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Abstract
Background Let-7 microRNA is an important regulator of cellular ageing and tissue senescence. The objective of this study is to evaluate the expression of let-7a/let-7b/let-7c microRNAs in human age-related cataracts.
Aim To evaluate the correlation among the severity of lens opacity, the level of let-7a/let-7b/let-7c microRNA expression and patient age in the context of age-related cataracts.
Methods The authors evaluated the mRNA level of let-7a/let-7b/let-7c microRNA in lens epithelia obtained from 174 eyes with age-related cataracts. The authors also recorded the patient age and the severity of lens opacity as classified according to the modified version of the Lens Opacities Classification System version III.
Results Let-7b microRNA expression was demonstrated to be positively associated with patient age (R=0.472; p<0.001). A positive correlation was also observed between higher N, C and P cataract scores and higher expression of let-7b microRNA in patients with age-related cataracts (p<0.001). However, no significant correlation was observed between the let-7a and let-7c microRNA expression levels and either the severity of lens opacity or the patient age.
Conclusion These findings suggest that microRNAs play a role in age-related cataracts. A local let-7b microRNA increase may represent a risk factor in the formation of age-related cataracts.
- Lens and zonules
- retina
- stem cells
- imaging
- neovascularisation
- degeneration
- eye lids
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Introduction
MicroRNAs are a large group of short (18–25 nucleotide), non-coding, small RNA molecules transcribed by RNA polymerase II or, in some cases, by RNA polymerase III.1 Most microRNA-mediated regulation occurs at the post-transcriptional level, and is mediated via the near-perfect or partial complementarity of the microRNA to the coding region or the 3′untranslated region of the target mRNA. The binding of the two RNA leads to translational repression and/or mRNA degradation and, therefore, to the precise regulation of gene expression.1 MicroRNAs are emerging as important regulators of cell proliferation,2 cancer formation,3–6 stress responses,7 cell apoptosis8 and senescence.8–11 The let-7 microRNA has been originally discovered in the Caenorhabditis elegans and subsequently detected as the first known human microRNA.12 The function of let-7 microRNAs is well known to control multiple targets for regulating cell proliferation and differentiation as required for proper tissue development and tumour suppression.3–6 Let-7 family members have been found to be downregulated during tumour progression, and the overexpression of let-7 microRNAs substantially reduces the tumour burden.4–6 Recently, more evidence has pointed to the let-7 microRNA as a regulator of cellular ageing and tissue senescence.9–11 Poor self-renewal abilities are detected in ageing stem cells, which are associated with let-7 microRNA upregulation.11 Elevated let-7 microRNA expression in ageing cells is associated with reduced levels of high mobility group AT-hook 2 (Hmga2), which functions in cellular proliferation and differentiation during development.11 Let-7 microRNA overexpression is also detected in senescent fibroids and aged skeletal muscles.9 10 However, there is still little information about the role that microRNAs play in modulating the ocular ageing process.
Cataract is a major contributing factor of blindness and accounts for 47.8% of the roughly 37 million cases of blindness in the world.13 Although multiple predisposing factors, such as diabetes mellitus, drug use, ultraviolet exposure and other ocular diseases affect the cataractogenesis, in most cases, cataract is caused by ageing alone.14 15 Recently, let-7 members have been found to be important regulators of dedifferentiation during lens regeneration in the adult newt.16 17 The overexpression of let-7 microRNA may dysregulate genes related to cellular proliferation or cell cycle control in aged tissue.10 However, the role of let-7 microRNA in ocular ageing such as cataract formation remains to be further elucidated.
The objective of this study was to evaluate the expression of let-7a/let-7b/let-7c microRNAs in lens epithelia, derived from patients with cataracts, in various age groups and in various stages as well as with various types of cataracts. To further investigate the correlation between patient age and the severity of lens opacity, we used the classification of Lens Opacities Classification System version III (LOCSIII) to evaluate and analyse the influence of let-7a/let-7b/let-7c expression levels and age in patients requiring cataract surgeries.
Methods
Lens epithelium samples were obtained from 174 eyes between January 2008 and December 2010. The mean age of patients was 72.44 years ±0.82 (SE) (range, 55–91 years). This study followed the tenets of the Declaration of Helsinki. All patients had a complete preoperative ophthalmologic examination. Only those who had no ocular disease other than age-related cataracts were included. Patients who had previously undergone ocular surgery or those with diabetes were not included in the study. All samples were collected with the informed consent of the patients. Lens epithelium samples were obtained by intact continuous curvilinear capsulorhexis, with care taken to avoid vascular contact or damage to the iris and other intraocular structures.
