| Objective:To observe the mRNA and protein expressions and methylation level of Klotho gene in lens epithelial cells (LECs) in normally transparent crystalline lens and in age-related cataract (ARC), analyze the relationship between Klotho gene and ARC, and explore the roles of the epigenetic changes of the Klotho gene in regulating the development of ARC. Totally 90 subjects were divided into three groups:young adult group with normally transparent crystalline lens (aged 18-30 years) (n=30,30 eyes), middle-aged group with ARC (aged 40-49 years) (n=30,30 eyes), and elderly group with ARC (aged 67-85 years) (n=30,30 eyes). The LECs were collected through curvilinear capsulorhexis. The mRNA expression of Klotho gene was observed using reverse transcription-polymerase chain reaction (RT-PCR). Its protein expression in LECs was detected using immunohistochemical (IHC) staining method. Methylation-specific PCR (MSP) was applied to detect the methylation level of the target gene. The decreased mRNA expression of Klotho gene was reversely correlated with the age (P<0.01). IHC showed the Klotho protein was mainly expressed in the cellular membrane and cytoplasm of LECs. The expressions were strongly positive in the young adult group, with the expression sites evenly distributed. The expressions were weakly positive in the middle-aged group, with the sites mainly distributed 4mm -5mm away from the center of the anterior lens capsule. The expressions were negative in the elderly group. Comparisons of the positive rates (100.0%,36.7%, and 0.0% in young adult group, middle-aged group, and elderly group, respectively) among these three groups showed statistical differences (P<0.05). MSP showed that the Klotho gene was highly methylated in the elderly group and weakly methylated in the middle-aged group, whereas no methylation was detected in the young adult group, with the rate of methylation being 3.3%,56.7%, and 93.3% in the young adult group, middle-aged group, and elderly group (P<0.05). The Klotho gene and protein are positively expressed in the LECs of normal individuals. Its promoter can be methylated with age, resulting in Klotho gene silencing as well as the down-regulated expression or non-expression of the Klotho protein. Such epigenetic changes can affect the biological activities of LECs. However, the relationship between Klotho gene and ARC awaits further investigations.Age-related cataract (ARC), sometimes also termed "senile cataract", is the most common type of cataract and typically occurs with aging. Clinically it is mainly manifested as painless progressive loss of vision and can be divided into three types:nuclear, cortical, posterior subcapsular, with the cortical ARC being the most common type. As the population ages and people live longer, the incidence of ARC increases annually, accounting for 40.6% of low vision or blindness cases. Currently there are no drugs that have been shown to be effective to treat or prevent ARC. The only possible treatment is surgical removal of the cloudy lens. However, the charges and complications of the surgery carry with it a tremendously heavy financial and emotional cost. Klotho is a recently-discovered anti-aging gene by Kuro-O M et al, with its expression level closely related with age. The Klotho protein can adjust aging and aging-related diseases by regulating the metabolisms of vitamin D, calcium, and phosphorus, protecting cardiovascular system, and affecting the cell structures and immune function. Additionally, Klotho-knockout mice early demonstrated behaviors and pathophysiological changes (e.g. short lifespan, hearing loss, and clouding of the cornea) resembling human aging. Genetically-based remedial treatment using the Klotho gene can resolve these symptoms of premature aging. In our previous experiment, the biological activity of lens epithelial cells (LECs) decreased with age and aging could induce cataract, suggesting that the development of ARC might be related with the Klotho gene.The epigenetic studies have explored the changes in heritable gene expression or cell phenotypes due to certain mechanisms without changing the DNA sequences. These changes may include DNA methylation, RNA interference, and histone modification. Research has shown that dietary supplements and changes in the environmental exposures can change the epigenetic features of the gene expressions in mice and thus affect the coat color, body weight, and susceptibility to tumorigenesis.When human beings are under different environmental exposure conditions, the DNA content and genomic distribution of 5-methyl cytosine showed no significant difference between young twins but did exert significant differences between elderly twins. Quite a few epigenetic studies have been performed on ARC. Zhou et al found the hypermethylation of crystallin alpha A (CRYAA) gene promoter in the in vitro cultured LECs obtained from ARC patients was associated with the increased biological activities of LECs,which sheds new light on research on the pathogenesis of cataract from the perspective of epigenetics. Abnormal methylation of the Klotho gene promoter has been found in patients with tumor, arteriosclerosis, and/or renal failure. However, whether the Klotho gene is expressed on human Lens epithelia and whether the changes in its epigenetical traits remain unclear.In our current study, by harvesting specimens