Repression of miR-764-5p by enhancing IGF1R and cochaperone CHIP expression in mesenchymal stem cells (MSCs) regulates cardioprotective effects in Aging-Spontaneously Hypertensive rats model

doi:10.21203/rs.3.rs-3605059/v1

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Repression of miR-764-5p by enhancing IGF1R and cochaperone CHIP expression in mesenchymal stem cells (MSCs) regulates cardioprotective effects in Aging-Spontaneously Hypertensive rats model

https://doi.org/10.21203/rs.3.rs-3605059/v1

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Age-associated cardiovascular disease (CVD) progression is marked by increased misfolded proteins and reduced growth factor receptor activity. Evidence links the co-chaperone CHIP and insulin-like growth factor-1 receptor (IGF1R) to stem cell dynamics and function through miR-764-5p in rat adipose-derived stem cells (rADSCs) remains largely unknown. We observed that short-term hypoxia (6 h) downregulated miR-764-5p in rADSCs, while normoxia conditions led to miR-764-5p upregulation, targeting the 3' UTR region of IGF1R and STUB1/CHIP. qRT-PCR confirmed altered mRNA expression. Overexpression of anti-miR-764-5p enhanced rADSC survival via CHIP and IGF1R upregulation, while miR-764-5p mimic increased ROS generation and apoptosis. HIF1α transcription factor downregulated miR-764-5p under short-term hypoxia. Administering rADSCs^{anti−miR−764−5p} in aging-spontaneously hypertensive rats (SHR) via tail-vein injection demonstrated cardioprotective effects, reducing cardiac hypertrophy, fibrosis, and apoptosis and it could be the potential to act as a regenerative medicine. In conclusion, suppressing miR-764-5p enhances IGF1R expression and CHIP activity in rADSCs, mitigating cardiac hypertrophy and remodeling in the aging-SHR model.

Heart failure is a major cause of mortality and morbidity worldwide. Various factors, such as high blood pressure, aging, diabetes, coronary heart diseases, and obesity, contribute to heart dysfunction. Pathological hypertensive conditions are induced by pressure overload resulting in cardiac remodeling further which leads to functional impairment of the heart ¹. Currently, age-associated hypertension is a major challenge in mortality, however, stem cell therapy is one of the possible approaches in biomedical research² and in cardiovascular diseases³. Mesenchymal stem cells (MSCs) have the potential property to differentiate into different cell types and have the self-renewal ability ⁴. These cells have been isolated from different tissue sources such as bone marrow, adipose tissue, brain, Wharton’s Jelly, and umbilical cords ⁵. The microenvironment helps to maintain the survival and proliferation of stem cells in which, hypoxia is one of the conditions that can regulate stem cell dynamics. In addition, short-term hypoxia (STH) has various beneficial effects on cell biogenesis including metabolism, proliferation, migration, differentiation, and apoptosis ⁶.

MicroRNAs (miRNAs) play a key role in epigenetic regulation ^7,8. The previous study indicates the involvement of these large number of small non-coding RNAs (miRNAs) regulates mesenchymal stem cell functions ⁹. miR-764-5p has been shown to inhibit the expression of co-chaperone carboxyl-terminus of HSP70 interacting protein (CHIP) encoded in STIP1 homology and U-box containing protein 1 (STUB1) gene ¹⁰ and this co-chaperone CHIP has maintained various beneficial effects on stem cell functions¹¹. Hypoxia-inducible factor (HIF)1α, is a transcription factor that regulates various protein activities, contains a DNA binding region for its transcriptional activity and an oxygen-dependent degradation (ODD) domain for its protein stability ¹². HIF is mostly regulated by low oxygen concentrations and majorly impacts stem cell biology by determining their niche ¹³. The low oxygen tension can help to induce proliferation which can increase the cell number. This property mostly depends upon the time of culture, oxygen tension, and hypoxia preconditions. Researchers have used a different range of oxygen tensions (1%, 3%, 5%, 10%, and 21%) whereas, few researchers have used a single defined oxygen tension ¹⁴. MSCs with high Insulin-like growth factor-1 receptor (IGF1R) expression display strong multipotent or pluripotent properties ¹⁵. IGF1R is a cell surface tyrosine kinase receptor that can bind to the cognate ligand IGF1 and IGF2 to activate its downstream pathway that promotes cell proliferation, migration, survival, and differentiation ¹⁶.

In this study, we used anti-miR-764-5p to nullify the effect of miR-764-5p mimic expression and upregulate CHIP activity and IGF1R stability in rADSCs under normoxia conditions to determine its effects on age-associated hypertensive conditions. Several studies have been reported that stem cell survival is reduced after transplantation into the appropriate host ^17,18. Hence, maintenance of its viability is an important focus in stem cell treatment. This study mainly focuses on anti-miR-764-5p in rADSCs for stem cell dynamics which can enhance the cardiac function in the aging-SHR rat model after the transplantation.

