miR-196a Promotes Proliferation of Mammary Epithelial Cells by Targeting CDKN1B

Simple Summary The physiological status of a dairy cow’s udder is significantly impacted by heat stress (HS). Therefore, improving the physiological function of dairy cow udders under HS conditions is important to improve animal welfare and dairy production efficiency. In the present study, we found that HS inhibited the proliferation of bovine mammary epithelial cells (BMECs) and significantly downregulated the expression of endogenous miR-196a. Furthermore, overexpression of miR-196a could alleviate the inhibitory effect of HS on BMEC proliferation. Additional data confirmed that miR-196a promoted BMEC proliferation. Subsequently, we identified CDKN1B as a target gene of miR-196a. The effect of miR-196a on BMEC proliferation was reversed by CDKN1B. In summary, we found that miR-196a could promote BMEC proliferation by targeting CDKN1B. These findings can provide a reliable reference for alleviating the inhibition of BMEC proliferation caused by HS. Abstract Heat stress (HS) has become one of the key challenges faced by the dairy industry due to global warming. Studies have reported that miR-196a may exert a role in the organism’s response to HS, enhancing cell proliferation and mitigating cellular stress. However, its specific role in bovine mammary epithelial cells (BMECs) remains to be elucidated. In this study, we aimed to investigate whether miR-196a could protect BMECs against proliferation arrest induced by HS and explore its potential underlying mechanism. In this research, we developed an HS model for BMECs and observed a significant suppression of cell proliferation as well as a significant decrease in miR-196a expression when BMECs were exposed to HS. Importantly, when miR-196a was overexpressed, it alleviated the inhibitory effect of HS on cell proliferation. We conducted RNA-seq and identified 105 differentially expressed genes (DEGs). Some of these DEGs were associated with pathways related to thermogenesis and proliferation. Through RT-qPCR, Western blotting, and dual-luciferase reporter assays, we identified CDKN1B as a target gene of miR-196a. In summary, our findings highlight that miR-196a may promote BMEC proliferation by inhibiting CDKN1B and suggest that the miR-196a/CDKN1B axis may be a potential pathway by which miR-196a alleviates heat-stress-induced proliferation arrest in BMECs.


Introduction
With the ongoing shifts in the global climate, HS has emerged as a significant challenge for the dairy cattle production industry [1].The physiological state of the udder significantly influences milk productivity under HS [2].Changes in the number or functional capacity of mammary epithelial cells correlate with reductions in milk production.HS is observed to inhibit cell growth and alter structural proteins, membrane permeability, and metabolism within these cells [3].Concurrently, some studies have reported adverse effects of HS on the viability and cell cycle phase of BMECs [4].
In a previous HS experiment with rabbits, our team identified 23 differentially expressed miRNAs, including miR-196a, between tissues under HS and a control condition [5].This information suggests that miR-196a, identified in the study, could play a role in modulating cellular physiological changes during HS.Previous research has shown that miR-196a can enhance the proliferation of cancer cells by targeting FOXO1 [6].miR-196a's ability to downregulate HOXA5 was found to stimulate nonsmall lung cancer and gastric cancer cell proliferation [7,8].In breast cancer, UBE2C was identified as a new target gene of miR-196a, and its upregulation by miR-196a promoted cell proliferation [9].In swine, miR-196a was found to promote cell proliferation in immature porcine Sertoli cells by binding the 3 UTR of RCC2 and ABCB9 [10].Meanwhile, miR-196a was also found to be potentially involved in regulating fat deposition and muscle differentiation and development in cattle [11,12].In addition, miR-196a has been recognized for enhancing cellular stress resistance [13].Nevertheless, the regulatory function of miR-196a in BMECs remains elusive.Thus, exploring the potential roles of miR-196a in BMECs is a valuable endeavor.
Modifications in cell cycle progression play a pivotal role in regulating cellular proliferation.In eukaryotic cells, the progression of the cell cycle is overseen by the sequential expression of cell cycle proteins.These proteins activate the transcription of specific genes corresponding to different cycle-dependent kinases (CDKs), thereby promoting cell cycle progression [14].Additionally, cell cycle progression is influenced by CDK inhibitors.Overexpressing these genes might hinder cell proliferation [15].Among these inhibitors, CDKN1B stands out.An upregulation of CDKN1B has been observed to inhibit cell proliferation.Numerous studies have shown that CDKN1B is integral to the cellular response to HS [16].CDKN1B, being an oxidative-stress-related gene, becomes upregulated during HS.This upregulation is vital for the selective elimination of protein aggregates caused by HS, thus effectively reducing cellular proteotoxic stress [17].
Our objective for this study was to investigate whether miR-196a could protect BMECs against proliferation arrest induced by HS and explore its potential underlying mechanism.
In the present study, we measured a significant downregulation in miR-196a expression under HS compared to BMECs cultured at 37 • C (control).Further functional analyses suggested that miR-196a can act as a promoter of BMEC proliferation.A luciferase reporter assay corroborated that miR-196a directly targets CDKN1B in BMECs.Moreover, data from the rescue experiment demonstrated that miR-196a promotes BMEC proliferation by binding to CDKN1B.

