miR-363 Alleviates Detrusor Fibrosis via the TGF-β1/Smad Signaling Pathway by Targeting Col1a2 in Rat Models of STZ-Induced T2DM.

Dysregulated expression of microRNAs (miRNAs or miRs) has been implicated in the pathophysiology of type 2 diabetes mellitus (T2DM). However, their underlying role in the complication of detrusor fibrosis remains poorly understood. Therefore, this study aimed to examine the potential functional relevance of miR-363 in detrusor fibrosis of rats with streptozotocin (STZ)-induced T2DM through the predicted target gene collagen type I alpha 2 (Col1a2). Immunohistochemical analysis found an increase in the positive expression of collagen type III alpha 1 (Col3a1) and Col1a2 in detrusor tissues, where miR-363 expression was decreased. Next, gain- and loss-of-function experiments were performed to clarify the effects of miR-363 and Col1a2 on the activities of bladder detrusor cells. Of note, binding affinity between miR-363 and Col1a2 was verified by a dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Upregulated miR-363 inhibited Col1a2 expression, which led to increased expression of B-cell lymphoma 2 (Bcl-2) and Smad7 and accelerated cell viability, along with decreases in cell apoptosis and Col3a1, Bcl-2-associated X protein (Bax), transforming growth factor (TGF)-β1, and Smad4 expressions. In conclusion, miR-363 upregulation reduces detrusor fibrosis in rats with STZ-induced T2DM through suppression of the TGF-β1/Smad signaling pathway by targeting Col1a2. Therefore, our study provided further insights for the development of new therapeutic targets for T2DM.

Introduction

Diabetes mellitus is a common metabolic disorder with a high glycemic phenotype, with two main types, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), with T2DM accounting for more than 90% of patients with diabetes. T2DM develops secondary to complicated interactions between lifestyle and genetic modifications, with some of the risk factors including easily measurable phenotypic features such as obesity, insulin resistance and secretion dysfunction, increased glucagon secretion, and impaired glucose renal reabsorption. Urinary bladder dysfunction is one of the complications of diabetes caused by alterations in the detrusor smooth muscle. A previous study provided evidence that diabetes attenuates urothelial modulation of detrusor contractility and spontaneous activity. In this study, T2DM was induced by streptozotocin (STZ) in rats, which is a cytotoxic drug that damages nuclear DNA as well as cell membranes and destroys β-pancreatic cells, which produce insulin. The key involvement of microRNAs (miRNAs or miRs) in diabetes and its complications, which is by regulating multiple biological pathways, has been highlighted in several studies.6, 7, 8

miRNAs are small, noncoding RNA molecules that regulate a variety of biological processes by binding to the 3′ untranslated regions (UTRs) of protein-coding messenger RNAs (mRNAs). Accumulating evidence has demonstrated the functionality of miRNAs in various biological processes, including cell cycle, cell proliferation, differentiation, and apoptosis., Previous experiments have implicated noncoding RNA molecules, including miRNAs, as important biomarkers in the regulation of hyperglycemia, and that they are associated with diabetic nephropathy. miR-363 expression has been recently reported to be dysregulated in cancers, indicating its potential involvement in oncogenesis and resistance to medicines; miR-363 promotes cisplatin-induced apoptosis by targeting Mcl-1 in breast cancer. miR-363 is also involved in type 1 diabetes and therefore can be used as a candidate biomarker for diabetes. miR-363 has been reported as a potent promoter of the epithelial-mesenchymal transition (EMT), a pathological process that is involved in a variety of diseases, including cancer and fibrosis, partially through transforming growth factor β (TGF-β) downregulation. TGF-β is a multifunctional polypeptide growth factor that regulates cell growth, proliferation, and migration and is involved in collagen formation in diabetic nephropathy and glomerulosclerosis. TGF-β can activate the collagen type I alpha 2 (Col1a2) gene, indicating the possibility of co-regulation. Smad7 inhibits Col1a2 expression, whereas Smad3 upregulates it, both of which occur through the Smad signaling pathway. Therefore, based on the aforementioned data, we conducted this study in order to investigate the potential roles of miR-363 in detrusor fibrosis of rats with STZ-induced T2DM, along with the involvement of Col1a2 and the TGF-β1/Smad signaling pathway.

Results

Successful T2DM Model Establishment

After model establishment, the successful model establishment rate was 83.33%, and the modeling indices are shown in Table 1. Compared with normal rats, rats with STZ-induced T2DM had lower body weight (Figure 1A), increased urinary volume and fasting blood glucose (Figure 1B), and increased insulin plasma levels (p < 0.05) (Figure 1C), with glucose occurring in the urine. These findings were indicative of successful establishment of the rat model.

Table 1

Comparisons of Rats in the Normal and T2DM Groups after Model Establishment

Group	n	Body Weight (g)	Length (cm)	Fasting Blood Glucose (mmol/L)	Insulin Plasma Level (mmol/L)	Urinary Volume (mL)	Urinary Glucose
Normal group	20	231.90 ± 10.15	18.15 ± 1.20	5.13 ± 0.56	15.85 ± 2.98	32.18 ± 10.43	0
T2DM group	20	203.50 ± 9.40∗	17.50 ± 1.30	17.80 ± 2.23∗	24.87 ± 3.82∗	73.54 ± 16.28∗	10.32 ± 2.01∗

T2DM, type 2 diabetes mellitus. ∗p < 0.05 versus the normal group.