directly from clinical settings, we observe the expressions of Klotho gene and functional protein in LECs in young adult group and two ARC groups (middle-aged group and elderly group) and detected the methylation level of its gene promoter. We found the methylation level of Klotho gene increased with age, resulting in low and even no expression of the Klotho gene and its protein. Thus, the epigenetic modifications of the Klotho gene may be involved in the development of ARC. These findings can serve as new clues in the further studies on targeted therapies.Materials and methods:Specimen collection:A total of 60 inpatients (60 diseased eyes) who underwent ARC surgeries for their cortical cataract from Henan Institute. September 2013 to October 2014 were enrolled in this study. Disease histories including systemic conditions (e.g. diabetes, hypertension, and heart disease) and eye diseases (e.g. iridocyclitis, glaucoma, eye injuries, as well as chronic ocular radiation exposure) were excluded before the surgery. In addition,30 healthy subjects were also enrolled. A total of 90 subjects were divided into three groups:young adult group with normally transparent crystalline lens (aged 18-30 years; mean:25.00 years) (n=30,18 men and 12 women; 30 eyes), middle-aged group with ARC (aged 40-49 years; mean:45.33 years) (n=30, 17 men and 13 women; 30 eyes), and elderly group with ARC (aged 67-85 years; mean:75.53 years) (n=30,15 men and 15 women; 30 eyes). Before the surgery, the gender and age of the patients as well as the degree and type of lens opacity were examined and recorded by the same physician. The phacoemulsification for cataract was performed by the same surgeon, during which the curvilinear capsulorhexis (5.5mm in diameter) was conducted routinely. The anterior lens capsule of normal young adults were obtained from the Eye Bank of Henan Institute of Ophthalmology after corneal transplantation. With the same method, the transparent anterior lens capsules were used as the control group, in which all the anterior lens capsules were rinsed in PBS buffer to remove the blood, iris pigment, and intraocular tissues including lens cortex and vitreous body. All the sampling procedures were approved by the Medical Ethics Committee of the hospital. All the obtained specimens were immediately stored in a-80 ℃ refrigerator for further usage.Main reagents and instruments:The main reagents included Trizol kit (Invitrogen, USA), high-capacity cDNA reverse transcription kit (Applied Biosystems, USA), real-time fluorescent quantitative PCR kit (SYBRPremixEx Taq PCR), SYBR premix (Takara), and EZ DNA Methylation-Gold kit (Zymo Research, USA). The primary antibody (goat anti-human Klotho antibody) and the secondary antibody (donkey anti-goat antibody) were purchased from Santa. The 1,3-dimethyl-5-acetyl-barbituric acid (DAB) was purchased from Sigma (USA).Equipment:The PCR equipment included the Applied Biosystems 7300 RealTime PCR System and 2720 Thermal Cycler. Gel imaging analyzer (Alpha Innotech, San Leandro,CA,USA, Fluor Chem Q) was also employed..RT-PCR:The harvested anterior lens capsule was rapidly added into an Eppendorf tube. After having been rinsed with 10ml PBS, the samples were centrifuged to deposit 5×105 cells. Add cell lysis solution after discarding the supernatant. Centrifuge for 5min and then absorb liquids. Dry up the RND for lmin, and then add diethypyrocarbonate (DEPC) solution; the mixture was then stored at-80 ℃. Reverse transcription & cDNA synthesis:The range of the rate is between 0.6 and 1.19. Primer design:After the human Klotho gene sequence was obtained from the Internet-based GenBank (Klotho ID:16591; Klotho mRNA:NM-013823.1), we designed the specific primer using the Primer Premier software (version 5.0):sense: 5’-ACCTGGTGGCGCACAC, anti-sense:5’-TTGGCAAACCAACCTAGTACA; GAPDH:sense:5’-GAAGGTGAAGGTCGGAGT, anti-sense: 5’-GAAGATGGTGATGGGATTTC.The conditions for reverse transcription reaction were as follows:20℃, 10min; 42℃,60min to produce complementary DNA (cDNA) via reverse transcription. Terminate the reaction by heating at 70℃ for 10 min. Finally, store the product in a-20℃ refrigerator for further usage. Assessment of RT-PCR:The routine PCR products underwent agarose gel electrophoresis at appropriate concentrations. If image analysis showed that the target lanes were clear and without any mixed lanes, the primer design and synthesis were interpreted as correct, and the fluorescence quantitative PCR could be performed. Real-time fluorescence quantitative PCR:The amplification was performed on the fluorescence quantitative PCR system using the TaqMan probes synthesized by ABI company. Reaction system for Klotho-PCR gene reaction:10 x PCR buffer solution (Mg2+free) 3.0μl; MgCl2 (25mmol/L) 1.8μl; dNTP (25mmol/L) 0.36μl; upstream primer (10μmol/L)1.0μl; downstream primer (10μmol/L) 1.0μl; and Taq enzyme (5U/μl). Solubility curve analysis and agarose gel electrophoresis were performed to verify the amplification. The test was repeated three times for each sample to obtain an mean. The changes in the Klotho mRNA expression in different groups were calculated using the 2-△△Ct method.Immunohistochemical (IHC) staining.The harvested anterior lens capsule specimen was rinsed with PBS, and then smear the specimen on a slide, dry it, and stain it using the ABC method. The cells were inoculated with 1:200 goat