IGF2BP2 inhibits invasion and migration of clear cell renal cell carcinoma via targeting Netrin-4 in an m6A-dependent manner.

Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma, often leads to a poor prognosis due to metastasis. The investigation of N6-methyladenosine (m6A) methylation, a crucial RNA modification, and its role in ccRCC, particularly through the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), revealed significant insights. We found that IGF2BP2 was notably downregulated in ccRCC, which correlated with tumor aggressiveness and poor prognosis. Thus, IGFBP2 has emerged as an independent prognostic factor of ccRCC. Moreover, a strong positive correlation was observed between the expression of IGF2BP2 and Netrin-4. Netrin-4 was also downregulated in ccRCC, and its lower levels were associated with increased malignancy and poor prognosis. Overexpression of IGF2BP2 and Netrin-4 suppressed the invasion and migration of ccRCC cells, while Netrin-4 knockdown reversed these effects in ccRCC cell lines. RNA immunoprecipitation (RIP)-quantitative polymerase chain reaction validated the robust enrichment of Netrin-4 mRNA in anti-IGF2BP2 antibody immunoprecipitates. MeRlP showed significantly increased Netrin4 m6A levels after lGF2BP2 overexpression. Moreover, we found that IGF2BP2 recognized and bound to the m6A site within the coding sequence of Netrin-4, enhancing its mRNA stability. Collectively, these results showed that IGF2BP2 plays a suppressive role in the invasion and migration of ccRCC cells by targeting Netrin-4 in an m6A-dependent manner. These findings underscore the potential of IGF2BP2/Netrin-4 as a promising prognostic biomarker and therapeutic target in patients with ccRCC metastasis.

Morphine suppresses the immune function of lung cancer by up-regulating MAEL expression.

BACKGROUND: Patients with cancer rely on morphine for analgesia, while studies have indicated morphine can induce immunosuppression in cancer. Therefore, investigating the immunosuppressive roles and molecular mechanism of morphine on lung cancer progression is imperative. METHODS: Lactate dehydrogenase (LDH) release assay was used to determine the cytotoxicity of morphine to lung cancer cells. The percentage of CD4+ and CD8+ T cells was detected by flow cytometry. In addition, Maelstrom (MAEL), Nrf2, and PTEN were determined by western blot and RT-qPCR. Immune factors programmed death-ligand 1 (PD-L1), transforming growth factor (TGF-ß), interleukin (IL)-10, and IL-2 were determined by western blot and ELISA assay. RESULTS: Morphine increased the levels of PD-L1, TGF-ß, and IL-10, while decreased IL-2 level. Morphine enhanced MAEL expression in A549 cells and H460 cells. Morphine up-regulated Nrf2 and down-regulated PTEN, and morphine-induced MAEL up-regulation was reversed by PTEN. However, MAEL silencing inhibited the enhanced effects of morphine on cell viability and proliferation of A549 cells. Furthermore, morphine treatment reduced the LDH release and the percentage of CD8+ T cells, and increased the ratio of CD4+/CD8+ T cells and tumor weight. Meanwhile, MAEL silencing reversed the effects of morphine on immune factors (PD-L1, TGF-ß, IL-10, and IL-2), the percentage of CD8+ T cells, and the ratio of CD4+/CD8+ T cells. CONCLUSION: Morphine activated MAEL in lung cancer cells by Nrf2/PTEN pathway and regulated the immune factors, thereby promoting tumor immune escape.

Long non-coding RNA HCG22 inhibits the proliferation, invasion and migration of oral squamous cell carcinoma cells by downregulating miR-425-5p expression.

Long non-coding RNA (lncRNA) HLA complex group 22 (HCG22) is known to be involved in the occurrence and development of cancer; however, its role in oral squamous cell carcinoma (OSCC) remains unclear. Therefore, the main aim of the present study was to investigate the role and mechanisms of action of lncRNA HCG22 in OSCC cells. The expression levels of lncRNA HCG22 and microRNA (miR)-425-5p in OSCC cells were assessed using reverse transcription-quantitative PCR analysis. Cell proliferation was detected using Cell Counting Kit-8 and colony formation assays. In addition, the expression levels of cell proliferation-related proteins, p27, cyclin E and cyclin-dependent kinase 2, were detected by western blot analysis. The cell invasive ability was detected by Transwell assay, while the cell migratory ability was detected via a wound healing assay. The expression levels of the invasion- and migration-related proteins, MMP2 and MMP9, were measured by western blot analysis. The targeted association between lncRNA HCG22 and miR-425-5p was verified by RNA immunoprecipitation and dual-luciferase reporter assays. The results revealed that lncRNA LCG22 was expressed at low levels, while miR-425-5p was highly expressed in OSCC cell lines, based on bioinformatics analysis. The overexpression of lncRNA HCG22 inhibited the proliferation, invasion and migration of OSCC cells. Moreover, lncRNA HCG22 and miR-425-5p were found to have a direct targeted association, and lncRNA HCG22 inhibited cell proliferation, invasion and migration by targeting miR-425-5p. Collectively, the findings of the present study demonstrated that lncRNA HCG22 may inhibit the proliferation, invasion and migration of OSCC cells by downregulating miR-425-5p expression.

miR-129-5p inhibits gemcitabine resistance and promotes cell apoptosis of bladder cancer cells by targeting Wnt5a.

