Human Milk-Derived Levels of let-7g-5p May Serve as a Diagnostic and Prognostic Marker of Low Milk Supply in Breastfeeding Women

Low milk supply (LMS) is associated with early breastfeeding cessation; however, the biological underpinnings in the mammary gland are not understood. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally downregulate gene expression, and we hypothesized the profile of miRNAs secreted into milk reflects lactation performance. Longitudinal changes in milk miRNAs were measured using RNAseq in women with LMS (n = 47) and adequate milk supply (AMS; n = 123). Relationships between milk miRNAs, milk supply, breastfeeding outcomes, and infant weight gain were assessed, and interactions between milk miRNAs, maternal diet, smoking status, and BMI were determined. Women with LMS had lower milk volume (p = 0.003), were more likely to have ceased breast feeding by 24 wks (p = 0.0003) and had infants with a lower mean weight-for-length z-score (p = 0.013). Milk production was significantly associated with milk levels of miR-16-5p (R = −0.14, adj p = 0.044), miR-22-3p (R = 0.13, adj p = 0.044), and let-7g-5p (R = 0.12, adj p = 0.046). Early milk levels of let-7g-5p were significantly higher in mothers with LMS (adj p = 0.0025), displayed an interaction between lactation stage and milk supply (p < 0.001), and were negatively related to fruit intake (p = 0.015). Putative targets of let-7g-5p include genes important to hormone signaling, RNA regulation, ion transport, and the extracellular matrix, and down-regulation of two targets (PRLR and IGF2BP1/IMP1) was confirmed in mammary cells overexpressing let-7g-5p in vitro. Our data provide evidence that milk-derived miRNAs reflect lactation performance in women and warrant further investigation to assess their utility for predicting LMS risk and early breastfeeding cessation.


Introduction
Low milk supply is associated with early breastfeeding cessation. Nearly 40% of breastfeeding women cite concerns over low milk supply as a primary reason for not meeting their breastfeeding goals [1]. The etiology of suboptimal lactation is clearly multifaceted. Abnormal breast conditions and previous breast surgeries [2] are structural factors that contribute to suboptimal lactation. In addition, numerous social, psychological, and behavioral factors are associated with early breastfeeding cessation [3][4][5][6]. Several studies reported associations between maternal metabolic conditions such as malnutrition [7], excessive maternal fat mass [8], and gestational diabetes [9] and milk supply. Moreover, we and others determined that genetic variations in genes critical for mammary gland function (i.e., prolactin (PRL) [10], prolactin receptor (PRLR) [11], ZnT2 (SLC30A2) [12], and milk fat globule-epidermal factor 8 protein (MFGE8) [13]) are associated with the ability to produce milk, providing evidence that biological factors are also responsible for low milk supply.
MicroRNAs (miRNAs) are small, non-coding nucleic acid sequences (~22 nucleotides) that post-transcriptionally regulate gene expression by binding to specific mRNA targets and either inhibiting translation or promoting degradation, thus affecting corresponding protein expression. Strong evidence indicates miRNAs control normal physiology and miRNAs have been associated with various pathological states [14]. MiRNAs are found in all bodily fluids, including saliva, plasma, and urine [15,16], although milk is one of the richest sources of miRNAs in humans, containing~1400 mature miRNAs [17]. Many of these miRNAs are released by secreting mammary epithelial cells (MECs) in exosomes. Mammary gland miRNA profiles differentiate discrete stages of mammary gland development in rodents and dairy animals, and parallel gene expression required for ion transport, G protein signaling, translation, and intracellular protein transport, and oxidative phosphorylation [18][19][20]. Several previous studies suggested the profile of human milk miRNAs may reflect breast function [17,21]. Consistent with this hypothesis, we and others [22,23] previously showed that several maternal factors associated with milk production and composition (i.e., diet, genotype, preterm birth, and stage of lactation) are associated with the profile of milk-derived miRNAs, implicating milk miRNAs as bioreporters of lactation performance in humans [24]. Here we hypothesized that specific milk-derived miRNAs are associated with low milk supply, and using a genome-wide computational approach, identified milk-derived miRNAs that may serve as novel genetic drivers or reporters of low milk supply and confirmed regulation of two targets in MECs in vitro.