Adipose-derived stem cells (rADSCs) isolation and characterization

The rat adipose-derived stem cells (rADSCs) isolation and characterization were performed and described in our previous report ¹⁹. Briefly, the rADSCs cells were isolated from epididymal adipose tissue then minced and digested for 3 h at 37°C with collagenase type 2 (Col-II) (0.2% in PBS). After the digestion process, the solution was centrifuged, and the pellet was collected for cell culture. Further, cultured in DMEM low-glucose medium (Invitrogen, Carlsbad, CA) supplemented with 100 U/mL penicillin (Invitrogen), 10% FBS (Invitrogen), 100 mg/mL streptomycin (Invitrogen) and 2mM L-glutamine (Invitrogen) at 37°C with 5% CO₂.

Western Blot Analysis

The following method was carried out and reported in our previous report ²⁰. Briefly, the concentrations of the protein were determined using the Bradford method (Bio-Rad, USA). The proteins were separated by using 8–12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Belford, Massachusetts, USA). Membranes were kept in 5% blocking buffer and incubated with specific primary antibodies (listed in Supplementary Table S1) at 4°C overnight. Then, the protein signals were measured using HRP-conjugated secondary antibodies (Santa Cruz, CA, USA) and Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). The immunoblots were performed by Alphamager2200 digital imaging system (Digital Imaging System, Commerce, CA, USA).

RNA extraction and qRT PCR analysis

Total RNA from cell cultures and left ventricular heart tissue were extracted and purified by using the RNA isolation- Zymo Research Quick-RNA™ MiniPrep kit (Irvine, CA, USA) as per the manufacturer’s instructions. Further, the RNA purity was quantified by Nanodrop instrument (260/280 = 1.8). The microRNA expression profile underwent analysis using qRT-PCR on the CFX96™ Quantitative Real-Time system by Bio-Rad, following the protocol provided by the manufacturer of the Mir-X™ microRNA first-strand synthesis (cDNA) kit (TaKaRa, Ohtsu, Japan). MicroRNA forward primers was received from PROTECH, Taipei, Taiwan and complementary reverse primer strands were supplied within the Mir-X™ microRNA first-strand synthesis (cDNA) kit. The forward and reverse primers of interested genes (listed in Supplementary Table S2) and the qRT PCR experiment were performed using SYBR Green PCR Master Mix (Bio-Rad, CA, USA) as per the manufacturer’s instructions, employing a total reaction volume of 20 µL. The cycle number at which the reaction crossed the threshold cycle (Ct) was determined for each gene. The 2^ΔCt equation was employed to measure the relative amount of each gene to U6 rRNA, where ΔCt = (Ct _{interested gene} – Ct _U6). The relative quantification was done by normalization to U6. All reactions were done in triplicate.

Plasmids and construction of reporter plasmids

The CHIP wild-type plasmid was gifted by Dr. Jeng-Fan Lo in Yang-Ming Medical University, Taipei, Taiwan ²¹. The lentiviral shLV and shHIF1α plasmids were purchased from National RNAi Core (Academia Sinica, Taipei, Taiwan). JetPRIME Nanotechnology-based Transfection Reagent (JetPRIME, Strasbourg, France) was used for transfection for 24 h. The reporter assays for the 3′ UTR of the STUB1 and IGF1R plasmids (both wild type and mutant) were constructed using the annealing method with an insert (listed in Supplementary Table S2) containing the restriction site for XhoI and SalI sites of pmiRGLO vector, confirmed by colony PCR and restriction digestion. Purification of plasmid DNA was done by using the Qiagen-midi plasmid purification kit (Qiagen, Hilden, Germany) and further verified by sequencing and restriction digestion process.

Luciferase reporter assay

The following assay was performed and mentioned in our previous report ²². Briefly, the plasmid was constructed with the 3′ UTR region (listed in Supplementary Table S2) of the two genes, STUB1 and IGF1R, both wild and mutant types using the pmirGLO vector (Promega, Madison, WI, USA). Twelve well plates were used for the co-transfection of the pmiRGLO-3′ UTR followed by a scramble or miR-764-5p mimic. Luciferase activities were identified using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA). For promoter assay, the promoter-luciferase miR-764-5p construct, PGL4.1, was purchased from Promega (Madison, WI, USA). The shRNA or shHIF1α were transfected into rADSCs cells by using JetPRIME^® reagents (Polyplus-transfection, France) as per the manufacturer’s instructions after 24 h transfection. Relative luciferase activity was quantified after normalized with Renilla luciferase in rADSCs. The sample was analyzed and each experiment was done at least three times.