OBJECTIVES: Gemcitabine resistance is a major obstacle for effective treatment of bladder cancer. This study was aimed to investigate the potential role of miR-129-5p in the development of gemcitabine resistance in bladder cancer cells and its underlying mechanism. METHODS: The IC50 for gemcitabine in 20 bladder cancer cells was first profiled from Genomics of Drug Sensitivity in Cancer. miR-129-5p level and gene mRNA expression were detected using quantitative real-time PCR (qRT-PCR). Cell viability, apoptosis, and gene protein level were assessed by MTT, flow cytometry, and Western blot, respectively. Regulatory relationship between Wnt5a and miR-129-5p was determined using luciferase reporter assay. RESULTS: We found that down-regulated miR-129-5p level contributed to gemcitabine resistance in bladder cancer cells and tissues. We also observed restoration of miR-129-5p could significantly increase cell sensitivity to gemcitabine and promote cell apoptosis. Mechanism analysis revealed that Wnt5a is a direct target gene of miR-129-5p and knock-down of Wnt5a reversed gemcitabine resistance. CONCLUSIONS: Taken together, our findings indicate that miR-129-5p and Wnt5a may be novel therapeutic targets for overcoming gemcitabine resistance in bladder cancer treatment.

Function of Hevea brasiliensis NAC1 in dehydration-induced laticifer differentiation and latex biosynthesis.

MAIN CONCLUSIONS: HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene ß-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.

Puerarin ameliorates cognitive deficits in streptozotocin-induced diabetic rats.

Previous research has indicated that Diabetes is a high risk of learning and memory deficits. Puerarin, an isoflavonoid extracted from Kudzu roots, has been reported to possess antioxidant, anti-inflammatory, anti-apoptotic and anti-diabetic properties which are useful in the treatment of various diseases. Recently, Puerarin was found to have the effects on learning and memory performances in humans and animal models. However, up to now, there is no detailed evidence on the effect of Puerarin on diabetes-associated cognitive decline (DACD). In this study, we designed to assess the effects of Puerarin on diabetes-associated cognitive decline (DACD) using a streptozotocin (STZ)-injected rat model and exploring its potential mechanism. Diabetic rats were treated with Puerarin (100 mg/kg per d) for 7 days. The learning and memory function was evaluated by morris water maze test. The acetylcholinesterase (AChE), choline acetylase (ChAT), oxidative indicators [malondialdehyde (MDA) and superoxide dismutase (SOD)] and inflammatory cytokine (TNF-a, IL-1ß and IL-6) were measured in hippocampus by using corresponding commercial kits. mRNA and Protein levels of Bcl-2 were analyzed by RT-PCR and Westernblot. The results showed that supplementation of Puerarin improved the learning and memory performances compared with the STZ group by the morris water maze test. In addition, Puerarin supplement significantly prevented AChE and MDA activities, increased ChAT and SOD activities, and alleviated the protein level of TNF-α, IL-1ß and IL-6 in the hippocampus compared with the STZ group. Moreover, the pretreatment with Puerarin also significantly increased the Bcl-2 expression. It is concluded that Puerarin possesses neuroprotection to ameliorate cognitive deficits in streptozotocin-induced diabetic rats by anti-inflammatory, antioxidant and antiapototic effects.

Wogonoside Shows Antifibrotic Effects in an Experimental Regression Model of Hepatic Fibrosis.