Heat Stress Exposure
The temperature of 42 • C applied to the HS model was based on a previous study conducted in our laboratory [18].Firstly, BMECs were seeded at a density of 1.25 × 10 6 /well in 6-well plates or 5 × 10 4 /well in 96-well plates and cultured in a Thermo Fisher Scientific incubator (Thermo Fisher Scientific, San Jose, CA, USA) at 37 • C and 5% CO 2 environment until reaching a cell density of 50-60%.At this point, cells were divided into two groups: the control group continued to be cultured in the same incubator, while the HS group was transferred to another incubator at 42 • C and maintained under 5% CO 2 .We collected both groups of cells at time points of 0, 6, 12, and 24 h.

Transfection
We used a Lipofectamine™ 3000 transfection kit (Invitrogen, Carlsbad, CA, USA) to transfect miR-196a mimic (mimic), miR-196a inhibitor (inhibitor), miR-NC, inhibitor-NC (I-NC), PCDNA, and PCDNA-CDKN1B at a cell density reaching 50-60%.After 6 h, the culture medium was replaced with fresh medium.The RNA oligos were synthesized by Sangon Biotech Co., Ltd.(Shanghai, China).The miRNA mimic is a chemically synthesized mature double-stranded miRNA that enhances endogenous miRNA function.It consists of a sequence corresponding to the target miRNA mature body sequence and a complementary sequence to the miRNA mature body sequence.The miRNA inhibitor is a chemically synthesized mature single-stranded miRNA with methoxy modification designed to specifically target and efficiently inhibit endogenous miRNA activity in cells, enabling functional deficiency research on specific miRNAs.miR-NC and I-NC served as negative controls, respectively.Table S1 shows detailed RNA oligo sequences.
The RT-qPCR conditions included an initial step at 95 • C for 5 min, followed by 40 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 15 s, and extension at 72 • C for 20 s.We performed a thermal denaturation cycle to determine dissociation curves for verifying the specificity of PCR amplification.Each sample was tested in triplicate.All quantitative primers were designed using Primer 5.0 and synthesized by Tsingke Biotechnology (Beijing, China).Table S2 provides details on primers for RT-qPCR.The CQ values of β-actin and U6 small nuclear RNA were used as internal references to calculate gene mRNA expression levels and the relative abundance of miR-196a, respectively.The relative expression levels of genes and miR-196a were calculated using the 2 −∆∆Ct method.

Western Blotting
Total cellular protein was collected from BMECs using the ProteinExt ® mammalian total protein extraction kit (TransGene Biotech, Beijing, China, #DE101-01).The protein concentration of each sample was determined using a Bradford protein assay kit (Novoprotein, Shanghai, China, #PA001-01A) according to the manufacturer's instructions.For electrophoresis, 30 µg of protein from each sample was loaded onto a 12% SDS-PAGE gel.The corresponding primary antibodies were incubated with the membranes at 4 • C for 8 h, followed by a 2 h incubation with secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), Zen Bioscience, Chengdu, China).The membranes were washed three times and developed using chemiluminescence in a Touch Imager Pro (e-BLOT Life Science Co., Ltd.Shanghai, China).The primary antibodies PCNA, CDKN1B, and β-actin were purchased from Proteintech (Wuhan, Hubei, China).β-actin served as the internal reference for normalization.

EDU Assay
BMECs were seeded in 96-well plates and transfected when the cell density reached approximately 60%.The Beyoclick EDU-555 cell proliferation assay kit (Beyotime Biotechnology, Shanghai, China, #C0075S) was used for the EDU assay following the manufacturer's instructions.After 36 h of transfection, an equal volume of EDU working solution was added to the wells and incubated for 1 h.DAPI (Solarbio, Beijing, China, C0065) was used to stain the cell nuclei and help observe the ratio of cell proliferation.The stained cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) with a magnification of 100 times, allowing the cell nuclei to appear blue and the proliferating cells to appear red.Photographs were captured using an inverted fluorescence microscope (Olympus, Tokyo, Japan) under identical visual field conditions.Subsequently, the images were analyzed using ImageJ 1.51 software (National Institutes of Health, Bethesda, MD, USA).

CCK-8 Assay
BMECs were seeded in 96-well plates and then transfected.After transfection, we added 10 µL of CCK-8 solution (Beyotime Biotechnology, Shanghai, China, #C0037) to each well and incubated them for 2 h.The optical density (OD) value at 450 nm was measured using a Varioskan LUX spectrophotometer (Thermo Scientific, Waltham, MA, USA).The obtained absorbance values were subsequently plotted and analyzed using GraphPad Prism 8 software (GraphPad, San Diego, CA, USA).

