Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-014-2692-5
RETINAL DISORDERS
Comparisons of microRNA expression profiles in vitreous humor between eyes with macular hole and eyes with proliferative diabetic retinopathy Kazunari Hirota & Hiroshi Keino & Makoto Inoue & Hitoshi Ishida & Akito Hirakata
Received: 29 March 2014 / Revised: 2 June 2014 / Accepted: 3 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose MicroRNAs (miRNAs) are small noncoding RNAs which regulate the activities of target mRNAs. We compared the expression profiles of the miRNAs in the vitreous of eyes with macular hole (MH) to that in eyes with proliferative diabetic retinopathy (PDR). Methods Vitreous and whole blood samples were collected from four patients with MH and from four patients with PDR. We assayed for 168 miRNAs in the vitreous and serum samples by the microRNA PCR Panel method. Results The mean number of miRNAs expressed in the vitreous was 63 (55–69) in eyes with MH and 86 (65–117) in eyes with PDR. The mean number of miRNAs expressed in the serum was 162 (159–167) in the MH patients and 142 (115– 160) in the PDR patients. Twenty-six miRNAs were expressed in the vitreous of both MH and PDR eyes. Although there was no significant difference in the levels of 20 of the 26 (73 %) miRNAs expressed in both MH and PDR eyes, six of 26 miRNAs (24 %) (hsa-miR-15a, hsa-miR320a, hsa-miR320b, hsa-miR-93, hsa-miR-29a, and hsa-miR-423-5p) were expressed significantly more highly in PDR eyes. In addition, the mean fold changes of three miRNAs, hsa-miR-23a, hsamiR-320a, and hsa-miR-320b, in the vitreous to serum were significantly higher in the PDR group than in the MH group. Conclusions The expression of several miRNAs related to angiogenesis and fibrosis was expressed significantly higher in the vitreous of eyes with PDR. Further studies are needed to
K. Hirota (*) : H. Keino : M. Inoue : A. Hirakata Department of Ophthalmology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan e-mail: [email protected] H. Ishida Department of 3rd Internal Medicine, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
understand the role played by the miRNAs in the biological function of the eye. Keywords microRNA . Vitreous humor . Proliferative diabetic retinopathy . Macular hole
Introduction Micro-RNAs (miRNAs) are non-coding, 21–24 nucleotide long, RNAs that have recently emerged as important posttranscriptional regulators of gene expression. They play critical roles in proliferation, differentiation, apoptosis, immunity, and angiogenesis [1–5]. MiRNAs have been found in the serum, plasma, and other body fluids such as urine and saliva [6, 7]. Recent studies have shown that some miRNAs are present in the vitreous of the eye, and the expression of microRNA-155 was elevated more significantly in the vitreous of eyes with uveitis than eyes with vitreoretinal lymphoma [8, 9]. However, the number of studies comparing the type and level of miRNA expression in different ocular diseases is limited. Diabetic retinopathy (DR) is an example of a disease that is a major cause of vision reduction [10]. In particular, proliferative diabetic retinopathy (PDR) which is caused by retinal ischemia and angiogenesis in the retina and vitreoretinal interface is mainly responsible for the blindness in eyes with DR. Although panretinal photocoagulation and vitrectomy are the main treatments for PDR, anti-vascular endothelial growth factor (VEGF) treatments have emerged in recent years as another treatment option [11]. However, anti-VEGF therapy has its limitations, e.g., recurrences of the neovascularization can occur in a few months after the anti-VEGF injection, systemic side-effects such as cerebrovascular or cardiovascular events can develop, endophthalmitis related to the intravitreal injections can also develop, and the anti-VEGF injections can alter the normal retinal vascular structure and reduce the
Graefes Arch Clin Exp Ophthalmol
retinal and choroidal blood flow in monkey eyes [12, 13]. Therefore, it is necessary to search for other therapeutic methods to treat the neovascularization in eyes with PDR. To accomplish this, we need to determine the pathophysiology of the vitreous of eyes with PDR in more detail. MiRNAs have been detected in the vitreous of eyes with different types of retinal diseases [8, 9], but the differential expression profiles of miRNAs in different retinal diseases have not been determined. Thus, the aim of this study was to compare the miRNA expression profiles in the vitreous of eyes with macular hole (MH) to that of eyes with PDR. A MH is considered to be a non-inflammatory/non-proliferative disease, while PDR is associated with inflammation and proliferation caused by angiogenesis. We shall show that the vitreous contained some miRNAs that were present in both types of eyes, and also that several miRNAs related to the regulation of angiogenesis and fibrosis were expressed at significantly higher levels in the vitreous of eyes of PDR patients. We discuss the potential roles of the miRNA in the vitreous of eyes with PDR.
