Mir-15b and mir-322 inhibit setd3 expression to repress muscle cell differentiation

ABSTRACT SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the

transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown.

Here, we report that two important microRNAs (miRNAs) could repress _SETD3_ and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and

characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of _SETD3_ through directly targeting the 3’-untranslated region of _SETD3_ gene. Functionally,

overexpression of miR-15b or miR-322 leads to the repression of endogenous _SETD3_ expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322

derepresses endogenous _SETD3_ expression and facilitates myoblast differentiation. In addition, knockdown _SETD3_ in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated

differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during

muscle cell differentiation, which in turn affects _SETD3_ expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate

microRNAs fates, thereby controlling _SETD3_ expression and eventually determining skeletal muscle differentiation. SIMILAR CONTENT BEING VIEWED BY OTHERS KAP1-ASSOCIATED TRANSCRIPTIONAL

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SKELETAL MUSCLES Article Open access 10 February 2021 INTRODUCTION Skeletal muscle differentiation is a complex process orchestrated by a family of myogenic regulatory factors (MRFs),

including MyoD, myogenin, MRF4, and Myf51,2. Expression of MyoD and Myf5 in the initial stages of differentiation induces expression of myogenin and muscle-specific transcription factors

MEF2, whereas myogenin and MRF4 are expressed in the late stages of differentiation to activate the myogenic program by induction of muscle gene expression and silence of cell cycle-related

gene expression2,3,4. Moreover, the functional interplay between key myogenic transcriptional factors and additional regulators is also critical for determining muscle cell fate and

myotube/myofibers formation2,5,6. MicroRNAs (miRNAs) modulate gene expression at the post-transcriptional level either by promoting mRNA degradation or inhibiting translation through

complementary targeting 3’ untranslated regions (3’-UTRs) of specific mRNAs2,6. Many studies have demonstrated that miRNAs participate in skeletal muscle differentiation. The muscle-specific

miRNAs, miR-206, miR-1, and miR-133, are abundantly expressed during skeletal muscle differentiation, and promote muscle differentiation by inhibition specific transcription

repressors7,8,9,10. In addition, many non-muscle specific miRNAs also regulate muscle differentiation by post-transcriptional mechanisms that affect the presence and functions of the

myogenic factors, either positively or negatively. Our previous work focused on studying the biological roles of SETD3, which has been reported as a histone H3 Lys4 and Lys36

methyltransferase11. But very recent two studies clearly demonstrated that SETD3 is an actin-specific histidine methyltransferase12,13. We have shown that SETD3 is a cell-cycle regulated

protein, and abnormal high level of SETD3 would lead to liver tumorigenesis14. A previous study has suggested that SETD3 is capable to interacting with MyoD and synergistically binding to

the promoter of several muscle-related genes, thereby promoting muscle cell differentiation11. Knockdown of _SETD3_ markedly impairs the differentiation processes, indicating its important

role in muscle differentiation. However, how SETD3 is regulated during this process is completely unknown. In this study, we hypothesized that _SETD3_ gene is post-transcriptionally

repressed by miRNAs. We uncovered that miR-15b and miR-322 could repress _SETD3_ expression by targeting the 3′-UTR region in skeletal muscle cells. Furthermore, we revealed that two known

transcription factors, E2F1 and FAM3B, could regulate miR-15b or miR-322 expression, respectively, during muscle cell differentiation. Thus, our results established a regulatory network

between transcription factors, miRNAs, and an epigenetic modifier SETD3, which highlights a protein-microRNA involved cascade regulatory mechanism during skeletal muscle differentiation.

