| IntroductionAge-related cataract (ARC) is the main cause of blindness worldwide. Cataract surgery is still the only therapeutic approach. With the aging of the population in today’s society, the demand for cataract surgery will increase year by year, which will bring a huge economic burden. Therefore, it is important to explore the pathogenesis of ARC and to find an effective intervention in the early disease process. Aging, including lens aging, i.e. cataract formation is a complex process. Various environmental outside and metabolic factors inside the body play an important role in its pathogenesis. It has been proved that epigenetic patterning is modified by environmental exposures and may be a mechanistic link between environmental risk factors and the development of disease. Thus, epigenetic may plays an important role in the pathogenesis of ARC. Epigenetics pertains to heritable alterations in gene expression that do not involve modification of the underlying genomic DNA sequence, the mechanisms of which mainly including DNA methylation, histone modifications and micro RNA (microRNA) are single-stranded non-coding RNA molecules that contribute to post-transcriptional downregulation of gene expression. They bind complementary regions of target mRNAs, most commonly at the 3’untranslated region (3’-UTR). MicroRNA plays important roll in regulating cell growth, differentiation, proliferation, apoptosis, stress and biological processes such as aging. In previous years, microRNA has been one of the hotspots in tumor and blood, but its role in eye disease cause less attention. Recently studies of microRNA in eye disease have been initially launched. For the first time, it is reported that miRNA expression profiles differ between the central epithelium of transparent and cataractous lens samples, suggesting that the miRNAs that are differentially expressed may play roles in lenticular development and cataractogenesis. Based on the current status of research, the present study will focus on the specific microRNA molecular mechanisms involved in the pathogenesis of ARC, by screening and validating of microRNA expression changes in vivo, and exploring the specific molecular mechanisms of microRNA induced lens epithelial cells functional changes in vitro. Meanwhile, we are trying to explore whether there is a relationship between microRNA abnormal expression and its promoter DNA methylation and whether they are co-interacting in ARC.Part I Levels of MicroRNA-34a expression and degree of its methylation in ARCObjectives To investigate the expression changes of microRNA-34a (miR-34a) and the methylation status of its promoter in age-related cataract (ARC).Methods RT-PCR was used to detect and evaluate the expression level of miR-34a and miR-933 in 30 lens capsules of ARC versus 18 transparent lens capsules. The most significantly expressed microRNA differed in the ARC and the control group was selected for the study. Pyrosequencing was performed in 12 lens capsules of ARC and 8 transparent lens capsules of control to evaluate the degree of DNA methylation in miR-34a promoter region.Results The expression of miR-34a was significantly higher in cataract group than in the transparent lens group (P<0.05), no significant difference were found in the expression level of miR-933 (P= 0.69); promoter methylation of miR-34a had no significant difference between the two groups (P= 0.152).Conclusions miR-34a was upregulated and involved in the pathogenesis of ARC.Part Ⅱ Functional analysis in microRNA-34a regulating apoptosis in human lens epithelial cellsObjective To study the functional changes in miR-34a induced apoptosis in HLECs.Methods The double-stranded miR-34a mimics and negative control mimics were transfected into cultured human lens epithelial cell line SRA01/04. After 24 to 72 hours of transfection, apoptosis of the transfected cells were evaluated by FACS and caspase3/7 activity detection. Western Blot was performed to look for the upstream activated target protein. Mitochondrial membrane potential change was detected by JC-1 staining and observed under fluorescence microscopy. The mitochondria outer membrane function was evaluated by the content of cytochrome C (Cyto C) in mitochondria and in cytoplasm by using WB. Total RNA of the transfected cells was extracted to analyze the expression of the mitochondrial respiratory chain-related gene by qRT-PCR. DCFH-DA probe was used to detect the extent of accumulation of ROS.Results 48 hours after transfection the early and late apoptosis of SRA01/04 increased significantly (P<0.05) in miR-34a overexpressed group compared with the control group. Also, the expression of activated caspase3/7 was significantly higher (P <0.05) in miR-34a overexpressed HLECs. JC-1 staining showed decreased mitochondrial membrane potential in miR-34a overexpression HLECs. WB showed that the content of Cyto C were significantly greater in the cytoplasm than that within the mitochondria, indicating the mitochondrial outer membrane function damaged with increased permeability. Meanwhile, the expressions of mitochondrial respiratory chain-related gene NDUFS8, COX5b and ATP5al were significantly decreased, suggesting that mitochondrial respiratory electron transport chain dysfunction. DCF fluorescent in miR-34a was significantly higher than that in control cells, suggesting that over expression of miR-34a caused the production of oxygen free radicals and the accumulation of intracellular ROS induced oxidative stress.Conclusion The overexpression of miR-34a induced HLECs apoptosis through mitochondrial dysfunction.Part III Mechanism research on MicroRNA-34a regulating human lens epithelial cells apoptosisObjective To study the mechanism of miR-34a regulating HLECs apoptosis on transcriptional level.Method The targets of miR-34a was predicted by bioinformatics website including, Pictar, TargetScan and MiRanda. Validation of candidate genes expression in HLECs was carried out by absolute quantitative PCR. Relative quantitative PCR and WB were used to detect the predicted gene expression in SRA01/04 transfected with miR-34a mimics and that with negative control mimics. The wild type and mutant type of potential targets 3’UTR reporter plasmids were constructed and then were co-transfected with miR-34a mimics or negative control mimics into HEK293-T cells. Dual-luciferase reporter assay (Luciferase) was used to confirm the targets. Furthermore, the target genes knockout was carried out by transfecting target genes si-RNA into SRA01/04. Evaluation of apoptosis by flow cytometry, caspase3/7 activity and detection of caspase-9 by WB were performed to evaluate apoptosis and mitochondrial function change.Results Four of the candidate genes, Notch 1, Notch2, Jag1 and Jag2 were selected, all of which located in the Notch pathway and expressed in the lens epithelium. Notch 1 and Notch2 serve as critical receptor genes in this pathway. The expression of Notch 1 and Notch2 displayed by PCR and WB revealed significantly down regulated in miR-34a transfected HLECs compared to the control group. The 3’UTR reporter activities of Notch 1and Notch2 were significantly inhibited in miR-34a overexpressed group (P<0.05), suggesting miR-34a could directly inhibit Notch-1 and Notch-2 protein expressions by binding to their 3’UTR. After transfecting si-Notchl and si-Notch2 into HLECs to know down the target genes, flow cytometry and caspase3/7 activity assay found no significant increase of apoptotic cells or caspase3/7 activity in Notch 1 knocked-down group (P= 0.276), but found significant increase of early and late apoptotic cells (P<0.05) and significant increase of caspase3/7 activity (P<0.05) in Notch2 knocked-down group. In addition, mitochondrial apoptotic marker protein caspase-9 increased significantly in Notch2 knocked-down HLECs.Conclusion Over expression of miR-34a may induce HLECs apoptosis by inhibiting Notch2. |
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