Materials and methods Patients Vitreous and blood serum samples were collected from four patients with MH (two men and two women, ages 57 to 75; MH group) and four patients with PDR (two men and two women, ages 40 to 67 years; PDR group). All of the patients underwent vitrectomy at the Kyorin Eye Center between April 2011 and March 2013. We excluded patients with systemic disease such as hypertension, diabetes mellitus, autoimmune diseases, and cancer in the MH group. In the PDR group, patients with diabetes mellitus and hypertension were included; however, patients with other systemic diseases were excluded. Patients with history of ocular diseases and ocular surgeries were excluded from both groups. The procedures used in this study conformed to the tenets of the Declaration of Helsinki, and an informed consent was obtained from each patient after an explanation of the purpose and possible consequences of the procedures. This study was approved by the Kyorin University Hospital Research Ethics Committee. Collection of samples Peripheral blood (7 ml) was collected from the patients before surgery, and immediately centrifuged at 3,500 rpm for 7 min to isolate the blood serum. All surgeries were performed by one experienced vitreoretinal surgeon. Before beginning the surgery, one 25-G trocar was inserted 3 mm posterior to the corneal limbus by a transconjunctival one-step incision.
Vitreous (1.0 ml) was collected before the infusion, and it was immediately centrifuged at 400×g (1,500 rpm) for 10 min to remove any cell debris. All samples were preserved at -80 ° C until assayed. Sample preparation The frozen vitreous and serum were thawed on ice and centrifuged at 3,000 × g for 5 min at 4 ° C. Total RNA was extracted from the supernatants using the Qiagen miRNeasy® Mini Kit (Qiagen, GmbH, Hilden, Germany). A 200 μl aliquot of the vitreous and serum samples was transferred to new microcentrifuge tubes, and 750 μl of Qiazol mixture containing 0.94 μg of MS2 bacteriophage RNA was added to the serum. The solution was thoroughly mixed and incubated for 5 min, followed by the addition of 200 μl chloroform. The solution was mixed, incubated for 2 min, and centrifuged at 12,000 × g for 15 min in a microcentrifuge at 4 ° C. The upper aqueous phase was transferred to a new microcentrifuge tube and 100 % ethanol was added. The contents were mixed thoroughly, and 700 μl of the sample was transferred to a Qiagen RNeasy® Mini spin column in a collection tube followed by centrifugation at 15,000 × g for 30 s at room temperature. The Qiagen RNeasy® Mini spin column was rinsed with 700 μl Qiagen RWT buffer and centrifuged at 15,000 × g for 1 min at room temperature, followed by another rinse with 500 μl Qiagen RPE buffer and centrifuged at 15,000 × g for 1 min at room temperature. The contents of the Qiagen RNeasy® Mini spin column were transferred to new collection tubes and centrifuged at 15,000 × g for 2 min at room temperature. The contents of the Qiagen RNeasy® Mini spin column was transferred to a new microcentrifuge tube, and the lid was left uncapped for 1 min to allow the column to dry. Then, 50 μl of RNase-free water was added, and the total RNA was stored in a freezer at −80 ° C. MicroRNA real-time qPCR Total RNA (6 μl) was reverse-transcribed using the miRCUR Y LNA Universal RT microRNA PCR, polyadenylation, and cDNA synthesis kit (Exiqon, Vedbaek, Denmark). Each RT was performed once including an artificial RNA spike-in (UniSp6). The cDNA was diluted 50× (22.5 μl cDNA reactions+1102.5 μLlwater) and assayed in 10 μl PCR reactions according to the protocol for miRCURY LNA Universal RT microRNA PCR (Exiqon). Each microRNA was assayed once by qPCR on the microRNA Ready-to-Use PCR, Human Serum/Plasma Focus microRNA PCR Panel, V1 (Exiqon). Negative controls excluding the template from the reversetranscription reaction were performed and profiled like the samples. The amplification was performed in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) in 384 well plates. The amplification curves were analyzed by
Graefes Arch Clin Exp Ophthalmol
the Roche LC software (ver. 1.5) to determine the Cp by the 2nd derivative method and for melting curve analysis.
significantly different between the two groups, (Fig. 3a, b), the number of miRNAs expressed in the serum was significantly higher than in the vitreous of both groups (Fig. 3c, d).
