Lizhi Xu

Alterations in microRNA expression linked to microcystin-LR-induced tumorigenicity in human WRL-68 Cells.

Microcystin-LR (MC-LR) is a cyclic heptapeptide that acts as a potent hepatotoxin and carcinogen. However, the mechanism of its carcinogenic action remains undetermined. In this study, MC-LR was used to induce the malignant transformation of the WRL-68 cell line. Alterations in microRNA (miRNA) expression in the transformed cell were analyzed to determine the role of miRNAs in MC-LR-induced carcinogenesis. Cultured WRL-68 cells (labeled 25MC10) were continuously exposed to a low concentration (10 μg/L) of MC-LR for 25 passages. Compared with the mock-treated parental cells, the induced 25MC10 cells exhibited a higher growth rate, resistance to serum-induced terminal differentiation, and tumorigenicity in a nude mouse xenograft test. A pilot miRNA expression array analysis was conducted on the 25MC10 cells, followed by validation of select miRNAs by RT-PCR. We found that the onco-miRNAs miR-21 and miR-221 displayed upregulated expression while the liver-specific miR-122 was downregulated. These results suggest that chronic MC-LR exposure alters the miRNA expression profile of WRL-68 cells and causes phenotypic transformation. We propose that characteristic miRNA alterations could be used as molecular targets for the development of environmental water monitoring methods.

Tectorigenin inhibits the in vitro proliferation and enhances miR-338* expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis

UNLABELLED Tectorigenin is one of the main components in rhizomes of Iris tectorum, which is traditionally used to treat disorders such as hepatic cirrhosis caused by fibrosis. Idiopathic pulmonary fibrosis (IPF), one of the most common interstitial lung diseases, is caused by accumulation of fibroblasts in lungs. AIM OF THE STUDY In this work we sought to examine the effects of tectorigenin on pulmonary fibroblasts in the IPF animal model and investigated the molecular mechanism (microRNA regulation) of tectorigenin treatment. MATERIALS AND METHODS A well-known animal disease model of pulmonary fibrosis in rat was established by intratracheally instilling of bleomycin. In vitro cultured pulmonary fibroblasts in bleomycin-treated rats and in controls were treated with or without tectorigenin. Comparative analyses of cell proliferation, apoptosis and cell cycle of pulmonary fibroblasts in bleomycin-treated rats and in controls were performed. Expression of miR-338* and its candidate gene LPA1 related to IPF of tectorigenin-treated pulmonary fibroblasts in bleomycin-treated rats were further investigated. RESULTS Tectorigenin significantly inhibited the proliferation of pulmonary fibroblasts in bleomycin-treated rats but not in controls. However, no altered cell cycle and apoptosis of pulmonary fibroblasts in bleomycin-treated rats and in controls was observed after tectorigenin treatment. Tectorigenin remarkably enhanced miR-338* expression of pulmonary fibroblasts in bleomycin-treated rats and downregulated LPA1 in the protein level. CONCLUSIONS Tectorigenin inhibits the proliferation of pulmonary fibroblasts in vitro and enhances miR-338* expression, which might in turn downregulate LPA1. This indicates a potential inhibitory role of tectorigenin on the pathogenesis of IPF.

Mucin 5B Promoter Polymorphism Is Associated with Susceptibility to Interstitial Lung Diseases in Chinese Males

The variation of G>T in the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) and familial interstitial pneumonia (FIP) in Caucasians, but no information is available regarding this variant in the Chinese population. We recruited 405 patients with interstitial lung diseases (ILD), including 165 IPF patients and 2043 healthy controls, for genotyping the MUC5B gene in the Chinese population. One hundred three patients with pneumonia and 360 patients with autoimmune diseases (ADs) were recruited as disease controls. Our results indicated that the prevalence of the minor allele (T) of the polymorphism rs35705950 in healthy Chinese subjects was approximately 0.66%, which was lower than that described in the Caucasian population. The frequencies of the T allele were 3.33% and 2.22% in IPF and ILD patients, respectively, and these values were significantly higher than those of healthy controls (P = 0.001, OR = 4.332 for IPF, and P = 0.002, OR = 2.855 for ILD). A stratified analysis showed that this variant in MUC5B associated with the risk for ILD mainly in older male Chinese subjects. No difference was observed between patients with pneumonia, AD patients, and healthy controls.