Statistical Analysis
All data were analyzed using Prism 8 software (GraphPad, San Diego, CA, USA).Statistically significant difference between the two groups was determined using Student's t-test.For multiple comparisons, the one-way analysis of variance (ANOVA) method was employed.The results are presented as the mean ± SEM, and each independent experiment was performed in triplicate.Statistical significance was indicated by p < 0.05.

Heat Stress Downregulates miR-196a Expression and Suppresses BMEC Proliferation
A prior study highlighted elevated expression levels of miR-196a in the mammary gland compared to other tissues [26].To delve into the tissue-specific expression of miR-196a, we performed RT-qPCR to assess the expression patterns of miR-196a across various cow tissues, including the heart, liver, spleen, lung, kidney, adipose, mammary gland (mammary epithelial cells versus stroma), and skin.Figure 1A illustrates that miR-196a expression differed across these tissues.Specifically, miR-196a exhibited high expression in the kidney and mammary gland, moderate expression in the skin and adipose tissue, and minimal expression in the heart and ovarian tissues.These findings suggest that miR-196a may play an important regulatory function in the mammary glands of dairy cows.One of our previous studies suggested that miR-196a may be involved in regulating the alteration of the physiology of the animal organism under HS [5].We established an HS model using BMECs to investigate whether miR-196a regulates the mammary gland's One of our previous studies suggested that miR-196a may be involved in regulating the alteration of the physiology of the animal organism under HS [5].We established an HS model using BMECs to investigate whether miR-196a regulates the mammary gland's physiological response to HS in dairy cows.RT-qPCR was employed to determine the expression patterns of HSPB1 and HSP90AA1 under HS conditions at 42 • C. Notably, Figure 1B,C shows that the expression levels of both HSPB1 and HSP90AA1 were substantially elevated in BMECs exposed to 42 • C compared to the initial culture stage (0 h).Additionally, HSPB1 and HSP90AA1 expression levels rose with prolonged exposure to HS at intervals of 0, 6, and 12 h.Subsequently, we assessed fluctuations in miR-196a expression under HS at 0, 6, 12, and 24 h using RT-qPCR.We identified a significant decline in miR-196a expression post HS exposure, with its expression being the least at 24 h.The miR-196a expression in the control group showed no significant change (Figure 1D).Furthermore, we noticed a pronounced reduction in BMECs' proliferative activity during HS (Figure 1E).Nevertheless, increasing miR-196a expression appeared to alleviate the suppressive effect of HS on cellular proliferation (Figure 1F).

mRNA-Seq and Bioinformatics Analysis Were Used to Explore the Potential Functions of miR-196a
To investigate the biological function of miR-196a in BMECs, we conducted transcriptome profiling on BMECs transfected with miR-196a mimic and miR-NC.We constructed six cDNA libraries with three replicates for each group (mimic and miR-NC) and sequenced them using the Illumina NovaSeq platform (Illumina, San Diego, CA, USA).The sequencing produced 150 bp paired-end reads, yielding over 4.2 million raw reads for each sample.After discarding low-quality and unclear reads, a minimum of 97.55% of raw reads remained as clean reads in every sample.These clean reads were aligned to the Bos taurus reference genome, with mapping rates ranging from 96.02 to 97.08% in individual samples (Table S3).These outcomes indicate the sequencing data's reliability and suitability for subsequent bioinformatics processing.
To pinpoint genes with altered expression due to miR-196a overexpression, we identified 105 DEGs (Table S4).These included 48 upregulated genes, such as MMP3, EDN1, ATP5ME, NDUFAF8, and FGF21, and 57 downregulated genes, such as FOXO1, GADD45A, and SOCS2, using padj < 0.05 and |log2foldchange|≥ 1 as criteria (Figure 2A).The genes ATP5ME, NDUFAF8, and FGF21 are reportedly associated with cellular energy metabolism and thermogenesis, while FOXO1, GADD45A, and SOCS2 are vital in regulating cell proliferation.Five DEGs were randomly selected based on the following criteria: |log2foldchange| > 1, padj < 0.05, FPKM > 50, and read count > 20.Their expression was verified with RT-qPCR to confirm the RNA-seq results' reliability, which showed a similar trend to that of sequencing (Figure 2B).For a deeper understanding of DEG roles, we conducted GO and KEGG enrichment analyses.We categorized the 105 DEGs into three primary groups: biological process (BP), cellular component (CC), and molecular function (MF) (Table S5). Figure 2C shows the top 10 GO terms from each category impacted by miR-196a overexpression.Most DEGs were grouped under the regulation of multicellular organism processes (BP), extracellular regions (CC), and signaling receptor binding (MF).Additionally, the DEGs were annotated across 171 pathways in the KEGG analysis (Table S6). Figure 2D presents the top 20 KEGG pathways influenced by miR-196a overexpression.Most DEGs were enriched in cellular senescence and transcription misregulation in cancer, followed by nine equal-sized pathways, such as TNF signaling and JAK-STAT signaling.Notably, numerous studies have linked many top 20 pathways, including calcium signaling, cAMP signaling, P53 signaling, JAK-STAT signaling, and others, to cell proliferation regulation [27][28][29][30][31].These data suggest that miR-196a's primary role in BMECs may be to regulate cell proliferation.