The type and severity of cataracts were graded and recorded according to the modified version of the LOCSIII. Six slit-lamp images were used for grading nuclear colour (NC) and nuclear opalescence (NO). Five retroillumination images were used for grading cortical (C) cataracts and posterior subcapsular (P) cataracts. Each scale on the LOCSIII is a decimalised scale ranging from 0.1 (a completely clear or colourless lens) to 5.9 (upper value on the C and P scales indicating complete opacification of the cortex or posterior capsule) and 6.9 on the NO and NC scales (indicating advanced opacification and brunescence of the nucleus). The control lens epithelium sample with LOCSIII score in C1, N1 and P1 was obtained from 24 eyes undergoing vitrectomy for epiretinal membranes. The mean age of the 24 control patients was 50.17 years ±0.84 (SE) (range, 40–54 years). Agreement in the assessment of cataract grading was noted by at least five individuals.
MicroRNA quantitative RT-PCR
The levels of let-7a/let-7b/let-7c microRNAs were determined by quantitative real-time reverse transcriptase (RT)-PCR as previously described.16 Briefly, a mirVana PARIS kit (Ambion, Austin, Tx, USA) was used to isolate microRNAs from the total RNA according to the manufacturer's instructions. The mature let-7a sequence was (5′ to 3′) UGAGGUAGUAGGUUGUAUAGUU. The mature let-7b sequence was (5′ to 3′) UGAGGUAGUAGGUUGUGUGGUU. In addition, the mature let-7c sequence was (5′ to 3′) UGAGGUAGUAGGUUGUAUGGUU. For microRNA detection, qRT–PCR was performed using TaqMan miRNA assays (Applied Biosystems) with specific primer sets (Let-7a, AB assay ID: 000377; Let-7b, AB assay ID: 000378; Let-7c, AB assay ID: 000379). All reagents and protocols were from Applied Biosystems. Detection was performed using a 7900HT fast real-time PCR system using RNU6B (RNA, U6 small nuclear 2, AB assay primer ID: 001093) as internal controls. The conditions for amplification were as follows: one cycle at 94°C for 2 min, followed by 50 cycles at 94°C for 20 s, 57°C for 20 s and 70°C for 20 s. MicroRNA-specific qRT-PCR was done in triplicate and repeated three times on the ABI Prism 7700 Sequence Detector system (Applied Biosystems). We defined the level of let-7a/let-7b/let-7c mRNA in control patients as 1.
Western blot analysis
Selected samples were fixed, washed once in cold phosphate-buffered saline, scraped, lysed with extraction buffer, and centrifuged at 10 000 rpm for 10 min to remove insoluble material. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, California, USA). Sample extracts in buffer were placed in boiling water for 5 min and then separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. After electrophoresis, the gel was transferred onto a polyvinylidene difluoride membrane for immunoblotting. The membrane was blocked by incubation in non-fat milk at room temperature for 0.5 h and incubated with Hmga2 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) for 16 h at 4°C, washed five times with tris-buffered saline tween-20, and incubated at room temperature with horseradish peroxidase-conjugated secondary antibody for 2 h. The membrane was washed six times with tris-buffered saline tween-20, and specific bands were made visible by chemiluminescence (ECL, Santa Cruz).
Statistical methods
For statistical analysis, patient age and the let-7a/let-7b/let-7c levels were expressed as the mean±SE. The correlations among the LOCSIII score, age and the level of let-7a/let-7b/let-7c mRNA were analysed by one-way analysis of variance (ANOVA), followed by the post hoc Bonferroni's test. The correlations between the LOCSIII score and age were analysed by the Pearson correlation coefficient and the unpaired Student t test. The correlations between the LOCSIII score and the level of let-7a/let-7b/let-7c expression were analysed by the Pearson correlation coefficient and the unpaired Student t test. A p value of <0.05 was considered statistically significant. All analyses were performed using SPSS V.12.0.
Results
The relationship between the severity of nuclear (N) cataract (NO and NC) and age at the time of surgery showed significant correlation between higher grades of N cataracts and older age (one-way ANOVA, p<0.001) (figure 1). There was a significant correlation between higher grades of C cataracts and older age (one-way ANOVA, p<0.001) (figure 1). In P cataracts, a significant correlation was also revealed between the higher LOCSIII score of P cataract and older age (one-way ANOVA, p<0.001) (figure 1). Older patients showed higher LOCSIII scores in N, C and P cataracts, and significant correlations were observed between age and N cataracts (Pearson correlation coefficient R=0.540; p<0.001), between age and C cataracts (R=0.623; p<0.001) and between age and P cataracts (R=0.542; p<0.001).
Grading according to the Lens Opacities Classification System version III (LOCSIII) and the age of patients at the time of cataract surgery. The LOCSIII scores for nuclear (N), cortical (C) and posterior subcapsular (P) cataracts were significantly higher in older patients (*p<0.001).
