miR-30a-3p Regulates Autophagy in the Involution of Mice Mammary Glands

The mammary gland undergoes intensive remodeling during the lactation cycle, and the involution process of mammary gland contains extensive epithelial cells involved in the process of autophagy. Our studies of mice mammary glands suggest that miR-30a-3p expression was low during involution compared with its high expression in the mammary glands of lactating mice. Then, we revealed that miR-30a-3p negatively regulated autophagy by autophagy related 12 (Atg12) in mouse mammary gland epithelial cells (MMECs). Restoring ATG12, knocking down autophagy related 5 (Atg5), starvation, and Rapamycin were used to further confirm this conclusion. Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the involution of the mammary glands of mice, which was indicative of alteration in mammary remodeling. Taken together, these results elucidated the molecular mechanisms of miR-30a-3p as a key induction mediator of autophagy by targeting Atg12 within the transition period between lactation and involution in mammary glands.


Introduction
The evolutionary origin of the mammary gland dates to the Carboniferous period nearly 310 million years ago [1].The mammary glands of mammals are highly conserved in terms of structure, development, and nutritional composition, and the postnatal stage of the mammary gland development cycle involves pregnancy, lactation, and involution [2].The weaning of offspring regulates the development of the mammary gland from lactation to involution, which leads to remodeling processes and the regression of mammary tissue.Cessation of suckling and milk accumulation triggers this transformation [2].The involution process of the mammary gland includes extensive epithelial cell death, extracellular matrix remodeling, and recruitment of immune cells [3].These events are regulated by various factors such as hormones, cytokines, noncoding RNAs, etc. Mammary involution of rodents consists of two main phases: the reversible phase (0-2 days) and the irreversible phase (8-18 days) [4].Zinc transporter 2 regulates lysosomal-mediated cell death during lactation to a reversible phase of involution [5].Estrogen promotes involution by exacerbating inflammation, cell death, and adipocytes' repopulation in BALB/c mice [6].
Autophagy and apoptosis play important roles in cell death and removal during the mammary involution of mice, and autophagy mediates efferocytosis and suppresses inflammation in this process [7].However, the specific roles of autophagy in cell survival or death in mammary involution have remained debatable [8].Autophagy plays a key role in the transition from reversible to irreversible involution and regulates mammary gland involution by restraining apoptosis-driven irreversible changes [9].Cows exposed to heat stress had extended involution due to delayed autophagy signaling, which impaired overall mammary gland turnover during the dry period [10].Signal transducer and activator of transcription 3 (STAT3) target genes p55α/p50α regulated autophagy and cell death during the mammary involution of mice [11].
The expression of microRNAs (miRNAs) has time-and tissue-specific characteristics, and miRNAs are differentially expressed in the mammary gland at different developmental stages [12,13].The miR-145 level increased in the lactating mammary gland of dairy cows, and miR-145 regulated fatty acid metabolism by targeting forkhead box protein O1 to affect sterol regulatory element-binding protein 1 activity in bovine mammary epithelial cells [14].Our previous works had shown that let-7g-5p decreased in the mammary glands of mice during pregnancy and regulated mammary cells' differentiation and function by targeting protein kinase C alpha [15].In addition, we showed that miR-142-3p decreased in the mammary glands of lactating mice and regulated milk synthesis and the structure of prolactin receptor-mediated multiple signaling pathways [16].
In mice mammary glands, miR-30a-3p was downregulated during involution compared with its level during lactation.We revealed that miR-30a-3p negatively regulated autophagy by Atg12 in MMECs.Restoring ATG12, knocking down Atg5, starvation, and rapamycin were used to further confirm this conclusion.Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the mammary involution of mice, which was indicative of alteration in mammary remodeling.These results elucidated the molecular mechanisms of miR-30a-3p's regulation of mammary involution.

miR-30a-3p Was Downregulated during Involution Compared with Lactation in Mice Mammary Glands
In previous research, miRNA microarray was used to compare the miRNA expression profiles in mouse mammary glands of four developmental stages [12]: virgin, pregnancy, lactation, and involution (Figure 1A).We found that miR-30a-3p was most significantly downregulated during involution compared with during lactation (Figure 1B).The result of quantitative real-time PCR (qRT-PCR) showed that expression of miR-30a-3p was also decreased in the involution of the mammary glands of mice (Figure 1C; p < 0.001).

