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MiRNA-92a protects pancreatic B-cell function by targeting KLF2 in diabetes mellitus
Biochemical and Biophysical Research Communications 500 (2018) 577e582
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MiRNA-92a protects pancreatic B-cell function by targeting KLF2 in diabetes mellitus Wenyi Wang a, Jian Wang a, Meiling Yan b, Jiechun Jiang c, Ailin Bian a, * a
International Medical Center, Tianjin First Central Hospital, Tianjin, China Department of Pharmacy, Tianjin First Central Hospital, Tianjin, China c Clinical Laboratory, Tianjin First Central Hospital, Tianjin, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2018 Accepted 12 April 2018
Aims: diabetes mellitus is one of the most common metabolic diseases worldwide characterized by insulin resistance and pancreatic b cell dysfunction. miRNA plays an important role in DM. In previous studies, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. The aim of the study was to investigate the role of miRNA-92a in pancreatic b cell dysfunction. Methods: Apoptosis, proliferation, insulin secretion and cell survival rate were detected to evaluate the function of miRNA-92a. Results: we found that miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation. Moreover, we identify the KLF2 as direct target of miRNA-92a, suggesting that miRNA-92a may function through regulating KLF2. Conclusion: Altogether, we verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: miRNA-92a Diabetes Apoptosis KLF2
1. Introduction Diabetes mellitus (DM) is characterized as high blood glucose conditions and has been identified as the most threaten disease in the world [1]. DM can not only lead to serious health problem, but also caused heavy social economic burdens. Despite significant progress has been put into the clinical treatment and scientific research, the mechanism of DM remains poorly understood [2]. Pancreatic b cell dysfunction has been proved to serve a significant role in the progress and development of DM [3]. Hyperglycemia plays an important role in inducing pancreatic b cell apoptosis and insulin secretion [4]. Pancreatic b cells are specifically sensitive to hyperglycemia and dysfunctional pancreatic b cell can contribute to serious DM symptoms [5]. In present clinical treatment, it is important to maintain the normal glucose conditions and homeostasis. Previous studies have concentrated on how to protect and improve functional b cells to regulate glucose homeostasis [6]. However, the exact mechanisms of the dysfunctional
* Corresponding author. International Medical Center, Tianjin First Central Hospital, Tianjin, China. E-mail address: [email protected] (A. Bian).
b cells remain unknown. In recent years, new evidence estimated that approximately 95% of human genome transcripts are non-coding RNAs [7]. miRNA, a specific non coding RNA, 18e25 nucleotides in length, has been proved to associated with various disease, such as heart development [8], diabetes [9], obesity [10], tumor development [11]. miRNA could negatively regulate their target gene by directly binding to their 30 -UTR regions to induce mRNA deregulation and translational repression [12]. Previous studies have revealed that miRNA may play an important role in pancreatic function. For example, miR-375 was demonstrated to negatively regulate glucose-stimulated insulin secretion by regulating the expression of myotrophin and phosphoinositide-dependent protein kinase 1 [13]. The evidence all pointed the importance of miRNAs in disease aetiology. In previous study, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity [14]. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. The aim of this study was to elucidate the biological functions and potential target of miRNA92a in pancreatic b cells and to evaluate its role in a high-glucose environment. Our results showed that miRNA-92a could inhibit
https://doi.org/10.1016/j.bbrc.2018.04.097 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Taken together, we verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. 2. Materials and methods
2.5. Cell survival rates assay Cardiomyocytes were digested for 3 min and then pipetting completely. Trypan bule staining was used to assess the cell survival rates. Cell proliferation was detected using a CCK8 assay kit (Dojindo, Japan). Cells were seeded (1000 cells per well) into 96well plates, and CCK8 was added 0, 24, 48, 72 and 96 h later for 1 h, after which the OD values were recorded.
