Long non-coding RNA Loc105611671 promotes the proliferation of ovarian granulosa cells and steroid hormone production upregulation of CDC42

Granulosa cells (GCs) are essential for follicular development, and long non-coding RNAs (LncRNAs) are known to support the maintenance of this process and hormone synthesis in mammals. Nevertheless, the regulatory roles of these lncRNAs within sheep follicular GCs remain largely unexplored. This study delved into the influence of a Loc105611671, on the proliferation and steroid hormone synthesis of sheep ovarian GCs and the associated target genes in vitro. Cell Counting Kit-8 (CCK-8) gain-of-function experiments indicated that overexpression of Loc105611671 significantly boosted GCs proliferation, along with estrogen (E2) and progesterone (P4) levels. Further mechanistic scrutiny revealed that Loc105611671 is primarily localized within the cytoplasm of ovarian granulosa cells and engages in molecular interplay with CDC42. This interaction results in the upregulation of CDC42 protein expression. Moreover, it was discerned that increased CDC42 levels contribute to augmented proliferation of follicular granulosa cells and the secretion of E2 and P4. Experiments involving co-transfection elucidated that the concurrent overexpression of CDC42 and Loc105611671 acted synergistically to potentiate these effects. These findings provide insights into the molecular underpinnings of fecundity in ovine species and may inform future strategies for enhancing reproductive outcomes.


Introduction
GCs proliferation and growth are pivotal to the process of follicular development (1).This dynamic sequence begins with oocyte growth, followed by the recruitment of GCs (2,3).As they progress toward ovulation, GCs undergo morphological changes and proliferate (4).As the predominant cell type within the follicle, GCs not only regulate their own proliferation but also contribute to follicle development by synthesizing hormones (E 2 , P 4 ) (5, 6) and growth factors (7,8).The process begins with the transformation of cholesterol, during which the STAR protein plays a pivotal role in translocating cholesterol to the inner mitochondrial membrane.At this juncture, the enzyme P450scc (Cyp19a1 gene product) metabolizes cholesterol into pregnenolone (9).E 2 production within GCs is facilitated by the enzymatic actions of P450arom aromatase and 17β-HSD (10).With the development and maturation of the follicle, E 2 levels rise, consequently stimulating GCs proliferation and differentiation.This not only impacts the quality and maturation of the oocyte but also plays a role in follicular evolution (11,12).Furthermore, E 2 facilitates the production of P 4 by activating the P4 receptor, thus influencing GC proliferation and follicular development (13).The concentration of P 4 fluctuates throughout the stages of follicular development, peaking during the maturation phase (14).
LncRNAs are a diverse class of RNAs over 200 bp in length, including intronic, intergenic, and antisense variants (15).These lncRNAs are known to play essential regulatory roles in mammalian reproduction, participating in cell proliferation (16), apoptosis (17), follicle development (18), oocyte maturation (19), and steroid hormone synthesis (20), underscoring their significance in reproductive biology.Recent studies indicate that lncRNAs contribute to reproductive processes by interacting with proteins and other RNAs.For example, lncRNA PVT1 induces GC apoptosis by upregulating Foxo3a levels (21), while lncRNA RP11-552 M11. 4 collaborates with BRCA2 to stimulate GC proliferation and prevent apoptosis (22).Despite these findings, research on mammalian reproduction has largely cantered on the discovery of novel lncRNAs (23)(24)(25)(26), with limited investigation into the specific functions and mechanisms of lncRNAs, particularly in sheep ovarian GCs.
Our prior study revealed differential expression of Loc105611671 in Qira black sheep during the pre-estrus and estrus phases (27), suggesting it may influence sheep reproductive capacities by regulating GC functions.Yet, the exact role of Loc105611671 in sheep follicular GC regulation is still to be elucidated.To this end, we probed the impact of Loc105611671 on GC proliferation and steroid hormone secretion by developing an in vitro cultured follicular GC model.Our study is designed to elucidate the complex regulatory mechanisms lncRNAs exert on sheep follicular development.Concurrently, it may lay the groundwork for identifying novel therapeutic approaches to reproductive disorders such as polycystic ovarian syndrome (PCOS), a condition that can result in ovulatory failure.