Figure 1

Successful T2DM Model Establishment

(A) Body weight of rats in the normal and T2DM groups. (B) Blood glucose of rats in the normal and T2DM groups. (C) Insulin plasma levels over time in rats in the normal and T2DM groups. (D) Bladder pressure over time in rats in the normal and T2DM groups. ∗p < 0.05 versus the normal group.

Rats with STZ-Induced T2DM Showed Enhanced Maximum Bladder Capacity, Static Bladder Pressure, and Compliance

The urodynamic indicators of rats in the normal and T2DM groups after 3 weeks of modeling are shown in Table 2. Compared with normal rats, rats in the T2DM group presented with increased maximum bladder capacity, static bladder pressure and compliance (p < 0.05), and decreased maximum bladder pressure (p < 0.05) (Figure 1D).

Table 2

Comparisons of the Urodynamic Indicators of Rats in the Normal and T2DM Groups after 3 Weeks of Modeling

Group	n	Maximum Bladder Capacity (mL)	Static Bladder Pressure (kPa)	Maximum Bladder Pressure (kPa)	Compliance (mL/kPa)
Normal group	20	1.81 ± 0.13	0.58 ± 0.04	5.12 ± 0.56	0.65 ± 0.05
T2DM group	20	3.32 ± 0.46∗	0.97 ± 0.10∗	2.93 ± 0.30∗	1.35 ± 0.12∗

T2DM, type 2 diabetes mellitus. ∗p < 0.05, compared with the normal group.

Rats with STZ-Induced T2DM Presented Morphological Variations of Bladder Detrusors

Hematoxylin and eosin (H&E) staining (Figure 2A) revealed that the transverse and longitudinal detrusors in the normal group were arranged regularly and closely. The gap was filled with connective tissues, and the muscles were observed to have more nerve tracts. Smooth muscle cells were arranged in a regular and compact structure. However, in the T2DM group, muscle fascicles had a disorderly, loose, and fractured arrangement. The muscle gap was evidently swollen with edema.

Figure 2

H&E staining and Masson’s Trichrome Staining of Bladder Detrusor Tissues of Rats with STZ-Induced T2DM

(A) H&E staining of bladder detrusor tissues of rats with STZ-induced T2DM (original magnification, ×400). (B) Masson’s trichrome staining of bladder detrusor tissues of rats with STZ-induced T2DM (original magnification, ×400). The arrowhead indicates the broken or disordered muscle bundle.

In the normal group, Masson’s trichrome staining (Figure 2B) demonstrated that the detrusor was stained red, collagenic fiber was stained blue, and the transverse and longitudinal detrusors were arranged regularly and closely in the normal group. The gap was abundant with connective tissues, and the smooth muscle cells were arranged in a regular and compact structure. However, compared with the normal rats, T2DM rats exhibited thickened bladder tissues, increased collagen deposition, obvious fibrosis, and disordered muscle fascicles with muscle cell atrophy.

Damaged Ultrastructure in Rats with STZ-Induced T2DM

Transmission electron microscope (TEM) (Figure 3) revealed that the bladder detrusor cells in the normal group were arranged in an orderly manner, and had regular cell nuclei, with small amounts of mitochondria and mild edema in the cytoplasm. Compared with the normal group, the T2DM group had a wider gap between detrusor cells. The detrusor cells presented with variable shapes and sizes, with an uneven distribution and irregular arrangement. Numerous depressions were observed, and the mitochondria were swollen and ruptured, with physalides present in the cytoplasm. The above results demonstrated that rats with STZ-induced T2DM undergo ultrastructural changes.

Figure 3

Rats with STZ-Induced T2DM Have Damaged Ultrastructure of Detrusor Cells as Observed by TEM

Scale bars, 500 nm.

Rats with STZ-Induced T2DM Exhibited Higher Expression of Col1a2

The cause of detrusor fibrosis is irregular proliferation of detrusor tissue, and the main components of detrusor fibrosis are Col1a2 and Col3a1. Therefore, immunohistochemistry was conducted to determine the expression of Col1a2 and Col3a1. The immunohistochemistry results shown in Figure 4 suggested that the brownish-yellow granules of Col3a1 and Col1a2 were more numerous in the T2DM group than those observed in the normal group. The expression of Col3a1 and Col1a2 was much higher in the T2DM group than that in the normal group (p < 0.05).

Figure 4

Rats with STZ-Induced T2DM Exhibit Higher Expression of Col3a1 and Col1a2 in Bladder Detrusor Tissues as Detected by Immunohistochemistry

(A) Immunohistochemical staining (original magnification, ×200) of detrusor tissues in the normal and T2DM groups. The arrowhead indicates yellow granules with Col3a1- or Col1a2-positive expression. (B) Quantitative analysis for the expression of Col3a1 and Col1a2 in the normal and T2DM groups. ∗p < 0.05 versus the normal group.

Poor Expression of miR-363 and High Expression of Col1a2 in Rats with STZ-Induced T2DM

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot analysis were conducted to detect the expression of miR-363 and Col1a2 in rats with STZ-induced T2DM. The results depicted in Figure 5 show that compared with the normal group, the T2DM group presented with decreased miR-363 expression and decreased expression of B cell lymphoma 2 (Bcl-2) and Smad7 (p < 0.05), whereas the expression of Col1a2, Col3a1, Bcl-2-associated X protein (Bax), TGF-β1, and Smad4 was increased (p < 0.05). These findings provided evidence that miR-363 and TGF-β1/Smad participated in tissue fibrosis associated with T2DM.