Effect of miR-196a on Cell Proliferation of BMECs
To assess miR-196a's impact on BMEC proliferation, we employed the CCK-8 assay to gauge cellular viability.We observed a marked enhancement in cell viability in BMECs treated with the miR-196a mimic relative to miR-NC (Figure 3A).On the contrary, BMECs transfected with the miR-196a inhibitor relative to the I-NC group were significantly decreased in cell viability (Figure 3B).Furthermore, upon conducting the EDU assay, we determined that the rate of EDU-positive cells in the mimic group was significantly higher than in the miR-NC group (Figure 3C,D).Meanwhile, the proportion of EDU-positive cells was considerably diminished in BMECs treated with the miR-196a inhibitor compared to the I-NC group (Figure 3E).Additionally, we analyzed the relative expression levels of proliferation-associated genes, namely, PCNA, CDK2, and MCM3, in BMEC post-transfection with miR-196a mimic, miR-196a inhibitor, miR-NC, and I-NC.PCNA, CDK2, and MCM3 transcript abundance increased significantly when miR-196a was overexpressed.Conversely, inhibiting miR-196a markedly reduced their transcript levels (Figure 3F-H).miR-196a's role in promoting BMEC proliferation was further substantiated by assessing the protein expression of PCNA (Figure 3I).PCNA protein levels were either substantially increased or decreased in BMECs transfected with a miR-196a mimic or inhibitor

Effect of miR-196a on Cell Proliferation of BMECs
To assess miR-196a's impact on BMEC proliferation, we employed the CCK-8 assay to gauge cellular viability.We observed a marked enhancement in cell viability in BMECs treated with the miR-196a mimic relative to miR-NC (Figure 3A).On the contrary, BMECs transfected with the miR-196a inhibitor relative to the I-NC group were significantly decreased in cell viability (Figure 3B).Furthermore, upon conducting the EDU assay, we determined that the rate of EDU-positive cells in the mimic group was significantly higher than in the miR-NC group (Figure 3C,D).Meanwhile, the proportion of EDUpositive cells was considerably diminished in BMECs treated with the miR-196a inhibitor compared to the I-NC group (Figure 3E).Additionally, we analyzed the relative expression levels of proliferation-associated genes, namely, PCNA, CDK2, and MCM3, in BMEC posttransfection with miR-196a mimic, miR-196a inhibitor, miR-NC, and I-NC.PCNA, CDK2, and MCM3 transcript abundance increased significantly when miR-196a was overexpressed.Conversely, inhibiting miR-196a markedly reduced their transcript levels (Figure 3F-H).miR-196a's role in promoting BMEC proliferation was further substantiated by assessing the protein expression of PCNA (Figure 3I).PCNA protein levels were either substantially increased or decreased in BMECs transfected with a miR-196a mimic or inhibitor compared to cells with negative controls, respectively.Collectively, these findings suggest that miR-196a enhances BMEC proliferation.
compared to cells with negative controls, respectively.Collectively, these findings suggest that miR-196a enhances BMEC proliferation.

CDKN1B Is a Direct Target Gene of miR-196a in BMECs
To investigate the underlying mechanisms through which miR-196a modulates the proliferation of BMECs, we used TargetScan Human 8.0 and miRDB to computationally predict potential target genes regulated by miR-196a (Table S7).By integrating these predictions with downregulated genes identified in our RNA-seq analysis, CDKN1B emerged

