MicroRNAs (miRNAs or miRs), a group of small non-coding RNAs, have been demonstrated to play key roles in various physicological processes and diseases, including diabetes, the most common metabolic disorder. However, the underlying mechanisms remains largely unknown. In this study, we aimed to investigate the role of miR-19a-3p in diabetes. The results of RT-qPCR demonstrated that the level of miR-19a-3p was significantly decreased in the diabetic patients, and that the decreased miR-50a-5p level was significantly associated with a high concentration of blood glucose. miR-19a-3p mimic was further used to transfect pancreatic β cells, and we found that the overexpression of miR-19a-3p promoted cell proliferation and insulin secretion, while it suppressed the apoptosis of pancreatic β cells. Suppressor of cytokine signaling 3 (SOCS3) was further identified as a direct target gene of miR-19a-3p, and its protein level was significantly decreased following the overexpression of miR-19a-3p. Moreover, the siRNA-induced downregulation of SOCS3 also enhanced cell proliferation and insulin secretion, while it inhibited the apoptosis of pancreatic β cells. In addition, the overexpression of SOCS3 reversed the effects of miR-19a-3p overexpression on cell proliferation, insulin secretion and on the apoptosis of pancreatic β cells, which further indicates that SOCS3 acts as a downstream effector in the miR-19a-3p-mediated function of pancreatic β cells. Finally, the level of SOCS3 was increased in diabetic patients, and inversely correlated with the miR-19a-3p level, suggesting that the downregulation of miR-19a-3p leads to the upregulation of SOCS3, which contributes to the dysfunction of pancreatic β cells. On the whole, the findings of this study suggest that miR-19a-3p plays an important role in β cell function, and that the miR-19a-3p/SOCS3 axis may become a potential therapeutic target for diabetes.
Diabetes mellitus, which is a complex metabolic disease, is characterized by increased blood glucose levels, which are caused by the lack of insulin production or resistance to insulin (
MicroRNAs (miRNAs or miRs), a class of non-coding RNAs, 18–25 nucleotides in length, are able to suppress gene expression by targeting the complementary regions of mRNAs and inhibiting protein translation (
miR-19a-3p, a member of the miR-17-92 miR cluster, has been previously found to be a regulator of the expression of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis (
In this study, we aimed to examine the role of miR-19a-3p in diabetes. We found that miR-19a-3p was significantly downregulated in diabetic patients, and that it enhanced cell proliferation and insulin secretion, while it inhibited the apoptosis of pancreatic β cells by directly targeting suppressor of cytokine signaling 3 (SOCS3). Accordingly, we suggest that miR-19a-3p may serve as a potential candidate for the clinical management of diabetes.
This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Chongqing, China), and was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study subjects prior to enrollment. Blood samples from patients with T2D (n=45) and normal subjects (n=20) were collected from the First Affiliated Hospital of Chongqing Medical University from November, 2012 to February, 2014. Patients with serious liver or kidney diseases, malignancy and acute heart failure were excluded.
The glucose levels in the blood samples were determined by the routine laboratory method. The blood glucose levels were examined at the Department of Clinical Laboratory of our hospital using the Glucose Assay kit (BioVision, San Francisco, CA, USA), according to the manufacturer's instructions. Plasma SOCS3 levels were analyzed by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions using the SOCS-3 ELISA kit (Ybiotech, Shanghai, China). Briefly, an equal amount of protein (50
The pancreatic β cell lines, INS-1 and MIN6, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM glucose and 15% fetal bovine serum (FBS) (both from Life Technologies, Grand Island, NY, USA) and 5.5 mM 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA). INS-1 and MIN6 cells were collected and then suspended using DMEM with 10% FBS. The cells were then seed into a 6-well plate (300,000 cells per well), and cultured to 70%–80% confluence.
miR-negative control (miR-NC), miR-19a-3p mimic (both from GenePharma, Shanghai, China), siRNA targeting SOCS3 (SOCS3 siRNA), non-specific siRNA (NC siRNA), the pc-DNA3.1-SOCS3 plasmid and pc-DNA3.1 vector (NC), and Lipofectamine 2000 were diluted with OPTI-MEM (both from Life Technologies). The diluted Lipofectamine 2000 was added to the respective diluted plasmid, miR, or siRNA. Following incubation at room temperature for 20 min, the above mixture was added to the cell suspension, which was then incubated at 37°C, 5% CO2 for 6 h. Subsequently, the transfection mixture was replaced by DMEM with 10% FBS. Untransfected cells were used as controls. Following transfection for 48 h, the following assays were conducted:
Total RNA was extracted from the cells using TRIzol reagent (Life Technologies). RT-qPCR was used to examine the relative miR-19a-3p expression using the mirVana™ real-time RT-PCR microRNA detection kit (Life Technologies), in accordance with the manufacturer's instructions. U6 was used as an internal reference. The specific primers for miR-19a-3p and U6 were purchased from Genecopoeia, Guangzhou, China. The relative mRNA expression of SOCS3 was detected by quantitative PCR (qPCR) using the standard SYBR-Green RT-PCR kit (Takara, Otsu, Japan) in accordance with the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. The specific primers for SOCS3 were as follows: forward, 5′-ATGGTCACCCACAGCAAGTTT-3′ and reverse, 5′-TCCAGTAGAATCCGCTCTCCT-3′. The specific primers for GAPDH were as follows: forward, 5′-TGGCCTTCCGTGTTCCTAC-3′ and reverse, 5′-GAGTTGCTGTTGAAGTCGCA-3′. The relative expression level was quantified using the the 2−ΔΔCt method.