2.1. Cell culture and transfection 2.6. Caspase 3 activity assay Min-6 mouse pancreatic b cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's Modification of Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) in 5% CO2 at 37 C. Min-6 cells were treated with 25 mmol/L glucose for further experiment as previously described [15]. miRNA-92a mimics, miRNA-92a inhibitor and controls were constructed by Shanghai GenePharma (Shanghai, China). Min-6 cells were transfected with 50 nM miRNA-92a mimic, miRNA-92a inhibitor or NC miRNAs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. pcDNA-KLF2 transfection was used to perform overexpression experiment. The empty pcDNA3.1 (Invitrogen, Shanghai, China) was used as control. The protocol was same as miRNA-92 transfection. 2.2. Gene detection and western blotting Total RNA was extracted from Min-6 cells using TRIzol reagent (Thermo Fisher Scientific). The purity of RNA was determined by measuring the absorbance ratio of 260/280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) Reverse transcription of RNA was carried out using a PrimeScript™ RT reagent Kit with gDNA eraser (RR047A; Takara, Tokyo, Japan), and cDNA was performed by qRT-PCR using SYBR® Premix Ex Taq™(RR420A; Takara, Tokyo, Japan). The data were normalized using GAPDH levels and further analyzed by the 2DDCT method. All the primers used for qPCR are listed in the Table S1. Total protein was isolated from Min-6 cells and solubilized using RIPA lysis buffer containing proteinase inhibitor (Sigma, USA). Concentrations of total protein were determined using a BCA assay kit (Pierce, Rockford, IL, USA). Total protein samples (30 mg) were analyzed by 10% SDS-PAGE gel (120 V, 120 mins) and transferred to polyvinylidene difluoride (PVDF) membrane (300 mA, 90 min). After 1 h blocking using milk, the membranes were incubated with primary antibodies against KLF2 (1:1000, Abcam, MA, USA), Caspase3 (1:1000, Abcam, MA, USA), Notch (1:1000, Abcam, MA, USA) at 4 C overnight. Immunopositive bands were analyzed using a FluorChem M system (ProteinSimple, San Jose, CA, USA). 2.3. Insulin secretion The cells were seeded in a 96-well plate and cultured for 24 h. ELISA was used to determine the insulin level. Total insulin content was measured after sonication of cells in acid ethanol (2% H2SO4), followed by 3 cycles, and then centrifuged for 5 min at 10,000 g. The supernatant was used to further measure the insulin level. 2.4. ROS detection The cells were seeded in a 6-well plate and cultured for 24 h. The cells were stained with DCFH-DA (10 mmol/L, Beyotime, Shanghai, China) at 37 C for 30 min and the intracellular ROS was detected using fluorescence microscopy (BX61, Olympus). The results were analyzed by Image J software (version 1.48, USA).
Total protein was isolated from Min-6 cells and solubilized using RIPA lysis buffer containing proteinase inhibitor (Sigma, USA). Concentrations of total protein were determined using a BCA assay kit (Pierce, Rockford, IL, USA). The supernatant were mixed with reaction buffers using Caspase-3 activity kits (caspase-3, Ac-DEVDpNA) and incubated at 37 C for 2 h. The Caspase-3 activity was detected by the fluorescence level. 2.7. Luciferase reporter assay The 30 -UTR of KLF2, with wild-type or mutant (mut) binding sites for miRNA-92a, was amplified and cloned into the pGL3 vector (Promega, Madison, WI, USA) to generate the plasmid pGL3-wtKLF2-30 -UTR or pGL3-mut-KLF2-30 -UTR. HEK 293 cells were used to perform the luciferase reported assay, and the miRNA-92a mimics or inhibitor and the KLF2 vector were transfected using Lipofectamine 2000 reagent. Luciferase activity was analyzed using DualLuciferase system following the manufacture's protocol. 2.8. Statistical analysis SPSS 13.0 was used to calculate all the values (means ± standard error of the mean (SEM)). Statistical analyses were analyzed with Student's t-test. The statistical significance was P < 0.05. 3. Results 3.1. Bioinformatics information of miRNA-92a High-glucose induced dysfunctional pancreatic b cell model is one most commonly used diabetes model. Firstly, we examined the expression level of miRNA-92a in HG conditions. miRNA-92a was decreased under HG conditions in a time dependent manner (Fig. 1A). To evaluate the role of miRNA-92a in pancreatic b cell, we constructed the miRNA-92a mimics to verify the function of miRNA-92a. The expression of miRNA-92a was significantly increased compared with control (Fig. 1B). Furthermore, transcripts of miRNA-92a could be detected in multiple tissues in adult mice (Fig. 1C). Assessment of the distribution indicated that miRNA-92a is mainly expressed in pancreas and lungs, suggesting that miRNA92a may have an important function in pancreas. 3.2. MiRNA-92a attenuated HG-induced apoptosis in Min-6 cells To investigate the role of miRNA-92a in HG conditions, caspase3 activity was measured to assess the apoptosis effect. We found that caspase3 activity was increased in HG group, whereas miRNA-92a group showed opposite effect. miRNA-92a could inhibit the HGinduced caspase3 activity (Fig. 1D). In addition, we also detected the protein level of caspase3 through western blotting. Interestingly, the cleaved caspase3 band was significantly decreased compared with control group (Fig. 1E). All the evidence emphasizes the anti-apoptosis effect of miRNA-92a.
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Fig. 1.