CDKN1B Is a Direct Target Gene of miR-196a in BMECs
To investigate the underlying mechanisms through which miR-196a modulates the proliferation of BMECs, we used TargetScan Human 8.0 and miRDB to computationally predict potential target genes regulated by miR-196a (Table S7).By integrating these predic-tions with downregulated genes identified in our RNA-seq analysis, CDKN1B emerged as a promising candidate gene targeted by miR-196a (Figure 4A).The RT-qPCR and Western blotting results indicated that overexpression of miR-196a markedly diminished CDKN1B expression levels, whereas suppressing miR-196a elevated the relative abundance of CDKN1B expression (Figure 4B,C).We conducted dual-luciferase reporter assays to confirm the binding sites.These assays revealed that co-transfection with pmirGLO-WT and miR-196a mimic significantly reduced luciferase activity compared to co-transfection with pmirGLO-WT and miR-NC.Additionally, no significant difference in luciferase activity was observed between groups co-transfected with pmirGLO-MUT, irrespective of miR-196a mimic or miR-NC presence (Figure 4E).Furthermore, the Western blotting result indicated an increase in CDKN1B protein expression levels under heat stress, which was contrary to the expression trend observed for miR-196a under such conditions.These findings validate CDKN1B as a direct target gene of miR-196a.
Animals 2023, 13, x FOR PEER REVIEW 10 of 17 as a promising candidate gene targeted by miR-196a (Figure 4A).The RT-qPCR and West ern blotting results indicated that overexpression of miR-196a markedly diminished CDKN1B expression levels, whereas suppressing miR-196a elevated the relative abun dance of CDKN1B expression (Figure 4B,C).We conducted dual-luciferase reporter assays to confirm the binding sites.These assays revealed that co-transfection with pmirGLO WT and miR-196a mimic significantly reduced luciferase activity compared to co-trans fection with pmirGLO-WT and miR-NC.Additionally, no significant difference in lucifer ase activity was observed between groups co-transfected with pmirGLO-MUT, irrespec tive of miR-196a mimic or miR-NC presence (Figure 4E).Furthermore, the Western blot ting result indicated an increase in CDKN1B protein expression levels under heat stress which was contrary to the expression trend observed for miR-196a under such conditions These findings validate CDKN1B as a direct target gene of miR-196a.

CDKN1B Reverses the Promotive Effect of miR-196a Overexpression on BMEC Proliferation
To investigate the role of CDKN1B as a target gene in miR-196a-mediated BMEC pro liferation, BMECs were co-transfected with either a miR-196a mimic or miRNA-NC along with either a PCDNA or PCDNA-CDKN1B plasmid.The CCK-8 and EDU assays

CDKN1B Reverses the Promotive Effect of miR-196a Overexpression on BMEC Proliferation
To investigate the role of CDKN1B as a target gene in miR-196a-mediated BMEC proliferation, BMECs were co-transfected with either a miR-196a mimic or miRNA-NC along with either a PCDNA or PCDNA-CDKN1B plasmid.The CCK-8 and EDU assays demonstrated that the OD value at 450 nm and the proportion of proliferating cells in the group co-transfected with the miR-196a mimic and PCDNA rose significantly compared to the miR-NC+PCDNA group (Figure 5A-C).This increase implies that miR-196a overexpression markedly enhances BMECs' proliferative capacity.However, miR-196a's promotion of BMEC proliferation was substantially negated when CDKN1B was overexpressed (Figure 5A-C).Furthermore, RT-qPCR data for proliferation-associated genes, namely, PCNA, CDK2, and MCM3, revealed an upregulation when the miR-196a mimic was overexpressed and a downregulation when cells were co-transfected with the miR-196a mimic and PCDNA-CDKN1B (Figure 5D-F).The Western blotting results indicated that overexpression of CDKN1B significantly increased its protein expression level.And overexpression of miR-196a significantly enhanced the protein expression level of PCNA, but this promoting effect was counteracted by CDKN1B (Figure 5G).These findings suggest that CDKN1B may reverse the proliferative effect of miR-196a on BMECs.demonstrated that the OD value at 450 nm and the proportion of proliferating cells in the group co-transfected with the miR-196a mimic and PCDNA rose significantly compared to the miR-NC+PCDNA group (Figure 5A-C).This increase implies that miR-196a overexpression markedly enhances BMECs' proliferative capacity.However, miR-196a's promotion of BMEC proliferation was substantially negated when CDKN1B was overexpressed (Figure 5A-C).Furthermore, RT-qPCR data for proliferation-associated genes, namely, PCNA, CDK2, and MCM3, revealed an upregulation when the miR-196a mimic was overexpressed and a downregulation when cells were co-transfected with the miR-196a mimic and PCDNA-CDKN1B (Figure 5D-F).The Western blotting results indicated that overexpression of CDKN1B significantly increased its protein expression level.And overexpression of miR-196a significantly enhanced the protein expression level of PCNA, but this promoting effect was counteracted by CDKN1B (Figure 5G).These findings suggest that CDKN1B may reverse the proliferative effect of miR-196a on BMECs.