The cells were lysed in the protein lysis buffer [50 mM Tris/HCl, pH 8.0, 250 mM NaCl, 1% NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% sodium dodecylsulfate, 1% PMSF and 1X phosphatase inhibitor cocktail]. The protein concentration was determined using the BCA Protein assay kit (Pierce Chemical, Rockford, IL, USA). Protein (60
The cells were seeded in a 96-well plate and cultured overnight. The cells were then treated with basal glucose (3.3 mmol/l) or stimulatory glucose (16.7 mmol/l) for 1 h. Subsequently, the insulin level was measured by ELISA. In brief, the cells in each well were sonicated in acid ethanol, followed by 3 freeze/thaw cycles, and then centrifuged for 5 min at 10,000 x g. The supernatant was used to measure the insulin level by ELISA as described above.
The cells (2×103) in each group (miR-NC, miR-19a-3p mimic, SOCS3 siRNA, NC siRNA, miR-19a-3p mimic + SOCS3, ormiR-19a-3p mimic + NC vector) were plated into a 96-well plate and cultured for 1 to 5 days at 37°C with 5% CO2. Subsequently, 20
The cell apoptotic levels were examined using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. The cells in each group were re-suspended in 1X binding buffer solution with Annexin V-FITC and PI and incubated for 15 min at room temperature in the dark. The apoptotic rate was determined using the EPICS Altra flow cytometer (Beckman Coulter, Brea, CA, USA).
Bioinformatics analysis was conducted to predict the putative targets of miR-19a-3p using TargetScan 4.2 online software (
The wild-type (WT) of SOCS3 3′-UTR was constructed by PCR and inserted into the pMIR-REPORT miRNA Expression Reporter vector (Ambion, Carlsbad, CA, USA). The mutant type (MT) of SOCS3 3′-UTR was constructed using the Easy Mutagenesis system kit (Promega, Madison, WI, USA), in accordance with the manufacture's instructions, and inserted into the pMIR-REPORT miRNA Expression Reporter vector. 293 cells (Cell Bank of the Chinese Academy of Sciences) were plated in 96-well plates, and co-transfected with the WT SOCS3-3′UTR or MT SOCS3-3′UTR plasmid (300 ng), and miR-NC or miR-19a-3p mimic (100 nM), using Lipofectamine 2000, in accordance with the manufacture's instructions. The dual-luciferase reporter assay system (Promega) was used to determine the activity of
The data of at least 3 independent experiments are expressed as the means ± SD. GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis. The differences were analyzed using the Student's t-test or one-way ANOVA. The association between the miR-19a-3p level and the blood glucose or SOCS3 levels was analyzed using Spearman's correlation coefficient. A value of P<0.05 was considered to indicate a statistically significant difference.
To reveal the function of miR-19a-3p in diabetes, we first conducted RT-qPCR to examine the plasma miR-19a-3p level in diabetic patients and normal subjects. Our data demonstrated that the miR-19a-3p level was significantly decreased in the blood of diabetic patients, when compared with that of normal subjects (
The glucose-responsive pancreatic β cell lines, INS-1 and MIN6, were used in our
As miRs function by regulating the expression of their target genes, we then used TargetScan software to analyze the putative target genes of miR-19a-3p. SOCS3 was predicated to be a potential target gene of miR-19a-3p with evolutionary conservation (
As the overexpression of miR-19a-3p led to a significant decrease in the protein level of SOCS3, we investigated whether the downregulation of SOCS3 has similar effects as miR-19a-3p overexpression in pancreatic β cells. SOCS3 siRNA was used to transfect the INS-1 and MIN6 cells, and the protein level of SOCS3 was found to be signifi-cantly decreased following transfection (
Finally, we examined the plasma SOCS3 level using ELISA. We found that the SOCS3 level was significantly increased in the blood samples of diabetic patients compared with those of normal subjects (
Diabetes poses one of the most severe threats to human health worldwide, as it can induce high incidence rates of a number of complications. In recent years, various miRs, such as miR-375, miR-7, miR-124a2, miR-195, miR-126, miR-9, miR-96 and miR-34a have been reported to regulate the expression of a variety of genes that participate in the regulation of cell proliferation and apoptosis, as well as glucose-stimulated insulin secretion, and thus may play a role in pancreatic development and diabetes (
In the present study, the data from RT-qPCR demonstrated that the miR-19a-3p level was significantly decreased in the blood of diabetic patients compared with that of normal subjects. Moreover, a significant inverse correlation was observed between the plasma miR-19a-3p level and the blood glucose concentration among the diabetic patients, suggesting that the dysregulation of miR-19a-3p is associated with the progression of diabetes. INS-1 and MIN6 are glucose-responsive murine pancreatic β cell lines. Therefore, to further reveal the role of miR-19a-3p in pancreatic β cell function, the INS-1 and MIN6 cells were transfected with miR-19a-3p mimic. We found that the overexpression of miR-19a-3p promoted the proliferation and insulin secretion, while it suppressed the apoptosis of INS-1 and MIN6 cells, which further supports the notion that miR-19a-3p plays a role in diabetes, consistent with the clinical data.
