MiR-326 Knockdown Increases BetaB2-crystallin Expression In The Lens Delaying The Process Of Lens Opacification By Targeting Fibroblast Growth Factor 1

Age-related cataract is one of the most common causes of blindness in the elderly population,accounting for almost half of all vision loss.With the increase of the aging population,the incidence of age-related cataract has been greatly increasing.The main clinical characteristic of cataract is the presence of lens opacification.Therefore,the identification of the key steps in the pathogenesis of cataract that contributes to lens opacification is important for developing a new therapeutic strategy to delay or substantially prevent the progress of cataract.Crystal protein in mammalian lens,mainly divided into three categories such as α、b、g.Beta-crystallin B2(CRYBB2 or beta B2)is encoded by the CRYBB2 gene,and is the most abundant crystallin in the β family of crystallin proteins expressed in the lens.The normal structure and water solubility of Crybb2 is important for maintaining the refraction and transparency of the lens.Thus,the Crybb2 is more suitable as a target protein for age-related lens function studies.So if we can find out the the upstream miRNA which could regulate the expression of Crybb2,we may delay the process of lens opacity.MiRNAs are a group of endogenous small non-coding RNAs that are 20 to 25 nucleotides in length.MiRNAs regulate m RNA translational interference or degradation by binding to the 3′-untranslated regions of target m RNAs.MiRNAs can regulate many cellular processes such as proliferation,differentiation,metabolism and apoptosis.Furthermore,miRNAs are specifically expressed in many ocular tissues,including the cornea,lens and retina.It has been reported that miRNAs promote the occurrence and development of lens opacity by regulating the expression of crystallins.Therefore,it is important to explore whether and how miRNAs to regulate the expression of Crybb2 are important for early prevention and treatment of senile cataract.In the present study,we investigated the effect of miR-326 on the expression of Crybb2 in lens epithelial cells and elucidate the therapeutic effect of miR-326 in selenium cataract model mice.We found that miRNA-326、miR-204-5p、miR-491-5p、miR-330-5p are related with bB2.MiR-326 and miR-204-5p expression were significantly downregulated in KO mice,compared with WT mice,especially miR-326.Then we found that knockdown miR-326 upregulated the expression of Crybb2,inhibited apoptosis,and promoted proliferation in lens epithelial cells and Selenium cataract model rat by directly targeting fibroblast growth factor 1(FGF1).Our finding suggests that the inhibition of miR-326 is a promising therapeutic target in cataract treatment.Section1 Screening of micro RNAs in the lens of WT and beta-crystallin B2(Crybb2)knockout miceObjective: To screen with large differences micro RNAs in beta B2 in the lens of WT and beta-crystallin B2(CRYBB2)knockout mice.Methods: 1.MiRNA extraction,c DNA synthesis and quantitative RT-PCR(q RT-PCR).Total RNA was extracted from the lens tissues of WT and CRYBB2 KO mice using Trizol reagent.RNA was reverse transcribed into c DNA using the Prime Script RT Reagent Kit,according to manufacturer’s instructions.2.MiRNA screening and prediction target genes.The possible miRNA binding sites on CRYBB2 was predicted by bioinformatics databases including Pic Tar,miRBase and Target Scan algorithms.Result: 1.MiR-326,miR-204-5p,miR-330-5p and miR-491-5p were predicted to bind to CRYBB2.2.MiR-326 and miR-204-5p,especially MiR-326 expression were significantly downregulated in Crybb2-/-mice,compared with WT mice.Conclusion: The expression of miR-326 and miR-204-5p were significantly downregulated in Crybb2-/-mice,compared with WT mice,especially miR-326.The miR-326 is used to study the effect of micro RNA on the expression of Crybb2 crystal protein in HLEC-B3 cells.Section2 Micro RNA-326 knockdown increases beta B2-crystallin expression in the lens by targeting fibroblast growth factor 1Objective: To investigate the downstream target gene and regulatory pathway of the regulation of Crybb2 by miR-326.Methods: 1.The downstream target gene of miR-326 was identified by luciferase activity assay.2.Cell transfection.After HLEC-B3 cells were treated with sh-FGF1 or sh-FGF1 control(FGF1 [-]-NC)or FGF1 or FGF1 control(FGF1 [+]-NC),cells were treated with miR-326 antagomir or miR-326 antagomir control(miR-326[-]-NC)for 72 hours.Western blot and immunocytochemistry.were used to compare the changes of Crybb2 expression between the two groups and the negative control group.3.HLEC-B3 cells were treated with 200μM H2O2 for 24 hours,and the apoptosis rate was observed by flow cytometry.Result: 1.FGF1 was the target of miR-326.2.FGF1 can effectively regulate the expression of Crybb2 in lens epithelial cells.3.Knockdown of miR-326 increased the expression of Crybb2 by upregulating FGF1.4.MiR-326 antagomir alleviated H2O2-induced apoptosis of HLEC.Conclusion: We found that the miR-326-FGF1-Crybb2 axis played an important role in human lens epithelial cells.Knockdown of miR-326 upregulated Crybb2 expression via the upregulation of FGF1.In the third part,the expression of Crybb2 in the lens can be increased by the inhibition of miR-326 through the selenium-type cataract model rat,and finally retard the progress of cataract.Section3 Knockdown of miR-326 inhibited the progression of cataractObjective: To investigate the role of miR-326 in the regulation of Crybb2 expression and delay the progress of cataract in the lens of selenium-deficient cataract model rat.Methods: The cataract model in rats was established by the subcutaneous injection of sodium selenite.Nine-day-old SD rats were subcutaneously injected with sodium selenite(25 μmol/kg)once every other day for three times.The appearance of lens opacity indicated the successful creation of cataract.Four cataract rats were used to investigate the effect of miR-326 on cataract.The left eye of each mouse was treated with eye drops containing miR-326 antagomir control(control group),and the right eye was treated with eye drops containing miR-326 antagomir(treatment group).After the model was established,the rats received antagomir treatment two times a day for seven days with a total amount of 1 nmol.The maximal diameter changes of the cataract plaques were detected using vernier cursor.The drug absorption in the lens was detected by q RT-PCR,and the expression of FGF1 and Crybb2 in the lens was detected by western blot.Result: 1.The white punctuate was observed in the middle of the nuclear area one day after the cataract model was established,and was quickly extended into the cortex.2.MiR-326 antagomir treatment reduced the progression of the white spot volume of the lens.Conclusion: knockdown of miR-326 upregulated the expression of Crybb2 by targeting FGF1,and delay the progress of cataract which providing a new way for the early prevention and treatment of cataract.