Flow cytometry analysis for apoptosis, cell cycle, and ROS detection

For the analysis of apoptosis, flow cytometry was performed by using the apoptosis detection kit (Annexin V-FITC; BD, Biosciences, USA) according to the manufacturer’s instructions. The cells were collected and double-stained with Annexin V-FITC and PI staining (BD, Biosciences, USA). Flow cytometry was carried out using the FACS Canto™ system (BD, Biosciences, USA) at the FACS Core Facility, China Medical University, Taiwan. Finally, cells were obtained and gated. For cell cycle analysis, the cells were exposed to two different conditions, normoxia or short-term hypoxia (1% O₂) for 6 h. After the treatments, the cells were collected by trypsinization and washed with 1X PBS. Next, the following cells were kept in ice-cold ethanol (70%) overnight at 4°C, followed by a wash with 1X PBS three times and then stained with PI (10 mg/mL) in PBS and RNase A (0.5 mg/mL) for 15 min at RT. Finally, the cells were again washed with PBS, and flow cytometry was performed by using FACS Canto™. The cells were gated with DNA content labeled cells. The cells for ROS analysis also underwent the same flow cytometry using DCFDA (Invitrogen, Thermo Fisher Scientific-US) staining. Cells were incubated with 10µM fluorescein, carboxy-H₂DCFDA (C400) for 30 min. Then analyzed by flow cytometry. All experimental cycles, n = 10,000.

Co-immunoprecipitation (Co-IP) assays

The Co-IP experiment was performed by using rADSCs cell lysates obtained with the protein G magnetic beads (Millipore) as per the manufactures’ instruction. After cells were harvested and lysed in 20 mM Tris-HCl (pH 7.5), 1% NP-40, 150 mM NaCl, and 1 mM EDTA. A total of 400 µg cell lysate was taken for quantification and incubated with anti-CHIP (sc-66830) overnight at 4°C. Further, the immunoprecipitated proteins were eluted from the beads at 95°C for 8 min and analyzed by SDS-PAGE. The following separated proteins were transferred onto a PVDF membrane and the result was observed after being probed with the specific antibodies.

Transwell migration assay

The migration assay was performed by using transwell cell culture chambers (8 µm pore size; SPLInsert™ hanging 24-well plate). For migration assay, 2 × 10⁴ cells were seeded in the upper chamber with a serum-free medium then exposed to normoxia or STH (6 h) conditions. In the lower chamber, the medium contains 10% FBS for chemoattractant property. After 24 h, the non-migrating cells were removed from the upper face by cotton swabs, and the migrating cells were stained with crystal violet staining with air-dried in the lower face and further cells were counted after images were taken by using 20x magnification by using Olympus microscope (Tokyo, Japan).

Cell viability assay

The following assay was performed and mentioned in our previous report²³. In brief, the colorimetric assay was done to analyze the viability of the cells on the mechanism based on the conversion of MTT reagent (3–4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) into the final product formazan to produce the blue color formation. After seeding the cells (1 × 10⁴ cells/well), the rADSCs were exposed to normoxia or STH (6 h) then incubated in DMEM with 10% FBS. Further, the MTT (0.5 mg/mL) reagent was added to the stem cells for 4 h and incubate at 37°C. Then cells were mixed well with DMSO and kept for 10 min. The cell viability was quantified at OD 550 nm wavelength by using an automated microplate reader (Bio-Tek, Winooski, VT, USA).

Docking studies

The docking study between the transcription factor HIF1α and the miR-764-5p promoter region was performed using GOLD (Genetic Optimization of Ligand Docking) software, based on genetic GA ²⁴ and the open-source transcription factor prediction tool (http://www.ifti.org/). The docking was performed using the best and most energetically favorable conformation of the HIF1α transcription factor protein database (PDB) structure (1H2K), which was chosen and downloaded from the PDB database (https://www.rcsb.org/). The docking study score was calculated by using this formula:

(GoldScore = S (hbext) + S (vdwext) + S (hb int) + S (vdw int).

Immunofluorescence staining

The following methods were described in our earlier report ²⁵. Briefly, cell fixation was done by using 4% paraformaldehyde for 15 min at RT and permeabilization by using 0.1% Triton X-100 for 15 min at RT before staining with a specific antibody. Next, the seeded equal number of cells (1 × 10⁵ cells/well) were thoroughly washed with 1X PBS three times and incubated with the secondary antibody, Alexa Fluro 488 goat anti-rabbit IgG secondary antibodies, or Alexa Fluro 594 goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Then images were captured using fluorescent microscopy (Olympus, Tokyo, Japan).