Data analyses The data were internally calibrated by UniSp3 IPC using GenEx software (ver.5) (Exiqon). Assays must be Cp
RETINAL DISORDERS
Comparisons of microRNA expression profiles in vitreous humor between eyes with macular hole and eyes with proliferative diabetic retinopathy Kazunari Hirota & Hiroshi Keino & Makoto Inoue & Hitoshi Ishida & Akito Hirakata
Received: 29 March 2014 / Revised: 2 June 2014 / Accepted: 3 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose MicroRNAs (miRNAs) are small noncoding RNAs which regulate the activities of target mRNAs. We compared the expression profiles of the miRNAs in the vitreous of eyes with macular hole (MH) to that in eyes with proliferative diabetic retinopathy (PDR). Methods Vitreous and whole blood samples were collected from four patients with MH and from four patients with PDR. We assayed for 168 miRNAs in the vitreous and serum samples by the microRNA PCR Panel method. Results The mean number of miRNAs expressed in the vitreous was 63 (55–69) in eyes with MH and 86 (65–117) in eyes with PDR. The mean number of miRNAs expressed in the serum was 162 (159–167) in the MH patients and 142 (115– 160) in the PDR patients. Twenty-six miRNAs were expressed in the vitreous of both MH and PDR eyes. Although there was no significant difference in the levels of 20 of the 26 (73 %) miRNAs expressed in both MH and PDR eyes, six of 26 miRNAs (24 %) (hsa-miR-15a, hsa-miR320a, hsa-miR320b, hsa-miR-93, hsa-miR-29a, and hsa-miR-423-5p) were expressed significantly more highly in PDR eyes. In addition, the mean fold changes of three miRNAs, hsa-miR-23a, hsamiR-320a, and hsa-miR-320b, in the vitreous to serum were significantly higher in the PDR group than in the MH group. Conclusions The expression of several miRNAs related to angiogenesis and fibrosis was expressed significantly higher in the vitreous of eyes with PDR. Further studies are needed to
K. Hirota (*) : H. Keino : M. Inoue : A. Hirakata Department of Ophthalmology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan e-mail: [email protected] H. Ishida Department of 3rd Internal Medicine, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
understand the role played by the miRNAs in the biological function of the eye. Keywords microRNA . Vitreous humor . Proliferative diabetic retinopathy . Macular hole
Introduction Micro-RNAs (miRNAs) are non-coding, 21–24 nucleotide long, RNAs that have recently emerged as important posttranscriptional regulators of gene expression. They play critical roles in proliferation, differentiation, apoptosis, immunity, and angiogenesis [1–5]. MiRNAs have been found in the serum, plasma, and other body fluids such as urine and saliva [6, 7]. Recent studies have shown that some miRNAs are present in the vitreous of the eye, and the expression of microRNA-155 was elevated more significantly in the vitreous of eyes with uveitis than eyes with vitreoretinal lymphoma [8, 9]. However, the number of studies comparing the type and level of miRNA expression in different ocular diseases is limited. Diabetic retinopathy (DR) is an example of a disease that is a major cause of vision reduction [10]. In particular, proliferative diabetic retinopathy (PDR) which is caused by retinal ischemia and angiogenesis in the retina and vitreoretinal interface is mainly responsible for the blindness in eyes with DR. Although panretinal photocoagulation and vitrectomy are the main treatments for PDR, anti-vascular endothelial growth factor (VEGF) treatments have emerged in recent years as another treatment option [11]. However, anti-VEGF therapy has its limitations, e.g., recurrences of the neovascularization can occur in a few months after the anti-VEGF injection, systemic side-effects such as cerebrovascular or cardiovascular events can develop, endophthalmitis related to the intravitreal injections can also develop, and the anti-VEGF injections can alter the normal retinal vascular structure and reduce the
Graefes Arch Clin Exp Ophthalmol
retinal and choroidal blood flow in monkey eyes [12, 13]. Therefore, it is necessary to search for other therapeutic methods to treat the neovascularization in eyes with PDR. To accomplish this, we need to determine the pathophysiology of the vitreous of eyes with PDR in more detail. MiRNAs have been detected in the vitreous of eyes with different types of retinal diseases [8, 9], but the differential expression profiles of miRNAs in different retinal diseases have not been determined. Thus, the aim of this study was to compare the miRNA expression profiles in the vitreous of eyes with macular hole (MH) to that of eyes with PDR. A MH is considered to be a non-inflammatory/non-proliferative disease, while PDR is associated with inflammation and proliferation caused by angiogenesis. We shall show that the vitreous contained some miRNAs that were present in both types of eyes, and also that several miRNAs related to the regulation of angiogenesis and fibrosis were expressed at significantly higher levels in the vitreous of eyes of PDR patients. We discuss the potential roles of the miRNA in the vitreous of eyes with PDR.