Analysis of hMLH1 Missense Mutations in East Asian Patients with Suspected Hereditary Nonpolyposis Colorectal Cancer

Purpose: Germ line mutations in the DNA mismatch repair gene hMLH1 are a frequent cause of hereditary nonpolyposis colorectal cancer and about one-third of these are missense mutations. Several missense mutations in hMLH1 have frequently been detected in East Asian patients with suspected hereditary nonpolyposis colorectal cancer, but their pathogenic role has not been extensively assessed. The aim of this study was to perform functional analyses of these variants and their association with gastrointestinal cancer in East Asians. Experimental Design: Altogether, 10 hMLH1 variants were analyzed by yeast two-hybrid and coimmunoprecipitation assays. Results: The carboxyl-terminal replacements Q542L, L549P, L574P, and P581L in hMLH1 resulted in complete loss of activity in both yeast two-hybrid and coimmunoprecipitation tests and thus might be considered as pathogenic. The amino-terminal variants S46I, G65D, G67R, and R217C did not affect complex formation with hPMS2 in coimmunoprecipitation, but partly or fully lost their activity in yeast two-hybrid assay, and we suggested that these variants might reduce the efficiency of the heterodimer to go into the nucleus and thus the mismatch repair function might be blocked or reduced. The V384D and the Q701K variant resulted in the interaction of hMLH1 with hPMS2 at reduced efficiency and might raise the gastrointestinal cancer risk of the mutation carriers. Conclusions: This work availably evaluated the functional consequences of some missense mutations not previously determined in the hMLH1 gene and might be useful for the clinical diagnosis of hereditary gastrointestinal cancer, especially in East Asians.

A haplotype variation affecting the mitochondrial transportation of hMYH protein could be a risk factor for colorectal cancer in Chinese

BackgroundThe human MutY homolog (hMYH), a DNA glycolsylase involved in the excision repair of oxidative DNA damage, is currently studied in colorectal cancer (CRC). We previously demonstrated a haplotype variant c.53C>T/c.74G>A of hMYH (T/A) increasing the risk for gastric cancer in Chinese. However, most investigations on correlation between hMYH and CRC are conducted in Western countries and the underlying mechanism has been poorly understood.MethodsTo determine whether the haplotype T/A variant of hMYH was related to colorectal carcinogenesis, we performed a case-control study in 138 colorectal cancer (CRC) patients and 343 healthy controls in a Chinese population. Furthermore, the C/G for wild-type, C/A or T/G for single base variant and T/A for haplotype variant hMYH cDNAs with a flag epitope tag were cloned into pcDNA3.1+ vector and transfected into cos-7 cell line. Their subcellular localizations were determined by immunofluorescence assay.ResultsIt was found that the frequency of haplotype variant allele was statistically higher in CRC patients than that in controls (P = 0.02, odds ratio = 5.06, 95% confidence interval = 1.26 – 20.4). Similarly, significant difference of heterozygote frequency was indicated between the two groups (P = 0.019), while no homozygote was found. In addition, immunofluorescence analysis showed that hMYH protein with haplotype T/A variation presented in both nucleus and mitochondria, in contrast to the wild-type protein only converging in mitochondria. However, neither of the single missense mutations alone changed the protein subcelluar localization.ConclusionAlthough preliminarily, these results suggest that: the haplotype variant allele of hMYH leads to a missense protein, which partly affects the protein mitochondrial transportation and results as nuclear localization. This observation might be responsible for the increased susceptibility to cancers, including CRC, in Chinese.

Mitochondrial modulation is involved in the hepatoprotection of Limonium sinense extract against liver damage in mice.