Animal experiments and design

The eight weeks of normotensive WKY (Wistar Kyoto rats) and SHR (Spontaneously Hypertensive rats) were purchased from BioLasco Taiwan Co., Ltd, Taipei, Taiwan. All animal protocols were approved by the Animal Care and Use Committee of Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (108-IACUC-71), Taiwan. The animals were housed in an animal care unit at 24 ± 2°C and regularly provided with food and water. After 9 months, the aging rats (n = 7) were grouped into five categories: (A) WKY, (B) SHR, (C) SHR tail-vein administered with rADSCs that transfected with constructed plasmid of scramble (SHR-rADSCs^scramble), (D) miR-764-5p mimic (SHR-rADSCs^{mir−764−5p−mimic}), or (E) antagomir (SHR-rADSCs^{anti−mir−764−5p}) for 24 h. The 4 weeks of consecutive treatment with the cells (1 x 10⁷ cells/rat/week) through intravenous tail-vein injection, were followed by echocardiography and sacrifice by terminal anesthesia. The hearts were then collected and stored at -80°C.

Immunohistochemistry

The heart tissue sections were dried overnight at 58°C and deparaffinized by using xylene followed by a hydration method in a series of ethanol. The activity of endogenous peroxidase was stopped by using H₂O₂ (3%) for 10 min followed by a rinse with ddH₂O for 15 min, then the tissue sections were kept with citrate buffer in the microwave for 15 min and further cooled to RT for 30 min. After that, the slides were incubated by using a blocking buffer (i.e. 2.5% horse serum) for 15 min. Next, a specific primary antibody anti-BNP (Bioss, bs-2207R) at a 1:100 dilution ratio was added for 2h. The slides were washed thoroughly with 1X PBS and further incubated with the secondary antibody for 30 min at RT. After this step, the tissue slides were again washed with 1X PBS and treated with streptavidin-HRP conjugate for 30 min. Subsequently, the processed slides were washed with 1X PBS three times and chromogenic substrate DAB (diaminobenzidine) was added and followed by a 1 to 3 min incubation at RT. Finally, the slides were washed three times in PBS, air-dried, and mounted with VectaMount (H-5000). The images were taken using a microscope (Olympus, Tokyo, Japan).

Heart tissue for TUNEL assay

The following method is described in the previous reports ²⁰. Briefly, the TUNEL + cells were identified by using TUNEL reagent (Roche Molecular Biochemical, Mannheim, Germany) detected by brilliant green color by using wavelength range 515–565 nm, and cell nuclei were counterstained with DAPI further identified by blue color at 454 nm. Finally, the images were captured by using an Olympus DP 74 fluorescent microscope (Tokyo, Japan).

Statistical Analysis

The evaluation of statistics was quantified by mean ± standard error of the mean (SEM). The P-value of p < 0.05 was considered statistically significant. Statistical data was performed in repeated independent experiments. Data processed by using Graph Pad Prism5 statistical software (San Diego, CA, USA) with unpaired student’s t-test for two groups and one-way ANOVA for multiple comparisons followed by post hoc Tukey’s Honestly Significant Difference tests.

miR-764-5p downregulation under STH (6 h) conditions in rADSCs

MicroRNAs suppress the target gene expression by targeting their 3′ untranslated region (UTR)²⁶ and here, we investigated the role of miR-764-5p and its target proteins expression in rADSCs. The microarray analysis indicated that the higher expression of miR-764-5p under normoxia (red box) and lower in expression under STH (6 h) (green box) conditions (Fig. 1a). The qRT-PCR analysis indicated the significant (**p < 0.01) upregulation of miR-764-5p under normoxia conditions compared to STH (6 h) conditions in rADSCs (Fig. 1b) which were consistent with the microarray analysis. Furthermore, to identify the target gene of miR-764-5p, we initially used the open-source bioinformatics prediction websites ((miRbase (http://www.mirbase.org/), TargetScan (http://www.targetscan.org/vert_71/), and miRWalk (http://mirwalk.umm.uni-heidelberg.de/)) for primary screening, the results confirmed that the 3′ UTR region of STUB1 and IGF1R were targeted by miR-764-5p, as represented in the schematic diagram (Fig. 1c; Supplementary Fig. 1a). Next, to investigate the expression analysis of miR-764-5p in a dose-dependent manner in rADSCs. The data revealed that a concentration of 40 nM of miR-764-5p mimic was expressed significantly (***p < 0.001) by qRT-PCR analysis (Fig. 1d). The illustrated image indicated the miR-764-5p and its 3′ UTR region of the two target genes STUB1 and IGF1R and their mutant forms (Fig. 1e). The reporter assay was significantly (*p < 0.05) suppressed by the presence of the 3′ UTR of both the STUB1 and IGF1R genes, however, the activity was reversed when the 3′ UTR was mutated (Fig. 1f), suggesting that the 3′ UTR of STUB1 and IGF1R was inhibited by endogenous miR-764-5p. Collectively, these results indicate that under normoxia conditions miR-764-5p upregulates and inhibits the expression of STUB1 and IGF1R in rADSCs.

