LncRNA HCP5 Promotes ADSC Adipogenic Differentiation via Its Sponging of miR-27a-3p and a Concomitant Increase in PPAR γ Expression

Current studies have shown that lncRNA plays an essential regulatory role in the physiological metabolism of cells. lncRNA HCP5 promotes proliferation and invasion in tumor cells and regulates differentiation in some stem cells. Our study was designed to determine the effects of HCP5 on the adipogenic differentiation of adipose-derived stem cell (ADSCs) and explore its underlying molecular mechanisms. ADSCs are known for their pluripotent differentiation potential and ready availability, making them promising intermediates for cell-based tissue damage repair. Our data revealed that HCP5 expression was elevated in differentiation-induced ADSCs, and that its overexpression promoted adipogenic differentiation in these cells. Then we predicted and then con�rmed the effects of targeted interaction between HCP5 and miR-27a-3p. We also predicted and con�rmed that miR-27a-3p demonstrated some binding a�nity for PPAR γ and then used this information to design a gain-and-loss-of-function experiment to verify that HCP5 regulates ADSCs adipogenic differentiation via its regulation of miR-27a-3p and PPAR γ . These experiments showed that HCP5 overexpression promotes adipogenic differentiation in ADSCs, which was impaired by the upregulation of miR-27a-3p. Overexpression of miR-27a-3p inhibited PPAR γ expression, and overexpression of HCP5 restored this inhibitory effect. Finally, transfection with PPAR γ siRNAs reduced the adipogenic promotion due to HCP5 overexpression in these cells. Taken together our data suggest that the HCP5/miR-27a-3p/PPAR γ axis may be a major regulator of adipogenic differentiation in ADSCs, which might be the probable molecular mechanism underlying the effects of HCP5 in these cells.