Discussion
HS is described as a physiological state where an animal cannot adequately dissipate heat, thereby disrupting its body's thermal equilibrium [32].Studies have documented that milk production diminishes when dairy cows are exposed to HS [33].Reduced milk yield during HS can be attributed partly to nutritional intake and management considerations [34,35].Furthermore, several studies have highlighted the significance of udder physiology status as a key determinant affecting milk production during HS [2,36].In dairy cows, milk production hinges on the secretory activities of mammary epithelial cells [37].HS adversely affects transcription, gene translation, and shifts in the BMEC cycle [1,[38][39][40].Thus, alleviating the inhibition of BMEC proliferation due to HS is pivotal for bolstering the productivity of dairy cows under such conditions.
A previous study emphasized significantly elevated miR-196a expression in the mammary gland compared to other tissues, which is consistent with our findings [26].Therefore, miR-196a may play a crucial regulatory role in BMECs.To further investigate the potential role of miR-196a in BMECs' response to HS, an HS model was established for BMECs.The upregulation of heat shock proteins (HSPs) serves as a well-established biomarker for detecting heat stress in animals [41].The expression levels of HSP90AA1 and HSPB1 in BMECs under HS were evaluated.We also noticed a significant decrease in miR-196a expression in BMECs under HS exposure.Previously, miR-196a was proven to enhance cancer cell proliferation by targeting FOXO1 and HOXA5 [6][7][8].Thus, miR-196a may play a role in cell proliferation under HS.Interestingly, miR-196a overexpression appeared to alleviate HS-induced proliferation inhibition.
mRNA-seq was used to gain preliminary insights into the potential functional roles of miR-196a in BMECs.The mRNA-seq analysis yielded 105 differentially expressed genes based on the thresholds of padj < 0.05 and |log2foldchange| ≥ 1. MMP3, EDN1, and FGF21 exhibited significant upregulation, while FOXO1, GADD45A, and SOCS2 showed significant downregulation.These genes are known to be closely linked to cellular proliferation [42][43][44][45][46][47].The identified DEGs were categorized using the GO and KEGG databases.In the GO enrichment analysis, most of the DEGs were ascribed to GO terms associated with categories such as immune response, ion transport, mitochondrial inner membrane, and receptor regulator activity.
Within the top 20 KEGG pathways, seven pathways were associated with regulating cell proliferation.These pathways included the calcium signaling pathway, cAMP signaling pathway, p53 signaling pathway, JAK-STAT signaling pathway, FoxO signaling pathway, TNF signaling pathway, and transcriptional misregulation in cancer.All were found to be significantly enriched.Numerous studies have identified that the activation or suppression of these signaling pathways can either enhance or inhibit cell proliferation [27][28][29][30]48,49].The enrichment analysis results suggest that miR-196a may play a role in regulating the proliferation of BMECs.However, the specific regulatory mechanisms through which miR-196a modulates cell proliferation via these pathways remain unknown and require further elucidation through future experiments.
Several DEGs, such as ATP5ME, NDUFAF8, and FGF21, were significantly enriched in the thermogenesis pathway.These genes are established participants in controlling mitochondrial energy metabolism [44,50,51].Given that many studies have posited their connection to cell thermogenesis [52], miR-196a may play a role in thermal balance regulation in BMECs during HS.The intricate underlying mechanisms require more detailed exploration.
To investigate miR-196a's role in BMEC proliferation, we assessed cell viability and proliferation capabilities using CCK-8 and EDU assays.Both assay outcomes revealed that cells transfected with miR-196a mimic exhibited a significant increase in proliferation compared to cells transfected with miR-NC.Conversely, suppressing miR-196a dampened BMECs' proliferation.Corroborating this, Shaoxuan et al. [10] found that miR-196a overexpression markedly improved the proliferation of immature porcine Sertoli cells.miR-196a inhibition notably curtailed their proliferation capacity, consistent with our findings.A separate study on laryngeal cancer further underscored miR-196a's role in modulating cell proliferation [53].
One common method to evaluate cell proliferation is to monitor the expression of proliferation-associated genes.Proteins such as the proliferating cell nuclear antigen (PCNA) and minichromosome maintenance (MCM) are conventional markers for cell proliferation, expressed solely during cell division [54].Another notable protein, cyclin-dependent kinase 2 (CDK2), belongs to the cell cycle protein kinase family and promotes cell proliferation when activated [55].The RT-qPCR data indicated a significant upregulation of these genes in the group treated with miR-196a mimic.Suppressing miR-196a led to a pronounced downregulation of these genes.
To understand the mechanism through which miR-196a enhances BMEC proliferation more accurately, we integrated a list of downregulated genes based on the thresholds of padj < 0.05 and log2foldchange < 0 identified by mRNA-seq with findings from TargetScan Human 8.0 and miRDB.CDKN1B emerged as a potential target gene of miR-196a in this integrated analysis, especially given the significant role of CDKN1B in cell proliferation [15].Subsequent tests using RT-qPCR, Western blotting, and dual-luciferase reporter assays confirmed this relationship, establishing CDKN1B as a target gene of miR-196a.To confirm the role of CDKN1B as a target gene influencing the effect of miR-196a on BMEC proliferation, BMECs were co-transfected with mimic, miR-NC, PCDNA, and PCDNA-CDKN1B vector.We determined that the proliferation-enhancing effect of miR-196a on BMECs could be reversed by CDKN1B through RT-qPCR, Western blotting, and EDU assay.These results suggest that miR-196a promotes BMEC proliferation by targeting CDKN1B.CDKN1B is a member of the Cip/Kip family and plays a crucial role as a cell cycle regulator, primarily involved in inhibiting or decelerating cell division [56,57].CDKN1B can induce cell cycle arrest specifically in the G0/G1 phase [58].In the normal progression of the cell cycle, CDKN1B levels are elevated during the G0/G1 phase.However, upon mitogenic stimulation, CDKN1B undergoes rapid degradation, facilitating the activation of Cdk2/cyclinE and Cdk2/cyclinA complexes to promote further progression through the cell cycle [59].miR-196a can facilitate CDK activity and promote the proliferation of BMECs by downregulating CDKN1B.Past studies have revealed that one miRNA can influence the expression of various genes and, conversely, a single gene can be modulated by multiple miRNAs.Previous evidence also suggests that miR-196a promotes cell proliferation by targeting FOXO1 and HOXA5 [6,8].The interaction between miR-196a and CDKN1B may play a crucial role in regulating BMEC proliferation.
Our study revealed that HS can reduce miR-196a expression and that increasing miR-196a levels can alleviate HS-induced suppression of cell proliferation.In our research, we identified CDKN1B as a target of miR-196a.Interestingly, several groups have reported that CDKN1B is directly involved in the cellular response to HS [16,17].Given the established significance of the miR-196a and CDKN1B interaction in regulating BMEC proliferation, it would be worth investigating whether miR-196a mediates cell proliferation responses to HS via this specific pathway.These findings not only provide a foundation for further comprehensive study of miR-196a function in BMECs but also offer a novel insight into identifying potential targets for alleviating heat-stress-induced proliferation arrest in BMECs (Figure 6).