Beneficial effects of STH (6 h) on adipose-derived stem cells (rADSCs)

Hypoxia is involved in the proliferation of MSCs ²⁷. Next, we sought to investigate whether short-term hypoxia (STH) is better for stem cell survival and proliferation than normoxia conditions. In addition, the minimum oxygen concentration (1%) was used to mimic the microenvironment that may regulate stem cell biology. The cell cycle analysis showed the synthesis phase (S-phase), which is responsible for DNA replication, was significantly (*p < 0.05) increased under hypoxia (6 h) compared to normoxia conditions (Fig. 2a). Our data revealed that the 6 h of STH had a more significant effect than normoxia by the upregulation of HIF1α, VEGF, and bcl2 protein expressions and CHIP, pIGF1R, and pAKT protein expressions (Fig. 2b, c). Moreover, an increase in S-phase significantly acts to upregulate the migration rate (*p < 0.05 vs normoxia) by lowering the cell death (**p < 0.01 vs normoxia) (Fig. 2d; Fig. 2e, Supplementary Fig. 1b). Furthermore, the immunoblot results consistent with qRT-PCR analysis by upregulation of HIF1α, IGF1R, and STUB1 mRNA expression under STH (6 h) conditions than other hypoxia time periods and normoxia conditions (Fig. 2f). The red color deposition of lipid droplets during STH (6 h) indicated a decrease in lipid formation and adipogenesis compared to the normoxia and long-term hypoxia (24 h) by retaining their stemness (Supplementary Fig. 1c). Next, the characterization of stem cell surface markers such as CD90 positive marker and CD45 negative marker indicated the confirmation of stemness by flow cytometry analysis in rADSCs (Supplementary Fig. 1d) and also additional stem cell surface markers characterized in our previous report ²⁸,²³. Taken together, short-term hypoxia induces migration rate, cell viability with higher S-phase and target proteins expression compared to normoxia and long-term hypoxia conditions in rADSCs.

Effects of miR-764-5p mimic on IGF1R and co-chaperone CHIP expressions

Importantly, the immunoblot results indicated that the miR-764-5p mimic at 40 nM concentration inhibited the expression significantly of IGF1R (**p < 0.01), CHIP (**p < 0.01) and also reduced the expression of their associated proteins such as pIGF1R, pAKT, pERK1/2, HSP70, and HSP90 levels remarkably. However, the expression was reversed by the miR-764-5p antagomir treatment (Fig. 3a-c). The schematic diagram represents the STUB1 gene encoding the CHIP co-chaperone and its important domains for their different functions (Fig. 3d). The immunoblot results which was concordance with qRT-PCR results (Fig. 3e, f). Co-chaperone CHIP can stabilize and nuclear translocate the HSF1 after the interaction in H9c2 cells ²¹. We further investigated whether a similar mechanism takes place in rADSCs. Interestingly, the data showed a stronger interaction between CHIP and HSF1 during CHIP wild-type (CHIP^WT) overexpression, however, miR-764-5p transfection decreased the interaction between CHIP and HSF1 with reduced the stabilization of HSF1 confirmed by co-IP analysis (Fig. 3g), indicating that the CHIP-HSF1 complex formation is similar in both H9c2 cells and rADSCs, respectively. As depletion of CHIP can no longer stabilize IGF1R ²⁹, its overexpression rescued the effect of mir-764-5p and induced stabilization that leads to enhance the cell survival. Intriguingly, the data revealed that the expression of IGF1R (**p < 0.01) was enhanced with mimic transfection followed by CHIP^WT plasmid overexpression whereas the anti-mir-764-5p treatment also exhibited remarkably similar effects, however, the mimic inhibited both IGF1R and CHIP expressions by immunoblot analysis (Fig. 3h). This data confirms that miR-764-5p inhibits IGF1R and CHIP under normoxia conditions whereas anti-miR-764-5p and CHIP^WT plasmid overexpression rescue the adverse effects that enhances the target protein expression in rADSCs.

