly, several models elaborating functions of nuclear miRNAs at transcriptional gene silencing (TGS) and transcriptional gene activation (TGA) have been constructed. MiRNA targets and binds nascent RNA transcripts, gene promoter, or enhancer regions to exert different effects via recruiting additional epigenetic and (or) transcriptional factors [39]. For example, miR-483-5p promotes IGF2 transcription by targeting the P3 5 UTR and promoter, and enriching enhancer marks such as H3K4me3 and H3K27ac [16].
Given the close relationship between gene enhancer and promoter [45,46], we speculate that most likely enhancer-related miRNA-target pair, including enhancer-related miRNA or enhancer-related target, play a critical role in the activation of miRNAs on its target. Currently, the enhancer-related miRNAs are classified into the super-enhancers (SEs)-miRNAs (SEs neighbor cell-type-specific abundant miRNAs) that boost pri-miRNA processing [47] and the enhancer-overlapped-miRNAs (transcribed from enhancer region) that activate their neighboring gene in cis, as well as global genes in trans [15]. Here, we validated that goat miR-193b-3p induced IGF2BP1 through the complementary binding on its 3 UTR. Furthermore, goat miR-193b-3p was partially nucleus-distributed and promoted IGF2BP1 hnRNA. In addition, both pre-miR193b-5p and 3p could activate gene expression and promote its neighboring genes' expression, especially in GM MuSCs. Nevertheless, in mouse C2C12 myoblasts, miR-193b-3p negatively regulated IGF2BP1, which may be ascribed to the absence of H3K27ac and H3K4Me3 modification on the mouse miR-193b-3p gene locus. Additionally, the differed character between goat myoblasts and immortalized C2C12 could potentially affect their effect on miR-193b-3p function, similar to the inconsistent apoptotic responses between cell lines and primary culture cells [48].
These suggest that goat miR-193b-3p is most likely similar to hsa-193b-3p, which works as an enhancer-overlapped miRNA in the nucleus.

Ethics Declarations
All experimental protocols in this study were strictly conducted following the Regulations for the Administration of Affairs Concerning Experimental Animals by China Ministry of Science and Technology and entirely approved by the Animal Care and Use Committee, Sichuan Agricultural University (No. DKY-20190035).

Animals and Samples Collection
A total of 12 goat fetuses and kids aged 45, 60, and 105 days of gestation and 3 days postnatal (n = 3 per stage; female) were randomly chosen and humanely sacrificed at the Jianzhou Big-Eared goats farm (Chengdu, China). Samples from the heart, liver, lung, spleen, kidney, and longissimus dorsi (LD) muscle were quickly collected and snap-frozen in liquid nitrogen for further study.
For the gain and loss function study, when cells were at 80~90% confluence, the GM was replaced with DMEM supplemented with 10% FBS. Then, cells were transfected using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) with interfering RNA (siCtrl, siIGF2BP1, in-Ctrl, or in-193b-3p) or overexpression plasmid (pCtrl or pIGF2BP1) and chi-miR-193b-3p mimic (mi-193b-3p, RiboBio, Guangzhou, China) at indicative concentrations, according to the manufacturer's direction. The transfected cells were kept in GM or replaced with DM. Cells were collected for RNA/protein assay at 48 h, stained with EdU at indicative time points, or with MyHC (Myosin Heavy Chain) antibody at 7th d post differentiation.
Given that culturing cells are easily contaminated by mycoplasma, we monitored mycoplasma contamination based on previously reported protocols [57]. Briefly, cell culture supernatant was collected and detected using TransDetect ® PCR Mycoplasma Detection Kit (TRansgen Biotech, Beijing, China) to ensure cells were mycoplasma-free before transfection (Supplementary Figure S2C).
To provide solid interfering results for IGF2BP1, three small interfering RNA, including siRNA1, CAGCTCCTTTATGCAGGCT; siRNA2, CTCCTTTATGCAGGCTCCA; and siRNA3 CTTTATGCAGGCTCCAGAG, targeting goat IGF2BP1 mRNA, were designed and synthesized in RiboBio (Guangzhou, China). We transfected them separately into MuSCs at two concentrations (50 nM and 100 nM) and then quantified IGF2BP1 mRNAs. The results suggest that IGF2BP1 levels are efficiently decreased at 100 nM for each siRNA, thus in the following experiment, we pooled them at 100 nM concentration. In addition, the miR-193b-3p miRNA mimic (mi-193b-3p) and control (mi-Ctrl), as well as inhibitor (in-193b-3p) and control (in-Ctrl) were ordered from RiboBio (Guangzhou, China).

