| BackgroundWith the increasing population and the arrival of aging society, visual impairments caused by cataract are more than before. This problem has seriously affected people's health and quality of life. Cataract becomes the world's leading cause of blindness, and in China it is listed as the first cause of blindness, but its pathogenesis is still not completely clear.Previous studies have found that the occurrence of cataract results from interaction of a variety of factors. Some common factors include oxidative damage and age-related degeneration of LECs, gene mutation and radiation injury of crystallin, excessive stimulation of glucose, galactose disbolism and lipid peroxidation injury, etc. As a result of these factors on long-term stimulation to lens, lens epithelial cells (LECs) have been injured at different levels. Then it causes the apoptosis of LECs, and lens vary from transparent to opaque, so that their functions are affected. The pathway of light reaching to retina through vitreum is affected, which results in visual impairments.Age related cataract (ARC), also known as senile cataract, an age-related disease, is currently the main reason for the visual impairments and blindness of the elder. The reason of ARC is mainly that with age increasing, phacoscotasmus occurs in the lens affected by some above-mentioned factors. With China's growing population and aging problems, the number of ARC patients is increasing. Previous studies have shown that apoptosis of LECs plays an important role in its pathogenesis. The proliferation and differentiation of epithelial cells in equator of lens form new lens fibers. Then these lens fibers move to the center of lens--that is nuclei lentis. During this process, the organelles and the nucleus of lens fiber cells continuously degrade, and eventually disappear. Only transparent cytoplasm remains and it forms the nuclei lentis. There is almost no protein in it. Over time, the hardness of lens is increased. With the increase of age, aging is inevitable. Humans cannot completely prevent the aging of organs, including the aging of the lens. But with the development of science, we are trying to take some measures to delay the aging of lens. For ARC, if we used topical drugs as early intervention, we could prevent cataract, maintain transparent lens, reduce the degree of ARC, improve the quality of the ARC patients'lives to a larger degree. The main current treatment is to remove the opaque lens and implant intraocular lens to make patients'vision recover through cataract phacoemulsification or extracapsular cataract extraction. But this technology is still a traumatic cataract treatment, and the effect of traditional medicine in the clinical application is not very good. Therefore, further research for the pathogenesis of cataract will provide new ideas for the treatment of age-related cataract.ObjectiveTo further explore the pathogenesis of ARC, in this study miRNA microarray is adopted in screening miRNA expression profile of LECs of ARC and normal LECs. Fluorescence quantitative analysis of pathogenesis of hsa-miRNA-15a regulating the apoptosis of LECs and overexpression detection of cell culture are used to further clarify the apoptosis of ARC causing LECs, and lay the foundation for genetic treatment for cataract.MethodsThere are three parts in this research:Part 1 miRNA microarray is adopted in screening miRNA expression profile of LECs of ARC and normal LECs. miRNA expression profile of LECs is preliminarily screened to provide researching foundation for the study of pathogenesis of miRNA participating the apoptosis of LECs.This study adopts miRNA microarray, and 5 cases of ARC lens anterior capsule and 5 cases of normal lens anterior capsule are chosen to detect the expression of miRNAs in two kinds of LECs. Among these miRNAs, those whose differences are greater than 2 times and are relevant to cataract are screened.Part 2 The expression of hsa-miRNA-15a and its target gene bcl-2 and mcl-1 are detected in of ARC and normal LECs. The change of hsa-miRNA-15a and its target gene--bcl-2 and mcl-1 are studied in the occurence of ARC to further elucidate their roles in the pathogenesis of cataract.This study selects 60 cases of ARC patients, including 20 cases of cortical cataract,20 cases of nuclear cataract and 20 cases of posterior subcapsular cataract (they are labeled as group A, group B, and group C), and 20 cases of normal lens anterior capsule as the control group, using RT PCR to detect the expression of hsa-miRNA-15a-5p and hsa-miRNA-15a-3p in control group and three different types of LECs of ARC; With the same method, half lens anterior capsule is detected by RT-PCR, and the other half by Western blot. These two methods are used to detect the expression of their target gene bcl-2 and Mcl-1 in control group and three different types of