Knockdown of HIF1α regulates miR-764-5p expression under STH (6 h) conditions

Hypoxia Inducible Factor (HIF)-1α, which has a key role for stem cell survival under low oxygen concentrations ³⁰. As HIF1α is a transcription factor, we knocked down it under STH conditions and checked the miR-764-5p expression in rADSCs. Surprisingly, we found that the knockdown of HIF1α in a dose-dependent manner significantly (**p < 0.01) enhanced the expression of miR-764-5p under STH (6 h) conditions (Fig. 4a). Further, the immunoblot confirmed that the HIF1α expression was downregulated in the siRNA transfected cells in a dose-dependent manner (Fig. 4b). Based on the siHIF1α results, we sought to investigate the knockdown of HIF1α by shRNA. As expected, similar results were found. Immunoblot confirmed that the HIF1α expression was significantly suppressed (**p < 0.01) (Fig. 4c). Inhibited HIF1α by shRNA transfection showed a significant enhancement (**p < 0.01) of miR-764-5p expression and suppressed (*p < 0.05) the target genes STUB1 and IGF1R in rADSCs (Fig. 4d). The open-source transcription factor binding analysis further confirmed that HIF1α can bind to premature miR-764-5p promoter region, and this was consistent with our in-silico analysis using PDB structures (Supplementary Fig. 2a, b). Furthermore, transcriptional factor, HIF1α knockdown upregulated the promoter activity significantly in a dose-dependent manner (*p < 0.05) after co-transfected with miR-764-5p promoter plasmid in STH (6 h) conditions (Supplementary Fig. 2c). In addition, the co-culture analysis indicated a significant increase in cell size (**p < 0.01) with Ang II in H9c2 cells regulated by the rADSCs transfected with the miR-764-5p mimic as well as the scramble. However, the size of the H9c2 was significantly reduced (**p < 0.01) by the anti-miR-764-5p transfection was confirmed by the rhodamine-phalloidin staining (Supplementary Fig. S1e). Thus, the result confirms that the key regulator of hypoxia, i.e. the HIF1α transcription factor, plays a major role in the regulation of miR-764-5p, which further regulates the target genes.

Effects of miR-764-5p on ubiquitination and reactive oxygen species (ROS) generation

miR-764-5p inhibited the mRNA expression of IGF1R and CHIP, both are important for cell survival and inhibit apoptosis, however, using anti-mir-764-5p significantly decreased apoptosis rate (*p < 0.05) and increased the survival rate of rADSCs. Intriguingly, CHIP^WT plasmid also reduced the apoptosis rate significantly (**p < 0.01) after co-transfected with mimic (Fig. 5a, b). ROS is a key component that regulates oxidative damage in the cell³¹. miR-764-5p targets CHIP, which is an E3 ubiquitin ligase, the inhibition of these proteins can induce the accumulation of damaged and unfolded proteins leading to the generation of ROS inside the cell. The attenuation of the CHIP co-chaperone by miR-764-5p caused ROS generation elevation in comparison to scramble, however, the inhibitor (*p < 0.05) and CHIP^WT (**p < 0.01) significantly reduced this effect by miR-764-5p in rADSCs (Fig. 5c, d). Inhibition and mutation of CHIP can cause various problems with compounding misfolded protein inside the cells. The overexpression of CHIP^WT enhanced the ubiquitination in comparison to miR-764-5p challenge and CHIP^WT with miR-764-5p mimic, which nullified the effects of miRNA and induced the proteostasis effects (Fig. 5e, f). These results confirm that the inhibition of CHIP and IGF1R by the miR-764-5p mimic induces ROS generation and apoptosis in rADSCs.

Effects of rADSCs overexpressed with antagomir on cardiac function in aging-SHR model

To investigate the treatment of MSCs on cardiac hypertrophy in hypertensive rats, the enlarged heart size in the SHR and the SHR administered with rADSCs^{mir−764−5p} rats were determined (Fig. 6a). Importantly, the upper panel indicated the reduced intercalating space and lower panel with blue color formation indicated the less collagen deposition and fibrosis formation in WKY and the SHR-rADSCs^{anti−mir−764−5p} groups (Fig. 6b) and hypertrophic marker BNP expression (Supplementary Fig. 2d). As shown the Table 1, LVIDd and LVIDs, heart weight and WHW/Tibia length ratio of the SHR and SHR-rADSCs^{mir−764−5p} groups were significantly higher whereas the SHR-rADSCs^{anti−mir−764−5p} group exhibited a significantly reversed effect which was concordance with (Fig. 6c). Interestingly, the SHR-rADSCs^scarmble injected heart sizes that showed a reduction in hypertrophy due to stem cells alone effects but not consistent, whereas the SHR-rADSCs^{anti−mir−764−5p} injected rat’s heart sizes were consistently reduced (Fig. 6d). We observed a remarkable increase in the expression of IGF1R, pIGF1R, HSF1 and also decreased expression of hypertrophic markers (Fig. 6e). Further, the mRNA expression of IGF1R and p53 in heart tissue significantly increased in the same groups, respectively (Fig. 6f, g) and with lower in apoptosis rate in WKY and the SHR-rADSCs^{anti−mir−764−5p} group (Fig. 6h; Supplementary Fig. 2e). In addition, there was a significant (*p < 0.05) increase in the LV in the EF (%) and FS (%) in SHR-rADSCs^{anti−mir−764−5p} whereas the normotensive WKY rats showed a greater significant (**p < 0.01) effects as compared to other groups (Fig. 6i, j). Thus, the data confirms that SHR-rADSCs^{anti−mir−764−5p} transplantation may play a key role in attenuation of hypertrophy, fibrosis, apoptosis, and augmentation of cardiac function in the aging-SHR model.

