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Purpose: To investigate the effect of 3-methyladenine (3-MA) and starvation on the expression of matrix metalloproteinase (MMP-2) in patients with primary open-angle glaucoma. Methods: Primary TM cells were cultured and divided into three groups. The control group was treated with a normal medium, the 3-MA group was stimulated with 3-MA, and the starvation group received nutrient depletion by replacing the normal media with Earle's balanced salt solution. Cellular mRNA and protein were measured at different 3-MA concentrations and starvation time periods. The level of autophagy was accessed by monodansylcadaverine fluorescent staining and expression of specific autophagy-related genes, light chain 3 (LC3), and Beclin1. The effects of 3-MA and starvation on cell proliferation were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay kit. The mRNA and protein expression of LC3-II, Beclin1, and MMP-2 were measured by reverse transcription-polymerase chain reaction and western blot, respectively. Results: Compared to the control group, starvation significantly upregulated LC3-II and Beclin1 in TM cells after 3 h of stimulation, which peaked at 6 h and 9 h, respectively. Increased MDC-labeled cells were also observed. Starvation downregulated the expression of MMP-2. On the contrary, 3-MA suppressed the activation of autophagy, as shown by the marked downregulation of LC3-II and Beclin1. The expressions of MMP-2 were higher in the 3-MA group compared to the control group, reaching a peak at a concentration of 5 mM. Conclusion: Autophagy may be involved in the pathogenesis of POAG via regulating the expression of MMP-2 and, subsequently, the deposition of the extracellular matrix.
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AIM: To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension. METHODS: Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48h after perfusion respectively, to investigate the activation of autophagy following ischemia-reperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot. RESULTS: The number of retinal ganglion cells (RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer (GCL), and p62 protein was expressed in the nerve fiber layer (NFL), GCL, inner plexiform layer (IPL), inner nuclear layer (INL) and outer plexiform layer (OPL). In comparison to the control group, the expression level of LC3- II was higher in all the experimental groups (P<0.05), with the peak expression at 12h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control (P<0.05, except for group A), with the peak level occurred 24h after reperfusion. CONCLUSION: Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3- II and p62 expression in the retina. Autophagy flux is damaged 12h after reperfusion, potentially resulting in further loss of RGCs.
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Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-ß catalytic subunit (ß-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.
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Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.