Figure 5

qRT-PCR and Western Blot Analysis Show That miR-363 Is Downregulated, whereas Col1a2 Is Highly Expressed in Bladder Detrusor Tissues of Rats with STZ-Induced T2DM

(A) Expression of miR-363, and mRNA expression of Col1a2 and TGF-β1/Smad signaling pathway-related genes between the normal and T2DM groups as determined by qRT-PCR. (B) Western blots of Col1a2 and TGF-β1/Smad signaling pathway-related proteins in the normal and T2DM groups. (C) Comparisons of Col1a2 and TGF-β1/Smad signaling pathway-related proteins in the normal and T2DM groups. ∗p < 0.05 versus the normal group; n = 20.

Col1a2 Was a Target Gene of miR-363

Following online analysis using the microRNA.org website, a specific binding region was observed between the 3′ UTR sequences of Col1a2 gene and miR-363, suggesting that Col1a2 is a target gene of miR-363 (Figure 6A). miR-363 mimic and negative control (NC) were respectively co-transfected with luciferase reporter vectors containing pCol1a2-wild-type (WT) (Col1a2-WT sequence without mutation treatment) and pCol1a2 mutant (MUT) (Col1a2 sequence with directional mutation of the binding site between miR-363 and Col1a2) into detrusor cells to perform a dual-luciferase reporter gene assay. The results from the dual-luciferase reporter gene assay (Figure 6B) showed decreased luciferase signal for WT-Col1a2/miR-363 in the pCol1a2-WT and miR-363 mimic co-transfected group, when compared to the co-transfected NC group (p < 0.05), while there was no significant difference in the pCol1a2-MUT and miR-363 mimic co-transfected group (p > 0.05). Furthermore, we conducted a RIP assay to validate the binding between miR-363 and Col1a2. The results showed that miR-363 and Col1a2 expressions were more enriched in the anti-Ago2 group when compared with the anti-immunoglobulin G (IgG) group (Figure 6C). These results indicated that miR-363 could specifically bind to Col1a2 gene.

Figure 6

Col1a2 Is a Target Gene of miR-363

(A) Predicted binding sites of miR-363 on the 3′ UTR of Col1a2 gene. (B) Luciferase activity in the miR-363 mimic and NC groups. pCol1a2-WT is the recombinant luciferase vector for Col1a2 without mutation, and pCol1a2-MUT is the recombinant luciferase vector for Col1a2 with directional mutation of miR-363 and Col1a2. (C) The binding of miR-363 to Col1a2 confirmed by RIP assay. On the left, immunoprecipitation of the RNA-induced silencing complex (Ago2-RISC) using the Ago2 antibody in 293T cells transfected with miR-NC or miR-363 inhibitor. IgG was used as a NC and β-actin was used as an internal control; in the middle, miR-363 expression incorporated into RISC was detected by qRT-PCR, with U6 used as an internal control; on the right, Col1a2 expression incorporated into RISC was detected by qRT-PCR, with β-actin used as an internal control. ∗p < 0.05 versus the NC group or the IgG group. The experiment was repeated three times.

Overexpressed miR-363 Downregulated Col1a2 by Inhibiting the TGF-β1/Smad Signaling Pathway

The results from qRT-PCR and western blot analysis (Figure 7) revealed that compared with the normal group, the blank and NC groups had increased relative expression of Col1a2, Col3a1, Bax, TGF-β1, and Smad4, while relative expression of miR-363, Bcl-2, and Smad7 was decreased (all p < 0.05). No significant differences were observed between the blank group and NC group (p > 0.05). Compared with the blank and NC groups, miR-363 expression was decreased in the miR-363 inhibitor + small interfering RNA (siRNA)-Col1a2 group, whereas the expression of other genes exhibited no significant difference (p > 0.05). The miR-363 mimic group had increased miR-363 expression, whereas the SB431542 and siRNA-Col1a2 groups had no significant changes (p > 0.05). The miR-363 mimic and siRNA-Col1a2 groups presented with increased expression of Bcl-2 and Smad7 but lower expression of Col1a2, Col3a1, Bax, TGF-β1, and Smad4 (all p < 0.05). The miR-363 inhibitor group had increased expression of Col1a2, Col3a1, Bax, TGF-β1, and Smad4 but decreased expression of miR-363, Bcl-2, and Smad7 (all p < 0.05). The miR-363 mimic + SB431542 group exhibited elevated miR-363 expression but reduced expression of Col1a2, Col3a1, Bax, TGF-β1, and Smad4 (all p < 0.05). Compared with the miR-363 mimic group, expression levels of miR-363, Col1a2, Col3a1, Bax, TGF-β1, and Smad4 were all lower (all p < 0.05). The aforementioned findings indicated that the high expression of miR-363 could result in decreased Col1a2 through the inhibition of the TGF-β1/Smad signaling pathway, thus alleviating the incidence of T2DM.

Figure 7

miR-363 Inhibits the TGF-β1/Smad Signaling Pathway by Regulating Col1a2 in Bladder Detrusor Cells

(A) MiR-363 expression and mRNA expression of Col1a2 and TGF-β1/Smad signaling pathway-related genes as determined by RT-qPCR. (B) Western blots of Col1a2 and TGF-β1/Smad signaling pathway-related proteins in each group. (C) Comparisons of Col1a2 and TGF-β1/Smad signaling pathway-related proteins in each group. #p < 0.05 versus the blank and NC groups. The experiment was repeated three times.

