Abstract:

Objective To investigate the effects of Myocilin point mutation(c.A1348T: p.N450Y, Myoc-N450Y) on the expression of extracellular matrix (ECM) protein in human primary trabecular meshwork cells (HTMC) , so as to explore the mechanism of Myoc-N450Y causing glaucoma. Design Experimental study. Participants Human primary trabecular meshwork cells. Methods The experiment was divided into three groups: nine samples in total, empty vector group (Empty), wild type myoc (Myoc-WT) group and Myoc-N450Y group. Lentivirus mediated overexpression method was used to overexpress Myoc-WT gene or Myoc-N450Y gene in HTMC. Western Blot and qRT-PCR were used to quantify the expression of ECM protein collagenⅠ (COL Ⅰ), collagen Ⅳ (COL Ⅳ), fibronectin (FN) and matrix metalloproteinase (MMP) related gene matrix metalloproteinase 2 (Mmp2), matrix metalloproteinase 9 (Mmp9) in Myoc-WT or Myoc-N450Y group. Lentiviral vectors of Myoc-WT and Myoc-N450Y were constructed. Lentivirus expressing Myoc-WT or Myoc-N450Y was packaged and the multiplicity of infection (MOI) of HTMC was determined. HTMC was infected by Myoc-WT lentivirus particles or Myoc-N450Y lentivirus particles. Western Blot and qRT-PCR were used to detect the overexpression of Myoc gene and the effect of N450Y mutation on the expression of Col Ⅰ, Col Ⅳ, Fn, Mmp2 and Mmp9. Main Outcome Measures The expression of Col Ⅰ, Col Ⅳ, Fn, Mmp2 and Mmp9. Results The results of double enzyme digestion and sequencing showed that lentivirus vectors overexpressing Myoc-WT or Myoc-N450Y was successfully constructed, and the MOI in HTMC was 5. Western Blot and qRT-PCR results showed that protein level and mRNA level of Myoc gene were highly over-expressed in Myoc-WT and Myoc-N450Y groups. The relative expression level of Myoc gene in Empty group, Myoc-WT group and Myoc-N450Y group was 1.00±0.02, 28.88±1.28, 22.06±0.47 (F=1020.02，P<0.001）. The relative mRNA expression level of Col Ⅰ, Col Ⅳ and Fn in Myoc-N450Y group was 2.08±0.07, 1.76±0.08,2.63±0.06, significantly higher than that of Myoc-WT group (0.93±0.04, 0.95±0.09, 0.89±0.06) (all P<0.001). Compared to the expression in Myoc-WT group, the protein level of COL Ⅰ and FN were significantly upregulated in Myoc-N450Y group. The relative mRNA levels of Mmp2 and Mmp9 in Myoc-N450Y group were 0.32±0.02 and 0.33±0.15, which was significantly lower than that of Myoc-WT group (1.01±0.03 and 0.95±0.03) (all P<0.001). Conclusion Lentiviruses overexpressing Myoc gene in HTMC were successfully prepared, and it was proved that overexpression of Myoc-N450Y promoted the expression of Col Ⅰ, Col Ⅳ, Fn and inhibit the expression of Mmp2 and Mmp9 in HTMC. Therefore, we speculate that Myoc-N450Y may be involved in the occurrence and development of glaucoma by regulating the expression of ECM protein-related genes. (Ophthalmol CHN, 2019, 28: 374-380)

Key words: myocilin, glaucoma, human primary trabecular meshwork cells, extracellular matrix