Table 1

Echocardiography and cardiac morphological assessments of cardiac function.
	WKY (n = 7)	SHR groups (n = 7)
				rADSCs
			Scramble	miR-764-5p mimic	Anti-miR-764-5p
LVIDd(mm) LVIDs (mm) Body weight (g)	6.57 ± 0.31 3 ± 0.33 383.62 ± 9.97	8.2 ± 0.18^## 5.32 ± 0.21^## 376.44 ± 7.5	7.07 ± 0.35 4.3 ± 0.33 379.17 ± 5.62	7.5 ± 0.2^## 4.75 ± 0.16^## 374.7 ± 5.89	6.87 ± 0.25* 3.7 ± 0.21* 380.47 ± 5.39
Tibia length (mm)	41.06 ± 0.29	39.15 ± 0.30	40.64 ± 0.38	40.23 ± 0.27	40.83 ± 0.26
WHW (mg) LVHW (mg) WHW/Tibia length(mg/mm)	1245.28 ± 23.58 878.34 ± 24.96 29.69 ± 0.50	1353.14 ± 25.40^# 1035.4 ± 23.51^## 33.96 ± 0.92^#	1317.71 ± 22.85 989.2 ± 19.95^# 32.58 ± 0.80	1352.2 ± 16.42^# 1003.31 ± 17.24^## 33.21 ± 0.82^#	1266.37 ± 18.88 917.06 ± 18.32 31.32 ± 0.76
Notes: Left ventricular internal diameter end diastole (LVIDd); Left ventricular internal diameter end systole (LVIDs); Whole heart weight (WHW); Left ventricular heart weight (LVHW). Values are presented as mean ± SEM (n = 7). ^#p < 0.05, ^##p < 0.01, compared to WKY group; *p < 0.05 compared to SHR group.

In this study, MSCs transplantation in the SHR model revealed a protective role of rADSCs^{anti−mir−764−5p} in regulating cardiac hypertrophy and heart failure. Anti-miR-764-5p suppressed the miR-764-5p mimic in rADSCs by accelerating IGF1R and STUB1 gene expression. Moreover, cardiac remodeling and hypertrophy indicated the reversal effects by overexpressed rADSCs with antagomir when transplanted in the aging-SHR rats. The previous study has demonstrated that MSCs transplantation can be a successful therapy for heart diseases ^32,33. The role of IGF1R is well known in stem cell survival mechanisms. In addition, STUB1 also plays an important role in the ubiquitination of misfolded and damaged proteins in stem cell dynamics. In this study, we found that miR-764-5p can inhibit the expression of IGF1R and STUB1 to bind its 3′ UTR region. Here, the data indicated that the miR-764-5p inhibited the expression of CHIP encoded STUB1 and IGF1R mRNA and protein expressions. Earlier reports on STH (6 h) induced the expression of different survival and hypoxia-related markers by immunoblot analysis³⁴. In concordance with the earlier report, we found the enhanced mRNA expression of IGF1R, HIF1α, and STUB1 marker by qRT PCR analysis. Next, the co-chaperone CHIP also regulates HSF1 by maintaining its stability and facilitating nuclear translocation in H9c2 cells, which could protect the cells from apoptosis ²¹. However, the role of the CHIP-HSF1 complex is unclear in stem cells. We found that the miR-764-5p downregulated CHIP expression and its binding affinity towards HSF1. However, the overexpression of CHIP^WT upregulated the binding affinity in rADSCs.