Overexpression of miR-363 or siRNA-Col1a2 Elevated Cell Viability

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 8) demonstrated that compared with the normal group, the remaining groups had reduced cell viability (all p < 0.05). There was no significant difference in cell viability among the blank group, NC group, and miR-363 inhibitor + siRNA-Col1a2 group (p > 0.05). Compared with the blank and NC groups, the miR-363 mimic, SB431542, siRNA-Col1a2, and miR-363 mimic + SB431542 groups were observed to have accelerated cell viability, and the miR-363 mimic + SB431542 group showed increased cell viability compared with the miR-363 mimic group, but the miR-363 inhibitor group had reduced cell viability (all p < 0.05). Therefore, the overexpression of miR-363 or silencing of the Col1a2 gene could enhance the viability of detrusor cells in T2DM. Moreover, the combined function of miR-363 overexpression and inhibition of the TGF-β1/Smad signaling pathway could promote the viability of detrusor cells in T2DM.

Figure 8

Overexpression of miR-363 or Col1a2 Silencing Elevates Cell Viability in Bladder Detrusor Cells, as Detected by the MTT Assay

∗p < 0.05 at each time point versus the normal group; #p < 0.05 at each time point versus the blank and NC groups. The experiment was repeated three times.

Upregulated miR-363 or siRNA-Col1a2 Inhibited Cell-Cycle Progression and Reduced Apoptosis

Propidium iodide (PI) staining (Figures 9A and 9B) revealed that compared with the normal group, more G0/G1 phase-arrested cells were noted in the remaining groups while there were less S phase-arrested cells (all p < 0.05). There was no significant difference in the cell cycle among the blank, NC, and miR-363 inhibitor + siRNA-Col1a2 groups (p > 0.05). Compared with the blank and NC groups, the miR-363 mimic, SB431542, miR-363 mimic + SB431542 and siRNA-Col1a2 groups showed fewer G0/G1 phase-arrested cells but more S phase-arrested cells (all p < 0.05). The miR-363 mimic + SB431542 group presented with reduced G0/G1 phase-arrested cells but increased S phase-arrested cells as compared with the miR-363 mimic group (p < 0.05). The miR-363 inhibitor group had more G0/G1 phase-arrested cells but fewer S phase-arrested cells (all p < 0.05). Annexin V-fluorescein isothiocyanate (FITC)/PI double staining (Figures 9C and 9D) revealed that in comparison to the normal group, the remaining groups had increased apoptosis rates (all p < 0.05). There was no significant difference in the apoptosis rate among the blank, NC, and miR-363 inhibitor + siRNA-Col1a2 groups (p > 0.05). Compared with the blank and NC groups, the miR-363 mimic, SB431542, miR-363 mimic + SB431542, and siRNA-Col1a2 groups had decreased apoptosis rates (p < 0.05), and the apoptosis rate in the miR-363 mimic + SB431542 group was lower than in the miR-363 mimic group, but the miR-363 inhibitor group had an increased apoptosis rate (p < 0.05). These results suggest that overexpression of miR-363 and silencing of Col1a2 could result in the inhibition of the apoptosis of bladder detrusor cells in rats, and the combined function of miR-363 overexpression and inhibition of the TGF-β1/Smad signaling pathway could inhibit cell apoptosis of detrusor cells in T2DM.

Figure 9

Upregulated miR-363 or Col1a2 Silencing Promotes Cell Cycle Progression and Reduces Apoptosis in Bladder Detrusor Cells, as Detected by Flow Cytometry

(A) Flow cytometric analysis of cell cycle distribution. (B) Quantitative analysis for the cell cycle in the seven groups. (C) Flow cytometric analysis of apoptosis conditions. (D) Quantitative analysis for the apoptosis rate in the seven groups. ∗p < 0.05 versus the normal group; #p < 0.05 versus the blank and NC groups. The experiment was repeated three times.

Discussion

Hyperglycemia induces myogenic and neurogenic variations and compensatory hypertrophy caused by polyuria in the early stage of DM, whereas the accumulation of oxidative stress results in decompensation of bladder tissues and occurs in the late stage. Increasing evidence has highlighted miRNAs as blood biomarkers for early prevention of DM development and as tools for gene-based therapy to treat both T1DM and T2DM. The present study explored the potential effect of miR-363 in detrusor fibrosis of rats with STZ-induced T2DM. The results revealed that the miR-363-mediated inhibition of Col1a2 suppressed the activation of the TGF-β1/Smad signaling pathway, promoted cell viability, inhibited cell apoptosis, and reduced detrusor fibrosis in rats with STZ-induced T2DM.

Our study demonstrated that compared with normal rats, rats with STZ-induced T2DM presented with increased blood glucose. Pre-existing evidence has indicated that STZ is commonly used in the establishment of diabetic rat models, as it is a cytotoxic drug that can destroy islet β cells, resulting in an increase in blood glucose. The amount of STZ injected correlates to the degree of diabetes., T2DM is an increasingly prevalent disease that is associated with numerous adverse complications that can lead to lower urinary tract symptoms, partly due to decreased interstitial cells in the detrusor and lamina propria. A previous study indicated that bladder weight and pressure were increased secondary to hyperplasia and hypertrophy of detrusor smooth muscle, in addition to other components of the bladder wall, and that diuresis induced by DM can activate DNA synthesis and cell proliferation in bladder tissues. In addition, rats with STZ-induced T2DM were observed to have abnormal detrusors and bladder dysfunction. Diabetic bladder dysfunction is a common feature of T2DM that occurs as a result of a sensory disturbance and abnormal detrusor reflex in its early stage, which then develops into bladder detrusor contraction and decreased bladder emptying in the later stage., A prior study showed that the excessive activity of the detrusor continues to increase without detrusor decompensation in patients with diabetic bladder dysfunction.