RESULTS SETD3 IS REQUIRED FOR C2C12 CELL DIFFERENTIATION Previous study suggested that SETD3 regulates muscle differentiation11. To confirm this, we first generated a monoclonal SETD3

antibody to detect endogenous SETD3 protein. This anti-SETD3 antibody specifically recognizes the SETD3 protein, as detected SETD3 signal was diminished when _SETD3_ gene was knocked out in

Hela S3 cells and overexpression of SETD3 constructs from either human or mouse species in the _SETD3_ knockout cell line displayed specific bands (supplementary Fig. S1a). In addition, this

anti-SETD3 antibody also recognizes endogenous SETD3 in C2C12 mouse myoblast cells, and knockdown of mouse _SETD3_ by stable expression of two different sh_SETD3_ constructs exhibited

significant reduction of SETD3 level, indicating its specificity and species reactivity against mouse homolog SETD3 as well (supplementary Fig. S1a). Next, to examine whether SETD3 is

required for cell differentiation, C2C12 cells was induced by cultured in the differentiation medium (DM), and expression of _SETD3_ in both transcriptional levels and protein levels were

examined. Consistent with previous results, transcription levels of several key regulatory factors including _MYF5, MYOG, TNNT2/Troponin_, and _MYH1/MYHC_ were gradually increased during

differentiation, with a similar trend of _SETD3_ expression, indicating cell differentiation occurred (Fig. S1b)2. Intriguingly, we found that the protein levels of SETD3 displayed an

increase at the early stage of differentiation, but showed a reduction when MHC protein was significantly accumulated, which may suggest a complicated regulatory mechanism of SETD3 involved

in muscle differentiation (Fig. S1c). To rule out the possibility that the reduction of SETD3 protein level at the late stage of differentiation is due to our home-made antibody recognition

issue, a commercial available antibody was utilized to examine SETD3 protein levels, and a similar expression pattern of SETD3 protein was observed (Fig. S1c). In addition, both antibodies

were verified using two different synthesized siRNA oligos targeting _SETD3_, which confirmed the specificity of both antibodies (Fig. S1d). Consistent with previous report that knockdown of

_SETD3_ severely slows muscle cell differentiation based on the observation of cell morphology and differentiation gene expression11, we also observed knockdown of _SETD3_ remarkably

delayed cell differentiation, based on the divergence of cell morphology (Fig. S1e, f). Moreover, the protein levels of MHC as well as the mRNA levels of various differentiation markers were

significantly reduced compared to the control cells during the progression (Fig. S1g, h). Therefore, our data support that SETD3 is required for C2C12 muscle cell differentiation.

IDENTIFICATION OF MIRNAS THAT MIGHT AFFECT _SETD3_ EXPRESSION We are interested in how _SETD3_ levels are regulated at post-transcriptional levels during cell differentiation. Thus, we

attempted to identify whether miRNAs might regulate expression of _SETD3_. To this end, the 3′ end of untranslated region (3′-UTR, nt 1786-2541) of mouse _SETD3_ gene was selected for

searching potential miRNAs using miRanda and TargetScan softwares15,16. Based on the predicted scores, we obtained several potential miRNAs and the top 5 candidates were selected (Fig. 1a).

Interestingly, the binding regions of these five potential candidates are nearly identical, which are located from nt 1872 to nt 1894 in the 3′-UTR of _SETD3_ gene. To identify which miRNAs

might regulate _SETD3_ expression, we first cloned the full-length (756 nt) 3′-UTR of the mouse _SETD3_ gene and inserted into the downstream of a dual-luciferase reporter construct17. After

the reporter construct was transfected into 293 T cells, we observed that only miR-15b or miR-322, but not other tested miRNAs, inhibited luciferase activity compared with the control

construct (Fig. 1b). MiR-410 has been known to be not involved in regulation of _SETD3_ expression, which served as a negative control. To further confirm this, a short 3′-UTR sequence (23 

nt) that only contains the predicted binding sites shared by all the 5 miRNAs were inserted into the downstream of a dual-luciferase reporter construct. Again, we found that only miR-15b and

miR-322 showed repressive effect towards luciferase activity (Fig. 1c). Of note, miR-15b and miR-322 share the same 3′-UTR region of _SETD3_ gene, but with a slight difference in the seed

region (Fig. 1d). Moreover, these two miRNAs are highly conserved among different species, suggesting their intrinsic function (Fig. 1e). MIR-15B AND MIR-322 DIRECTLY TARGETED THE 3′-UTR OF