AIM OF THE STUDY Limonium sinense (Girard) Ktze is a Chinese folk medicine used to treat fever, hemorrhage, hepatitis, and other disorders. The present research focused on the protective effects of L. sinense extracts (LSE) against liver damage. MATERIALS AND METHODS In this study the extract from the root of Limonium sinense was used. Aminotransferase activity detection, electron microscopy, mitochondrial function evaluation, RT-PCR and western blot were used to evaluate the hepatoprotection of LSE in LPS/d-GalN-intoxicated mice. RESULTS Pretreatment with 100, 200 or 400mg/kg LSE significantly blocked the increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels induced by treatment with LPS plus d-GalN (LPS/d-GalN). Ultrastructural observation by electron microscopy showed reduced hepatocyte nuclear condensation and less lipid deposition. The decrease in both the mitochondrial membrane potential (14.6%) and sensitivity to mitochondrial swelling induced by Ca(2+) (45.9%) observed in the liver of LPS/d-GalN-treated mice were prevented by pretreatment with LSE. In addition, different doses of LSE increased both the transcription and the translation of voltage-dependent anion channels (VDAC), which was down-regulated by LPS/d-GalN treatment. CONCLUSIONS In summary, LSE protects livers against LPS/d-GalN-induced damage, possibly by mitochondrial mechanisms related to increased expression of VDAC.

Cyanobacteria-blooming water samples from Lake Taihu induce endoplasmic reticulum stress in liver and kidney of mice

To investigate whether endoplasmic reticulum (ER) stress was involved in apoptosis induced by cyanobacteria-blooming water, healthy male ICR mice were fed with water samples from cyanobacteria-blooming regions of Lake Taihu (China), including Meiliang Bay (M1 and M2), central lake region (H), macrophyte-dominated Xukou Bay (X), and tap water (control group) for three consecutive months. Hepatic and renal mRNA and protein expression of ER stress signaling molecules were measured with quantitative real-time PCR and western blotting. Compared to macrophyte-dominated and control water samples, cyanobacteria-blooming water changed hepatic ER stress signaling molecules. M1 water treatment increased the mRNA and protein levels of glucose regulation protein 78 (GRP78) and C/EBP homologous protein (CHOP), and decreased the mRNA levels of B-cell lymphoma 2 (Bcl-2). M2 water treatment up-regulated GRP78 mRNA and protein expression, whereas H water treatment up-regulated mRNA and protein expression of GRP78 and caspase-12. Cyanobacteria-blooming water exposure also changed mRNA and protein expression of ER stress signaling molecules in the kidneys. M1 water exposure up-regulated GRP78 mRNA and protein expression and CHOP mRNA expression, whereas M2 water treatment up-regulated caspase-12 and Bcl-2 mRNA expression. M1 and M2 cyanobacteria-blooming water exposure significantly increased relative liver weights, and induced hepatic cell apoptosis. However, cyanobacteria-blooming water treatment did not change kidney weights, and did not induce renal apoptosis compared to macrophyte-dominated and control water samples. Hence, cyanobacteria-blooming water induces hepatic apoptosis via ER stress, and ER stress may play an important role in the apparent anti-apoptotic effects on renal cells exposed to cyanobacteria-blooming water.

Microcystin-LR altered mRNA and protein expression of endoplasmic reticulum stress signaling molecules related to hepatic lipid metabolism abnormalities in mice.