Participants
This longitudinal cohort study involved a convenience sample of 221 women, ages 19-42 years. Mothers of full-term, singleton infants (37-42 weeks gestation) who planned to breastfeed at least six months were eligible. Exclusion criteria and the study design were previously reported [13]. Participants were dichotomized into two groups: low milk supply (LMS) or adequate milk supply (AMS) as we have previously reported. Ultimately, 47 women with LMS were compared against mothers with AMS (n = 123) who either maintained exclusive breast feeding throughout the duration of the study or reported formula introduction for reasons other than "decreased or low breastmilk production" (Figure 1).

Participant Characteristics
Participant characteristics were collected via electronic surveys administered by research staff at enrollment. Survey responses were confirmed through review of the medical record where possible. The following medical and demographic characteristics were collected: maternal age, maternal race, parity, pre-pregnancy body mass index, tobacco use, maternal educational level, marital status, duration of previous breastfeeding, infant gestational age, infant sex, and infant birth weight as previously reported [13]. No women reported the use of galactagogue supplements.
Maternal nutrition was assessed alongside each milk collection (at 1, 4, 16 wks) through electronic administration of the Dietary Screener Questionnaire (DSQ), developed as part of the National Health and Nutrition Examination Survey (NHANES) [25]. Published guidelines were used to compute consumption of fruit, vegetables, dairy, added sugars, and calcium. Infant feeding characteristics were collected through electronic administration of the modified Infant Feeding Practices survey (IFP), and as we have previously reported, milk production was approximated based on maternal report of pumping volumes and infant feeding practices [13]. Mothers who were unable to estimate milk production volumes (n = 57/340 data-points; 16%) were excluded from analyses of milk production. For mothers who reported infant weaning (or failed to provide a milk sample due to low milk supply; n = 25), a milk production volume of 0 oz/day was assigned. Infant weight and length were abstracted from the medical record at birth and four weeks post-delivery. For each infant, the change in weight-for-length Z-score was calculated at each time point using standardized curves from the World Health Organization. to breastfeed at least six months were eligible. Exclusion criteria and the study design were previously reported [13]. Participants were dichotomized into two groups: low milk supply (LMS) or adequate milk supply (AMS) as we have previously reported. Ultimately, 47 women with LMS were compared against mothers with AMS (n = 123) who either maintained exclusive breast feeding throughout the duration of the study or reported formula introduction for reasons other than "decreased or low breastmilk production" (Figure 1). Figure 1. CONSORT Diagram. Research staff screened 2487 mother-infant dyads, approached 359 eligible dyads, and obtained consent from 221 dyads. There were 180 mothers that provided at least one milk sample and completed sufficient longitudinal surveys to determine whether they experienced low milk supply (LMS) or adequate milk supply (AMS). There were 4 or 6 mothers excluded from each group for excessive infant weight gain or weight loss (defined as a change in weight-for-length (WfL) Z-score > 2.0), which suggested milk production may have been over-or under-estimated. This left 47 mothers with LMS, and 123 mothers with AMS. Of the 123 mothers with AMS, 48 introduced formula into their infant's diet prior to 12 months for reasons other than concerns about milk supply.

Milk Collection
One milk sample was collected from each mother at 1, 4, and 16 wks post-delivery (or until breastfeeding ceased). The 170 mothers provided 453 milk samples: 170 samples in the first wk post-delivery, 158 samples 4 wks post-delivery, and 125 samples 16 wks post-delivery. Maternal milk (1-5 mL) was manually expressed from a sterilized nipple surface into RNAse-free tubes prior to feeding (i.e., fore-milk). Foremilk samples were exclusively collected from the same breast to minimize confounding impacts of fore-and hind-milk differences [17]. Samples were immediately transferred to −20 • C, underwent one freeze-thaw cycle for aliquoting, and stored at −80 • C.

RNA Processing
Milk was skimmed by centrifugation for 20 min at 4 • C at 800 rpm and the lipid fraction was used for RNA extraction [21,26]. RNA was purified, sequenced, and analyzed as previously described [26,27]. The 30 miRNA features with the most robust expression (present in raw counts >10 in all 453 samples) were quantile normalized and mean-center scaled.