_SETD3_ GENE To validate whether miR-15b and miR-322 indeed targeted the 3′-UTR of _SETD3_ gene, the predicted seed sequences of the short 3′-UTR in the luciferase reporter were mutated, and

luciferase assays were performed as described above (Fig. 2a). We noticed that, when the reporter construct containing triple repeats of the short 3′-UTR of _SETD3_, the luciferase activity

was repressed by both miR-15b and miR-322 more efficiently than the one containing a single copy of the short 3′-UTR of _SETD3_. In contrast, the luciferase activity remained invariable

after the seed sequences in the 3′-UTR were mutated, compared to the control sample (Fig. 2b, c). When the indicated luciferase reporter constructs were transfected into cells that stably

express pri-miR-15b or pri-miR-322, the luciferase activities were repressed by these miRNAs, but not by the empty luciferase reporter vector (Fig. 2d, e). In contrast, cotransfection of the

indicated miRNA inhibitors with the luciferase reporter constructs into cells, the luciferase activities were enhanced compared to the control, suggesting their repressive roles of miRNAs

in _SETD3_ gene expression (Fig. 2f). Therefore, these results provided clear evidence showing that miR-15b or miR-322 can directly target the 3′-UTR of _SETD3_ gene in vitro. MIR-15B AND

MIR-322 REPRESS _SETD3_ EXPRESSION THROUGH BINDING TO THE 3′-UTR OF _SETD3_ Next, we further explore whether miR-15b and miR-322 could repress _SETD3_ expression in vivo. Five different

miRNA sense oligos were transfected into C2C12 cells and SETD3 transcriptional and protein levels were examined by real-time quantitative PCR (RT-qPCR) and Western blot analyses.

Consistently, only miR-15b or miR-322 remarkably reduced _SETD3_ levels (Fig. 3a). Furthermore, we observed that transfection of the wild-type miRNA oligos, but not the miRNA-15b or

miRNA-322 mutants in which either the seed sequences or the non-seed sequences have been changed, was able to affect SETD3 protein levels dramatically (Fig. 3b–d). The inert effect of

miRNA-322 mutant 2 on _SETD3_ might result from its non-essential role of targeting 3′-UTR of _SETD3_ gene (Fig. 3d). As expected, overexpression of primary miR-15b or miR-322 construct in

turn inhibited SETD3 protein levels (Fig. 3e). In addition, after antisense oligos of miR-15b or miR-322 (miRNA inhibitor) were transfected into C2C12 cells, both the transcriptional levels

and the protein levels of SETD3 markedly increased compared to the control transfection (Fig. 3f, g). Alternatively, small guide RNA (sgRNA) was utilized to investigate the impact on _SETD3_

expression. The two sgRNAs were able to decrease expression level of miR-15b or miR-322, respectively (Fig. 3h). Consistently, SETD3 protein levels were increased by sgRNA knockdown of

miRNAs, suggesting _SETD3_ may be the target of the two miRNAs in C2C12 cells (Fig. 3i). Consistent with previous reports that _CCNE1_ was also targeted by miR-15b and miR-322, the encoded

Cyclin E1 protein levels were moderately increased with knockdown of these two miRNAs18,19,20,21. Importantly, the effect of two sgRNAs on _SETD3_ were not due to off-target effect, as

coexpression of sgRNAs and their corresponding miRNA mimics compromised an accumulation of SETD3 caused by transfection sgRNAs alone (supplementary Fig. S2). Taken together, we conclude that

miR-15b and miR-322 can directly target the 3′-UTR of _SETD3_ gene, which lead to inhibition of _SETD3_ expression. MIR-15B AND MIR-322 REPRESS MYOBLAST DIFFERENTIATION Since both miR-15b

and miR-322 can repress _SETD3_ expression, we want to determine how these two miRNAs are expressed during muscle cell differentiation. To do this, we first utilized a database published

previously and analyzed the expression levels of these miRNAs in various mouse tissues22. Consistently, the expression level of miR-206, a muscle-specific miRNA, is enhanced over 2000 fold

in mouse muscle compared with that in mouse embryonic stem (ES) cells10. Interestingly, the expression levels of miR-15b and miR-322 are significantly decreased in mouse muscle compared to

those in ES cells (33-fold or 8-fold reduction respectively) (Fig. 4a and supplementary Table S1). These two miRNAs are comparably expressed in other tissues, suggesting that they are not