To explore the effects of microcystin-LR (MC-LR), a hepatotoxin, on the incidence of liver lipid metabolism abnormality, and the potential molecular mechanisms of action, healthy male Balb/c mice were intraperitoneally injected with MC-LR at doses of 0, 5, 10, and 20 μg/kg/d for 14 days. Hepatic histopathology and serum lipid parameters of mice were determined, and the changes of mRNA and protein expression of endoplasmic reticulum (ER) stress signaling molecules related to the lipid metabolism abnormalities in the livers of mice were investigated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting, respectively. The results indicated that 5-20 μg/kg/d MC-LR altered serum lipid parameters and caused hepatic steatosis. MC-LR treatment at 10 or 20 μg/kg/d changed mRNA and protein expression of ER stress signaling molecules, including upregulation of mRNA and protein expression of activating transcription factor 6 (ATF6), pancreatic ER eukaryotic translation initiation factor 2α (eIF-2α) kinase (PERK), and eIF-2α. MC-LR exposure at 10 or 20 μg/kg/d also altered mRNA and protein expression of downstream factors and genes of ER stress signaling pathways, including the downregulation of sterol regulatory element binding protein 1c (SREBP-1c) and fatty acid synthase (FASn), and upregulation of acetyl-coenzyme A carboxylase α (ACACA) and glycogen synthase kinase 3β (Gsk-3β). Our results reveal that ER stress plays a significant role in hepatic lipid metabolism abnormalities in mice exposed to MC-LR.

Microcystis bloom containing microcystin-LR induces type 2 diabetes mellitus

Epidemiological data from Lake Taihu showed significantly higher incidences of type 2 diabetes mellitus (T2DM) than in other areas of China. This may be related to the occurrence of a Microcystis bloom in Lake Taihu in the summer and autumn every year. The objective of this study is to investigate whether the contaminated water from the Microcystis bloom and the derivative pollutant microcystin-LR (MC-LR) can explain the higher incidences of T2DM. Healthy male mice were fed with water from different regions of Lake Taihu, and were either acutely or chronically exposed to MC-LR through oral administration or intraperitoneal injection. Serum lipid profiles were determined, and the effects on T2DM-related gene expression and insulin receptor signaling pathway were investigated. Intraperitoneal glucose tolerance (IPGTT) and insulin resistance (IRT) tests were implemented, and the functions of pancreatic islet and β-cell were also evaluated. The results showed that both water sampled from the region with a Microcysis bloom and those containing MC-LR altered the serum glucide and lipid profiles in mice after exposure. The exposure to a Microcysis bloom water affected the expression T2DM-related genes: up-regulated the mRNA levels of FASn, ACACA, G6pc, LPL, and Insig2, and down-regulated the mRNA level of PEPCK and Gsk-3β. Both acute and chronic exposure of MC-LR, even at very low concentrations (1 μg/L), impaired the insulin receptor signalling pathway and induced hyperinsulinemia and insulin resistance in mice. In this study, the most important intracellular target of MC-LR was found to be hetapocellular mitochondria. Thus, exposure to Microcystis bloom water containing microcystin-LR can induce the incidence of T2DM, by impairing the function of mitochondria by microcystin-LR. The study suggests a review of the risk assessment concerning 1 μg/L MC-LR as the reference dose in surface water.

The differential effects of microcystin-LR on mitochondrial DNA in the hippocampus and cerebral cortex

Microcystin-LR (MC-LR) is the most abundant toxicant among microcystin variants produced by cyanobacteria. MC-induced toxicity is broadly reported to pose a threat to aquatic animals and humans and has been associated with the dysfunction of some organs such as liver and kidney. However, MC-induced neurotoxicity has not been well characterized after long-term exposure. This study was designed to investigate the neurotoxic effects after chronic oral administration of MC-LR. In our trial, C57/BL6 mice received MC-LR at 0, 1, 5, 10, 20 and 40 μg/L in drinking water for twelve months. Our data demonstrated that mitochondrial DNA (mtDNA) damage was evident in the damaged neurons as a result of chronic exposure. Histopathological abnormalities and mtDNA damage were observed in the hippocampus and cerebral cortex. Furthermore, MC-LR exerted distinct effects on these two brain regions. The hippocampus was more susceptible to the treatment of MC-LR compared with the cerebral cortex. However, no strong relationships were observed between the genotoxic effects and exposure doses. In conclusion, this study has provided a mtDNA-related mechanism for underlying chronic neurotoxicity of MC-LR and suggested the presence of differential toxicant effects on the hippocampus and cerebral cortex.