Isolation and culture of GC
During the peak breeding season (August to October), healthy sheep ovaries from animals aged 1 to 1.5 years were sourced from a local abattoir in Shihezi, Xinjiang Uygur Autonomous Region, China.Mature dominant follicles were carefully selected, their follicular fluid aspirated and collected into Petri dishes containing (Dulbecco's modified Eagle's medium/nutrient mixture F-12 [DMEM/F12] (Gibco, France) medium.Oocytes were meticulously picked using a mouth pipette.The GCs were then transferred to erythrocyte lysis buffer to eliminate any red blood cells.The pelleted cells were washed twice with DMEM/F12 medium, cultured into Petri dishes enriched with 10% fetal bovine serum [FBS] (Gibco, France), 100 IU/mL penicillin, and 100 μg/mL streptomycin) aseptic culture at 37°C in a 5% CO 2 atmosphere.After 48 h, non-adherent cells were removed by gentle medium replacement.

Quantitative reverse transcription-polymerase reaction (qRT-PCR)
Total RNA was extracted from cells using the TRIzol (Invitrogen, USA) assay.After the RNA samples were reverse transcribed into cDNA using the TransScript ® First-Strand cDNA Synthesis SuperMix kit (Transgen, China) for quality assessment, they were assayed for gene expression by using the Perfectstart Green qPCR SuperMix PCR kit (Transgen, China) according to the user's manual and a Roche Light Cycler 480 (Roche, Switzerland) to detect gene expression.The housekeeping gene, GAPDH, was used as an internal reference.Data were analyzed using the 2 -ΔΔCt method.All primers used in this study are listed in Supplementary Table S1.

Plasmid construction and transfection of GCs
GCs were cultured to 60-70% confluence and then transfected, or co-transfected, with Lipofectamine 2000 (Invitrogen, USA) for 72 h.The overexpression construct for Loc105611671 was synthesized by cloning the full-length sequence into the EcoRI/BamHI sites of the pCDNA3.1-EGFPvector (denoted as LV-Loc105611671).Similarly, the CDC42 overexpression plasmid was produced by inserting the CDC42 coding sequence into the same sites of the pCDNA3.1-EGFPvector (denoted as LV-CDC42).An empty pCDNA3.1-EGFPvector served as the control (denoted as LV-EGFP).The primers utilized for cloning are listed in Table 1.

RNA-protein pull-down assay
To synthesize biotinylated transcripts, we used the T7 in vitro transcription kit mMESSAGE mMACHINE ® Kit (cat.AM1344, Invitrogen, USA) for transcription; the amplification primers are listed in Table 1.After purification using the RNeasy Mini Kit (cat.74,104, QIAGEN, Germany), biotinylated RNA was added to the cell lysates.After treatment with RNase-free DNase I, the biotin-labeled Loc105611671 was denatured for 3 min at 95°C, incubated on ice for 1 min, and rested at room temperature for 30 min to restore the secondary structure of the RNA.The RNA was then incubated with Streptavidin Magnetic Beads (cat.21,344, Thermo, USA) for 1 h at room temperature and stirred in a clean test tube to form a magnetic bead-RNA mixture.Next, the extracted total GCs protein was added to the magnetic bead-RNA mixture and incubated for 1 h at room temperature with rotation to generate the magnetic bead-RNAprotein complexes.After washing and elution, RNA-bound proteins were collected and subsequently separated using SDS-PAGE elution and silver staining.

Cell proliferation analysis
For CCK-8 analysis (Transgen, China), post-transfection pellet cells were inoculated into 96-well plates (5 × 10 3 cells per well).At 24, 48, and 72 h, 10 μL of CCK-8 reagent was added to each well and then cultured for 3 h.All experiments were performed in triplicate.The Frontiers in Veterinary Science 03 frontiersin.orgabsorbance of each well was then measured at 450 nm using an enzyme marker (Thermo Fisher, USA).

Determination of steroid hormone concentrations by ELISA
At the indicated time points, post-transfection cell culture supernatants were collected using sheep enzyme E 2 (sensitivity: less than 1.0 pg./mL, specificity: no cross-reactivity with other soluble structural analogs, reproducibility: intra-plate coefficient of variation less than 9%, inter-plate coefficient of variation less than 11%, cat.JL17550), P 4 (sensitivity: less than 0.1 ng/mL, specificity: no crossreactivity with other soluble structural analogs, repeatability: intraplate coefficient of variation less than 9%, inter-plate coefficient of variation less than 11%, cat.JL22308) ELISA was determined by absorbance at 450 nm by an enzyme labeller (Thermo Fisher, USA).Sheep E 2 ELISA kit and P 4 ELISA kit were purchased from Jianglai Biotechnology Co Ltd. (Jianglai, China).