Introduction
Stem cells have the potential for pluripotent differentiation and have promising research prospects in tissue damage repair.This study aimed mainly to nd the mechanisms of stem cell differentiation at the molecular level.Mesenchymal stem cells (MSCs), pluripotent cells derived from the mesoderm, can differentiate into multiple tissues, including bone, cartilage, fat, muscle, and tendons, and have been increasingly mentioned in studies related to tissue repair.Experimental MSCs have been isolated from various sources, including umbilical cords, bone marrow, and adipose tissues [1].Of these, adiposederived stem cells (ADSCs), which are extracted from adipose tissues, are among the most commonly used because they are easily extracted with minimal invasion or tissue damage and can be obtained in reasonably large quantities.Animal models of ischemic disease have shown that ADSCs function to promote angiogenesis and reconstruction [2], and the transplantation of ADSCs has demonstrated excellent therapeutic outcomes when applied to studies treating both peripheral and central nerve injury models [3,4].ADSCs also play an essential role in repairing tissues such as skeletal and smooth muscle [5,6].There has been an explosion of interest in ADSC differentiation in the treatment of tissue damage, with several of these interventions even entering the clinical evaluation stage [7].However, the mechanisms underlying ADSC differentiation are poorly de ned.A study of the mechanism of ADSC adipogenic differentiation contributes to the better application of ADSCs in tissue repair .
Long non-coding RNAs (lncRNA) are a class of non-coding RNAs that are more than 200 nucleotides in length and are capable of participating in multiple cellular and physiological processes, often playing essential regulatory roles in many diseases [8].Cao et al. found that lncRNA RMRP promotes bladder cancer cells proliferation and invasion [9], whereas Liao et al. suggested that lncRNA is involved in the proliferation and differentiation of various in ammatory cells and the secretion of several in ammatory factors [10].lncRNA MEG3 regulates apoptosis of adipose-derived stem cells [11].Several reports also suggest that these lncRNAs are involved in the regulation of stem cell differentiation.MALAT1 promotes BMSC differentiation into endothelial cells [12], whereas PRNCR1 affects the osteogenic differentiation of MSCs by modulating CXCR4 expression [13].HCP5 (human leukocyte antigen (HLA) Complex P5) is also a type of lncRNA.Recent studies have focused on its regulation of cancer cells where it promotes proliferation and invasion [14][15][16][17], and HCP5 also regulates the differentiation of some cells.HCP5 has recently been reported to facilitate epithelial-mesenchymal transition in colorectal cancer [18] and was linked to osteoblastic differentiation of BMSCs, in a study evaluating osteoporosis [19,20].Chen et al.'s study on childhood obesity showed that HCP5 promotes adipogenic differentiation [21], but the molecular mechanisms underlying this regulation remain unknown.miRNAs are a class of endogenous single-stranded non-coding RNA molecules that are 20-22 nucleotides in length and known to regulate the expression of their target genes via their 3′ untranslated regions.miRNAs play a regulatory role in many cellular and physiological processes, such as in self-repair, signal transduction, and cell differentiation [22].In experiments using both human and mouse-derived BMSCs, miRNA-130a promoted osteogenic differentiation and inhibited adipogenic differentiation [23].In vitro experiments conducted by Chen et al. showed that mir-363 inhibits both mitosis and adipose differentiation in ADSCs [24], whereas Kim et al.'s in vivo studies identi ed miRNA-27a as a repressor of lipogenic differentiation [25].Guo et al. found that miRNA-27a-3p binds speci cally to PPARγ (peroxisome proliferator-activated receptor) and suppresses its expression [26], which is signi cant since PPARγ, a nuclear hormone receptor, acts as the major transcription factor for adipogenic differentiation [27].Although the targeted regulatory relationship between miRNA-27a-3p and PPARγ has been reported, the mechanism by which miRNA-27a-3p affects adipogenic differentiation in ADSCs and its regulation of PPARγ requires further veri cation.
The theory of ceRNA (competing endogenous RNAs)-mediated regulation, rst proposed by Salmena et al. in 2011, suggests that various RNA transcripts interact and effect a complex regulatory network in cells.This has been con rmed in several studies and has gradually become a research hotspot [28].lncRNAs can regulate the development and progression of multiple diseases when they act as ceRNAs [29], with many of these effectors acting as an miRNA sponge, allowing for complex dynamic regulation of the target gene [30].We speculated that there might be a ceRNA relationship between HCP5 and miRNA-27a-3p.In this study, we experimentally veri ed the regulatory relationship between HCP5, miRNA-27a-3p and PPARγ to further discuss the molecular mechanism of ADSCs adipogenic differentiation.

Methods And Materials
Cell culture and differentiation Human ADSCs were purchase from the Cell Bank (Shanghai Institutes, China) for Biological Sciences and were cultured in DMEM/F12 1:1 medium (Gibco; Thermo Fisher Scienti c, Inc.USA) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scienti c, Inc.USA).Cells were incubated in a humidi ed chamber with a temperature of 37 ℃ and a CO 2 concentration of 5%.Adipogenic differentiation was induced in the cells were induced by incubating them in DMEM/F12 medium supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 5 μg /mL insulin at 37 ℃ for 2 days.This medium was then replaced with DMEM/F12 supplemented with 10% FBS and 5 μg /mL insulin every two days.

Transfection
Human HCP5 sequence (GenBank, NR_040662.1)was synthesized and subcloned into a pcDNA3.1 vector (Genepharma, Shanghai, China) for transfection, with the empty vector acting as a vehicle control.
Interactions between HCP5 and PPARγ were evaluated using co-transfections of the HCP5 vector (or NC vector) and PPARγ siRNAs (or siRNA NC).All cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and all subsequent experiments were completed 48 h post-transfection.