anti-human Klotho antibody at room temprature overnight, and then treated with the second antiboy (donkey anti-goat antibody,1:50) for 2 hours. Afterwards, it was treated in the DAB solution for 5min. Rinse with PBS to terminate staining, and then mount the cells in slides after drying. Judgment of the results:For each slice, five fields of view were randomly selected. The number of positively stained cells for every 50 cells was counted. The results were interpreted based on the percentage of the positively stained cells among these 50 cells:0, with the percentage of the positively stained cells≤5%; 1, with the percentage of the positively stained cells>5% but ≤25%; 2, with the percentage of the positively stained cells>25% but ≤50%; 3, with the percentage of the positively stained cells>50% but ≤75%; and 4, with the percentage of the positively stained cells >75% but <100%. The cell staining intensity was divided into 0 (negative),1 (yellowish granules),2 (brownish granules), and 3 (brown granules). A final score was calculated based on the staining intensity and the percentage of the positively stained cells. The scores were divided into 4 grades based on the product of these two figures:-,≤4;+,≤8;++,<12; and +++,=12, among which "-" means a negative Klotho expression, whereas "+", "++", and "+++" represent positive results.Methylation-specific polymerase chain reaction:Methylation-specific polymerase chain reaction (MSP) was applied to detect the Klotho mthylation level.DNA was extracted from the specimens in three groups. Then,500ng of DNA was treated with sulfite in strict accordance with the manual of EZ DNA Methylation-Gold Kit. With 1 ul of the treated DNA as the template, MSP was performed using the TaqGold DNA polymerase. The reaction system was as follows:12 predenaturation at 95 ℃ for lOmin, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, and extension at 72 ℃ for 30s. The amplification persisted for 35 cycles, with the final extension step extended to 5min at 72℃. The PCR product underwent 1.2% agarose gel electrophoresis and ethidium-bromide (EB) staining. The product lanes were observed on UV detector. The methylation and unmethylation primers were designed using the MethPrimer software. Klotho methylation (M) primer sense: 5’-GTCGTCGTTGTAGTTCGTTATC; anti-sense: 5’-CAACAAACGCCGATAATAACG. Klotho unmethylation (M) primer sense: 5’-TTGTTGTTGTTGTACTTTGTTATT; anti-sense: 5’-CCAACAAACACCAATAATAAC. Statistical analysis:Statistical analysis was implemented by using IBM SPSS 19.0 software package. The measurement data are expressed as mean ± standard deviations. One-way ANOVA was applied for inter-group comparisons; Welch test is applied in cases where the variances are not homogeneous. The count data are presented using frequencies and percentages, and their inter-group comparisons were performed using Pearson x2 test or Fisher’s exact test. The ranked data are presented using percentages and average ranks, and their inter-group comparisons were performed using Kruskal-Wallis test.Results:Klotho gene expression in lens epithelia:The expression of Klotho gen in the transparent lens epithelia was calculated using the 2-△△Ct method, which showed that the relative expression level of Klotho gene was significantly different among these three groups (Figure 1, Table 1, P<0.05).Klotho protein expression in LECs:The Klotho protein was homogeneously expression in the cytoplasm and cell membrane in the young adult group (Figure 2). In the middle-aged group, the Klotho protein was weakly expressed in the center of the anterior lens capsule; the color developed with DAB was uneven; and the cells had varied morphologies, showing pseudopod-like protrusions (Figure 3,5, and 6). The Klotho protein expression was negative in the elderly group (Figure 4). The differences among these three groups were significant (Table 1, P<0.05).Klotho gene methylation level:The percentage of Klotho gene methylation was 3.3%(1/30), 56.7%(17/30), and 93.3%(28/30) in the young adult group, middle-aged group, and elderly group. The percentage of Klotho gene methylation was significantly higher in the middle-aged group and elderly group than in the young adult group (Figures 7 and 8, Table 1, P<0.05). Correlation of Klotho gene methylation with Klotho gene expression and age:The methylation level was relatively low in the young adult group and high in the elderly group; comparisons among these three groups showed statistical significances (P<0.05).Conclusion:The cells used in our current study were directly harvested from human specimens, which avoided the interference of the in vitro culture. For the first time we found that the Klotho gene was expressed in the LECs of normal individuals, and its mRNA and protein expressions decrease with age. The Klotho gene is closely related with human aging and has been demonstrated to be an inhibitory factor of aging. The Klotho-knockout mice early demonstrated various changes (e.g. short lifespan, movement disorders, atherosclerosis, and osteoporosis) resembling human aging. Genetically-based remedial treatment using the Klotho gene can resolve these symptoms of premature aging. However, the lens opacity (which is a manifestation of aging) after Klotho gene depletion as well as the expression of Klotho gene in human eyeball have not been reported in literature. The Klotho protein has two forms: membrane