muscle-specific miRNAs. To this end, we then investigated the dynamic expression levels of miR-15b and miR-322 during C2C12 differentiation by RT-qPCR. MiR-1 was served as a positive

control, as its expression level has been reported to gradually increase during muscle differentiation8; whereas miR-16 was served as a negative control, as it does not target _SETD3_

demonstrated in our result (Fig. 1b, c). We observed that miR-15b levels were declined during C2C12 differentiation, which is inversely correlated with _SETD3_ levels. Intriguingly, miR-322

levels remained unchanged, which is consistent with the results that we analyzed using the public GEO database (NCBI, Gene Expression Omnibus, www.ncbi.nih.gov/geo) (Fig. 4b). Next,

undifferentiated C2C12 cells were transfected with synthetic miRNAs mimics, and treated with differentiation medium to induce myogenic differentiation for 4 days. RT-qPCR analysis showed

that endogenous _SETD3_ mRNA levels were decreased (Fig. 4c). Western blot analysis confirmed that the protein levels of differentiation markers, such as MHC and Myogenin, decreased upon

transfection of these two miRNAs mimics (Fig. 4d). Furthermore, immunofluorescence assays showed that myoblasts transfected with those miRNA mimics attenuated myoblast differentiation, as

visualized by a significant decrease in the number and size of myotubes (Fig. 4e, f). Of note, the nuclei numbers per fiber were dramatically decreased, illustrating the defect of myoblast

fusion into myotubes (Fig. 4g). Thus, we concluded that miR-15b and miR-322 may function in repression of myoblast differentiation. MIR-15B AND MIR-322 INHIBIT _SETD3_ TO REGULATE MUSCLE

CELL DIFFERENTIATION Next we determine whether reduction of miR-15b or miR-322 could derepress its negative role in muscle differentiation. Therefore, a pair of sgRNAs or miRNA inhibitors

that targeted miR-15b or miR-322, respectively, was transfected into C2C12 cells. After 4 days induction of myogenic differentiation, a significant increase of SETD3 levels accompanied with

elevated levels of myogenic markers MHC and Myogenin were observed (Fig. 5a, b). Furthermore, immunofluorescence assays showed that myoblasts transfected with miRNA inhibitors formed more

myotubes (Fig. 5c). Quantitative measurement of the numbers of myotubes and the nuclei numbers per fiber demonstrated that repression of miR-15b or miR-322 promotes myoblast differentiation

(Fig. 5d, e). To validate whether miR-15b or miR-322 affects myoblast differentiation via regulating _SETD3_, a rescue experiment was performed. C2C12 cells were transfected with siRNA

targeting _SETD3_. After 4 h treatment, the inhibitor of miR-15b or miR-322 was separately added into cells for additional 6 h. After continued to culture in fresh media for 24 h, cells were

induced to differentiation for additional 4 days. Inhibition of endogenous miR-15b or miR-322 increased _SETD3_ levels and facilitated cell differentiation; whereas knockdown of endogenous

_SETD3_ rescued miR-15b or miR-322 inhibitors-mediated cell differentiation, evaluated by MHC levels (Fig. 5f). This result suggested that miR-15b and miR-322 repress muscle cell

differentiation via inhibition of _SETD3_ expression. E2F1 AND FAM3B REGULATE _SETD3_ LEVELS THROUGH CONTROLLING MIR-15B OR MIR-322 EXPRESSION, RESPECTIVELY Next, we want to address how

expressions of miR-15b and miR-322 are regulated during muscle differentiation. Previous studies have demonstrated that the pivotal transcription factor E2F1 directly targets promoters of

miR-15 and miR-16 clusters and E2F1 inhibits myogenic differentiation23,24. Meanwhile, Zhang et al. recently reported that FAM3B inhibits miR-322 expression during high glucose induced

vascular smooth muscle cell proliferation25. These results prompt us to investigate whether E2F1 and FAM3B regulate expression of miR-15b and miR-322 during skeletal muscle differentiation,

respectively. To test this, we first examined the expression profiles of _E2F1_ and _FAM3B_. As expected, during myogenic differentiation process, _E2F1_ expression was reduced, whereas