Oil Red O staining
ADSCs were cultured in induction medium and then stained with oil-red O at 0, 2, 4, 6 and 8 days to evaluate adipogenic differentiation in response to changes in HCP5 expression.Next, we evaluated whether HCP5 regulates adipogenic differentiation in ADSCs via its interaction with miR-27a-3p.Therefore, we co-transfected ADSCs with pcDNA3.1-HCP5and miR-27a-3p mimics.We also cotransfected ADSCs with HCP5 shRNA and an miR-27a-3p inhibitor and then subjected the cells to 4 days of culture in the adipose induction medium.We then evaluated their adipogenic differentiation using oil red O staining.After removing the induction medium, the ADSCs were washed with PBS twice.They were xed with 4% paraformaldehyde for 30 min at 20 ℃, following which, the paraformaldehyde was removed by washing the cells twice in PBS.We then treated the cells with Oil Red O solution for 30 min and then washed the cells three times with PBS.After staining, plates were placed on a white background.Photos were taken to observe and compare the number of red-stained cells.

Luciferase reporter assay
We then validated these predictions using a dual-luciferase reporter assay using luciferase reporters with both the WT and mutant binding sequence from HCP5 and the WT and mutant binding sequences from PPARγ.A luciferase-based reporter assay was performed to evaluate the speci c interactions within our ceRNA network.We constructed two PGL3 (Promega, Madison, USA) reporter constructs.The wild-type (WT) HCP5 or the mutant (Mut) HCP5 sequence was cloned.We constructed a second similar set of reporters to evaluate miR-27a-3p binding of PPARγ, with the WT PPARγ or Mut PPARγ sequence inserted.
ADSCs were then cultured and transfected in a 96-well plate.Forty-eight hours after transfection, the cells were harvested, and the luciferase activity was measured using a Dual-Luciferase Reporter Assay kit (Promega, Madison, USA).These assays were performed in triplicate.

Statistical analysis
All continuous variables are described as the mean ± SEM, and all statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc.).Differences between groups were evaluated using an unpaired Student's t-test, and statistical signi cance was set at a P-value of < 0.05.All experiments were performed in triplicate.

HCP5 promotes adipogenic differentiation in ADSCs
On performing oil red O staining, we noted that increases in induction time led to increased oil red O staining in the treated adipocytes (Fig. 1A).Therefore, we examined the relationship between adipogenesis and HCP5 expression by quantifying HCP5 expression at each time point using qRT-PCR.The results of these assays showed that the expression levels of HCP5 gradually increased with increasing induction time (Fig. 1B).These results suggest that HCP5 expression may be linked to adipogenesis in these cells and support our hypothesis that this lncRNA plays an important role in the adipogenic differentiation of ADSCs.We then con rmed this by transfecting ADSCs with pcDNA3.1-HCP5and an empty vector control (NC) and comparing the oil red O staining from these two groups following 4 days of induction (Fig. 1C, D).We found more red-stained cells in the pcDNA3.1-HCP5group than in the NC group, suggesting that HCP5 plays a distinct role in promoting the adipogenic differentiation of ADSCs.

<Figure 1> Prediction and validation of the targeting effects of HCP5 and miR-27a-3p
We then explored the underlying mechanism of action for HCP5 in these cells.We predicted the targets of HCP5, which identi ed miR-27a-3p as an important potential target (Fig. 2A).In further predictions, we found that PPARγ is a potential target for miR-27a-3p (Fig. 2B), allowing us to begin to develop a potential regulatory axis.Dual-luciferase reporter assays using luciferase reporters showed that the uorescence activity of miR-27a-3p was signi cantly reduced in HCP5-Wt-transfected cells compared with that in HCP5-Mut cells (Fig. 2C, p<0.05) and that the uorescence activity of miR-27a-3p was signi cantly reduced in PPARγ-Wt compared to PPARγ-Mut (Fig. 2D, p<0.05).