RNA Extraction, Quality, Reverse Transcription, and Real-Time PCR (RT-qPCR)
Following the manufacturer's instructions, total RNAs were extracted from tissues or cultured cells using RNAiso Plus reagent (Takara, Dalian, China). To reduce DNA contamination, RNA samples were treated with DNase twice. Then, we measured their absorbances at 230, 260, and 280 nm using a Nano One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, samples with A260/A230 and A260/A280 ratio between 1.9 and 2.0 were kept, followed by monitoring their integrity on 1.5% agarose gel electrophoresis and concentration on a NanoDrop 2000c Spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA).
The qualified RNAs (~1 mg) were reversely transcribed into cDNA using the Prime-Script™ RT reagent Kit with gDNA Eraser or miRNA PrimeScript RT reagent Kit (Takara, Dalian, China) separately for mRNA or miRNA assay. Then, according to the manufacturer's guide, expression levels of target genes in these cDNA were accurately measured by using real-time PCR (RT-qPCR) on the Bio-Rad CFX96 system (Bio-Rad, Shanghai, China) with SYBR Premix Ex TaqTM II (Takara, Dalian, China). Each treatment was performed at least three independent times, and each sample triplicated in qPCR. Moreover, to enhance the accuracy, three housekeeping genes in goat (ACTB, Actin Beta; SDHA, Succinate Dehydrogenase Complex Flavoprotein Subunit A; PGK1, Phosphoglycerate Kinase 1) and mouse (ACTB; GAPDH; Hprt, Hypoxanthine Phosphoribosyltransferase 1) were used as an internal control. The 2 −∆∆Ct or 2 −∆Ct methods were employed to calculate the relative RNA levels of target genes.

Primers Designing and Validation
Primer pairs were designed using Primer Premier 5.0 and their specificities were screened via Primer-BLAST (https://www.ncbi.nlm.nih.gov/, accessed on 21 March 2019) with both goat and human Refseq as background, and then synthesized and PAGE purified (Sangon, Shanghai, China). Moreover, we validated their specificity using Sanger sequencing and quantified the amplification efficiency of each RT-qPCR primer pair (between 95~105%). The detailed information for primers was listed in Supplementary Table S1.

CCK-8 Assay
Cells were inoculated in 96-well plates with 2 × 10 3 cells per well initially (~30% confluency) and cultured in GM for 2 days, then transfected with different vectors according to protocols described above. Every 24 h, the absorbance value for each sample was measured at 450 nm by using a Microplate Reader (Model 680; Bio-Rad, Hercules, CA, USA), after adding 10 µL of Cell-Counting Kit-8 (CCK-8) reagents (Solarbio, Beijing, China) to the cells for 2 h.
Images were captured less than 5 h after hybridization, using Nikon DS-U3 in Nikon Eclipse Ti-SR, and then analyzed using the CaseViewer software version 2.3.0.

Nuclear-Cytoplasmic Fractionation
To accurately quantify target genes in the cytoplasm and nucleus separately, MuSCs proliferating for 2 d and differentiating for 7 d were fractionated into nuclear/cytoplasmic parts by using Nuclear/cytoplasmic fractionation and a Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) as described before [58]. In brief, the total RNA was separately extracted from both the supernatant (cytosol fraction) and pellet (nuclei), retro-transcribed into cDNA, and it quantified targeted genes using RT-qPCR. The nuclearenriched U6 and cytoplasm-enriched 18S were used as controls.

ActD Analysis
According to methods described previously [59], we analyzed the effect of miR-193b-3p on IGF2BP1 mRNA stability by using actinomycin D (ActD, Sigma, Shanghai, China) to block new mRNA synthesis. In general, MuSCs were cultured in 12-well (~1 × 105 cells per well) plates and separately transfected with miR-193b-3p mimic, mimic NC, inhibitor, and inhibitor NC for 24 h. Then, cells were treated with actinomycin D (5 µg/mL) and harvested at 0, 60, and 120 min. Each treatment contained at least three independent biological repetitions. The total RNA was extracted from each sample, and finally the remaining levels of IGF2BP1 mRNA were quantified using the canonical RT-qPCR method.

mRNA-seq and Bioinformatic Analyses
Library preparation and poly(A) selection mRNA-seq was performed at Novogene Company (Beijing, China). Briefly, using NEBNext ® UltraTM RNA Library Prep Kit Illumina ® (Illumina, San Diego, CA, USA), qualified total RNA samples (200 ng) extracted from cells transfected with miR-193b-3p mimic or control (n = 4 per group) were used to isolate the polyA fraction (mRNA), followed by fragmentation and generation of doublestranded cDNA. Libraries were sequentially evaluated by Qubit 2.0 Fluorometer and Agilent 2100 bioanalyzer. Then, qualified libraries were sequenced in Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) with a 2 × 150 bp pair-end.

Statistical Analysis
Unless stated otherwise, data presented here are shown as mean ± MSE from at least triplicated independent samples or animals. The unpaired two-tailed t-test in Graphpad Prism 7.0 was employed to evaluate the means' difference, with a significant threshold value set at *** p < 0.001, ** p < 0.01, * p < 0.05, and § p < 0.1.

Conclusions
We reported that miR-193b-3p induces proliferation and differentiation of goat MuSCs, phenocopying IGF2BP1 overexpressing. The enhancer-overlapped miRNA-193b-3p carries a dual function ability: activating its target gene within the nucleus, as well as repressing mRNA in the cytoplasm by directly targeting the 3 UTR of the IGF2BP1. Our research further extends the repertoire of miRNA functions.

Informed Consent Statement: Not applicable.
Data Availability Statement: The accession number for the raw RNA sequencing data reported here is NCBI BioProject: PRJNA665306.