Furthermore, bladder detrusor tissues of rats with STZ-induced T2DM presented with decreased miR-363 expression and increased expressions of Col1a2 and Col3a1. In a previous study, qRT-PCR revealed a significantly lower expression of miR-363-3p in renal cell carcinoma tissues. Moreover, miR-363 has been demonstrated to result in decreased expression in T2DM and may be used as a prognostic biomarker for T2DM. Col1a2 and Col3a1 have been found to be significantly increased in diabetic mice, which is consistent with our findings.

In addition, in STZ-induced T2DM rats, miR-363 could inhibit Col1a2 to contain the activation of the TGF-β1/Smad signaling pathway and thus contribute to proliferation while suppressing the apoptosis of detrusor cells. The abnormal expression of miRNAs has been proven to play crucial roles in the diagnosis, prognosis, and prediction of treatment outcomes of several diseases. In renal cell carcinoma, miR-363-3p is involved in cell proliferation, migration and apoptosis, and tumorigenesis, and the overexpression of miR-363-3p impairs proliferation of renal cancer cells. miRNAs often inhibit gene expression by directly binding to the 3′ UTR of a target mRNA; Col1a2 was identified as a target gene of miR-363 in this study, and its expression can be inhibited by miR-363. According to a previous study, there exists a positive correlation between Col1a2 and TGF-β1 in cardiac fibroblast cells. The TGF-β1/Smad signaling pathway can control the strength and timing of downstream signals and also mediate the fibrosis of animal organs. TGF-β1 can increase the expression of many fibrotic genes, including those expressing collagen extracellular matrix proteins and α-smooth muscle actin through the Smad2/3 signaling pathway. Among the various regulatory factors, an increased level of TGF-β1 has the potential to stimulate bladder fibrosis. The transcriptional activity of the Col1a2 gene in a mouse model of interstitial fibrosis induced by aristolochic acid has been investigated in a previous study. Carnosic acid (CA) inhibits the transcription of Col1a2 through SIRT1-regulated Smad3 deacetylation, and the activation of SIRT1 by CA involves the AMPKα1/SIRT1 signaling pathway in liver fibrosis. The TGF-β1/Smad signaling pathway is responsible for cardiac fibrosis in diabetic cardiomyopathy, and Shensong Yangxin capsules have been shown to suppress fibrosis and improve cardiac function by inhibiting this signaling pathway. The activation of SIRT1 led to the inhibition of the TGF-β signaling pathway via deacetylation of Smad3 and attenuated renal fibrosis in vivo in mouse models of chronic renal failure. In addition, the TGF-β1/Smad signaling pathway can sensitize Col1a2 gene through the Smad-independent signaling pathway. Moreover, miR-363 has been reported as a potent inducer of EMT, which is partially through the downregulation of TGF-β. These data indicated that the overexpression of miR-363 could diminish the biological functions of detrusor cells in T2DM through the blockage of the Col1a2-dependent TGF-β1/Smad signaling pathway.

In summary, the aforementioned findings suggested that the upregulation of miR-363 can inhibit Col1a2, which promotes viability and reduces apoptosis of detrusor cells in rats with STZ-induced T2DM through inhibition of the TGF-β1/Smad signaling pathway, which ultimately leads to the alleviation of detrusor fibrosis. These data provide new evidence regarding the functional actions of miR-363 in T2DM. Thus, therapeutic strategies aimed at restoring miR-363 function could potentially be of clinical use in the treatment and management of T2DM. However, further studies are required to explore the underlying mechanism by which Col1a2 affects mRNA transcription and protein activity of TGF-β1/Smads.

Materials and Methods

Ethics Statement

The experiment was conducted under the approval of the Ethics Committee of China-Japan Union Hospital of Jilin University in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

STZ-Induced T2DM Model Establishment

Fifty healthy male Sprague-Dawley (SD) rats weighing 200–250 g purchased from Shanghai SLAC Laboratory Animal (Shanghai, China) were enrolled in this study and raised in a laboratory environment with free access to water and food and natural light at 18°C–22°C, relative humidity of 40%–70%, and noise less than 50 dB. The rats were then divided into the T2DM group (n = 30) and the normal group (n = 20). The rat models of T2DM were established, provoked with STZ (S0130, Beijing Boai Technology & Trade, Beijing, China), through a single intraperitoneal injection of STZ (50 mg/kg). Prior to the operation, STZ was prepared in sodium citrate buffer (0.1 mol/L [pH 4.5]) and used up within 10 min. All rats were raised in separate cages in the same animal room. The normal group was injected with the same volume of sodium citrate solution. The normal rats were fed with a standard diet, while the rats in the T2DM group received a high-fat diet (40% of calories as fat, Beijing Huafukang Bioscience, Beijing, China). After 2 weeks of feeding, blood glucose, insulin plasma levels, urine glucose, and urine volume were measured. The model was successfully established if the blood glucose was ≥16.7 mmol/L and urine glucose was ≥2+. Finally, there were 25 successful models, out of which 20 rats were randomly selected as the T2DM group.