Subcellular localization
For nuclear and cytoplasmic RNA isolation, the nuclear and cytoplasmic fractions were collected and extracted using the nuclear/cytoplasmic separation kit (cat.HR0241, Biolab, China) according to the manufacturer's instructions.The expression of Loc105611671 and CDC42 in cytoplasmic and nuclear RNA of GCs was measured using qRT-PCR as mentioned above.XIST and ACTB were used as positive controls for the nucleus and cytoplasm, respectively.

Western blot analysis
Total proteins were extracted from the cells using RIPA lysis buffer (containing 1% PMSF) and then denatured by heating at 100°C.Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes.After blocking with 5% skimmed milk and incubation with primary and secondary antibodies, immunoreactive bands on the membrane were reacted with ECL solution and detected by chemiluminescence imaging using a Tennant chemiluminescence imager (Tanon, China).The primary antibodies used in this study were anti-GAPDH (1:5000, cat.GTX100118, GeneTex, USA), anti-CDC42 (1:1000, cat.GTX134588, GeneTex, USA) and goat an-tirabbit (1:10000, cat.YK2231, Y&K Bio, China).

Dual luciferase reporter gene assay
293 T cells were cultured in 96-well plates (5 × 10 3 cells per well).When the cell density reached 50-70%, the transfection plasmids were cotransfected individually into the cells.After 48 h of transfection, luciferase activity was measured using a dual luciferase reporter gene assay system (BioTek, USA).

Statistical analysis
All experimental data were analyzed using GraphPad Prism 8.0 software (GraphPad Inc) or SPSS 26.0 system (SPSS Inc).An independent samples t-test was used to analyze the statistical differences between the two groups.Each experiment was repeated three times.p-values less than 0.05 were considered statistically significant as follows: "*" p < 0.05, "**" p < 0.01, and "***" p < 0.001.

Overexpression of Loc105611671 enhances GCs proliferation
To elucidate the regulatory effects of Loc105611671 on GCs in vitro, we used overexpression plasmids to overexpress Loc105611671.The proliferation rate of GCs was analyzed using CCK-8 assay.The efficiency of Loc105611671 overexpression based on the pCDNA3.1-EGFPvector was confirmed using qRT-PCR (Figures 1A-C).The results demonstrated that Loc105611671 upregulation significantly enhanced GC proliferation at 24 h (0.747 ± 0.046 vs. 0.928 ± 0.026, p < 0.01) and at 48 h (1.088 ± 0.047 vs. 1.504 ± 0.032, p < 0.001) compared with the control.After 72 h (1.445 ± 0.204 vs. 1.617 ± 0.077, p > 0.05), the proliferation rates between the test and control groups began to align (Figure 1D).Additionally, the overexpression of Loc105611671 notably upregulated the mRNA expression levels of CDK1 and PCNA, both positive cell cycle regulators, while downregulating P21, a cell cycle inhibitor (Figure 1E).These results indicate that Loc105611671 plays a pivotal role in the proliferation of GCs.

Loc105611671 promotes E2 and P4 synthesis in GCs
To determine the effect of Loc105611671 expression on steroid hormone production, we performed ELISA to measure the E 2 and P 4

Gene
Primer sequence (5′-3′)  2C,D).Moreover, a marked upregulation was observed in the mRNA expression of STAR, Cyp11a1, and Cyp19a1, genes involved in steroidogenesis (Figure 2E).These data suggest that Loc105611671 plays an important role in steroid hormone synthesis.

RNA pull-down assay to identify Loc105611671 binding proteins
Long non-coding RNAs (lncRNAs) enact their biological roles through interactions with diverse biomolecules, and these interactions are often influenced by the RNA's subcellular localization.According to the iLoc-LncRNA database, 1  Loc105611671 is predominantly found in the cytoplasm, a finding 1 http://lin-group.cn/server/iLoc-LncRNA/home.phpfurther substantiated by nucleoplasmic separation experiments (Figures 3B,C).To explore the secondary structure of Loc105611671, we utilized the RNAfold tool, 2 which predicted Y-shaped structures in both minimally free energy (MFE) and centroid modes (Figure 3A).The cytoplasmic localization of Loc105611671, suggestive of its potential for protein interaction, prompted us to perform an RNA pull-down assay followed by mass spectrometry (Figure 3D), which identified 132 putative binding proteins (PBPs) (Figure 3E; Supplementary Table S2).
Further analysis of Loc105611671 PBPs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed enrichment in biological processes (BPs) such as metabolism, biological regulation, process regulation, and gene expression (Figure 4A).Molecular functions (MF) were predominantly associated with protein binding, nucleic acid binding, catalytic activity, RNA binding, and nucleotide binding (Figure 4B).Cellular components (CC) included intracellular organelles, the cytoplasm, and protein-containing complexes (Figure 4C).KEGG enrichment analysis identified 14 signaling pathways potentially linked to GC proliferation and hormone production, including PI3K-Akt signaling pathway, Wnt signaling pathway, GnRH signaling pathway, MAPK signaling pathway, oocyte meiosis and Cell cycle, etc. (Figure 4D).