3.3. MiRNA-92a inhibits insulin secretion and ROS release To further study the potential function of miRNA-92a, we detected the insulin secretion by ELISA. Insulin secretion and content in response to HG stimulus was increased in miRNA-92a groups (Fig. 2A and B). Subsequently, we detected the reactive oxygen species (ROS) production, which play an important role in oxidative stress. ROS production was significantly increased, whereas miRNA-92a group decreased (Fig. 2C), suggesting miRNA92a may attenuate oxidative stress. The CCK-8 assay was used to detect the proliferation of Min-6 cells that overexpressed miRNA92a over a 96 h-period (Fig. 2D). We also measured the cell survival rates using trypan staining. miRNA-92a group showed higher cell survival rates (Fig. 2E).
3.4. KLF2 is a direct target of miRNA-92a To further evaluate the molecular mechanism of miRNA-92a, the potential target of miRNA-92a was predicted using TargetScan software (www.targetscan.org). Interestingly, KLF2, which has been associated with diabetes [14], was certified to be the potential target of miRNA-92a (Fig. 3A). In order to confirm the direct binding relationship, luciferase reporter assay was performed. In KLF2-30 UTR-WT transfection group, after transfected with miRNA-92a mimics, the relative activity of luciferase significantly decreased compared with the mimics NC groups (Fig. 3B), whereas the relative activity of luciferase increased after transfected with miRNA92a inhibitor. However, there is no difference in the KLF2-30 -UTRmut transfection group, no matter transfected with miRNA-92a mimics or miRNA-92a inhibitor (Fig. 3B). We also demonstrated that the expression of KLF2 is inversely associated with miRNA-92a (Fig. 3C). To further confirm the result, we detected the protein level
of KLF2, which was decreased in the miRNA-92a mimics group, whereas increased in the miRNA-92a inhibitor group (Fig. 3D). Thus, miRNA-92a could negatively regulate the KLF2 expression.
3.5. MiRNA-92a protects pancreatic B-cell function by targeting KLF2 After confirming KLF2 was a direct target of miRNA-92a, we further elucidated whether the function of miRNA-92a is elaborated through downregulation of KLF2. Firstly, KLF2 overexpression vector was constructed. After co-transfected into min-6 cells, the insulin secretion, apoptosis activity, proliferation rates and cell survival rates were detected to assess the relationship between miRNA-92a and KLF2. Notably, the insulin secretion and content were decreased to a lower level compared with miRNA-92a group (Fig. 4A and B). Next, we measured the ROS production and caspase3 activity, which was slightly increased in the co-transfected group (Fig. 4C and D). CCK8 assay was used to analyze the proliferation rate. The proliferation rate was increased compared with miRNA-92a mimics group (Fig. 4E). We also detected the KLF2 level in HG conditions, the KLF2 level was significantly decreased in the miRNA-92a overexpressed group (Fig. 4F and G). Trypan staining was used to evaluate the cell survival rate, co-transfected group show a higher survival rate (Fig. 4H). To the end, caspase3 was detected to assess the apoptosis effect. Cleaved caspase3 band was significantly increased in the co-transfected group compared with miRNA-92a group in HG conditions (Fig. 4I and J). Recent studies have shown that KLF2 may function through Notch signaling pathway [16], so we detected the Notch level, which was decreased in KLF2 group, whereas increased in the co-transfected group (Fig. 4K and L), suggesting that miRNA-92a may function through Notch signaling pathway.
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Fig. 2.
Fig. 3.
4. Discussion
biological features and functions of miRNA-92a in pancreatic
b cells. Moreover, we also identify the possible mechanism of In previous study, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. In this study, we elucidated the
miRNA-92a and evaluate its role in a high-glucose environment. Our results showed that miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Taken together, we
W. Wang et al. / Biochemical and Biophysical Research Communications 500 (2018) 577e582
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Fig. 4.
verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. Considerable evidences indicate that miRNA play a critical role in progress and development in DM [17]. miRNA-29c could promotes the inflammatory response through regulating tristetraprolin (TTP) [18]. miR-19a-3p could promotes the proliferation and insulin secretion by targeting suppressor of cytokine signaling3 (SOCS3) [19]. In recent study, miRNA-185 could inhibit the pancreatic cells apoptosis [20]. Although many studies and efforts have been put into the field, the mechanisms and the key genes relevant with DM remains largely unknown. In our study, we identified the biological function of miRNA-92a, and found the potential binding target, KLF2. Interestingly, a negative correlation between miRNA-92a and KLF2 was detected, which indicated that miRNA-92a could work as a potential biomarker of DM. Our study suggests that miRNA-92a may be associated with the progression of DM. Recently, many new sights and efforts have already been carried out in the key genes and signaling pathways of DM [21]. To further identify the possible mechanism of miRNA-92a, bioinformatics analysis was performed to identify the target gene. KLF2 was hypothesized to be a potential target of miRNA-92a, which was further confirmed by luciferase reporter assay. The influence of miRNA-92a could be reversed by overexpression of
KLF2, suggesting that KLF2 was the direct target of miRNA-92a. In previous studies, the exendin-4 could promote the proliferation of pancreatic b cell by regulating PI3K/AKT signaling pathway [22]. miRNA-21 could activate the PI3K/AKT signaling pathway [23]. To further study the possible pathways that associated with DM, the effect of aberrantly expressed KLF2 on signaling pathway were investigated. Interestingly, the Notch level was slightly decreased in the KLF2 overexpression group. Our results suggested that Notch pathway was involved in the regulation of KLF2 in pancreatic b cells. In summary, we have elucidated the biological features and functions of miRNA-92a, and expound its possible mechanism in pancreatic b cells. miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Indeed, miRNA have been shown to be associated with various diseases, including cancer development, demonstrating potential applications as novel diagnostic biomarkers in clinical treatment. Understanding the precise role of miRNA-92a might ultimately shed new light for the therapeutics of DM. Acknowledgments This work was supported by Tianjin Municipal Commission of health and family planning (2014KY13, 15KG132).
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Conflicts of interest All authors read and approved the final manuscript. The authors declare no competing financial interests. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.097. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.04.097. References [1] J.J. Chamberlain, E.L. Johnson, S. Leal, et al., Cardiovascular disease and risk management: review of the American diabetes association standards of medical care in diabetes 2018, Ann. Intern. Med. (2018 Apr 3) [Epub ahead of print]. [2] X. Jin, D.D. Zhu, B.Z. Chen, et al., Insulin delivery systems combined with microneedle technology, Adv. Drug Deliv. Rev. (2018 Mar 28) [Epub ahead of print]. taux, The healthy diabetic cavefish conundrum, Nature 555 (2018) [3] S. Re 595e597. [4] C. Aguayo-Mazzucato, M. van Haaren, M. Mruk, et al., b cell aging markers have heterogeneous distribution and are induced by insulin resistance, Cell Metabol. 25 (2017) 898e910 e895. [5] F.M. Ashcroft, M. Rohm, A. Clark, et al., Is type 2 diabetes a glycogen storage disease of pancreatic b cells? Cell Metabol. 26 (2017) 17e23. [6] N.R. Johnston, R.K. Mitchell, E. Haythorne, et al., Beta cell hubs dictate pancreatic islet responses to glucose, Cell Metabol. 13 (2016) 389e401. [7] T. Derrien, R. Johnson, G. Bussotti, et al., The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression, Genome Res. 22 (2012) 1775. [8] Z. Cheng, Q. Zhang, A. Yin, et al., The long non-coding RNA uc.4 influences cell differentiation through the TGF-beta signaling pathway, Exp. Mol. Med. 50 (2018) e447.
[9] M. Wang, miR-433 protects pancreatic b cell growth in high-glucose conditions, Mol. Med. Rep. 16 (2017) 2604e2610. [10] N.L. Price, A.K. Singh, N. Rotllan, et al., Genetic ablation of miR-33 increases food intake, enhances adipose tissue expansion, and promotes obesity and insulin resistance, Cell Rep. 22 (2018) 2133e2145. [11] H. Yin, J. Ma, L. Chen, et al., MiR-99a enhances the radiation sensitivity of nonsmall cell lung cancer by targeting mTOR, Cell. Physiol. Biochem. 46 (2018) 471e481. [12] M. Awazawa, P. Gabel, E. Tsaousidou, et al., A microRNA screen reveals that elevated hepatic ectodysplasin A expression contributes to obesity-induced insulin resistance in skeletal muscle, Nat. Med. 23 (2017) 1466e1473. [13] Y. Chang, W. Yan, X. He, et al., miR-375 inhibits autophagy and reduces viability of hepatocellular carcinoma cells under hypoxic conditions, Gastroenterology 143 (2012) 177e187 e178. [14] I. Serr, R.W. Fürst, V.B. Ott, et al., miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) e6659ee6668. [15] F.Y. Wei, K. Nagashima, T. Ohshima, et al., Cdk5-dependent regulation of glucose-stimulated insulin secretion, Nat. Med. 11 (2005) 1104e1108. [16] F. Li, J. Sun, S. Huang, et al., LncRNA GAS5 overexpression reverses LPSinduced inflammatory injury and apoptosis through up-regulating KLF2 expression in ATDC5 chondrocytes, Cell. Physiol. Biochem. 