Urodynamic Testing

Three weeks after modeling, urodynamic testing was performed. The noses and mouths of the rats were covered with an ether tank. After the administration of light anesthesia, the limbs were fixed on the anatomical table, and a hypogastric mini-incision (approximately 1 cm) was made. The bladder tissue, prostate, and partial posterior urethra were separated. Subsequently, the urethra was separated and an approximately 0.5-cm cut was made in front of the prostate. An F2 pressure catheter was inserted into the bladder and connected to the Menuet urodynamic system (Nidoc 970C, Shanghai Siou Medical Instrument, Shanghai, China) using a pressure transducer. Finally, the maximum bladder capacity, pressure, and compliance were recorded. The experiment was conducted in triplicates.

Masson’s Trichrome Staining

On the 25th day of the experiment, the rats were euthanized. During the operation, the bladder muscle was cut from the top of bladder and fixed with 10% formaldehyde for 24 h. Following dehydration with 80%, 90%, and 100% ethanol and n-butanol, the samples were paraffin-embedded in a wax box at 60°C and serially sectioned (5 μm). The sections were spread at 45°C, baked for 1 h at 60°C, and dewaxed with xylene. The sections were then soaked in a gradient of alcohol (100%, 95%, 80%, and 70%) for 2 min each, followed by two washes with distilled water (5 min each). Then, the sections were stained according to the instructions of the Masson dye kit (Nanjing Jiancheng Biological Engineering, Nanjing, Jiangsu, China). Finally, sections were sealed with neutral gum, after which photographs of the sections were obtained with the use of a general optical microscope. Next, five high-power visual fields were randomly selected and the degree of detrusor fibrosis in the bladder layer of rats was observed. The experiment was conducted in triplicate.

H&E Staining

Rats were euthanized on the 25th day of the experiment. The bladder muscle was excised from the top of the bladder and fixed with 10% formaldehyde for 24 h. Following dehydration with 80%, 90%, and 100% ethanol and n-butanol, the samples were embedded in paraffin in a wax box at 60°C and subjected to serial sections (5 μm). The sections were then spread at 45°C, baked for 1 h at 60°C, and dewaxed with xylene. After hydration, H&E staining for the sections was carried out (Beijing Solarbio Science & Technology, Beijing, China), after which the sections were dehydrated using gradient alcohol, cleared with xylene, and mounted with neutral balsam. The morphology of the detrusor muscle was observed under a microscope (XP-330, Shanghai Bing Yu Optical Instrument, Shanghai, China) in more than five visual fields.

TEM

The bladder detrusor tissues of rats were obtained and trimmed to approximately 1-cm3 tissue blocks and fixed in 2.5% glutaraldehyde solution at a low temperature. They were then embedded and cut into ultrafine sections after osmium staining (120 nm). The ultrastructure of the detrusor tissues was observed using TEM (JEM-F200, JEOL, Tokyo, Japan). The experiment was conducted in triplicate.

Immunohistochemistry

The sections were conventionally dewaxed, hydrated, dewaxed by xylene I and xylene II for 10 min each, dehydrated with gradient alcohol (100%, 95%, 80% and 70%) for 2 min each, and washed twice (5 min each time on the shaking table) with distilled water. The sections were immersed in 3% H2O2 for 10 min and washed with distilled water again. After exposure to high pressure for antigen repair for 90 s, the sections were cooled at room temperature and washed with phosphate-buffered saline (PBS). Afterward, incubation was carried out with 5% bovine serum albumin (BSA) at 37°C for 30 min, followed by further incubation with rabbit anti-rat primary antibody (Col3a1 and Col1a2) (ab34710 and ab7778, respectively, Abcam, Cambridge, MA, USA) (1:100) at 4°C overnight. The following day, biotinylated goat anti-rabbit IgG (SF8-0.3, Beijing Solarbio Science & Technology, Beijing, China) (1:100) was added to the sections and incubated at 37°C for 30 min, after which it was immersed in streptavidin-biotin (SAB) solution, stained with diaminobenzidine (DAB), and counterstained with hematoxylin. The primary antibody was then replaced with PBS as NC. Positive cells were determined by the presence of brownish yellow stains. Five high-power fields were randomly selected, with 200 cells in each field counted, and the localization of positive cells, as well as the percentage of Col3a1 and Col1a2 positive cells to the total cells, was observed individually. Finally, the average values were calculated. The experiment was conducted in triplicate.

qRT-PCR

The detrusor tissues of the rats in the T2DM group (30 mg) and normal group (30 mg) were added to 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and ground into powder in liquid nitrogen. Total RNA was extracted using TRIzol, and the concentration and purity of the RNA were detected with the use of ultraviolet spectrophotometry (UV1901, Shanghai Aucy Technology Instrument, Shanghai, China). The RNA was reversely transcribed to cDNA (50 ng/μL) through reverse transcription with the PrimeScript RT reagent kit (Takara, RR047A, Beijing Think-Far Technology, Beijing, China) and stored at −80°C. Primers designed with Primer Premier 5.0 software were synthesized by Beijing Tsingke Biological Technology (Beijing, China). The primer sequences are displayed in Table 3. According to the two-step method, qRT-PCR was conducted using an ABI 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). U6 was used as an internal reference for miR-363 and β-actin was used as an internal reference for Col1a2, Col3a1, Bax, Bcl-2, TGF-β1, Smad4, and Smad7. The relative expression was determined using the 2−ΔΔCt method. The PCR for each gene of each sample was performed in triplicate. This method was also applicable for qRT-PCR detection in the cultured primary bladder detrusor cells and transfected cells in the T2DM and normal groups.