Materials and methods Patients Vitreous and blood serum samples were collected from four patients with MH (two men and two women, ages 57 to 75; MH group) and four patients with PDR (two men and two women, ages 40 to 67 years; PDR group). All of the patients underwent vitrectomy at the Kyorin Eye Center between April 2011 and March 2013. We excluded patients with systemic disease such as hypertension, diabetes mellitus, autoimmune diseases, and cancer in the MH group. In the PDR group, patients with diabetes mellitus and hypertension were included; however, patients with other systemic diseases were excluded. Patients with history of ocular diseases and ocular surgeries were excluded from both groups. The procedures used in this study conformed to the tenets of the Declaration of Helsinki, and an informed consent was obtained from each patient after an explanation of the purpose and possible consequences of the procedures. This study was approved by the Kyorin University Hospital Research Ethics Committee. Collection of samples Peripheral blood (7 ml) was collected from the patients before surgery, and immediately centrifuged at 3,500 rpm for 7 min to isolate the blood serum. All surgeries were performed by one experienced vitreoretinal surgeon. Before beginning the surgery, one 25-G trocar was inserted 3 mm posterior to the corneal limbus by a transconjunctival one-step incision.
Vitreous (1.0 ml) was collected before the infusion, and it was immediately centrifuged at 400×g (1,500 rpm) for 10 min to remove any cell debris. All samples were preserved at -80 ° C until assayed. Sample preparation The frozen vitreous and serum were thawed on ice and centrifuged at 3,000 × g for 5 min at 4 ° C. Total RNA was extracted from the supernatants using the Qiagen miRNeasy® Mini Kit (Qiagen, GmbH, Hilden, Germany). A 200 μl aliquot of the vitreous and serum samples was transferred to new microcentrifuge tubes, and 750 μl of Qiazol mixture containing 0.94 μg of MS2 bacteriophage RNA was added to the serum. The solution was thoroughly mixed and incubated for 5 min, followed by the addition of 200 μl chloroform. The solution was mixed, incubated for 2 min, and centrifuged at 12,000 × g for 15 min in a microcentrifuge at 4 ° C. The upper aqueous phase was transferred to a new microcentrifuge tube and 100 % ethanol was added. The contents were mixed thoroughly, and 700 μl of the sample was transferred to a Qiagen RNeasy® Mini spin column in a collection tube followed by centrifugation at 15,000 × g for 30 s at room temperature. The Qiagen RNeasy® Mini spin column was rinsed with 700 μl Qiagen RWT buffer and centrifuged at 15,000 × g for 1 min at room temperature, followed by another rinse with 500 μl Qiagen RPE buffer and centrifuged at 15,000 × g for 1 min at room temperature. The contents of the Qiagen RNeasy® Mini spin column were transferred to new collection tubes and centrifuged at 15,000 × g for 2 min at room temperature. The contents of the Qiagen RNeasy® Mini spin column was transferred to a new microcentrifuge tube, and the lid was left uncapped for 1 min to allow the column to dry. Then, 50 μl of RNase-free water was added, and the total RNA was stored in a freezer at −80 ° C. MicroRNA real-time qPCR Total RNA (6 μl) was reverse-transcribed using the miRCUR Y LNA Universal RT microRNA PCR, polyadenylation, and cDNA synthesis kit (Exiqon, Vedbaek, Denmark). Each RT was performed once including an artificial RNA spike-in (UniSp6). The cDNA was diluted 50× (22.5 μl cDNA reactions+1102.5 μLlwater) and assayed in 10 μl PCR reactions according to the protocol for miRCURY LNA Universal RT microRNA PCR (Exiqon). Each microRNA was assayed once by qPCR on the microRNA Ready-to-Use PCR, Human Serum/Plasma Focus microRNA PCR Panel, V1 (Exiqon). Negative controls excluding the template from the reversetranscription reaction were performed and profiled like the samples. The amplification was performed in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) in 384 well plates. The amplification curves were analyzed by
Graefes Arch Clin Exp Ophthalmol
the Roche LC software (ver. 1.5) to determine the Cp by the 2nd derivative method and for melting curve analysis.
significantly different between the two groups, (Fig. 3a, b), the number of miRNAs expressed in the serum was significantly higher than in the vitreous of both groups (Fig. 3c, d).
Data analyses The data were internally calibrated by UniSp3 IPC using GenEx software (ver.5) (Exiqon). Assays must be Cp