45 (2018) 1241e1251. [17] W. Ying, M. Riopel, G. Bandyopadhyay, et al., Adipose tissue macrophagederived exosomal miRNAs can modulate in vivo and in vitro insulin sensitivity, Cell 171 (2017) 372e384. [18] J. Guo, J. Li, J. Zhao, et al., MiRNA-29c regulates the expression of inflammatory cytokines in diabetic nephropathy by targeting tristetraprolin, Sci. Rep. 7 (2017) 2314. [19] Y. Li, T. Luo, L. Wang, et al., MicroRNA-19a-3p enhances the proliferation and insulin secretion, while it inhibits the apoptosis of pancreatic b cells via the inhibition of SOCS3, Int. J. Mol. Med. 38 (2016) 1515e1524. [20] J.S. Imam, K. Buddavarapu, J.S. Lee-Chang, et al., MicroRNA-185 suppresses tumor growth and progression by targeting the Six1 oncogene in human cancers, Oncogene 29 (2010) 4971e4979. [21] T. Thomou, M.A. Mori, J.M. Dreyfuss, et al., Adipose-derived circulating miRNAs regulate gene expression in other tissues, Nature 542 (2017) 450e455. [22] E. Candeias, I. Sebasti~ ao, S. Cardoso, et al., Brain GLP-1/IGF-1 signaling and autophagy mediate Exendin-4 protection against apoptosis in type 2 diabetic rats, Mol. Neurobiol. 55 (2017) 4030e4050. [23] X. Lu, C. Sun, D. Zheng, et al., The miR-21 attenuates hepatocyte hypoxia/ reoxygenation injury via inhibiting PTEN/PI3K/AKT signaling pathway, Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 33 (2017) 497e502.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MiRNA-92a protects pancreatic B-cell function by targeting KLF2 in diabetes mellitus Wenyi Wang a, Jian Wang a, Meiling Yan b, Jiechun Jiang c, Ailin Bian a, * a
International Medical Center, Tianjin First Central Hospital, Tianjin, China Department of Pharmacy, Tianjin First Central Hospital, Tianjin, China c Clinical Laboratory, Tianjin First Central Hospital, Tianjin, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2018 Accepted 12 April 2018
Aims: diabetes mellitus is one of the most common metabolic diseases worldwide characterized by insulin resistance and pancreatic b cell dysfunction. miRNA plays an important role in DM. In previous studies, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. The aim of the study was to investigate the role of miRNA-92a in pancreatic b cell dysfunction. Methods: Apoptosis, proliferation, insulin secretion and cell survival rate were detected to evaluate the function of miRNA-92a. Results: we found that miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation. Moreover, we identify the KLF2 as direct target of miRNA-92a, suggesting that miRNA-92a may function through regulating KLF2. Conclusion: Altogether, we verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: miRNA-92a Diabetes Apoptosis KLF2
1. Introduction Diabetes mellitus (DM) is characterized as high blood glucose conditions and has been identified as the most threaten disease in the world [1]. DM can not only lead to serious health problem, but also caused heavy social economic burdens. Despite significant progress has been put into the clinical treatment and scientific research, the mechanism of DM remains poorly understood [2]. Pancreatic b cell dysfunction has been proved to serve a significant role in the progress and development of DM [3]. Hyperglycemia plays an important role in inducing pancreatic b cell apoptosis and insulin secretion [4]. Pancreatic b cells are specifically sensitive to hyperglycemia and dysfunctional pancreatic b cell can contribute to serious DM symptoms [5]. In present clinical treatment, it is important to maintain the normal glucose conditions and homeostasis. Previous studies have concentrated on how to protect and improve functional b cells to regulate glucose homeostasis [6]. However, the exact mechanisms of the dysfunctional
* Corresponding author. International Medical Center, Tianjin First Central Hospital, Tianjin, China. E-mail address: [email protected] (A. Bian).
b cells remain unknown. In recent years, new evidence estimated that approximately 95% of human genome transcripts are non-coding RNAs [7]. miRNA, a specific non coding RNA, 18e25 nucleotides in length, has been proved to associated with various disease, such as heart development [8], diabetes [9], obesity [10], tumor development [11]. miRNA could negatively regulate their target gene by directly binding to their 30 -UTR regions to induce mRNA deregulation and translational repression [12]. Previous studies have revealed that miRNA may play an important role in pancreatic function. For example, miR-375 was demonstrated to negatively regulate glucose-stimulated insulin secretion by regulating the expression of myotrophin and phosphoinositide-dependent protein kinase 1 [13]. The evidence all pointed the importance of miRNAs in disease aetiology. In previous study, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity [14]. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. The aim of this study was to elucidate the biological functions and potential target of miRNA92a in pancreatic b cells and to evaluate its role in a high-glucose environment. Our results showed that miRNA-92a could inhibit
https://doi.org/10.1016/j.bbrc.2018.04.097 0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Taken together, we verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. 2. Materials and methods
2.5. Cell survival rates assay Cardiomyocytes were digested for 3 min and then pipetting completely. Trypan bule staining was used to assess the cell survival rates. Cell proliferation was detected using a CCK8 assay kit (Dojindo, Japan). Cells were seeded (1000 cells per well) into 96well plates, and CCK8 was added 0, 24, 48, 72 and 96 h later for 1 h, after which the OD values were recorded.