We also found that miR-764-5p was downregulated under STH conditions. Hypoxia is a major regulator of cell proliferation in stem cell biology ³⁵. Short-term hypoxia has a beneficial role in inducing the proliferation of stem cells ⁶. However, STH conditions and inhibition of this miR-764-5p, can rescue the mimic effect and induce cell survival with a high growth rate in rADSCs. We confirmed that miRNA can inhibit CHIP and IGF1R, which reduced cell survival and induced apoptosis by ROS generation under normoxia conditions. Therefore, this study is based on the therapeutic role of MSCs and antagomir-based stem cell therapy in the treatment of hypertensive conditions. In STH condition, the knockdown of HIF1α by using shHIF1α upregulated the miR-764-5p expression and its binding affinity to its promoter region further confirms that the transcription factor HIF1α plays a key role in regulating miR-764-5p expression. The MSCs have the property to secrete soluble growth factors and this ligand interacts with the receptor and induces its downstream signaling pathways regulating hypertrophic effects ³⁶. Consistent with these findings, the co-culture analysis of the stem cells and H9c2 showed a decrease in hypertrophic effects with the antagomir transfected with stem cells against Ang II challenged in H9c2 cells. However, the mimic and scramble did not exhibit such beneficial effects. Taken together the HIF1α plays a major role in regulating miR-764-5p mimic during STH conditions (Fig. 7) whereas in normoxia conditions antagomir inhibits the mimic and reduces the apoptosis by enhancing the cell survival in rADSCs.

Co-chaperone CHIP has been identified as an E3 ligase enzyme that responsible for proteasomal degradation of accumulated misfolded proteins by its E3 ligase activity ³⁷. In addition, its overexpression reduced ROS generation and oxidative stress. Although miR-764-5p inhibits the expression of CHIP, the CHIP co-chaperone overexpression enhanced the beneficial effects by regulating IGF1R stability and its expression by nullifying the mimic activity in rADSCs. Taken together, the antagomir and CHIP overexpression modulated the beneficial effect in stem cells by attenuating the miR-764-5p mimic effects in in vitro studies.

Our data clearly showed that the overexpressed stem cells with anti-miR-764-5p accelerate significantly cardiac function by decrease cardiac hypertrophy in the aging-SHR model. However, the miR-764-5p mimic-overexpressed stem cells did not exhibit such effects. The overexpression of antagomir in stem cells in the SHR rats maintained the CHIP co-chaperone and its IGF1R expression by inhibiting the mimic expression. Maintenance of stem cell microenvironments can promote the secretion of various growth factors that influence cardiac function in hypertensive conditions³⁸. In contrast, stem cell transplantation has a major challenge in maintaining viability after transplantation³⁹, however, the antagomir transfection in stem cells may enhance the viability that could be used to treat cardiac hypertrophic disorders in future aspects. Here, the stem cells with antagomir were rescued from the cardiac hypertrophic effects through inhibiting the miR-764-5p mimic in the SHR model. The overall results indicate that anti-miR-764-5p plays a major role in stem cell transplantation and its effects on the aging-SHR rats model modulate cardiac hypertrophy and heart failure. In conclusion, aging-SHR treated with rADSCs^{anti−mir−764−5p} accelerates their cardiac function in our in vivo study, by the inhibition of miR-764-5p in stem cells could be a therapeutic approach against cardiac hypertrophy. It can be a potential regenerative medicine for cardioprotective mechanisms in stem cell therapy.

Acknowledgments

This study was funded by China Medical University, and Asia University grants (CMU102-AISA-04-08), and Hualien Tzu Chi Hospital grant (IMAR110-01-13), Taiwan.

Conflicts of interest

The authors declare that they have no conflict of interest.

Author contributions

PB and CYH conceptualized and designed the study. WWK and CHK collected and assembled the data. DYH, CHD, and JD provided materials for the study. PB, MYC, and VP analyzed and interpreted the data. PB and MAS wrote the draft of the manuscript. WWK, MAS, and CYH reviewed and gave the final approval of the manuscript. WWK provided administrative support, CYH provided financial support.

Data availability statement

The data that supports the finding of this study are available from the corresponding author upon reasonable request.

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Repression of miR-764-5p by enhancing IGF1R and cochaperone CHIP expression in mesenchymal stem cells (MSCs) regulates cardioprotective effects in Aging-Spontaneously Hypertensive rats model

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Adipose-derived stem cells (rADSCs) isolation and characterization

Western Blot Analysis

RNA extraction and qRT PCR analysis

Plasmids and construction of reporter plasmids

Luciferase reporter assay

Flow cytometry analysis for apoptosis, cell cycle, and ROS detection

Co-immunoprecipitation (Co-IP) assays

Transwell migration assay

Cell viability assay

Docking studies

Immunofluorescence staining

Animal experiments and design

Immunohistochemistry

Heart tissue for TUNEL assay

Statistical Analysis

RESULTS

miR-764-5p downregulation under STH (6 h) conditions in rADSCs

Beneficial effects of STH (6 h) on adipose-derived stem cells (rADSCs)

Effects of miR-764-5p mimic on IGF1R and co-chaperone CHIP expressions

Knockdown of HIF1α regulates miR-764-5p expression under STH (6 h) conditions

Effects of miR-764-5p on ubiquitination and reactive oxygen species (ROS) generation

Effects of rADSCs overexpressed with antagomir on cardiac function in aging-SHR model

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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