Conclusions
Our study findings indicate that HS reduces miR-196a expression and that overexpressing miR-196a can alleviate the HS-induced inhibitory effect of BMEC proliferation.Additional research confirms that miR-196a can promote BMEC proliferation by suppressing CDKN1B expression.These results could provide an important theoretical basis for understanding the function of miR-196a in BMECs and offer a novel insight into identifying potential targets for alleviating HS-induced proliferation arrest in BMECs.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1,Table S1 provides the oligo sequence information of the miR-196a mimic and inhibitor; Table S2 provides the RT-qPCR primer sequences; Table S3 provides information on sequencing quality and reference genome alignment statistics; Table S4 shows differentially expressed genes between the miR-NC group (miR-NC_1, miR-NC_2, and miR-NC_3) and mimic group (mimic_1, mimic_2, and mimic_3); Table S5 shows the GO enrichment analysis of differentially expressed genes; Table S6 provides the KEGG pathway enrichment analysis of differentially expressed genes; and Table S7 provides a list of candidate target genes.

Conclusions
Our study findings indicate that HS reduces miR-196a expression and that overexpressing miR-196a can alleviate the HS-induced inhibitory effect of BMEC proliferation.Additional research confirms that miR-196a can promote BMEC proliferation by suppressing CDKN1B expression.These results could provide an important theoretical basis for understanding the function of miR-196a in BMECs and offer a novel insight into identifying potential targets for alleviating HS-induced proliferation arrest in BMECs.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani13233682/s1,Table S1 provides the oligo sequence information of the miR-196a mimic and inhibitor; Table S2 provides the RT-qPCR primer sequences; Table S3 provides information on sequencing quality and reference genome alignment statistics; Table S4 shows differentially expressed genes between the miR-NC group (miR-NC_1, miR-NC_2, and miR-NC_3) and mimic group (mimic_1, mimic_2, and mimic_3); Table S5 shows the GO enrichment analysis of differentially expressed genes; Table S6 provides the KEGG pathway enrichment analysis of differentially expressed genes; and Table S7 provides a list of candidate target genes.

Figure 3 .
Figure 3.Effect of miR-196a on BMEC cell proliferation.(A,B) We conducted a CCK-8 assay to detect the viability of BMECs transfected with a miR-196a mimic or miR-196a inhibitor, respectively (n = 5).(C-E) An EDU assay was performed to measure the ratio of proliferating cells after overexpression or suppression of miR-196a, respectively (n = 5).(F-H) Gene transcription levels for PCNA, CDK2, and MCM3 in BMECs were analyzed by RT-qPCR, with β-actin used as a reference (n = 9).(I) PCNA protein expression was analyzed by Western blotting (n = 3).β-actin was used as a reference.The data are presented as means ± SEM. * p < 0.05.

