Abstract:

Objective To study the influence of myocilin（MYOC） gene C1009del mutation（MYOC-C1009del） on the function of human trabecular meshwork cells（HTMC） in vitro and the pathway of nuclear factor-kappa B（NF-κB）.Methods Lentiviral vector pLVX-3FLAG-MYOC-EGFP（rLV-MYOC） and pLVX-3FLAG-MYOC-mut-EGFP（rLV-MYOC-mut） were constructed according to MYOC gene wild type and MYOC-C1009del. The lentiviral vectors were packaged and purified,and then were infected into HTMC. The blank HTMC and HTMC infected with empty plasmid were used as 2 groups of blank control. After 48 h of infection,the follow-up cell function assay was performed. Flow cytometry was used to determine HTMC apoptosis rate and cell cycle. Cell viability was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. The mRNA expressions of NF-κB and its inhibitor of NF-κB（IκB） were determined by fluorescence quantitation polymerase chain reaction（PCR）. Western blotting was used to determine the expressions of NF-κB and IκB at protein levels. Results HTMC infected with rLV-MYOC-mut showed a shortened G1 phase and prolonged S phase compared with those infected with rLV-MYOC（P<0.05）. However,there was no statistical significance in apoptosis and viability（P>0.05）. The mRNA expressions of NF-κB and IκB in HTMC infected with rLV-MYOC-mut were lower than those infected with rLV-MYOC（P<0.05）. At protein levels,the expressions of NF-κB in HTMC infected with rLV-MYOC-mut and with rLV-MYOC were the same,while the expression of IκB in HTMC infected with rLV-MYOC-mut showed downward trend（P<0.05）. ConclusionsMYOC-C1009del can affect HTMC cycle that may arrest in S phase and affect the normal function of HTMC. Although MYOC-C1009del can down regulate the expression of IκB,it does not lead to the occurrence of primary open-angle glaucoma by activating NF-κB pathway.

Key words: Myocilin gene, Nuclear factor-kappa B, Inhibitor of nuclear factor-kappa B, Primary open-angle glaucoma