LECs of ARC, and they are analyzed statistically.Part 3 Culturing human LECs. hsa-miRNA-15a is overexpressed in human LECs, and its effect on the apoptosis of LECs is detected to further clarify its pathogenesis.Human LECs are selected and cultured. hsa-miRNA-15a-5p and hsa-miRNA-15a-3p are transfected by RT-PCR to verify its expression in human cells culturing. Electron microscope is used to observe the morphological changes of the transfected cells. MTS, plate clone cell assay, Hoechst, TUNEL, AnnexinV-FTTC/PI apoptosis detection, wound healing and Transwell invasion and migration assay are used to observe its functional changes.Results1. Through the analysis of the results of the miRNAs microarray expression of LECs of ARC and normal LECs samples, up-regulated or down-regulated more than 2 times as the benchmark, and by screening, we can find that 181 miRNAs are up-regulated and 114 miRNAs down. Among these miRNAs,126 of them are up-regulated to more than 5 times; 51 of them down to more than 5 times.2. The results of RT-PCR shows that:hsa-miRNA-15a-5p and hsa-miRNA-15a-3p, are expressed a little in normal LECs, and are significantly increased in LECs of cortical cataract, nuclear cataract and posterior subcapsular cataract. In LECs of cortical cataract they are up to 5-8 times that in normal LECs, in nuclear cataract more than 10 times, and in posterior subcapsular cataract even more than 20 times. The results of statistical analysis show that comparative differences of the average relative expression between hsa-miRNA-15a-5p in cortical cataract, nuclear cataract, posterior subcapsular cataract and that in control group have significant statistical meaning (P<0.01); comparative differences of the average relative expression between hsa-miRNA-15a-3p in cortical cataract, nuclear cataract group, posterior subcapsular cataract and that in control group have significant statistical meaning (P<0.01). Real-time PCR and Western-blot test results show that: bcl-2 and mcl-1 in normal LECs have visible expression, but their expression are significantly decreased in that of cortical cataract, nuclear cataract, and posterior subcapsular cataract. Statistical analysis shows that comparative differences of the average relative expression between bcl-2 in cortical cataract, nuclear cataract, posterior subcapsular cataract and that in control group have significant statistical meaning (P<0.01); comparative differences of the average relative expression between mcl-1 in cortical cataract, nuclear cataract, posterior subcapsular cataract and that in control group have significant statistical meaning (P<0.01).3. By the classical Taqman probe method, HLE-B3 cells transfected with hsa-miR-15a-5p and hsa-miRNA-15a-3p are quantitatively analyzed. The results show that hsa-miRNA-15a-5p or hsa-miR-15a-3p are not detected in HLE-B3 cells maybe because their quantities are very low or do not exist. But in cells transfected with hsa-miRNA-15a-5p and hsa-miRNA-15a-3p, their expressions are both significantly increased. Cell forms show that cells transfected with these two miRNAs begin to change from 24h:cells become round and bigger, their contours become unclear, net structure reduces. MTS shows that after being transfected with the two types of miRNA, HLE-B3's growth is significantly inhibited. The maximum growth inhibition rate of hsa-miRNA-15a-3p on HLE-B3 reaches 57.13%, and that of hsa-miRNA-15a-5p reaches 44.60%. The results of plate clone show that hsa-miRNA-15a-5p and hsa-miRNA-15a-3p have significant inhibition on the formation and clone ability of HLE-B3 cells. The TUNEL apoptosis test results show that:HLE-B3 cells transfected with these two miRNAs are detected with obvious green fluorescence, indicating that these 2 microRNA cause the apoptosis of HLE-B3 cell. Annexin V-FITC/PI amphophil cell apoptosis test shows HLE-B3 cells after being transfected. The cell debris is more than that of the control group; the results of wound healing show that the lateral transferring ability of HLE-B3 cells transfected with hsa-miRNA-15a-5p is significantly inhibited, but that does not happen in hsa-miRNA-15a-3p transfected cells.Conclusion1. There are differences of expressions of miRNAs between in LECs of ARC and that in normal LECs, and these miRNAs may be involved in the occurrence of ARC.2. hsa-miRNA-15a-5p and hsa-miRNA-15a-3p in three different types of LECs of ARC expression are more than that of the normal LECs, while bcl-2 and mcl-1 are lower than that of the normal LECs. hsa-miRNA-15a-5p and hsa-miRNA-15a-3p may cause cell apoptosis by inhibiting expression of their target genes bcl-2 and mcl-1, resulting in ARC.3. hsa-miRNA-15a-5p and hsa-miRNA-15a-3p after their transfection of human LECs inhibit cell proliferation and clone to promote the LECs apoptosis, which starts the occurrence of ARC. |
PDF Full Text Request