Klotho and secreted Klotho. The secreted Klotho protein can regulate multiple signaling pathways, including the calcium channel TRPV5. In the FGF23 pathway, it can bind the basic fibroblast growth factor receptor (bFGFR) 1-4 to form a nitric oxide (NO) receptor, thus increasing NO synthesis. Meanwhile, by inhibiting the Wnt pathway, the Klotho protein can suppress cell apoptosis. However, the relationship between the Klotho gene and LECs remains unclear. Our current study demonstrated the expression of Klotho in the LECs of normal subjects, suggesting that Klotho may affect the activities of LECs and its abnormal expression may induce cataract.Cytologically, ARC is caused by the senescence and apoptosis of LECs. The LECs are tightly attached to the monolayer cells on the inner surface of the anterior capsule and meanwhile loosely connected with the lens fibers at the inferior side. The LECs have two major features inside human body:a) continuously differentiate into lens fiber cells; and b) form lens capsule. The cell has three definite areas:in the central region there are tightly aligned monolayer cells, under which there are continuously lined vesicle membrane. Previous studies have demonstrated that such a vesicle membrane contains type IV collagen and laminin that are similar to the human lens capsule basement membrane; thus, the formation or the tight relationship with LECs or the high-density proliferation of these cells is a key factor for the synthesis of lens capsule. In the equatorial and pre-equatorial regions, the LECs constantly undergo mitosis and differentiate into lens fibers, which exert pressure on the central region and form several optical plane structures under the slit lamp. The LECs in the central region of the anterior lens capsule are regular cuboidal epithelial cells and will not experience mitosis. The LEC layer is the most active site during the metabolism inside the lens. Using the glucose and oxygen, the LECs can produce energy to maintain the transport of carbohydrates and amino acids inside lens and provide the energy for forming proteins required by lens fibers in the equatorial region. Thus, it plays key roles in the growth, differentiation, and injury repair of lens. Therefore, the injury or age-related change of LECs will affect the physiological functions of lens fibers and cause lens opacity. Then, some questions still exist:does the Klotho gene exist in human LECs? Will the epigenetic changes of the Klotho gene affect the apoptosis of LECs? And, is it related with the development of cataract? These questions need to be addressed in experiments based on specimens directly harvested from human anterior lens capsule. In our current study, high methylation of Klotho gene DNA was popular among elderly patients with ARC; it resulted in the silencing of target gene expression and the unexpression of functional proteins. Interestingly, in the middle-aged group, the Klotho protein expression was located at the near-central region of the LECs, and no expression was seen in the central region. With increasing age, the morphologies of LECs changed, showing pseudopod-like changes, along with non-homogenous positive reactions. To our knowledge, no article has compared the expressions of the same protein between the central and peripheral regions of LECs. Also, our findings are consistent with the previous assumption that the LECs in the near-central area have genetic functions, while cells in the central region do not have such functions. Also, it was found that the Klotho gene and protein expressions are involved in the aging of LECs. DNA methylation is an epigenetic concept. DNA methylation affects gene expression. Also, DNA methylation is a reversible process, and the use of methylation inhibitors can reverse the Klotho gene inactivation caused by hypermethylation. Studies on CpG island hypermethylation can be used to screen and identify new genes and thus pave the way for the gene therapy for cataract. Thus, in our future studies, we will further elucidate this mechanism via in vitro cell culture and DNA demethylating drug-based interventions.Our study had some limitations:since the lens epithelia are composed of monolayer cells, the sampling range was limited at 5.5mm x 5.5mm to ensure the safety of surgical procedures; meanwhile, the specimens needed to be divided into three equal parts for RT-PCR, protein ICH tests, and gene methylation level measurement. As a result, there was no room for further laboratory tests such as Western blotting, calculation of cell apoptosis rate, and detection of relevant ion levels. Our group had consulted Dr. He Shi-kun from the Doheny Eye Institute at the University of Southern California/Keck School Of Medicine and was told that in vitro cell culture might be a solution. Accordingly, we have collected different human LEC lines from different sources for in vitro cultures, which will be applied in our future studies.In summary, the pathogenesis of ARC is a complex process involving multiple factors. The Klotho gene participates in the occurence and development of cataract, and its epigenetic change is one of the causes of senile cataract. Evidences in our current study elucidate the pathogenic mechanisms of ARC from the perspectives of aging and epigenetic regulation and may guide our future studies. |
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