Abstract 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. Keywords: dairy cow, heat stress, miR-196a/CDKN1B axis, proliferation arrest 1. Introduction With the ongoing shifts in the global climate, HS has emerged as a significant challenge for the dairy cattle production industry [[36]1]. The physiological state of the udder significantly influences milk productivity under HS [[37]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 [[38]3]. Concurrently, some studies have reported adverse effects of HS on the viability and cell cycle phase of BMECs [[39]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 [[40]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 [[41]6]. miR-196a’s ability to downregulate HOXA5 was found to stimulate nonsmall lung cancer and gastric cancer cell proliferation [[42]7,[43]8]. In breast cancer, UBE2C was identified as a new target gene of miR-196a, and its upregulation by miR-196a promoted cell proliferation [[44]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 [[45]10]. Meanwhile, miR-196a was also found to be potentially involved in regulating fat deposition and muscle differentiation and development in cattle [[46]11,[47]12]. In addition, miR-196a has been recognized for enhancing cellular stress resistance [[48]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 [[49]14]. Additionally, cell cycle progression is influenced by CDK inhibitors. Overexpressing these genes might hinder cell proliferation [[50]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 [[51]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 [[52]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. 2. Materials and Methods 2.1. Cell Culture BMECs were seeded and cultured in DMEM/F12 medium (Meilunbio, Dalian, China, MA0214) supplemented with 10% fetal bovine serum (FBS, Gibco, San Jose, CA, USA, 10099141C) and 1% penicillin–streptomycin (Solarbio, Beijing, China, P1400). They were kept in an incubator (Thermo Fisher Scientific, San Jose, CA, USA) at 37 °C and 5% CO[2]. For further experiments, the cells were subcultured or seeded in 6-, 12-, 24-, and 96-well plates (NEST, Wuxi, Jiangsu, China, 703002, 712002, 702002, 701002) until they reached about 80% cell density. 2.2. Heat Stress Exposure The temperature of 42 °C applied to the HS model was based on a previous study conducted in our laboratory [[53]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. 2.3. 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. [54]Table S1 shows detailed RNA oligo sequences. 2.4. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR) Total RNA and miRNA were extracted using TRIpure reagent (Aidlab Biotechnologies, Beijing, China #RN0102) and the RNAmisi micropurification kit (Aidlab Biotechnologies, Beijing, China, #RN0501), respectively, following the manufacturer’s instructions. The concentration and quantity of total RNA were measured using NanoDrop 2000 (Thermo Fisher Scientific, San Jose, CA, USA). Subsequently, mRNA and miRNA were reverse transcribed into cDNA using the HiScript III RT Super Mix for qPCR (+gDNA wiper) reagent kit (Vazyme, Nanjing, Jiangsu, China, #R323–01) and the stem-loop reverse transcription primer and miRNA 1st strand cDNA synthesis kit (by stem-loop) (Vazyme, Nanjing, Jiangsu, China, #MR101–02), respectively, following the instructions provided in the manual. ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China, #Q311–02) and miRNA Universal SYBR Master Mix (Vazyme, Nanjing, Jiangsu, China, #MQ101–02) were used to quantify mRNA and miR-196a expression, respectively. The Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, Hercules, CA, USA) was used for RT-qPCR reactions. 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). [55]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