Statistical Methods
Medical and demographic traits were compared between LMS and AMS groups using a student's t-test or chi-square test, as appropriate. Levels of miRNAs in the initial milk sample (at 1 wk) were compared between LMS and AMS groups using a Wilcoxon Rank Sum test. p-values were adjusted for multiple testing using the false detection rate (FDR) method, and values less than 0.05 were considered significant. Candidate miRNAs identified on Wilcoxon Rank Sum testing underwent the following secondary analyses: (1) Relationships between milk miRNA levels and milk production (oz/day) were assessed with Spearman Rank Correlation testing; (2) longitudinal changes in milk levels of miRNA candidates were assessed across 1, 4, and 16 wks post-delivery using a linear mixed model fit by restricted maximum likelihood (with each miRNA as the dependent variable, participant ID as the clustering variable, and maternal characteristics as covariates). Interactions between LMS/AMS group and time (wks post-delivery) were assessed with fixed effects omnibus tests; (3) the effects of modifiable maternal characteristics (nutrition, BMI, tobacco use) on milk levels of candidate miRNAs were assessed with a mixed model (with miRNA level was the dependent variable, participant ID as the clustering variable, and maternal characteristics as covariates); (4) the ability of candidate miRNA levels to differentiate participants at risk for LMS was assessed relative to maternal factors with a hierarchical logistic regression. LMS/AMS group served as the dependent variable, and candidate miRNAs without collinearity were used in a feed-forward model building approach. Sensitivity, specificity, and area under a receiver operator characteristic curve were reported; (5) physiologic functions of the candidate miRNAs were explored in DIANA miRPath v3 [28], through identification of putative mRNA targets (Tarbase algorithm). Enrichment of Kyoto Encyclopedia Genes and Genomes (KEGG) pathways was compared to that expected by chance using a Fisher's exact test with FDR correction.
For cell experiments, data represent mean ± SD. Statistical analysis was carried out using GraphPad Prism software (Version 9.0; GraphPad Prism Software, Inc., San Diego, CA, USA). Differences between means were determined by students t-test and were considered statistically significant at p-value < 0.05.

Longitudinal Changes in LMS-Related miRNAs
Linear mixed models were used to assess candidate miRNAs for changes in abundance over the course of lactation, while controlling for maternal age, education, and breastfeeding experience. Milk levels of let-7g-5p increased over the course of lactation (F = 21.4, p < 0.001), and there was a significant interaction between lactation period and group (F = 7.5, p < 0.001; Figure 3A). Levels of let-7g-5p generally increased in the AMS

Longitudinal Changes in LMS-Related miRNAs
Linear mixed models were used to assess candidate miRNAs for changes in abundance over the course of lactation, while controlling for maternal age, education, and breastfeeding experience. Milk levels of let-7g-5p increased over the course of lactation (F = 21.4, p < 0.001), and there was a significant interaction between lactation period and group (F = 7.5, p < 0.001; Figure 3A). Levels of let-7g-5p generally increased in the AMS group over time but remained stable in mothers with LMS. Levels of let-7a-5p (F = 15.7, p < 0.001), miR-22-3p (F = 71.2, p < 0.001), and miR-16-5p (F = 62.7, p < 0.001) also changed over the course of lactation but did not display an interaction between lactation stage and milk supply.

Predicting Low Milk Supply Status
Hierarchical logistic regression was used to assess the ability of milk miRNAs to predict low milk supply, relative to medical and demographic traits. The three maternal characteristics that differed between mothers with LMS and mothers with AMS (i.e., age, education, breastfeeding experience) accounted for 15.5% of the variance between groups, (X 2 = 31.0, p < 0.001), and accurately identified 35/47 mothers with AMS (74% sensitivity) and 80/123 mothers with AMS (65% specificity; AUC = 0.763). Addition of three miRNAs that lacked co-linearity (i.e., miR-22-3p, let-7a-5p, and let-7g-5p) accounted for an additional 8.1% of variance between groups (X 2 = 47.3, p < 0.001), and significantly improved the model (X 2 = 16.2, p = 0.001). The combined model displayed an AUC of 0.816.

Effect of Modifiable Maternal Characteristics on LMS-Related miRNAs
Mixed models were used to assess the effect of nutrition, BMI, and tobacco use on candidate miRNAs. Nutrition displayed an association with milk miRNA levels over the course of lactation. Lower milk levels of let-7g-5p were associated with higher maternal fruit consumption (F = 5.99, p = 0.015, Figure 3B). There was no interaction between fruit consumption and milk supply (F = 0.22, p = 0.63). Higher milk levels of miR-22-3p were associated with lower consumption of calcium (F = 7.31, p = 0.007) and dairy (F = 6.48, p = 0.011).

Predicting Low Milk Supply Status
Hierarchical logistic regression was used to assess the ability of milk miRNAs to predict low milk supply, relative to medical and demographic traits. The three maternal characteristics that differed between mothers with LMS and mothers with AMS (i.e., age, education, breastfeeding experience) accounted for 15.5% of the variance between groups, (X 2 = 31.0, p < 0.001), and accurately identified 35/47 mothers with AMS (74% sensitivity) and 80/123 mothers with AMS (65% specificity; AUC = 0.763). Addition of three miRNAs that lacked co-linearity (i.e., miR-22-3p, let-7a-5p, and let-7g-5p) accounted for an additional 8.1% of variance between groups (X 2 = 47.3, p < 0.001), and significantly improved the model (X 2 = 16.2, p = 0.001). The combined model displayed an AUC of 0.816.

Confirmation of PRLR and IGF2BP1 Regulation in Mammary Cells
To directly confirm the effect of elevated levels of let-7g-5p in MECs, cells were transiently transfected with a let-7g-5p mimic. Protein expression of two predicted mRNA targets, PRLR and IGF2BP1/IMP1, was measured 30 h later by immunoblot (Table S1). PRLR was selected for confirmation of its regulation by let-7g-5p in MECs because of its well-known importance in lactation, and IGF2BP1/IMP1 was selected to assess effects on a novel molecular target. Both the long and short forms of IGF2BP1/IMP1 were identified in HC11 cells [29], and both isoforms were significantly lower (p < 0.01) in cells transfected with 30 nM hsa-let-7g mimic compared to non-transfected cells ( Figure 5A-C). In addition, PRLR protein expression was significantly lower in cells transfected with 30 nM hsa-let-7g mimic (p < 0.05) compared to non-transfected cells ( Figure 5D,E). These results confirm that expression of PRLR and IGF2BP1/IMP1 are negatively regulated by let-7g-5p in MECs and provide evidence of two discrete molecular mechanisms that may underlie effects of let-7g-5p on lactation and milk supply. Hierarchical clustering showed that let-7a-5p and let-7g-5p had the most closely related physiologic targets, and miR-22-3p and miR-16-5p displayed the second highest physiologic relatedness (Figure 4). Due to the interaction between let-7g-5p, lactation stage, and milk supply, we further queried molecular pathways affected by this miRNA. Key KEGG pathways predicted to be downregulated by let-7g-5p (https://genome.jp, accessed on 2 November, 2022) include Metabolic, PI3K-Akt signaling, MAPK signaling, and JAK-STAT signaling pathways (Table 4), and include numerous genes associated with lactation traits such as those involved in hormone signaling (PRLR, INSR), extracellular matrix (COL1A1-2, COL3A1, COL4A1-3 and A6, COL5A2, COL14A1, COL27A1), zinc transport (SLC30A4), H + transport (ATP6V1G1, ATP6V1C1), and calcium transport (ATP2A2, ATP2B4). Moreover, the top mRNAs identified included several novel lactogenic targets involved in mRNA binding  in HC11 cells [29], and both isoforms were significantly lower (p < 0.01) in cells transfected with 30 nM hsa-let-7g mimic compared to non-transfected cells ( Figure 5A-C). In addition, PRLR protein expression was significantly lower in cells transfected with 30 nM hsalet-7g mimic (p < 0.05) compared to non-transfected cells ( Figure 5D,E). These results confirm that expression of PRLR and IGF2BP1/IMP1 are negatively regulated by let-7g-5p in MECs and provide evidence of two discrete molecular mechanisms that may underlie effects of let-7g-5p on lactation and milk supply.

Discussion
Here, we present a novel approach for identifying biological factors that underpin low milk supply in breastfeeding women. We established that the profile of milk-derived miRNAs reflects lactation performance in humans, and for the first time identified miRNAs associated with low milk supply. Importantly, we determined that let-7g-5p may serve as a potential regulon of breast function and predicted key genes involved in metabolism, hormone signaling, ion transport, mRNA binding, and tissue remodeling in the lactating mammary gland, supporting previous studies that have posited a role for let-7g-5p in breast function [30]. Importantly, bioinformatic prediction of PRLR and IGF2BP1/IMP1 as let-7g-5p targets was confirmed in MECS. We detected a significant interaction between let-7g-5p, milk volume, and lactation stage, suggesting that measurement of let-7g-5p levels in milk during early lactation may be useful in predicting risk for low milk supply. Interestingly, our data suggest the let-7g-5p regulon may be modifiable as there was a negative relationship between let-7g-5p levels and fruit intake in our population of breastfeeding women.
Milk is one of the richest sources of miRNAs in humans and contains~1400 mature miRNAs [17] and the profile of milk miRNAs has been proposed to reflect mammary gland function [17][18][19][20][21]. We found maternal estimates of daily milk production were indeed associated with levels of miR-16-5p, miR-22-3p, and let-7g-5p; however, only levels of let-7g-5p survived multiple testing correction. The let-7 family of miRNAs is one of the earliest discovered miRNA clusters and is conserved across species [31]. It is comprised of ten miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-98, and miR-202) and plays key roles in suppressing proliferation and differentiation [32] and is a key regulator of glucose metabolism [33]. Let-7g-5p levels were enriched in the milk of women with low milk supply, consistent with a putative role for let-7g-5p suppression in motivating proliferation, suppressing mammary gland differentiation [30] and affecting mammary gland metabolism. Importantly, let-7g-5p is conserved between human and mouse [31], suggesting the opportunity to use preclinical mouse models to understand mechanistic implications of let-7g-5p on MEC proliferation, differentiation, and milk production and secretion.
A critical gap in knowledge is how let-7g-5p is regulated in mammary epitheial cells. Let-7g is found on chromosome 3 in humans [31] and is post-transcriptionally repressed through binding of the RNA binding protein Lin28 to its terminal loop, thereby either inhibiting binding of Dicer and Drosha or re-routing pre-let-7g for degradation [34]. Thus, factors that affect Lin28 regulation would have major impacts on mammary gland function. One potential factor that regulates the Lin28/let-7 axis is inflammation [35]. Interestingly, inflammation is known to compromise lactation and milk supply [36,37], thus therapeutic strategies to reduce inflammation may be key to maximizing milk supply. Additionally, let-7g expression is repressed by DNA methylation [38], suggesting intake of methyl donors such as folate [39] and B 12 may play a role. Several studies suggest estrogen may regulate let-7g-5p; however, results are inconsistent [40,41]. Positive regulation by estrogen would be consistent with suppression of let-7g-5p levels upon estrogen withdrawal at partition and the subsequent activation of lactogenesis II. Given the importance of robust activation lactogenesis II in the maintenance of copious milk production going forward, identification of factors that regulate let-7g-5p is critical to our understanding of this regulon and its intriguing role in lactation [42].
Numerous mRNA targets of let-7g-5p associated with lactation traits include hormone signaling (PRLR, INSR) [43][44][45], extracellular matrix (COL1A1-2, COL3A1, COL4A1-3 and A6, COL5A2, COL14A1, COL27A1) [46], zinc transport (SLC30A4) [47], H + transport (ATP6V1G1, ATP6V1C1) [48], and calcium transport (ATP2A2, ATP2B4) [49], further implicating it as a regulatory hub for maintaining milk supply. Importantly, our study confirmed that PRLR expression was indeed regulated by let-7g-5p in MECs, providing direct evidence that let-7g-5p is a critical regulator of lactation. Three examples of novel targets of let-7g-5p that may have potential lactogenic implications include steriodogenic acute regulatory protein-related lipid transfer domain-containing protein 13 (STARD13), high mobility group AT-hook 2 (HMGA2), and insulin-like growth factor 2 messenger RNA-binding protein (IGF2BP1/IMP1). STARD13 serves as a Rho-GTPase activating protein that selectively regulates RhoA and cdc42 to inhibit actin assembly. STARD13 attenuation leaves RhoA constitutively active which inhibits Rac and thus inhibits motility [50]. To our knowledge, a role for STARD13 in mammary gland development or lactation has yet to be explored; however, this finding may have important implications for breast cancer detection as STARD13 is a tumor suppressor