Discussion
Postpartum mammary involution is a normal process and is accompanied by secretory epithelial cell death, alveolar regression, gland repopulation by adipocytes, and stroma remodeling [19,20].This process has received wide attention, since mammary involution has been involved in pregnancy-associated breast cancer and is associated witgh higher metastatic probability [21,22].A series of cell death modes have been identified in mammary involution, such as apoptosis, lysosomal permeabilization, and autophagy, but the mechanisms and significance of the different death modes are unclear [23].The level of estrogen receptor α is increased in virgin cows and decreased in lactating and involuting cows [24].Prolactin expression in rats is upregulated in the middle to late stages of pregnancy and then drops during the early stages of involution [25].STAT3 is activated by leukemia inhibitory factor in bovine mammary involution [26].Mammary gland protein-40 is catalytically inactivated during buffalo mammary gland involution [27].In summary, mammary involution is regulated by various signaling molecules.To further explore the mechanisms of involution, miRNA microarray was used to compare the miRNA expression profiles of mouse mammary gland in four developmental stages.We found that miR-30a-3p was most significantly downregulated during involution compared with lactation.
miRNAs are involved in the regulation of many biological processes, for example, cell proliferation, differentiation, apoptosis, and migration, by binding to specific sites of targets [28].Previous studies demonstrated that miR-30a-3p expression exerts tumorsuppressive functions in some forms of cancers [29][30][31], and miR-30a-3p represses growth, migration, and inflammatory response in rheumatoid arthritis fibroblastic synovial cells [32].We found that miR-30a-3p negatively regulated autophagy in MMECs and verified this conclusion by Baf A1 (autophagy inhibitor).
To further investigate the specific mechanism, TargetScanMouse 7.1 and microT-CDS (DIANA TOOLS) databases were used to predict the target of miR-30a-3p.We discovered that the potential binding target sites of miR-30a-3p were 3 -UTR of Atg12 mRNAs, which was consistent with the findings of some studies in the literature [31,33].The predicted binding sites of miR-30a-3p were 1407 and 1895 in ATG12 3 -UTR (NM_026217).We cloned the 1407, 1895, and 1407 + 1895 sites into pMIR-REPORT™ Luciferase vector to verify the effect of miR-30a-3p on Atg12.The results suggested that miR-30a-3p bound both 1407 and 1895 with Atg12 3 -UTR.ATG12 is an autophagy-related protein that contributes to proteasome degradation in the autophagy process [34].The conjugation of the ubiquitinlike protein ATG12 to a lysine residue of ATG5 associates noncovalently with ATG16, which is essential for autophagosome formation [35].Thus, restoring ATG12 and knocking down ATG5 were used, in our work, to validate the finding that miR-30a-3p inhibited autophagy in MMECs by ATG12.
Nutrient starvation induced a significant increase in autophagy by Ulk1 dephosphorylation and its subsequent dissociation from protein kinase AMP-activated catalytic subunit alpha 1 [36].We investigated the effect of starvation on the autophagy of MMECs and found that miR-30a-3p was decreased, Atg12 mRNA was increased, and autophagy was remarkably induced by starvation.However, upregulated miR-30a-3p and downregulated ATG12 reversed these tendencies.Rapamycin promotes autophagy markedly and has been used as a potent inducer of autophagy [37].Our works revealed that rapamycin treatment increased ATG12 expression and autophagy, but these tendencies were reversed by the overexpression of miR-30a-3p and by knocking down ATG12.Thus, miR-30a-3p regulated autophagy by ATG12; this was verified through starvation and rapamycin.
After weaning, alteration of mammary cytokine and prolactin levels trigger mammary gland involution [38].Lactating mice were forced to wean, miR-30a-3p was downregulated, ATG12 was upregulated, and autophagy was promoted in mice mammary glands.To further explore the effect of miR-30a-3p on involuting mammary glands, we upregulated miR-30a-3p levels in mammary involution.Experimental data showed that autophagy was inhibited and mammary acini and ducts were increased by the overexpression of miR-30a-3p.

Mice
BALB/c mice were purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China).Mammary tissues of female mice in different periods (virgin: 4, 5, 7 weeks; pregnant: 5, 13, 18 days; lactating: 3, 7, 13 days; involuting: 2, 5, 10 days) were collected, and six mice were used for each group.The mice were sacrificed, and mammary glands were harvested and frozen in liquid nitrogen for further analysis.All animal experiments were performed in accordance with guidelines and regulations of the Northeast Agricultural University Animal Care and Use Committee (2019-2, Harbin, China) and the Kunming University of Science and Technology Animal Ethics Committee (PZWH(DIAN)K2023-0026).

Quantitative Real-Time PCR (RT-PCR) Analyses
RNAs were isolated from mammary glands at different time points using Trizol (Invitrogen, Carlsbad, CA, USA).M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) was used to convert 100 ng RNA to cDNA.SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the miR-30a-3p and mRNA expressions, which normalized to the U6 or β-actin and were analyzed using the ∆∆ Ct method.

Cell Viability Assay
The cell proliferation test was based on the cell proliferation reagent CCK8 (DOJINDO LABORATORIES, Kamimashiki-gun, Kumamoto, Japan).After treatment, 10 µL of CCK8 reagent was added to the medium in each well of 96-well plate.The cells were incubated in a humidified atmosphere at 37 • C with 5% CO 2 in air for 1 h.The plate was shaken thoroughly for 10 s, and absorbance was read at 450 nm.The background absorbance was measured in wells containing only the culture medium and CCK8 solution.Cell proliferation data were obtained from at least three independent experiments with at least three wells in a separate 96-well plate.The mean optical density values corresponding to the untreated controls were taken as 100%.The results were expressed as the percentage of the optical density of treated cells relative to that of untreated controls.