2.1. Cell culture and transfection 2.6. Caspase 3 activity assay Min-6 mouse pancreatic b cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's Modification of Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) in 5% CO2 at 37 C. Min-6 cells were treated with 25 mmol/L glucose for further experiment as previously described [15]. miRNA-92a mimics, miRNA-92a inhibitor and controls were constructed by Shanghai GenePharma (Shanghai, China). Min-6 cells were transfected with 50 nM miRNA-92a mimic, miRNA-92a inhibitor or NC miRNAs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. pcDNA-KLF2 transfection was used to perform overexpression experiment. The empty pcDNA3.1 (Invitrogen, Shanghai, China) was used as control. The protocol was same as miRNA-92 transfection. 2.2. Gene detection and western blotting Total RNA was extracted from Min-6 cells using TRIzol reagent (Thermo Fisher Scientific). The purity of RNA was determined by measuring the absorbance ratio of 260/280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) Reverse transcription of RNA was carried out using a PrimeScript™ RT reagent Kit with gDNA eraser (RR047A; Takara, Tokyo, Japan), and cDNA was performed by qRT-PCR using SYBR® Premix Ex Taq™(RR420A; Takara, Tokyo, Japan). The data were normalized using GAPDH levels and further analyzed by the 2DDCT method. All the primers used for qPCR are listed in the Table S1. Total protein was isolated from Min-6 cells and solubilized using RIPA lysis buffer containing proteinase inhibitor (Sigma, USA). Concentrations of total protein were determined using a BCA assay kit (Pierce, Rockford, IL, USA). Total protein samples (30 mg) were analyzed by 10% SDS-PAGE gel (120 V, 120 mins) and transferred to polyvinylidene difluoride (PVDF) membrane (300 mA, 90 min). After 1 h blocking using milk, the membranes were incubated with primary antibodies against KLF2 (1:1000, Abcam, MA, USA), Caspase3 (1:1000, Abcam, MA, USA), Notch (1:1000, Abcam, MA, USA) at 4 C overnight. Immunopositive bands were analyzed using a FluorChem M system (ProteinSimple, San Jose, CA, USA). 2.3. Insulin secretion The cells were seeded in a 96-well plate and cultured for 24 h. ELISA was used to determine the insulin level. Total insulin content was measured after sonication of cells in acid ethanol (2% H2SO4), followed by 3 cycles, and then centrifuged for 5 min at 10,000 g. The supernatant was used to further measure the insulin level. 2.4. ROS detection The cells were seeded in a 6-well plate and cultured for 24 h. The cells were stained with DCFH-DA (10 mmol/L, Beyotime, Shanghai, China) at 37 C for 30 min and the intracellular ROS was detected using fluorescence microscopy (BX61, Olympus). The results were analyzed by Image J software (version 1.48, USA).
Total protein was isolated from Min-6 cells and solubilized using RIPA lysis buffer containing proteinase inhibitor (Sigma, USA). Concentrations of total protein were determined using a BCA assay kit (Pierce, Rockford, IL, USA). The supernatant were mixed with reaction buffers using Caspase-3 activity kits (caspase-3, Ac-DEVDpNA) and incubated at 37 C for 2 h. The Caspase-3 activity was detected by the fluorescence level. 2.7. Luciferase reporter assay The 30 -UTR of KLF2, with wild-type or mutant (mut) binding sites for miRNA-92a, was amplified and cloned into the pGL3 vector (Promega, Madison, WI, USA) to generate the plasmid pGL3-wtKLF2-30 -UTR or pGL3-mut-KLF2-30 -UTR. HEK 293 cells were used to perform the luciferase reported assay, and the miRNA-92a mimics or inhibitor and the KLF2 vector were transfected using Lipofectamine 2000 reagent. Luciferase activity was analyzed using DualLuciferase system following the manufacture's protocol. 2.8. Statistical analysis SPSS 13.0 was used to calculate all the values (means ± standard error of the mean (SEM)). Statistical analyses were analyzed with Student's t-test. The statistical significance was P < 0.05. 3. Results 3.1. Bioinformatics information of miRNA-92a High-glucose induced dysfunctional pancreatic b cell model is one most commonly used diabetes model. Firstly, we examined the expression level of miRNA-92a in HG conditions. miRNA-92a was decreased under HG conditions in a time dependent manner (Fig. 1A). To evaluate the role of miRNA-92a in pancreatic b cell, we constructed the miRNA-92a mimics to verify the function of miRNA-92a. The expression of miRNA-92a was significantly increased compared with control (Fig. 1B). Furthermore, transcripts of miRNA-92a could be detected in multiple tissues in adult mice (Fig. 1C). Assessment of the distribution indicated that miRNA-92a is mainly expressed in pancreas and lungs, suggesting that miRNA92a may have an important function in pancreas. 3.2. MiRNA-92a attenuated HG-induced apoptosis in Min-6 cells To investigate the role of miRNA-92a in HG conditions, caspase3 activity was measured to assess the apoptosis effect. We found that caspase3 activity was increased in HG group, whereas miRNA-92a group showed opposite effect. miRNA-92a could inhibit the HGinduced caspase3 activity (Fig. 1D). In addition, we also detected the protein level of caspase3 through western blotting. Interestingly, the cleaved caspase3 band was significantly decreased compared with control group (Fig. 1E). All the evidence emphasizes the anti-apoptosis effect of miRNA-92a.