Table 3

Primer Sequences for qRT-PCR

Gene	Sequence
miR-363	Forward: 5′-CCAATTGCACGGTATCCAT-3′
	Reverse: 5′-GTGCAGGGTCCGAGGT-3′
Col1a2	Forward: 5′-CAGCGGAGGAGGCTATGACTTT-3′
	Reverse: 5′-GGCGAGATGGCTTATTCGTTTT-3′
Col3a1	Forward: 5′-GTCCTGCTGGTCCTATTGGT-3′
	Reverse: 5′-CACCATTCTGCCCAGGAGCA-3′
Bax	Forward: 5′-CCAAGAAGCTGAGCGAGTGT-3′
	Reverse: 5′-TCACGGAGGAAGTCCAGTGT-3
Bcl-2	Forward: 5′-GGTGGTGGAGGAACTCTTCA-3′
	Reverse: 5′-GAGCAGCGTCTTCAGAGACA-3′
TGF-β1	Forward: 5′-CCAAGGAGACGGAATACAGG-3′
	Reverse: 5′-TGAGGAGCAGGAAGGGTC-3′
Smad4	Forward: 5′-TATCACTATGAGCGGGTTG-3′
	Reverse: 5′-GTGGGTAAGGATGGCTGT-3′
Smad7	Forward: 5′-ACGCTGTACCTTCCTCCG-3′
	Reverse: 5′-TCCTCCCAGTATGCCACC-3′
β-Actin	Forward: 5′-ACTGGCATTGTGATGGACTC-3′
	Reverse: 5′-CAGCACTGTGTTGGCATAGA-3′
U6	Forward:5′-GCTTCGGCAGCACATATACTAAAAT-3′
	Reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′

qRT-PCR, quantitative reverse transcription polymerase chain reaction; miR-363, microRNA-363; Col1a2, collagen type I alpha 2; Col3a1, collagen type III alpha 1; Bax, Bcl-2-associated X protein; Bcl-2, B cell lymphoma 2; TGF-β1, transforming growth factor β1.

Western Blot Analysis

Bladder detrusor tissues were obtained from the rats in the T2DM group and the normal group, ground into powder, preserved at a low temperature, washed twice with PBS, and added to protein lysis buffer (P0013, Beyotime Biotechnology, Shanghai, China). Following centrifugation at 25,764 × g for 20 min at 4°C, protein samples were prepared and the supernatant was collected. Total protein concentration was determined using a bicinchonininc acid (BCA) protein assay kit (P0012, Beyotime Biotechnology, Shanghai, China). A total of 50 μg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (BS5023001, Shanghai Bioscience Biological Technology, Shanghai, China). After blocking with 5% skim milk for 1 h at room temperature, the membranes underwent incubation with primary antibodies (1:200) (Col1a2, ab96723; Col3a1, ab7778; Bax, ab32503; Bcl-2, ab32124; TGF-β1, ab92486; Smad4, ab40759; Smad7, ab216428; and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]; Abcam, Cambridge, MA, USA). After three washes with Tris-buffered saline with Tween 20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary goat antibody (1:200) (BS13278, Shanghai Ke Xing Biochemical Reagent, Shanghai, China) for 1 h. After a rinse with TBST, enhanced chemiluminescence (ECL) (qdb-077, Shanghai Yan Hui Biotechnology, Shanghai, China) was used to visualize the results using X-ray film. The band intensities were quantified using a FluorChem Q imaging system (ProteinSimple, Santa Clara, CA, USA). The relative protein expression was regarded as the ratio of the gray value of the target band to internal reference GAPDH. The experiment was conducted in triplicates. The above method was also applicable for western blot analysis in cells.

Dual-Luciferase Reporter Gene Assay

A bioinformatics prediction website microRNA.org was used to predict the target genes of miR-363, after which a dual-luciferase reporter gene assay was used to validate whether Col1a2 was a direct target gene of miR-363. The full-length 3′ UTR of Col1a2 gene was cloned, and the PCR products were cloned to multiple cloning sites downstream of the pmirGLO Luciferase gene (E1330, Promega, Madison, WI, USA), which was named pCol1a2-WT. The binding sites in miR-363 and the target genes were predicted using bioinformatics for site-directed mutagenesis, and a pCol1a2-MUT vector was constructed. The Renilla luciferase vector pRL-TK (E2241, Promega, Madison, WI, USA) was used as an internal reference to adjust the cell number and transfection efficiency. Both miR-363 mimic and NC were transfected with luciferase reporter vector into detrusor cells to detect the luciferase activity. The experiment was conducted in triplicate.

Cell Treatment

The bladder detrusor tissues were extracted from three rats from the T2DM group and three rats from the normal group, washed with normal saline, placed in a clean and dry culture dish, and cut into tissue blocks. Following sterilization with PBS, the tissue blocks were moved to a centrifuge tube and centrifugation was carried out at 129 × g at room temperature for 5 min, followed by the removal of the supernatant. The tissues were detached with 0.5 mg/mL type IV collagenase solution (17101-015, Gibco, Grand Island, NY, USA) at 37°C for 15 min. The supernatant was extracted, and PBS was added to terminate detachment. The products were then mixed and filtered, and the tissues that were undetached were removed. The filtrate was then centrifuged at 129 × g for 5 min, with the supernatant removed, and the pellet was washed three times with PBS. After a final centrifugation, the cells were collected. The bladder detrusor cells in the T2DM and normal groups were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 15% fetal bovine serum (FBS) (22400089, Gibco, Grand Island, NY, USA). The cells were incubated in a six-well plate at a density of 2.5 × 107 cells/well at 37°C with 5% CO2 and saturated humidity. The medium was replaced every 2–3 days in accordance with cell growth, until 80%–90% cells settled at the bottom of the well to perform cell sub-culturing.