_FAM3B_ expression was gradually increased (Fig. 6a). Consistently, knockdown of the positive transcriptional regulator E2F1 remarkably repressed miR-15b expression, and consequently

increased _SETD3_ expression, but had no obvious impact on miR-322 expression (Fig. 6b). Meanwhile, knockdown of the negative regulator FAM3B promoted miR-322 expression, and consequently

reduced _SETD3_ expression, but had no effect on miR-15b expression (Fig. 6c). We speculate that E2F1 and FAM3B could regulate muscle differentiation by affecting _SETD3_ levels. Thus, SETD3

levels were examined in C2C12 cells transfected with HA-tagged E2F1 by immunoblotting. Interestingly, overexpression of E2F1 reduced SETD3 protein level (Fig. 6d). Similarly, when we

altered FAM3B levels by overexpression or siRNA knockdown FAM3B, SETD3 protein levels were accordingly changed as expected (Fig. 6e, f). Moreover, if E2F1 was inhibited by siRNA knockdown,

we observed much faster cell differentiation compared to the control cells after switching cells to the differentiation medium (Fig. 6g, h). In contrast, knockdown of FAM3B slowed down cell

differentiation compared to the control cells, as both SETD3 and Myogenin levels were obviously decreased in same differentiation conditions (Fig. 6i, j). Together, these data indicated that

E2F1 or FAM3B either positively or negatively regulates miRNAs, consequently affects SETD3 and muscle differentiation. DISCUSSION In this study, we uncover a novel function of miR-15b and

miR-322 in C2C12 differentiation beyond their roles in cancers26,27. Furthermore, we verify that a well-known transcription factor E2F1 is required for the reduction of miR-15b expression

and the upregulation of SETD3, thereby promoting myoblast transition to myotube formation. Meanwhile, we also illustrate that a negative transcription regulator FAM3B is upregulated during

this differentiation process, accompanied with decreased miR-322 level as well as increased SETD3 level (Fig. 7). These two parallel pathways of regulation of SETD3 expression highlight the

importance of a protein-miRNAs interplay network during skeletal muscle differentiation. MiRNAs have been elucidated to participate in almost every aspect of biology. For instance, the

miR-15a/16-1 and miR-15b/16-2 clusters have been shown to regulate cell cycle and apoptosis by targeting _CCND3_ or _CCNE1_24. MiR-15b has been shown to play roles in adipogenesis, lipid

metabolism, and modulating DNA damage response28,29,30. In addition, miR-15a and miR-15b also functions as tumor suppressors, especially in B-cell oncogenesis, suggesting their potential

clinical application26,31. It is worthy to note here that we uncovered miR-15b, but not miR-15a, regulates skeletal muscle differentiation, although these two miRNAs share very similar

sequences. We have provided clear evidence showing that the unique role of miR-15b in muscle differentiation (Figs. 1c and 3c). Given that miR-15a and miR-15b are located at different

chromosome loci, it is conceivable that these two miRNAs have distinct roles32. Unlike miR-15b, miR-322 has been indicated to regulate muscle differentiation as well as cardiomyocyte

specification33,34. As an X-chromosome miRNA, miR-322/-503 cluster specifically drives a cardiomyocyte program meanwhile inhibiting neural lineages33. MiR-322 can promote osteoblast

differentiation by downregulation of Tob2 and Tob2-regulated osteogenic genes34. Meanwhile, miR-322 represses muscle differentiation, as overexpression of miR-322 mimics dampened myotube

formation but promoted bone formation34. Consistently, we show here that inhibition of miR-322 significantly accelerate myotube formation, further confirming its negative role in myoblast

differentiation (Fig. 5). In contrast, miR-322 can promote cell cycle quiescence and differentiation by down-regulation of Cdc25A27. Despite of this, whether miR-322 represses muscle

differentiation has not been examined in that study. Our data have demonstrated that miR-322 indeed represses muscle differentiation. Nevertheless, why constant transcript levels of miR-322

are sustained during myoblast differentiation awaits further investigation. It is interesting to understand how expression of the two miRNAs themselves is regulated during muscle