<Figure 2>
We then con rmed that miR-27a-3p and PPARγ were involved in the adipogenic differentiation of ADSCs by examining their expression in the cells at days 0, 2, 4, 6 and 8 of induction.qRT-PCR showed that miR-27a-3p expression gradually decreased with increasing induction time (Fig. 2E) whereas western blotting showed that PPARγ expression gradually increased with increasing induction time (Fig. 2F).
We further con rmed whether the expression of HCP5 and PPARγ was associated with the expression of miR-27a-3p by transfecting a series of ADSCs with miR-27a-3p or NC mimics.qRT-PCR revealed that miR-27a-3p expression was signi cantly increased in ADSCs treated with the miR-27a-3p mimics compared to that in the NC mimic group (Fig. 2G, p<0.05).qRT-PCR also revealed that HCP5 expression was signi cantly lower in the miR-27a-3p mimic group compared to that in the NC group (Fig. 2H, p<0.05), con rming the interaction between these two transcripts.Western blotting revealed that PPARγ expression was signi cantly decreased in cells treated with the miR-27a-3p mimic compared to that in the NC group (Fig. 2I, p<0.05), con rming the interactions between miR-27a-3p and PPARγ.Taken together, these results suggest a direct regulatory interaction between HCP5, miR-27a-3p, and PPARγ in ADSCs.

<Figure 3>
HCP5 regulates adipogenic differentiation in ADSCs where it acts as a ceRNA Following the downregulation of HCP5, adipogenic differentiation in the HCP5 shRNA + inhibitor NC group was signi cantly decreased compared to that in the shRNA NC + inhibitor NC group.However, this decrease in adipogenic differentiation was weakened by the concomitant downregulation of miR-27a-3p (Fig. 4A).In contrast, upregulation of HCP5, as demonstrated by the pcDNA3.1-HCP5+ mimics NC group, promoted the adipogenic differentiation of cells compared to that in the pcDNA3.1 vector + mimic NC control.In contrast, upregulation of miR-27a-3p expression in the pcDNA3.1-HCP5+miR-27a-3pmimics group signi cantly impaired this effect compared with the pcDNA3.1-HCP5+ mimics NC group (Fig. 4B).

<Figure 4>
Then, we evaluated the competitive regulation of PPARγ effected by the interactions between HCP5 and miR-27a-3p by evaluating the changes in PPARγ expression in each group of cells using both westernblotting and immuno uorescence staining.The results revealed that PPARγ expression was signi cantly increased in the shRNA NC + miR-27a-3p inhibitor group compared to that in the shRNA NC + inhibitor NC group and in the HCP5 shRNA + inhibitor NC group compared to that in the shRNA NC + miR-27a-3p inhibitor group (Fig. 4C, D).In contrast, PPARγ expression was signi cantly reduced in the pcDNA3.1 vector + miR-27a-3p mimics group compared to that in the pcDNA3.1 vector + mimics NC group.However, the inhibitory effect of miR-27a-3p was signi cantly reduced following the upregulation of HCP5 as demonstrated by the comparison between the pcDNA3.1-HCP5+ miR-27a-3p mimics group and the pcDNA3.1 vector + miR-27a-3p mimics group (Fig. 4E, F).These results suggest that HCP5 acts as a ceRNA for miR-27a-3p and uses this mechanism to modulate PPARγ expression.
We then validated this regulatory network by co-transfecting ADSCs with pcDNA3.1-HCP5and PPARγ siRNA and then staining each group of cells with oil-red O to observe the extent of adipogenic differentiation.The number of adipogenic cells in the pcDNA3.1-HCP5+ PPARγ siRNA group was signi cantly lower than that in the pcDNA3.1-HCP5+ siRNA NC group (Fig. 4G, p<0.05), suggesting that PPARγ knockdown markedly reduced the HCP5 overexpression-induced promotion of adipogenic differentiation.These results support our hypothesis that HCP5 promotes ADSC adipogenic differentiation by promoting the expression of PPARγ.