Previous studies have mainly focused on the role of miR-19a-3p in human cancers. Yang
SOCS3 is a member of the SOCS family, which are negative regulators of cytokine signal transduction and inhibit the cytokine-induced activation of signal transducer and activator of transcription (Stat) signaling (
In conclusion, the findings of the present study demonstrate that miR-19a-3p is significantly downregulated in diabetic patients, and that its expression is inversely correlated with the blood glucose concentration. Moreover, our data indicate that miR-19a-3p significantly enhances the proliferation and insulin secretion, while it inhibits the apoptosis of pancreatic β cells (INS-1 and MIN6) by directly targeting SOCS3. Accordingly, miR-19a-3p may become a potential candidate for the treatment of diabetes. However, further studies are warranted to verify our findings.
(A) RT-qPCR was used to detect the miR-19a-3p level in the blood samples from type 2 diabetes patients (n=45) and normal subjects (n=20). (B) A significant inverse correlation was observed between the plasma miR-19a-3p level and the blood glucose concentration among patients with type 2 diabetes (n=45).
RT-qPCR was used to detect the miR-19a-3p level in (A) INS-1 and (B) MIN6 cells transfected with scramble miR mimic (miR-NC) or miR-19a-3p mimic. Untransfected INS-1 or MIN6 cells were used as a control. (C and D) Glucose-stimulated insulin secretion was then determined in each group. (E and F) MTT assay and (G and H) flow cytometry were then performed to examine cell proliferation and apoptosis. **P<0.01 vs. INS-1 or MIN6 untransfected cells (bars labeled as INS-1 or MIN6) or negative control (NC)-transfected cells.
(A and B) SOCS3 was predicated to be a potential target gene of miR-19a-3p with evolutionary conservation. (C and D) The wild-type (WT) SOCS3 3′-UTR containing the putative miR-19a-3p binding sites or the mutant type (MT) SOCS3 3′-UTR without the predicated miR-19a-3p binding sites were subcloned into the pMiR-REPORT miRNA Expression Reporter vector. (E) Luciferase reporter data indicated that co-transfection with the WT SOCS3 3′-UTR vector and miR-19a-3p mimic caused a significant decrease in luciferase activity; however, the luciferase activity was not altered in the 293 cells co-transfected with the MT SOCS3 3′-UTR vector and miR-19a-3p mimic. **P<0.01 vs. controls (untransfected cells or miR-NC-transfected cells). (F and G) The protein expression of SOCS3 was examined by western blot analysis in INS-1 and MIN6 cells transfected with negative control miR mimic (miR-NC) or miR-19a-3p mimic. **P<0.01 vs. INS-1 or MIN6 untransfected cells (bars labeled as INS-1 or MIN6) or negative control (NC)-transfected cells.
Western blot analysis was used to detect the protein expression of SOCS3 in (A) INS-1 and (B) MIN6 cells transfected with negative control miR mimic (miR-NC) or miR-19a-3p mimic. Untransfected INS-1 or MIN6 cells were used as controls. (C and D) Glucose-stimulated insulin secretion was then determined in each group. (E and F) MTT assay and (G and H) flow cytometry were then performed to examine cell proliferation and apoptosis. **P<0.01 vs. INS-1 or MIN6INS-1 or MIN6 untransfected cells (bars labeled as INS-1 or MIN6) or negative control (NC)-transfected cells.
Western blot analysis was used to detect the protein expression of SOCS3 in (A) INS-1 and (B) MIN6 cells transfected with miR-19a-3p mimic, or co-transfected with miR-19a-3p mimic and SOCS3 expression plasmid. (C and D) Glucose-stimulated insulin secretion was then determined in each group. (E and F) MTT assay and (G and H) flow cytometry were then performed to examine cell proliferation and apoptosis. **P<0.01 vs. cells transfected with miR-19a-3p and the negative control vector (bars labeled as miR-19a-3p).
(A) Enzyme-linked immunosorbent assay (ELISA) was used to detect the SOCS3 level in the blood samples from patients with type 2 diabetes (n=45) and normal subjects (n=20). (B) A significant inverse correlation was observed between the plasma miR-19a-3p level and blood glucose concentration among patients with type 2 diabetes (n=45).