Direct interaction between
Loc105611671 and CDC42 protein with positive regulatory effects KEGG analysis identified six proteins as candidate binding proteins, of which CDC42 was involved in signaling pathways significant in cell proliferation and hormone production (Supplementary Table S3).Subsequent functional queries of these RNA-binding proteins revealed that CDC42 participates in the regulation of cell proliferation, differentiation, and apoptosis.Therefore, CDC42, which showed the highest reliability, was selected for subsequent studies.Subsequently, we used the online tool IntaRNA 3 to predict a possible interaction between Loc105611671 and CDC42 3'UTR.This interaction was confirmed using a dual-luciferase reporter gene assay (Figure 5B).Nucleocytoplasmic separation of CDC42 demonstrated its presence in both the cytoplasm and nucleus, predominantly in the cytoplasm, providing additional support for the intracellular interaction with Loc105611671 (Figure 5A).
To investigate the promotion of GC proliferation and steroid hormone production through interaction with CDC42, we examined whether Loc105611671 would affect the expression of CDC42.Our findings revealed that overexpression of Loc105611671 significantly upregulated CDC42 protein and mRNA levels in GCs (Figures 5C,D).These outcomes indicate that Loc105611671 directly interacts with 3 http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jspCDC42, augmenting its expression, and suggesting that CDC42 is a downstream effector of Loc105611671.

Manipulation of CDC42 expression regulates GCs proliferation
CDC42 is highly expressed in the oocytes of activated follicles, and its overexpression promotes the growth of primordial follicles in mouse ovaries (28).We hypothesized that CDC42 would act as a crucial downstream regulator of Loc105611671 and play a role in the promotion of GC proliferation.To verify this hypothesis, we elevated CDC42 expression levels in GCs (Figures 6A-C).CCK-8 results showed that GCs proliferated at a faster rate after transfection with CDC42 than the controls did at 24 h (0.306 ± 0.038 vs. 0.388 ± 0.008, p < 0.05), (0.559 ± 0.044 vs. 0.683 ± 0.059, p < 0.05) 48 h, and 72 h (0.971 ± 0.101 vs. 1.458 ± 0.126, p < 0.01) (Figure 6D).In addition, CDC42 overexpression significantly increased the mRNA level of PCNA but had no significant effect on CDK1 expression, and the expression of the negative regulator of proliferation, P21, was significantly downregulated (Figure 6E).These results suggest that Loc105611671 plays a crucial role in follicular development by promoting GC proliferation.

Discussion
In this investigation, our findings indicate that Loc105611671 plays a crucial role in promoting the proliferation of sheep ovarian GCs and the biosynthesis of E 2 and P 4 .Loc105611671 enhances GC   GCs as the ovarian's primary functional units, are instrumental in follicular growth and maturation, offering nutritional support and secreting hormones imperative for follicular development (5, 7, 29).Notably, Loc105611671 was identified in the follicles of Qira Black sheep during estrus, implicating a regulatory effect on follicular development through GC modulation (27).Consequently, there is a compelling need for further research into Loc105611671 roles in GC proliferation and steroid hormone production.The functionality and mechanistic action of Loc105611671 in sheep GCs, however, have remained uncharted.Bridging this knowledge gap, we isolated GCs from ovine follicles to explore the impact of Loc105611671 overexpression on their proliferation and steroidogenesis.Functional assays revealed that elevated Loc105611671 levels not only stimulated GCs proliferation but also upregulated the cell cycle regulators CDK1 and PCNA and downregulated P21.Intriguingly, these effects attenuated after 72 h, potentially due to the dilution of exogenous genes amidst ongoing GCs division.Correspondingly, Loc105611671 overexpression significantly boosted E 2 and P 4 concentrations and the expression of STAR, Cyp11a1, and Cyp19a1, highlighting its role in promoting steroidogenesis.In conclusion, these findings suggest that Loc105611671 is an important regulator of follicular development.