Discussion
Several recent studies have shown that lncRNAs play various essential regulatory roles in many diseases and biological functions with many of the physiological activities of stem cells, including differentiation, regulated by these transcripts [31].Li et al. reported that MEG3 inhibits the adipogenic differentiation of ADSCs and promotes their osteogenic differentiation [32], while Liu et al. reported that TINCR regulation was critical to their adipogenic differentiation [33].HCP5 expression was upregulated in response to adipogenesis in ADSCs.Consequently we proposed that HCP5 is involved in the regulation of their adipogenic differentiation.We validated this hypothesis in an overexpression experiment, which showed that upregulation of HCP5 signi cantly increased adipogenic differentiation in the ADSCs.
We predicted that HCP5 could target miR-27a-3p, which is known to play a regulatory role in multiple physiological processes [34,35].miR-27a-3p promotes the osteogenic differentiation of MC3T3-E1 cells [36], and Shi et al. reported that miR-27a-3p is involved in adipogenic differentiation and obesity in humans [37].Our experiments revealed that adipogenesis decreased the expression of miR-27a-3p in ADSCs.The result suggested the involvement of miR-27a-3p in regulating the adipogenic differentiation of ADSCs and its downstream targets remained to be further explored.Our prediction experiments showed that miR-27a-3p targeted PPARγ, which is a signi cant regulator of adipogenesis [38,39] and whose levels were signi cantly increased in our adipogenic induction assay.Transfection with an miR-27a-3p mimic revealed that the expression of both HCP5 and PPARγ was inhibited by increased expression of this miRNA, con rming the likely interaction between these three effectors.
We then used overexpression and silencing assays to explore the regulatory effects of HCP5 on miR-27a-3p and PPARγ.Our data revealed that miR-27a-3p expression was signi cantly inhibited, and PPARγ expression was induced in response to increased HCP5 expression.However, HCP5 silencing induced a signi cant increase in miR-27a-3p expression and a signi cant decrease in PPARγ.This suggests that miR-27a-3p expression is negatively correlated with HCP5, while PPARγ expression is positively correlated with HCP5.We speculate that miR-27a-3p may be an intermediate mediator of HCP5 regulation of PPARγ.
Various recent studies have linked HCP5 to the regulation of various physiological activities such as proliferation, migration, invasion, and EMT.Moreover, reports suggest that this regulation is mediated by the fact that HCP5 acts as a gene sponge for effector miRNAs, thus regulating the expression of their downstream targets [40].We also found that silencing of HCP5 inhibited ADSCs adipogenic differentiation, while knockdown of miR-27a-3p restored this adipogenic effect.Moreover, we found that miR-27a-3p had an inhibitory effect on PPARγ expression, and that the overexpression of HCP5 restored its expression, while miR-27a-3p knockdown induced a marked increase in PPARγ.However, this upward trend was signi cantly reduced when HCP5 expression was downregulated.This result con rmed that the competitive binding of HCP5 to miR-27a-3p regulates ADSCs adipogenic differentiation likely via changes in its regulation of PPARγ.
To con rm this, we then co-transfected ADSCs with pcDNA3.1-HCP5(or pcDNA3.1 vector) and PPARγ siRNA (or siRNA NC), which revealed a clear promoting effect for HCP5 during adipogenesis that was signi cantly reduced in cells with reduced PPARγ.This result con rmed that HCP5 promotes adipogenesis by promoting the expression of PPARγ.
Taken together, our data con rm that HCP5 promotes adipogenic differentiation of ADSCs.Further investigations of its mechanism revealed that HCP5 acts as a ceRNA for miR-27a-3p, competitively inhibiting its expression and thereby regulating the expression of its downstream target gene, PPARγ.Stem cell differentiation relies on a series of complex regulatory networks, but our study is the rst to identify the HCP5/miR-27a-3p/PPARγ axis, which may be a critical mechanism in the regulation of adipogenic differentiation in ADSCs.Whether similar mechanisms exist in other stem cells remains to be (PPAR gamma).J Biol Chem.1995 Jun 2;270( 22 ):12953-6.40.Zou Y, Chen B. Long non-coding RNA HCP5 in cancer.Clin Chim Acta.2021 Jan;512:33-39.

Figures Figure 1
Figures