and Rac1 plays a major role in cancer cell motility [50]. HMGA2 is a group of small chromatin-associated proteins that act as an architectural transcription factor that directly binds to DNA and modulates the transcription of target genes; however, a putative role for HMGA2 in the breast requires exploration. IGF2BP1/IMP1 is a highly conserved RNA-binding protein that regulates RNA processing at several levels, including localization, translation, and stability. Key targets of IGF2BP1/IMP1 with potential consequences on lactation include β-actin, STAT3, c-myc, glutathione peroxidases, and several mitochondrial proteins [51]. Two IGF2BP1/IMP1 isoforms have been identified in MECs [29], a long isoform (~70 kDa) and a short isoform (~40 kDa) resulting from an Nterminal truncation with currently unknown function. Herein, we confirmed that let-7g-5p downregulated both isoforms, suggesting key molecular functions such as morphogenesis, oxidative stress, and ATP production are targets of the let-7g-5p regulon, which implicates IGF2BP1/IMPs as a novel molecular target for low milk supply.
While our genome-wide approach identified critical milk-derived miRNAs and predicted novel genes and molecular pathways for further exploration, an exciting finding from this study was that high milk levels of let-7g-5p during early lactation may be useful as a bioreporter of low milk supply and risk for early breastfeeding cessation. This observation warrants further study as >40% of women cite concerns over low milk supply as a primary reason for not meeting their breastfeeding goals [1] and early identification could inform interventions to improve breastfeeding success. One such intervention might include dietary modification, as intriguing findings suggest levels of let-7g-5p may be modifiable. For example, ursolic acid (UA) is a natural triterpene found in various fruits and vegetables that suppresses let-7g-5p [52] and there is a growing interest in UA because of its beneficial effects, which include pro-and anti-inflammatory, antioxidant, anti-apoptotic, and anti-carcinogenic effects [53]. Additionally, quercetin is a polyphenol ubiquitously present in certain fruits (e.g., apples and grapes) and vegetables (e.g., onions, kale, broccoli, lettuce, and tomatoes) that also has pro-and anti-inflammatory and antioxidant capacity, and let-7g-5p levels are positively associated with a quercetin-rich diet [54]. Further studies are required to reproduce our findings and determine how dietary polyphenols and secondary metabolites affect the let-7g-5p regulon in the mammary gland, which may eventually offer a therapeutic option with an evidence-based rationale for women with low milk supply.
There are several strengths of the present study: (1) a large sample size with longitudinal collections; (2) uniformity insample collection and processing; (3) high throughput sequencing; and (4) the use of mixed effects models to assess relative impacts of maternal characteristics. However, there are several limitations. The scatter in our data suggest that trends may be driven more heavily by samples at the fringe ends of the dataset, and additional studies are required to confirm our findings. The current cohort was mostly white and included only mothers delivering at term, which may limit generalizability of the findings. In addition, given our finding that let-7g-5p may be a potential regulator, further studies should include potential modifiers such as caloric intake, physical activity, quality of life, and comprehensive dietary analysis to better understand the role and regulation of let-7g-5p during lactation. Finally, our results (which include both exosomal and non-exosomal miRNA) may differ from studies focused solely on exosomal miRNAs [55] and while prior studies have demonstrated minimal miRNA differences across milk fractions [56,57], results from miRNAs in skim versus cellular fractions may differ.

Conclusions
In conclusion, the results of this study advance our collective understanding of the biological contributors to low milk supply by identifying miRNAs, novel molecular pathways, and new gene targets associated with poor milk production. Predicted transcripts highlight both classical and novel lactogenic targets, therefore future studies should interrogate consequences of these miRNAs on mammary gland function. Importantly, this study is the first to identify an interaction between milk levels of miRNAs, low milk supply, and early cessation of breastfeeding, and suggests that measuring milk levels of let-7g-5p during the first weeks after birth may be a useful tool in predicting risk for low milk supply and identifying women who need targeted lactation support.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu15030567/s1, Table S1: predicted mRNA targets.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The RNA sequencing data presented in the study are deposited in the Gene Expression Omnibus repository, accession number GSE192543. GEO repository link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE192543; 1 January, 2022.