The detrusor cells in the logarithmic growth phase were divided into a normal group (cells from normal rats), a blank group (cells from the T2DM rats without any transfection), a NC group (cells from the T2DM rats transfected with scrambled siRNA), an SB431542 group (cells from the T2DM rats transfected with the inhibitor of the TGF-β1/Smad signaling pathway), a miR-363 mimic group (cells from the T2DM rats transfected with miR-363 mimic), a miR-363 inhibitor group (cells from the T2DM rats transfected with miR-363 inhibitor), a siRNA-Col1a2 group (cells from the T2DM rats transfected with siRNA-Col1a2), a miR-363 inhibitor + siRNA-Col1a2 group (cells from the T2DM rats co-transfected with miR-363 inhibitor and siRNA-Col1a2), and a miR-363 mimic + SB431542 group (cells from the T2DM rats transfected with miR-363 mimic and the inhibitor of the TGF-β1/Smad signaling pathway). The cells were inoculated in a six-well plate. When cell density reached 70%–80%, the cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The miR-363 mimic, siRNA-Col1a2, miR-363 inhibitor, miR-363 inhibitor + siRNA-Col1a2, and NC lyophilized powders (Invitrogen, Carlsbad, CA, USA) were then centrifuged, dissolved in RNase-free ultrapure water, and diluted with 250 μL of serum-free minimum essential medium (MEM). Subsequently, 5 μL of Lipofectamine 2000 was diluted with 250 μL of serum-free MEM (Gibco, Grand Island, NY, USA) and mixed gently, and incubation was carried out at room temperature for 5 min. The mixtures were incubated at room temperature for 20 min and added to the cell culture well. After the cells were cultured at 37°C with 5% CO2 for 6–8 h, the medium was replaced with complete medium and cultured for 24–48 h for subsequent experiments.

MTT Assay

Detrusor cells at logarithmic growth phase were selected to prepare cell suspension (5 × 105 cells/mL) in MEM containing 10% FBS and inoculated in a 96-well plate. Each group had eight wells, with 100 μL in each well. The cells were cultured at 37°C with 5% CO2, after which the plate was removed at 24, 48, 72, or 96 h. Each well was cultured with 10 μL of 5 mg/mL MTT solution (Sigma-Aldrich, St. Louis, MO, USA) for another 4 h. The optical density (OD) values of each well were then detected at 490 nm using an automatic enzyme-labeling instrument (Bio-Rad, Hercules, CA, USA). The experiment was conducted in triplicates.

Flow Cytometry

After transfection for 48 h, the cells were collected, detached with 0.25% trypsin, and adjusted to a density of 1 × 106 cells/mL. The cells (1 mL) were centrifuged at 453 × g for 10 min for the removal of the supernatant. PBS was added at 2 mL per mL of cells. The cells were centrifuged again and fixed with pre-cooled 70% ethanol at 4°C overnight following the removal of the supernatant. The following day, the cells were washed twice with PBS. The cell suspension (100 μL) was supplemented with 50 μg of PI solution (containing RNase) (40710ES03, Shanghai Qian Chen Bioscience & Technologies, Shanghai, China) for 30 min with the avoidance of light exposure, and filtered with a 100-mesh nylon filter. Flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) was performed to record the cell cycle at 488 nm by red fluorescence. Cell apoptosis was detected using annexin V-FITC/PI double staining. The treated cells were cultured at 37°C with 5% CO2 for 48 h, washed twice with PBS, and re-suspended in 200 μL of binding buffer after centrifugation. Next, the cells were added to 10 μL of annexin V-FITC (ab14085, Abcam, Cambridge, MA, USA) and 5 μL of PI and mixed gently. After 15 min of reaction at room temperature under dark conditions, 300 μL of binding buffer was added to the cells, and cell apoptosis was detected with the use of flow cytometry at 488 nm. The experiment was conducted in triplicates.

RIP Assay

RIP assays were performed using the Magna RIP Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, the 293T cells following transfection were lysed in RIP lysis buffer, and then 100 μL of whole-cell extract was incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (ab32381, Abcam), positive control anti-β-actin antibody (ab179467, Abcam), or NC normal mouse IgG antibody (ab125900, Abcam). The samples were next incubated with proteinase K by shaking in order to detach the protein, after which the immunoprecipitated RNA was isolated. At last, miR-363 expression and Col1a2 mRNA expression in the precipitates were detected by qRT-PCR.

Statistical Analysis

Statistical analysis was conducted using the SPSS 21.0 statistical software (IBM, Armonk, NY, USA). All data were from three independent experiments and subjected to normality test. The measurement data were presented as mean ± standard deviation. Comparisons between two groups were analyzed using a t test. Data conforming to normality among multiple groups were analyzed using one-way analysis of variance (ANOVA), followed by a Tukey post hoc test. The data with skewed distribution were analyzed using Dunn’s multiple comparison post hoc test in a Kruskal-Wallis test. p < 0.05 was considered statistically significant.

Author Contributions

X.-F.L., S.-H.Z., G.-F.L., and S.-N.Y. designed the study. X.-F.L., S.-H.Z., and G.-F.L. collated the data, carried out data analyses, and produced the initial draft of the manuscript. X.-F.L. and S.-N.Y. contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

Conflicts of Interest

The authors declare no competing interests.