IntroductionThe aim of the study was to investigate the effect of CNRIP1 promoter methylation on the proliferative, invasive and migration potential of colorectal cancer cells, including its potential use for the early detection and prognostic assessment of colorectal cancer.Material and methodsQuantitative methylation-specific PCR (qMSP) was used to detect the methylation status of the CNRIP1 promoter region in peripheral blood samples drawn from patients with colorectal adenocarcinoma, benign colorectal adenoma, and matched healthy controls. Putative CpG methylation sites were then pyrosequenced. We subsequently suppressed CNRIP1 methylation within colon cancer cells via treatment with 5-azacytidine and overexpressed colon cancer cells by transfection with a CNRIP1-overexpression pcDNA3.0 plasmid. Thereafter, the CNRIP1 methylation status and mRNA and protein expressions levels were determined. Finally, the proliferative, invasive and migration abilities of cell lines were determined with the CCK-8 and Transwell cell assays.ResultsThere were differences in the methylation status at loci 2216, 2226, 2231, 2245, and 2254 within the promoter region of CNRIP1 between patients with colorectal adenocarcinoma, colorectal adenoma, and healthy volunteers. The methylation status of CpG sequence 2245 significantly correlated with tumor diameter, invasion depth, TNM stage, grade, and lymph node metastasis (p < 0.05). The proliferative, invasive and migration abilities of colon cancer cells treated with 5-azaC or transfected with a CNRIP1-overexpression plasmid were significantly impaired relative to negative controls (p < 0.05).ConclusionsThe methylation status at locus 2245 within the CNRIP1 promoter region has potential value for the early detection and prognostic evaluation of colorectal cancers. Demethylation of the CNRIP1 promoter or overexpression of CNRIP1 can reduce the proliferative and migration abilities of colon cancer cells.

Hutchinson‐Gilford progeria syndrome (HGPS) arises when a truncated form of farnesylated prelamin A accumulates at the nuclear envelope, leading to misshapen nuclei. Previous studies of adult Zmpste24‐deficient mice, a mouse model of progeria, have reported a metabolic response involving inhibition of the mTOR (mammalian target of rapamycin) kinase and activation of autophagy. However, exactly how mTOR or autophagy is involved in progeria remains unclear. Here, we investigate this question by crossing Zmpste24+/− mice with mice hypomorphic in mTOR (mTOR△/+), or mice heterozygous in autophagy‐related gene 7 (Atg7+/−). We find that accumulation of prelamin A induces premature aging through mTOR overactivation and impaired autophagy in newborn Zmpste24−/− mice. Zmpste24−/− mice with genetically reduced mTOR activity, but not heterozygosity in Atg7, show extended lifespan. Moreover, mTOR inhibition partially restores autophagy and S6K1 activity. We also show that progerin interacts with the Akt phosphatase to promote full activation of the Akt/mTOR signaling pathway. Finally, although we find that genetic reduction of mTOR postpones premature aging in Zmpste24 KO mice, frequent embryonic lethality occurs. Together, our findings show that over‐activated mTOR contributes to premature aging in Zmpste24−/− mice, and suggest a potential strategy in treating HGPS patients with mTOR inhibitors.

The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis. The endoplasmic reticulum (ER) stresses and unfolded protein response (UPR) are initiated to alleviate the accumulation of unfolded proteins against cell injury. The previous researches had demonstrated that fluoride induced ER stress in other cells or tissues. In this study, we determined the ER stress and UPR to investigate their roles in aberrant activation of fluoride-treated osteoblasts. The gene expression of bone markers and UPR factors in MC3T3-E1 cells treated with varying doses of fluoride administration was analyzed. Meantime, levels of glutathione and glutathione disulfide were tested by the ultraperformance liquid chromatography-tandem mass spectrometry applications. Our results indicated that a certain dose and period of fluoride administration induced cell proliferation and differentiation, and Runx2 was involved in the regulation of osteoblastic differentiation of MC3T3-E1 cells. Increase trend of Runx2 expression was consistent with change of marker of ER stress. Fluoride caused ER stress and stimulated UPR during the process of osteoblast maturation, while oxidative stress was also active in the occurrence of ER stress. These data indicated that ER stress and UPR were possibly involved in the action of fluoride on osteoblasts.

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Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis.