circular RNA circ-231 promotes protein biogenesis of TPI1 and PRDX6 through mediating the interaction of eIF4A3 with STAU1 to facilitate unwinding of secondary structure in 5' UTR, enhancing progression of human esophageal squamous cell carcinoma (ESCC).

Background: The nuclear cap-binding complex (CBC)-dependent translation (CT) is an important initial translation pathway for 5'-cap-dependent translation in normal mammal cells. Eukaryotic translation initiation factor 4A-III (eIF4A3), as an RNA helicase, is recruited to CT complex and enhances CT efficiency through participating in unwinding of secondary structure in the 5' UTR. However, the detailed mechanism for eIF4A3 implicated in unwinding of secondary structure in the 5' UTR in normal mammal cells is still unclear. Specially, we need to investigate whether the kind of mechanism in normal mammal cells extrapolates to cancer cells, e.g. ESCC, and further interrogate whether and how the mechanism triggers malignant phenotype of ESCC, which are important for identifying a potential therapeutic target for patients with ESCC. Methods: Bioinformatics analysis, RNA immunoprecipitation and RNA pulldown assays were performed to detect the interaction of circular RNA circ-231 with eIF4A3. In vitro and in vivo assays were performed to detect biological roles of circ-231 in ESCC. RNA immunoprecipitation, RNA pulldown, mass spectrometry analysis and co-immunoprecipitation assays were used to measure the interaction of circ-231, eIF4A3 and STAU1 in HEK293T and ESCC. In vitro EGFP reporter and 5' UTR of mRNA pulldown assays were performed to probe for the binding of circ-231, eIF4A3 and STAU1 to secondary structure of 5' UTR. Results: RNA immunoprecipitation assays showed that circ-231 interacted with eIF4A3 in HEK293T and ESCC. Further study confirmed that circ-231 orchestrated with eIF4A3 to control protein expression of TPI1 and PRDX6, but not for mRNA transcripts. The in-depth mechanism study uncovered that both circ-231 and eIF4A3 were involved in unwinding of secondary structure in 5' UTR of TPI1 and PRDX6. More importantly, circ-231 promoted the interaction between eIF4A3 and STAU1. Intriguingly, both circ-231 and eIF4A3 were dependent on STAU1 binding to secondary structure in 5' UTR. Biological function assays revealed that circ-231 promoted the migration and proliferation of ESCC via TPI1 and PRDX6. In ESCC, the up-regulated expression of circ-231 was observed and patients with ESCC characterized by higher expression of circ-231 have concurrent lymph node metastasis, compared with control. Conclusions: Our data unravels the detailed mechanism by which STAU1 binds to secondary structure in 5' UTR of mRNAs and recruits eIF4A3 through interacting with circ-231 and thereby eIF4A3 is implicated in unwinding of secondary structure, which is common to HEK293T and ESCC. However, importantly, our data reveals that circ-231 promotes migration and proliferation of ESCC and the up-regulated circ-231 greatly correlates with tumor lymph node metastasis, insinuating that circ-231 could be a therapeutic target and an indicator of risk of lymph node metastasis for patients with ESCC.

SIRT1 and SIRT5 activity expression and behavioral responses to calorie restriction.

To investigate the effects of calorie restriction (CR) on behavioral performance and expression of SIRT1 and SIRT5 in rat cerebral tissues. Beginning at 18 months of age, 60 rats were randomly divided into a CR group (n = 30) and a group that remained fed ad libitum (AL; n = 30). CR rats were restricted to a diet of 60% of their daily food consumption. After 6 months of CR, CR rats displayed a maximum 50% reduction in escape latency (AL 20 ± 0.3 s vs. CR 10 ± 0.2 s) and a 3.2 s decrease in time and distance to target when evaluated in Morris water maze tests. The levels of SIRT1 and SIRT5 protein in cerebral tissues of CR rats were elevated compared to AL rats (P < 0.05). CR retarded declines in cognitive ability and enhanced the expression of both SIRT1 and SIRT5 proteins in the cerebral tissue of CR rats compared with AL rats.

Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons.

To investigate the activity of senescence-associated beta-galactosidase (SA-beta-GAL) in the hippocampus of aging rats. Hippocampi of 6-, 18-, and 24-month-old rats were observed by histochemical staining for SA-beta-GAL and cytochemical staining for SA-beta-GAL in cultured hippocampal neurons. The activity of SA-beta-GAL doubled in hippocampal pyramidal cells of the CA3 region in rats between 6 and 18 months (14.57+/-2.74% vs. 31.66+/-14.12% SA-beta-GAL-positive, respectively), and reached 50.76+/-14.41% positive at 24 months. The activity of SA-beta-GAL also increased as a function of time upon prolonged culture of cultured hippocampal neurons with 95% of cells being SA-beta-GAL-positive at 20 days in vitro. Interestingly, no SA-beta-GAL-positive cells were found in neurons of the hippocampal dentate gyrus, a neurogenic region of the brain, at any age examined. SA-beta-GAL can be used as a senescence biomarker in determining senescent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging.

Increased intrinsic neuronal vulnerability and decreased beneficial reaction of macrophages on axonal regeneration in aged rats.

Previously we showed that macrophage activation in the eye by intravitreal application of zymosan increased retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve injury. It is known that the intrinsic ability of CNS neurons to survive and to regrow axons after optic nerve injury differs between developing and adult mammals. However, whether aged animals also differ in their ability to survive and regrow injured axons are not known. In this study we investigated whether the abilities of RGCs to survive and to regrow injured axons differed between rats aged 6-8, 60 and over 96 weeks, and whether macrophage responses in the eye were different at different ages. We found that the intrinsic viability of RGCs, as shown in vitro, was reduced in aged rats, but RGC viability after optic nerve injury in vivo was similar among rats of the different ages. The ability of RGCs to regrow injured axons into a peripheral nerve graft also remained similar between young and aged rats. Macrophage activation in the eye was confirmed to be beneficial and provided the basis for zymosan treatment-dependent RGC protection. However, reduced activation of macrophages in zymosan-treated eyes was seen in aged rats. Importantly, this reduced macrophage activation in aged rats led to a decreased level of RGC axonal regeneration when compared with that in young rats of the same treatment. Thus age influences the intrinsic viability of RGCs and the beneficial impact of macrophages on RGC axonal regeneration after optic nerve injury.

Evaluation of LOXL1 polymorphisms in primary open-angle glaucoma in southern and northern Chinese.

PURPOSE: The lysyl oxidase-like protein 1 (LOXL1) gene is strongly associated with exfoliation glaucoma, which is very rare in the Chinese population. The implicated LOXL1 polymorphisms have not been associated with primary open-angle glaucoma (POAG). In this study, we investigated three of the LOXL1 polymorphisms in POAG in a southern Chinese population of Hong Kong and northern Chinese from Beijing. METHODS: The Hong Kong group included 293 POAG patients and 250 controls, and the Beijing group included 169 POAG patients and 197 controls. LOXL1 single nucleotide polymorphisms (SNPs), rs1048661, rs3825942, and rs2165241, were genotyped by direct DNA sequencing. Individual association was analyzed using the chi(2) test, and haplotype-based association analysis was performed in WHAP. RESULTS: Each of the candidate SNPs was not statistically associated with POAG in either group (p>0.017, Bonferroni correction). Haplotype-based association analysis had identified a significant omnibus association (Omnibus chi(2)=18.16, p=0.00115) between these SNPs and POAG in the Hong Kong group. A minor haplotype (T-G-T) showed significant statistical association with POAG. It presented in 2.1% of cases and 0.4% of controls, conferring a 5.24 fold of increased risk to the disease (95% CI: 1.17-23.54, P(perm)=0.00108). However, this haplotype was absent in the Beijing group. CONCLUSIONS: Individual LOXL1 SNPs, rs1048661, rs3825942, and rs2165241, were not associated with POAG in the Chinese population. However, a minor haplotype T-G-T was found to be associated with the disorder in the southern Chinese. The low frequencies of the at-risk alleles at rs1048661 and rs2165241 may be one of the factors that led to the low prevalence of exfoliation syndrome in the general populations of the Chinese.

Metabolic assessment of the human pons by in vivo proton magnetic resonance spectroscopy.

OBJECTIVE: To determine the normal mean reference normal value for metabolic ratios in the pons of healthy adult Chinese subjects by using proton magnetic resonance spectroscopy (1HMRS). MATERIALS AND METHODS: Eighty healthy Chinese subjects, ranging in age from 21 to 60 years, were divided into four groups, each containing 20 subjects per decade. The pons of every subject was scanned on single-voxel 1HMRS by using the point-resolved proton spectroscopy sequence (PRESS) with echo time (TE)=144 ms and repetition time (TR)=1500 ms. RESULTS: The total mean ratios of N-acetylasparate/creatine-phosphocreatine (NAA/Cr), NAA/choline-containing compounds (Cho) and Cho/Cr in subjects ranging from 21 to 60 years were 2.13+/-0.07, 1.22+/-0.11 and 1.81+/-0.09 respectively. The highest metabolite ratios were seen in the 41-50 year group. There was no significant difference with respect to age or gender. CONCLUSIONS: The ratios of NAA/Cr, NAA/Cho or Cho/Cr in the pons did not correlate with the age or gender of healthy subjects.