Patients with age-related cataracts were further divided into three groups by age using 55 and 75 years as cut-off values; the older the patient, the higher the let-7b microRNA level. The let-7b microRNA level in lens epithelium samples was significantly increased in patients aged 55–75 years and in those over 75 years than in those younger than 55 years old (ANOVA post hoc Bonferroni's test, p<0.001) (figure 2A). The let-7b microRNA level was significantly increased in patients aged over 75 years than in those aged 55–75 years old (ANOVA post hoc Bonferroni's test, p<0.001) (figure 2A). A scatter plot of let-7b microRNA expression levels versus age at surgery in patients aged over 55 years demonstrated a moderate correlation between an older age and higher let-7b microRNA expression levels (Pearson correlation coefficient R=0.472; p<0.001, figure 2B). However, no statistically significant differences were observed between patient age and either the let-7a (p=0.1) or let-7c (p=0.058) microRNA level (figure 2A). There was also no significant correlation between patient age and the let-7a (p=0.158) or let-7c (p=0.083) microRNA level.
(A) Let-7a, let-7b and let-7c levels in the lens epithelia of the age-related cataract groups. Relative expression levels of let-7a, let-7b and let-7c microRNAs in lens epithelia obtained from cataract patients aged less than and over 55 years. The expression level of let-7b microRNA was significantly higher in cataract patients aged 55 years or older than in cataract patients younger than 55 years old (*p<0.001). (B) The relationship between age and the let-7b level in age-related cataract patients. Each dot indicated the let-7b level in one patient. Linear regression analysis showed a positive correlation between the let-7b level and age (R=0.472, p<0.001).
In N cataracts, a moderate correlation was found between the severity of N cataract and the let-7b microRNA level (Pearson correlation coefficient R=0.462; p<0.001, figure 3). There were also correlations between the severity of C cataract and the let-7b microRNA level (Pearson correlation coefficient R=0.432; p<0.001, figure 3), as well as between the severity of P cataract and the let-7b microRNA level (Pearson correlation coefficient R=0.345; p<0.001, figure 3). No significant correlations were found between the severity of lens opacity, including N, C and P types, and let-7a (N: p=0.735; C: p=0.099; P: p=0.627) or let-7c (N: p=0.053; C: p=0.055; P: p=0.197) microRNA levels (data not shown).
Relative expression of let-7b microRNA and the severity of nuclear, cortical and posterior subcapsular cataracts. Real-time PCR revealed that there was a significant association between higher expression of let-7b and higher grading of nuclear cataracts (R=0.462, p<0.001), cortical cataracts (R=0432, p<0.001) and posterior subcapsular cataracts (R=0.345, p<0.001). LOCSIII, Lens Opacities Classification System version III.
In order to evaluate the let-7b microRNA target genes, we measure the protein level of Hmga2 in some samples with LOCSIII score in N1C1P1 (control) and in N6C5P5 (dense cataract). Western blotting analysis confirmed the lower level of Hmga2 in samples with higher severity of lens opacity (figure 4).
Protein levels of high mobility group AT-hook 2 (Hmga2) in control and three cataract samples. Western blotting analysis revealed lower level of Hmga2 in cataract samples with Lens Opacities Classification System version III (LOCSIII) score in N6C5P5 as compared with the control sample with LOCSIII score in N1C1P1.
Discussion
Members of the let-7 microRNA family have many biological functions, and it is well known that dysregulation of let-7 microRNA induces the acceleration of cellular replication4 and development of systemic disorders such as cancer.5 6 The upregulation of let-7 microRNA dysregulates the expression of cellular proliferation-related genes, which leads to a senescence phenotype.10 In this study, we found that the let-7b microRNA level in the lens epithelium increased with age in patients with age-related cataracts. The expression of let-7b microRNA in the lens epithelia of cataract patients older than 85 years of age was approximately 1.15 times higher than that in patients aged between 55 and 64 years. We further investigated the association between let-7b microRNA expression in the lens epithelium and cataract subtypes and severity as classified by the LOCSIII. The level of let-7b microRNA expression was greater in lenses with more severe N, C or P cataracts. To the best of our knowledge, this is the first study to report that microRNAs in the lens epithelium may be involved in the development age-related cataracts and that let-7b microRNA may regulate the course of senescence in cataractous lenses.