Nucleoplasmic separation experiments have demonstrated that
Loc105611671 is enriched in the cytoplasm of GCs, suggesting its protein-binding capacity.This regulatory network of LncRNAs has been identified in the literature on GCs' functional mechanisms.For example, lncRNA ZNF674-AS1 affects GC proliferation by interacting with ALDOA (31).lnc-GULP1-2:1 was also found to bind directly to COL3A1 and affect GC proliferation by regulating COL3A1 expression and localization (32).In this study, we used RNA pull-down, mass spectrometry, and dual-luciferase gene assay experiments to further reveal that CDC42 directly binds to and interacts with Loc105611671 in GCs.To our knowledge, we are the first to report CDC42 acting as an lncRNA-binding protein in sheep GCs.However, understanding the detailed protein-binding motifs in Loc105611671 requires further characterization.
CDC42 is a member of the Rho GTPase family and is involved in various cellular functions and signaling pathways, including cell proliferation (33), apoptosis (34), and the cell cycle (35).In particular, CDC42 plays a crucial role in establishing mammalian oocyte polarity.For example, CDC42 deficiency disrupts oocyte maturation during development in vitro (36).The subcellular localization of CDC42 during primordial follicle activation is of interest.In dormant primordial follicles, CDC42 is specifically expressed in oocyte cytoplasm.When primordial follicles were activated, CDC42 expression on the oocyte membrane increased significantly (28).Furthermore, CDC42 interacts with epidermal growth factor EGF to improve primordial follicle activation in mice by regulating the PI3K signaling pathway (37).In this study, elevated levels of CDC42 were associated with enhanced cell proliferation, potentially contributing to improved GCs function.These findings are consistent with those in the literature on various animal species (33,38,39).As CDC42 can induce steroid hormone activity, an interactive relationship is implied between CDC42 expression and steroid hormone production (40,41).Therefore, in this study, we investigated steroid hormone synthesis.The upregulation of CDC42 resulted in a significant increase in E 2 and P 4 levels and STAR and Cyp19a1 expression, indicating that CDC42 promotes steroid hormone production in GCs.Subsequently, In vivo co-transfection experiments affirmed that Loc105611671 and CDC42 co-overexpression synergistically intensified GC proliferation and hormone secretion beyond their individual effects.
Unfortunately, although our findings have demonstrated that Loc105611671 plays a positive regulatory role in follicular granulosa cell proliferation through CDC42, the specific molecular mechanisms behind this phenomenon remain unknown.CDC42 is a crucial kinase within the MAPK signaling pathway and it influences a range of cellular behaviors.Therefore, our future research will concentrate on investigating how the Loc105611671-CDC42-regulated MAPK signaling pathway impacts follicular granulosa cells.

Conclusion
In conclusion, our study establishes that Loc105611671 enhances follicular granulosa cell proliferation and steroidogenesis through its interaction with CDC42.This novel mechanism of lncRNA-protein interaction deepens our understanding of the physiological roles played by lncRNAs in coordinating GCs function and the complex follicular maturation process.Moreover, these findings lay the groundwork for the development of innovative therapeutic strategies targeting reproductive diseases.

FIGURE 3 RNA
FIGURE 3 RNA pull-down captures Loc105611671 binding protein.(A) Secondary structure of Loc105611671 transcripts in MFE and Centroid modes predicted by the online tool RNAfold.(B) Loc105611671 subcellular localization in granule cells predicted by the online tool iLoc-LncRNA.(C) Subcellular localization of Loc105611671 (n = 3).(D) Silver-stained SDS-PAGE gels for proteins extracted from biotin-labeled Loc105611671 and antisense RNA, and two lanes for mass spectrometry.(E) Venn diagram of the results of mass spectrometry analysis.

FIGURE 4
FIGURE 4Bioinformatics analysis to identify proteins.GO analysis of Loc105611671 potential proteins (A-C).KEGG analysis of Loc105611671 potential binding proteins (D), The red pathway is more common in mammalian reproduction studies.

FIGURE 5
FIGURE 5 Loc105611671 interacts with CDC42 and positively regulates its protein and mRNA expression levels.(A) Subcellular localization of CDC42 was determined (n = 3).(B) The binding of Loc105611671 to the 3'UTR of CDC42 mRNA was demonstrated using the online IntaRNA tool and dual luciferase reproter gene assay (n = 3).(C,D) following Loc105611671 overexpression, significant increase in protein and mRNA expression levels of CDC42 was observed in GCs (n = 3).Data represent the mean ± SD. *p < 0.05, ***p < 0.001.

TABLE 1 PCR
Amplification primer information.