differentiation. Here we provide evidence showing that E2F1 or FAM3B regulates expression of miR-15b or miR-322, respectively, during this process: (1) expression of E2F1 and FAM3B are

dynamically altered from myoblast state to myotube formation; (2) E2F1 and FAM3B specifically control expression of the two miRNAs through directly targeting their corresponding promoters,

respectively; (3) knockdown of _FAM3B_ in C2C12 cells results in decreased _SETD3_ expression, which are correlated with repression of muscle cell differentiation (Fig. 6). Actually,

previous studies have shown many clues to support our findings. First, E2F1-mediated transcription plays an essential role in muscle differentiation and myogenesis23,35. E2F1 expression is

irreversibly downregulated during C2C12 myoblast differentiation, whereas overexpression of E2F1 promotes myoblast proliferation and represses myogenic differentiation23,36. Second, E2F

family members, including E2F1 and E2F3, can directly bind to the promoter of miR-15b-16-2, and positively regulate miRNA expression during cell proliferation24,37. In addition, FAM3B

protein is significantly increased during the proliferation and migration of vascular smooth muscle cells, accompanied with the inhibition of miR-322-5p, linking FAM3B to miR-322

regulation25. Moreover, luciferase reporter assay has been shown that FAM3B represses transcription of miR-322 by binding the promoter of miR-32225. Therefore, we at the first time

demonstrate that E2F1 and FAM3B can regulate SETD3 through two parallel miRNA regulatory pathways, and decipher a complex network during myogenic differentiation. Using cultured myoblast

cell system, our current studies convincingly demonstrate the function and regulation of miR-15b and miR-322 in myoblast differentiation. It will be important to determine whether the

repression of _SETD3_ by miRNAs contributes to skeletal muscle development and function. It will also be interesting to determine if miR-15b/miR-322 and SETD3 participate in skeletal muscle

degeneration/regeneration process as well as human muscular diseases, such as rhabdomyosarcoma. METHODS AND MATERIALS MIRNA PREDICTION MiRNAs potentially targeting the 3′-UTR of _SETD3_ gene

were predicted by the TargetScan (http://www.targetscan.org/mmu_71/) and the Miranda (http://34.236.212.39/microrna/home.do) websites. CONSTRUCTION OF PLASMIDS The E2F1 and FAM3B from human

cDNA library were transferred to pCS2-based Gateway vector containing 3xHA tag via LR reaction as described previously38. CELL CULTURE AND TRANSFECTION C2C12 mouse myoblasts were cultured

in growth medium (GM) — DMEM containing 20% fetal bovine serum (FBS) and maintained in a humidified incubator with 5% CO2 at 37 °C. For myogenic differentiation, when confluence was reached

to 80–90%, C2C12 cells were shifted into a differentiation medium — DMEM containing 2% horse serum (HS). 293 T cells were cultured in DMEM containing 10% FBS and maintained in a humidified

incubator with 5% CO2 at 37 °C. For miRNAs and plasmids transfection, when cells reached 60–70% confluence, the miRNAs or plasmids were transfected by the transfection reagent MAX according

to the manufacturer’s protocol. Cells were harvested in 36–48 h after transfection of plasmids or 48–96 h after transfection of siRNAs or miRNAs. Unless stated, 293T cells were only used for

the luciferase reporter assays; C2C12 cells were mainly used for cell differentiation experiments. The synthesized miRNA or siRNA sequences (GenePharma Com. from Shanghai) are below:

miR-15b mimics (WT): 5′- cagcagcacauaucagguuuaca-3′; miR-15b mutant 1: 5′-cucgucgacaucaugguuuaca-3′; miR-15b mutant 2: 5′-cagcagcacauguagguuuaca-3′; miR-322 mimics (WT):

5′-cagcagcaauucauguuuugga-3′; miR-322 mutant 1: 5′-cucgucguuuucauguuuugga-3′; miR-322 mutant 2: 5′-cagcagcaauuguaguuuugga-3′; miR-15b inhibitor: 5′-UGAA- CCAUGAUGUGCUGCUA-3′; miR-322

inhibitor: 5′-UCCAAAACAUGAAUUGCUGCUG-3′; si-mE2F1-1: 5′-ATCTGACCACCAAACGCTT-3′; si-mE2F1-2: 5′-GCCCTTGACTATCACTTTGGT-3′; si-mFAM3B-1: 5′-CAAACTGAAGGCTCAAGCAAA-3′; si-mFAM3B-2:

5′-GCACTCTCTACAACATCGAA-3′. WESTERN BLOT Cells were lysed by RIPA buffer and added the bromophenol blue loading buffer, and then the samples were boiled for 10 min and centrifuged at 12,000 

rpm for 5 min. The whole-cell lysate was separated into 8% SDS-acrylamide gels and transferred to PVDF membranes. After that, the membranes was blocks by 5% milk in TBST and probed with

primary antibodies including mouse SETD3 (3B3, generated by Wuhan Dia-An Company), rabbit polyclonal SETD3 (Abclonal, A8071), MyoD1 (Proteintech, 18943-l-AP), HDAC1 (Abclonal, A2238), Cyclin

E1 (Cell Signaling Technology, 20808 S), Myogenin (Abcam, ab124800; or Santa Cruz, D-10, sc-13137), MHC (Developmental Studies Hybridoma Bank, MF-20), β-Actin (Proteintech, 6008-I-Ig), and 

α-Tubulin (Sigma, T9026). For generation of mouse monoclonal SETD3 antibody, His-tagged full-length human SETD3 protein was expressed in _E. Coli_ and purified as described previously14.

Purified His-SETD3 proteins were immunizated into 5-8 weeks old Balb/C mice and boosted additional 4 times. After several steps including hybridoma production, screening, cloning, and

expanding the hybridomas, a subclone named 3B3 was validated and amplified followed the procedure described as before39. Membranes were further probed with horseradish peroxidase

(HRP)-conjugated secondary antibodies and the protein bands were visualized using chemiluminescence detection reagents. RNA EXTRACTION, REVERSE TRANSCRIPTION, AND REAL-TIME QUANTITATIVE PCR

Total RNA was isolated from C2C12 cells with TRIzol (Life technologies). The mRNA reverse transcription and real-time PCR were according to the manufacturer’s protocol (TIANGEN). The miRNA

reverse transcription and real-time PCR were using the Hairpin-itTM Real-Time PCR Kit (Shanghai GenePharma). The primer sequences used in RT-qPCR are available upon request. DUAL-LUCIFERASE

REPORTER ASSAYS The dual-luciferase reporter plasmid psiCHECK2 was generously gifted from Xiang-Dong Fu laboratory. The longer 3′-UTR fragment of _SETD3_ gene was amplified by PCR from cDNA

of C2C12 cells and cloned into psiCHECK2 vector’s downstream of the stop codon of Renilla luciferase gene. For Luciferase reporter assays, 20 nM miRNA and 10 ng plasmid were transfected into

293 T cells or C2C12 cells. After 24–48 h, cells were lysed and the luciferase activity was tested according to the manufacturer’s instructions (Promega). GENERATION OF _SETD3_ KNOCKDOWN

CELL LINE Short hairpin RNA fragments (shRNAs) of _SETD3_ containing 5′- CATCACCATGTTCCTTGTTAA-3′ (sh_SETD3_-1) or 5′-GCTGGAGATCA- GATTTACATT-3′ (sh_SETD3_-2) were cloned into plko.1 vector

using the restriction enzymes _EcoR_I and _Age_I (New England Biolabs). To obtain lentivirus, the knockdown plasmids were transfected into 293 T cells along with the helper plasmids pMD2G

and psPAX2 using the ratio of 2:1:1. Cell culture medium was changed after 12 h transfection and virus were harvested 24 h later with filter. Cells were seeded into a 12-well plate 1 day

before lentivirus infection. _SETD3_ knockdown cells will be harvested after 36–48 h. KNOCKDOWN OF MIRNAS BY CRISPR-CAS9 TECHNOLOGY We designed two sgRNAs each miRNA by the CRISPR Design

Tool (http://tools.genome-engineering.org) and inserted them into pSpCas9 (BB)-2A-Puro vector. The sgRNA sequences are below: miR-15b-sgRNA-1: 5′-AGTACTGTAGCAGCACATCA-3′; miR-15b-sgRNA-2:

5′-CAAACATAATACAACTGTGA-3′; miR-322-sgRNA-1: 5′-CCCTTCGGAGTCAACGAGGG-3′; miR-322-sgRNA-2: 5′-GCGCTGCAACACCCCTTCGT-3′. After CRISPR-Cas9 plasmids transfected and selected by puromycin for 2-3

days, C2C12 cells were harvested and _SETD3_ expression level was analyzed by western blot. PRI-MIRNA OVEREXPRESSION SYSTEM Pri-miRNA sequences were searched from UCSC Genome Browser

(http://genome.ucsc.edu) and a nucleotide segment containing mi-15b or mi-322 was cloned into pHAGE-CMV vector using the restriction enzymes _Not_I and _Xho_I (New England Biolabs). The

primers used for construction of pri-miRNA are as follows: pri-miR-15b forward (F): 5′-ATAAGAATGCGGCCGCGCCACCGGCATTG-ACTTAGACCATAATC-3′; pri-miR-15b reverse (R):

5′-CCGCTCGAGCACTACGCCAATATTTACGTG- 3′; pri-miR-322 forward (F): 5′-ATAAGAATGCGGCCGCGCCACCCTGAGGTAAGAGTCTCCTCC-3′; pri-miR-322 reverse (R): 5′-CCGCTCGAGGTGACCCTCACTAGACTAA-G-3′. 293 T cells

were infected and selected according to the lentiviral expression and packaging protocol described above. The packaged virus was used to infect C2C12 cells to generate pri-miRNA stably

expressed cell lines. IMMUNOFLUORESCENCE STAINING C2C12 cells were cultured on glass coverslips, induced to differentiation for 4 days, fixed with 4% paraformaldehyde for 10 min,

permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, blocked with 3% BSA solution, incubated with an primary antibody (for MHC: 1:50; for SETD3 1:100) at 4 °C overnight, incubated with a

secondary antibody at room temperature for 1 h. The coverslips were stained with DAPI and mounted. Immunofluorescence images were captured under a confocal laser-scanning microscope (Leica

SP8). STATISTICAL ANALYSIS For quantification of the western blot data, ImageJ software was used to measure the relative intensity of each band. Data are presented as mean ± standard

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Drs. Xi Zhou (Wuhan Institute of Virology, Chinese Academy of Sciences) and Yu Zhou (Wuhan University) for plasmids and technical help. We also thank Dr. Zhenji Gan (Nanjing University) for

discussion and Ms. Yumin Li and Hongguo Duan for technical assistant. This work was supported by the Major State Basic Research Development Program of China (2013CB910700 to H.N.D.), the

National Natural Science Foundation of China (31770843 and 31271369 to H.N.D.), and Wuhan University (2042018kf0217 to W.J. and H.N.D.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Hubei

Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, 430072, Wuhan, China Meng-Jie Zhao, Jun Xie, Wen-Jie Shu, Hong-Yan Wang, Jianping Bi & Hai-Ning Du * Hubei

Key Laboratory of Medical Information Analysis & Tumor Diagnosis and Treatment, 430074, Wuhan, China Jianping Bi * Medical Research Institute, School of Medicine, Wuhan University,

430071, Wuhan, China Wei Jiang Authors * Meng-Jie Zhao View author publications You can also search for this author inPubMed Google Scholar * Jun Xie View author publications You can also

search for this author inPubMed Google Scholar * Wen-Jie Shu View author publications You can also search for this author inPubMed Google Scholar * Hong-Yan Wang View author publications You

can also search for this author inPubMed Google Scholar * Jianping Bi View author publications You can also search for this author inPubMed Google Scholar * Wei Jiang View author

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J., Shu, WJ. _et al._ MiR-15b and miR-322 inhibit _SETD3_ expression to repress muscle cell differentiation. _Cell Death Dis_ 10, 183 (2019). https://doi.org/10.1038/s41419-019-1432-5

Download citation * Received: 26 December 2018 * Revised: 01 February 2019 * Accepted: 07 February 2019 * Published: 22 February 2019 * DOI: https://doi.org/10.1038/s41419-019-1432-5 SHARE

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