mRFP-GFP-LC3 Assay
The HC11 cells' culture medium serum concentration was reduced to 0.5% to leave the cells in a starvation state.Then, we obtained cell samples after 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h of cell starvation and analyzed the autophagic flux.Autophagic flux was measured in cells transfected with the autophagy tandem sensor mRFP-GFP-LC3.mRFP-GFP-LC3 adenovirus (MOI = 50; Hanbio, Shanghai, China) was transfected in 50% volume of the culture media for 2 h.After supplementing with an equal volume of media, cells were transfected for 2 h.Then, the culture media was changed to fresh DMEM for 48 h.Autophagy was observed under fluorescence microscopy (Leica, Am Leitz-Park, Wetzlar, Germany).Autophagosomes were labelled with green fluorescent protein (GFP) (green), and LC3 were labelled with monomeric red fluorescent protein (mRFP) (red).GFP is sensitive to acidity and green fluorescence of GFP decreased when autophagosome were fused with lysosome.The autophagic flux was evaluated by counting the number of GFP and mRFP puncta, and 15 cells were used for quantification in each group.

Immunofluorescence, Hematoxylin, and Eosin Staining
Female mice involuting for 1 day were infected with adenovirus expression vector AD-miR-30a-3p in the fourth mammary tissue and empty vector AD-NC was used as the control.Then, female mice lactating for 21 days were used as the lactation control.After 7 days, the mammary tissues of each group were made frozen sections.The mouse mammary gland tissues fixed in 10% formalin solution were sectioned (4 µM) after dehydration, cleaning, and paraffin embedding.The sections were flattened, pasted, and heated on blank glass slides.Histological evaluations were performed with hematoxylin and eosin (H&E) staining and observed with fluorescence microscopy (Leica, Am Leitz-Park, Wetzlar, Germany).

TEM
The fresh mice mammary gland tissue samples were obtained and quickly fixed, as previously described [15].Mammary gland tissue samples were fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.2) at 37 • C for 1 h, washed three times in buffer for 10 min, fixed in 1% osmium tetroxide for 1 h at 4 • C, and dehydrated through a graded series of ethanol.The samples were treated with propylene oxide, embedded, and polymerized for 72 h at 70 • C. Sections were cut on an ultramicrotome (Leica, Am Leitz-Park, Wetzlar, Germany) and stained with 5% uranyl acetate for 10 min, followed by 1 min in Sato's lead stain.TEM was performed on an FEI Tecnai G2 (Hillsboro, OR, USA).

Statistical Analysis
All experiments were performed three times.All statistical analyses used Student's t-tests or one-way analysis of variance (ANOVA, followed by Tukey's post hoc test).p < 0.05 was considered statistically significant.Statistical analyses were performed by GraphPad (Boston, MA, USA) Prism v.8.0.1 software.

Conclusions
In conclusion, miR-30a-3p regulated autophagy in involuting mice mammary glands by ATG12, and overexpression miR-30a-3p delayed mammary involution (Figure 9).To our knowledge, our results are the first to clarify the mechanism of miRNA in mammary involution.
glutaraldehyde in 0.1 M PBS (pH 7.2) at 37 °C for 1 h, washed three times in buffer for 10 min, fixed in 1% osmium tetroxide for 1 h at 4 °C, and dehydrated through a graded series of ethanol.The samples were treated with propylene oxide, embedded, and polymerized for 72 h at 70 °C.Sections were cut on an ultramicrotome (Leica, Am Leitz-Park, Wetzlar, Germany) and stained with 5% uranyl acetate for 10 min, followed by 1 min in Sato's lead stain.TEM was performed on an FEI Tecnai G2 (Hillsboro, OR, USA).

Statistical Analysis
All experiments were performed three times.All statistical analyses used Student's t-tests or one-way analysis of variance (ANOVA, followed by Tukey's post hoc test).p < 0.05 was considered statistically significant.Statistical analyses were performed by GraphPad (Boston, MA, USA) Prism v.8.0.1 software.

Conclusions
In conclusion, miR-30a-3p regulated autophagy in involuting mice mammary glands by ATG12, and overexpression miR-30a-3p delayed mammary involution (Figure 9).To our knowledge, our results are the first to clarify the mechanism of miRNA in mammary involution.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1.

Figure 1 .
Figure 1.miR-30a-3p was downregulated during involution compared with lactation in mice mammary glands.(A) Heat map illustrating the miRNAs' expression in mouse mammary gland of four developmental stages, picking up miRNAs with the top 30 ratio of lactation/involution. (B) miRNAs with the top 30 ratio of lactation/involution. (C) The expression of miR-30a-3p in mouse mammary gland of four developmental stages.Data are presented as mean ± SD, *** p < 0.001.

Figure 1 .
Figure 1.miR-30a-3p was downregulated during involution compared with lactation in mice mammary glands.(A) Heat map illustrating the miRNAs' expression in mouse mammary gland of four developmental stages, picking up miRNAs with the top 30 ratio of lactation/involution. (B) miRNAs with the top 30 ratio of lactation/involution. (C) The expression of miR-30a-3p in mouse mammary gland of four developmental stages.Data are presented as mean ± SD, *** p < 0.001.