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Fig. 1.
3.3. MiRNA-92a inhibits insulin secretion and ROS release To further study the potential function of miRNA-92a, we detected the insulin secretion by ELISA. Insulin secretion and content in response to HG stimulus was increased in miRNA-92a groups (Fig. 2A and B). Subsequently, we detected the reactive oxygen species (ROS) production, which play an important role in oxidative stress. ROS production was significantly increased, whereas miRNA-92a group decreased (Fig. 2C), suggesting miRNA92a may attenuate oxidative stress. The CCK-8 assay was used to detect the proliferation of Min-6 cells that overexpressed miRNA92a over a 96 h-period (Fig. 2D). We also measured the cell survival rates using trypan staining. miRNA-92a group showed higher cell survival rates (Fig. 2E).
3.4. KLF2 is a direct target of miRNA-92a To further evaluate the molecular mechanism of miRNA-92a, the potential target of miRNA-92a was predicted using TargetScan software (www.targetscan.org). Interestingly, KLF2, which has been associated with diabetes [14], was certified to be the potential target of miRNA-92a (Fig. 3A). In order to confirm the direct binding relationship, luciferase reporter assay was performed. In KLF2-30 UTR-WT transfection group, after transfected with miRNA-92a mimics, the relative activity of luciferase significantly decreased compared with the mimics NC groups (Fig. 3B), whereas the relative activity of luciferase increased after transfected with miRNA92a inhibitor. However, there is no difference in the KLF2-30 -UTRmut transfection group, no matter transfected with miRNA-92a mimics or miRNA-92a inhibitor (Fig. 3B). We also demonstrated that the expression of KLF2 is inversely associated with miRNA-92a (Fig. 3C). To further confirm the result, we detected the protein level
of KLF2, which was decreased in the miRNA-92a mimics group, whereas increased in the miRNA-92a inhibitor group (Fig. 3D). Thus, miRNA-92a could negatively regulate the KLF2 expression.
3.5. MiRNA-92a protects pancreatic B-cell function by targeting KLF2 After confirming KLF2 was a direct target of miRNA-92a, we further elucidated whether the function of miRNA-92a is elaborated through downregulation of KLF2. Firstly, KLF2 overexpression vector was constructed. After co-transfected into min-6 cells, the insulin secretion, apoptosis activity, proliferation rates and cell survival rates were detected to assess the relationship between miRNA-92a and KLF2. Notably, the insulin secretion and content were decreased to a lower level compared with miRNA-92a group (Fig. 4A and B). Next, we measured the ROS production and caspase3 activity, which was slightly increased in the co-transfected group (Fig. 4C and D). CCK8 assay was used to analyze the proliferation rate. The proliferation rate was increased compared with miRNA-92a mimics group (Fig. 4E). We also detected the KLF2 level in HG conditions, the KLF2 level was significantly decreased in the miRNA-92a overexpressed group (Fig. 4F and G). Trypan staining was used to evaluate the cell survival rate, co-transfected group show a higher survival rate (Fig. 4H). To the end, caspase3 was detected to assess the apoptosis effect. Cleaved caspase3 band was significantly increased in the co-transfected group compared with miRNA-92a group in HG conditions (Fig. 4I and J). Recent studies have shown that KLF2 may function through Notch signaling pathway [16], so we detected the Notch level, which was decreased in KLF2 group, whereas increased in the co-transfected group (Fig. 4K and L), suggesting that miRNA-92a may function through Notch signaling pathway.