Figure 3 .
Figure 3.Effect of miR-196a on BMEC cell proliferation.(A,B) We conducted a CCK-8 assay to detect the viability of BMECs transfected with a miR-196a mimic or miR-196a inhibitor, respectively (n = 5).(C-E) An EDU assay was performed to measure the ratio of proliferating cells after overexpression or suppression of miR-196a, respectively (n = 5).(F-H) Gene transcription levels for PCNA, CDK2, and MCM3 in BMECs were analyzed by RT-qPCR, with β-actin used as a reference (n = 9).(I) PCNA protein expression was analyzed by Western blotting (n = 3).β-actin was used as a reference.The data are presented as means ± SEM. * p < 0.05.

Figure 4 .
Figure 4. CDKN1B is a direct target gene of miR-196a in BMECs.(A) Venn diagram of target genes predicted by TargetScan Human 8.0 and miRDB overlapping with mRNA-seq downregulated gene (padj < 0.05, log2foldchange < 0).(B) The relative abundance of mRNA in the CDKN1B gene wa measured by RT-qPCR (n = 9).(C) CDKN1B protein expression was examined by Western blotting β-actin was used as an internal reference (n = 3).(D) The wild-type (WT) or mutant-type (MUT 3′UTR fragments of CDKN1B were inserted into dual-luciferase reporter vectors, respectively.(E The luciferase reporter assay was performed to verify the association between miR-196a and CDKN1B (n = 9).(F) Western blotting was performed to examine the expression level changes o CDKN1B under control and heat stress conditions.β-actin was used as an internal reference.The data are presented as means ± SEM. * p < 0.05, ns (no significance).

Figure 4 .
Figure 4. CDKN1B is a direct target gene of miR-196a in BMECs.(A) Venn diagram of target genes predicted by TargetScan Human 8.0 and miRDB overlapping with mRNA-seq downregulated genes (padj < 0.05, log2foldchange < 0).(B) The relative abundance of mRNA in the CDKN1B gene was measured by RT-qPCR (n = 9).(C) CDKN1B protein expression was examined by Western blotting.β-actin was used as an internal reference (n = 3).(D) The wild-type (WT) or mutant-type (MUT) 3 UTR fragments of CDKN1B were inserted into dual-luciferase reporter vectors, respectively.(E) The luciferase reporter assay was performed to verify the association between miR-196a and CDKN1B (n = 9).(F) Western blotting was performed to examine the expression level changes of CDKN1B under control and heat stress conditions.β-actin was used as an internal reference.The data are presented as means ± SEM. * p < 0.05, ns (no significance).

Figure 6 .
Figure 6.The potential mechanism of miR-196a alleviates cell proliferation inhibition caused by HS. miR-196a can promote BMEC proliferation by targeting CDKN1B at normal temperatures.miR-196a expression is downregulated during HS, which inhibits BMEC proliferation.However, increasing miR-196a levels can alleviate the suppressive effects of HS on cell proliferation.This process may occur through inhibition of CDKN1B expression by miR-196a.Solid lines indicate established regulatory mechanisms; dashed lines represent potential regulatory mechanisms.The upper arrow represents facilitation and the lower arrow represents inhibition.

Author
Contributions: G.C. and W.S. conceived and designed the study; G.C., W.S., Y.L. and M.L. collected data and conducted research; G.C. and W.S. carried out data analysis; G.C., W.S. and J.W. wrote the manuscript; S.L., W.S. and X.J. carried out funding acquisition.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Sichuan key research and development project on innovative breeding materials, methods, and new breeds of high-quality cattle, Key R&D Program of Sichuan Province, grant number 2021YFYZ0001; the National Modern Agricultural Industry Technology System, Sichuan Innovation Team, beef cattle industry technology research and

Figure 6 .
Figure 6.The potential mechanism of miR-196a alleviates cell proliferation inhibition caused by HS. miR-196a can promote BMEC proliferation by targeting CDKN1B at normal temperatures.miR-196a expression is downregulated during HS, which inhibits BMEC proliferation.However, increasing miR-196a levels can alleviate the suppressive effects of HS on cell proliferation.This process may occur through inhibition of CDKN1B expression by miR-196a.Solid lines indicate established regulatory mechanisms; dashed lines represent potential regulatory mechanisms.The upper arrow represents facilitation and the lower arrow represents inhibition.

Author
Contributions: G.C. and W.S. conceived and designed the study; G.C., W.S., Y.L. and M.L. collected data and conducted research; G.C. and W.S. carried out data analysis; G.C., W.S. and J.W. wrote the manuscript; S.L., W.S. and X.J. carried out funding acquisition.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Sichuan key research and development project on innovative breeding materials, methods, and new breeds of high-quality cattle, Key R&D Program of Sichuan Province, grant number 2021YFYZ0001; the National Modern Agricultural Industry Technology System, Sichuan Innovation Team, beef cattle industry technology research and development and integrated application positions in agricultural areas; the Sichuan innovation team of national modern agricultural industry technology system, grant number SCCXTD-2023-13; the Natural Science