In adult newts, investigations on lens regeneration have shown that the pigment epithelial cells of the dorsal iris dedifferentiate and then differentiate into lens cells after lentectony.16 17 The let-7 family is reported to be downregulated during the process of dedifferentiation in the dorsal iris.16 17 Specifically, the upregulation of let-7b will affect the proliferation of both dorsal and ventral pigment epithelial cells.16 These findings suggest that let-7 microRNA plays an important role in newt lens regeneration. Several studies have shown that increased let-7 microRNA expression is associated with cell ageing and tissue senescence. During nematode maturation, let-7 microRNA has a regulatory function and affects its longevity.18 These effects indicate that let-7 and other microRNAs are potential modulators of nematode ageing. Stem cells have self-renewing abilities, but this capacity declines with age. A significant increase in let-7 microRNA expression is reported to be associated with murine neural stem cell ageing and poor self-renewal abilities.11 In aged human tissues, the let-7 microRNA expression can be detected at higher levels. Uterine senescent fibroids in older women were observed to express high levels of let-7 microRNA.9 Functional and network analysis of microRNA expression profile in the aged human skeletal muscle has demonstrated that the level of let-7 microRNA is significantly elevated in older muscle biopsy samples.10 Let-7 microRNA targets genes in molecular networks that are involved in cell cycle control, and affects cellular proliferation and differentiation.10 Expression of let-7 microRNA may be a good indicator of impaired cell cycle function, possibly contributing to reduced cellular renewal in aged human tissues. During the embryonic stage, higher expression of stemness genes is associated with decreased levels of let-7 microRNA in undifferentiated cells.3 6 On the contrary, increased let-7 expression is detected during terminal cellular differentiation.19 The let-7 microRNA is an important regulator of cellular differentiation, tumour suppression and re-entry into the cell cycle.6 17 19 We have previously reported that the overexpression of let-7 negatively modulates the expression of stemness genes such as Oct4 and Nanog in tumour cells.6 During newt lens dedifferentiation and regeneration, the downregulation of let-7 gene expression16 17 and the overexpression of stemness factors20 have been observed. Tsonis et al found that increased let-7 expression was associated with lens differentiation and cellular proliferation.17 Our results demonstrated that human lens degeneration and more severe lens opacity are associated with higher let-7 expression, an interesting result similar to that of Tsonis et al. Many microRNAs, including let-7 microRNA, have been identified in mammalian lenses.21 22 The present study supports the notion that the upregulation of let-7 expression is associated with the formation of age-related cataracts. The level of let-7b microRNA expression was demonstrated to be positively correlated with the severity of lens opacity for the N, C and P types.
In humans, the let-7 family is composed of 10 let-7 microRNA members.23 Due to the limited amount of sample available per patient, it is difficult to evaluate the expression levels of all 10 mature let-7 sequences in human lens epithelia obtained through cataract surgery. Furthermore, microarray and RNA in situ hybridisation are also difficult to analyse in these samples. In the present study, the let-7b isoform increased expression with older patient age and higher severity of lens opacity. With respect to newt lens cell proliferation, Nakamura et al have found that let-7b in particular is upregulated in gain- and loss-of-function experiments using microRNA microarrays.16 Analysis of seven members of the let-7 family expression during stem cell ageing reveals that only let-7b exhibits a progressive increase in the expression level with age.11 Let-7b overexpression is shown to be inversely correlated with declining expression of Hmga2, which is an important transcription factor that is widely expressed in undifferentiated cells during embryogenesis.11 In general, let-7 family members usually work together as a group to significantly alter the gene expression or a common cellular function. Animal studies may be necessary to evaluate the expression levels of all let-7 family members in cataractous lenses. Our data indicate that let-7b microRNA expression is increased in the lens epithelia of patients with higher LOCSIII cataract scores and, therefore, let-7b microRNA expression may represent a novel biomarker of human lens ageing.
Conclusion
Although more studies are necessary to investigate the physiological function of let-7 microRNA, our study has demonstrated the increased expression of let-7 correlates with increased age and higher severity of lens opacity in human cataractous lenses. Our findings show that increased let-7 microRNA levels may lead to the damage to the lens epithelium, resulting in cataract formation. Further research is needed to determine the effect of the downregulation and to determine if such downregulation can be employed in the treatment of cataractogenesis.
References
Footnotes
This paper has not been presented at any conference.
Funding This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital. This study was supported by the National Science Council (98-2314-B-341-001-MY3, 99-2314-B-075-005- MY3), the Joint Projects of UTVGH (VGHUST VN100-03), Taipei Veterans General Hospital (C1-099/ER3-001), Taipei City Hospital (96/97/98/099XDAA00102/10001-62-042), Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-97-DR-25, SKH-8302-98-DR-26), Zuoying Armed Forces General Hospital (9502/9696/9719), Cheng-Hsin General Hospital (CHGH-100-01, 100-19, 100-20, 100-22, 100-40) and Yen-Tjing Ling Medical Foundation (96/97/98). Taiwan.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by Taipei City Hospital.
Provenance and peer review Not commissioned; externally peer reviewed.