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Fig. 2.
Fig. 3.
4. Discussion
biological features and functions of miRNA-92a in pancreatic
b cells. Moreover, we also identify the possible mechanism of In previous study, miRNA-92a could function as targets for innovative precision medicines to reduce T1D islet autoimmunity. However, the relationship between miRNA-92a and pancreatic b cell dysfunction remains unknown. In this study, we elucidated the
miRNA-92a and evaluate its role in a high-glucose environment. Our results showed that miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Taken together, we
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Fig. 4.
verified the function and mechanism of miRNA-92a and provide evidence that miRNA-92a may serve a potential candidate for the clinical treatment for DM. Considerable evidences indicate that miRNA play a critical role in progress and development in DM [17]. miRNA-29c could promotes the inflammatory response through regulating tristetraprolin (TTP) [18]. miR-19a-3p could promotes the proliferation and insulin secretion by targeting suppressor of cytokine signaling3 (SOCS3) [19]. In recent study, miRNA-185 could inhibit the pancreatic cells apoptosis [20]. Although many studies and efforts have been put into the field, the mechanisms and the key genes relevant with DM remains largely unknown. In our study, we identified the biological function of miRNA-92a, and found the potential binding target, KLF2. Interestingly, a negative correlation between miRNA-92a and KLF2 was detected, which indicated that miRNA-92a could work as a potential biomarker of DM. Our study suggests that miRNA-92a may be associated with the progression of DM. Recently, many new sights and efforts have already been carried out in the key genes and signaling pathways of DM [21]. To further identify the possible mechanism of miRNA-92a, bioinformatics analysis was performed to identify the target gene. KLF2 was hypothesized to be a potential target of miRNA-92a, which was further confirmed by luciferase reporter assay. The influence of miRNA-92a could be reversed by overexpression of
KLF2, suggesting that KLF2 was the direct target of miRNA-92a. In previous studies, the exendin-4 could promote the proliferation of pancreatic b cell by regulating PI3K/AKT signaling pathway [22]. miRNA-21 could activate the PI3K/AKT signaling pathway [23]. To further study the possible pathways that associated with DM, the effect of aberrantly expressed KLF2 on signaling pathway were investigated. Interestingly, the Notch level was slightly decreased in the KLF2 overexpression group. Our results suggested that Notch pathway was involved in the regulation of KLF2 in pancreatic b cells. In summary, we have elucidated the biological features and functions of miRNA-92a, and expound its possible mechanism in pancreatic b cells. miRNA-92a could inhibit apoptosis induced by high-glucose environment and increase the insulin secretion and proliferation by targeting KLF2. Indeed, miRNA have been shown to be associated with various diseases, including cancer development, demonstrating potential applications as novel diagnostic biomarkers in clinical treatment. Understanding the precise role of miRNA-92a might ultimately shed new light for the therapeutics of DM. Acknowledgments This work was supported by Tianjin Municipal Commission of health and family planning (2014KY13, 15KG132).
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Conflicts of interest All authors read and approved the final manuscript. The authors declare no competing financial interests. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.097. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.04.097. References [1] J.J. Chamberlain, E.L. Johnson, S. Leal, et al., Cardiovascular disease and risk management: review of the American diabetes association standards of medical care in diabetes 2018, Ann. Intern. Med. (2018 Apr 3) [Epub ahead of print]. [2] X. Jin, D.D. Zhu, B.Z. Chen, et al., Insulin delivery systems combined with microneedle technology, Adv. Drug Deliv. Rev. (2018 Mar 28) [Epub ahead of print]. taux, The healthy diabetic cavefish conundrum, Nature 555 (2018) [3] S. Re 595e597. [4] C. Aguayo-Mazzucato, M. van Haaren, M. Mruk, et al., b cell aging markers have heterogeneous distribution and are induced by insulin resistance, Cell Metabol. 25 (2017) 898e910 e895. [5] F.M. Ashcroft, M. Rohm, A. Clark, et al., Is type 2 diabetes a glycogen storage disease of pancreatic b cells? Cell Metabol. 26 (2017) 17e23. [6] N.R. Johnston, R.K. Mitchell, E. Haythorne, et al., Beta cell hubs dictate pancreatic islet responses to glucose, Cell Metabol. 13 (2016) 389e401. [7] T. Derrien, R. Johnson, G. Bussotti, et al., The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression, Genome Res. 22 (2012) 1775. [8] Z. Cheng, Q. Zhang, A. Yin, et al., The long non-coding RNA uc.4 influences cell differentiation through the TGF-beta signaling pathway, Exp. Mol. Med. 50 (2018) e447.
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