BACKGROUD: Wogonoside (WO), a flavonoid extracted from Huangqin, plays multiple physiological roles. However, it has remained elusive how WO regulates hepatic fibrogenesis until now. AIM: The purpose of the study was to investigate the potential protective effects of WO against liver fibrosis induced by carbon tetrachloride (CCl4). METHODS: In this study, male rats were randomly allocated into four groups: a control group, the CCl4 group, the CCl4 and WO (4 mg/kg) group, and CCl4 and WO (8 mg/kg) group. Hepatic fibrosis was induced by subcutaneous injection of CCl4 twice a week for a continuous 6-week period. Then the rats were intragastrically administrated with WO daily for 4 weeks before being killed. RESULTS: As expected, histopathological assessment, Masson trichrome staining, and Sirius red staining demonstrated that WO drastically ameliorated the hepatic fibrosis caused by CCl4. WO significantly attenuated the CCl4-induced upregulations of liver indices including alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, interleukin-1ß, IL-6, hexadecenoic acid and laminin in serum, as well as hydroxyproline, malondialdehyde and phosphatidylinositol 3-kinase (PI3K)/protein Kinase B(Akt)/mechanistic target of rapamycin (mTOR)/nuclear factor-kappa B signalings in liver. Meanwhile, WO also effectively recovered the depletions of superoxide dismutase, glutathione and IL-10. Furthermore, we evaluated the effects of WO on the alpha smooth muscle actin, type I collagen expressions, and PI3K/Akt/ mTOR/ribosomal protein S6 kinase 70 kDa (p70S6K) signaling in transforming growth factor (TGF-ß) stimulated hepatic stellate cell-T6 cells. CONCLUSIONS: These results suggested that WO had significant protective effects against liver fibrosis induced by CCl4.

RhoV mediates apoptosis of RAW264.7 macrophages caused by osteoclast differentiation.

Macrophages, a type of immune cell, are the precursors of osteoclasts, and have important roles in bone remodeling and the immune system. In the present study, the RAW264.7 cell line was used as a macrophage model in order to study the macrophage changes during osteoclastogenesis. Receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony­stimulating factor (M­CSF) induce the formation of osteoclasts from several precursor cells. Observation of RAW264.7 macrophage osteoclastogenesis under the induction of RANKL and M­CSF revealed that except the few RAW264.7 macrophages that were differentiated into osteoclasts, almost all undifferentiated RAW264.7 macrophages underwent apoptosis. BRL­3A cells have no differentiation ability, and RANKL and M­CSF treatments did not induce BRL­3A cell apoptosis. When osteoprotegerin (OPG) was used to completely inhibit the differentiation of RAW264.7 macrophages to osteoclasts, apoptosis did not occur amongst the RAW264.7 macrophages despite the action of RANKL and M­CSF. Rac1, RhoA and RhoV are apoptosis­associated genes in the Rho guanosine triphosphate (GTP)ase family. Their expression levels were detected using quantitative polymerase chain reaction (qPCR). During the process of osteoclast differentiation, the mRNA expression of RhoV was significantly upregulated, while apoptosis occurred in a large proportion of macrophages. However, when macrophage apoptosis was inhibited by OPG, RhoV expression was significantly downregulated. Conversely, Rac1 and RhoA expression did not vary in correspondence with the apoptotic rate of the RAW264.7 macrophages. In conclusion, differentiation of RAW264.7 macrophages into osteoclasts resulted in their apoptosis. OPG inhibited RAW264.7 macrophage differentiation into osteoclasts, and thereby inhibited the apoptosis of RAW264.7 macrophages. RhoV mediated the apoptosis of RAW264.7 macrophages during osteoclast differentiation.

Inhibition of osteoclast bone resorption activity through osteoprotegerin-induced damage of the sealing zone.

Bone remodeling is dependent on the dynamic equilibrium between osteoclast-mediated bone resorption and osteoblast-mediated osteogenesis. The sealing zone is an osteoclast-specific cytoskeletal structure, the integrity of which is critical for osteoclast-mediated bone resorption. To date, studies have focused mainly on the osteoprotegerin (OPG)­induced inhibition of osteoclast differentiation through the OPG/receptor activator of the nuclear factor kappa-B ligand (RANKL)/RANK system, which affects the bone resorption of osteoclasts. However, the effects of OPG on the sealing zone have not been reported to date. In this study, the formation of the sealing zone was observed by Hoffman modulation contrast (HMC) microscopy and confocal laser scanning microscopy. The effects of OPG on the existing sealing zone and osteoclast-mediated bone resorption activity, as well as the regulatory role of genes involved in the formation of the sealing zone were examined by immunofluorescence staining, HMC microscopy, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis and scanning electron microscopy. The sealing zone was formed on day 5, with belt-like protuberances at the cell edge and scattered distribution of cell nuclei, but no filopodia. The sealing zone was intact in the untreated control group. However, defects in the sealing zone were observed in the OPG-treated group (20 ng/ml) and the structure was absent in the groups treated with 40 and 80 ng/ml OPG. The podosomes showed a scattered or clustered distribution between the basal surface of the osteoclasts and the well surface. Furthermore, resorption lacunae were not detected in the 20 ng/ml OPG-treated group, indicating the loss of osteoclast-mediated bone resorption activity. Treatment with OPG resulted in a significant decrease in the expression of Arhgef8/Net1 and DOCK5 Rho guanine nucleotide exchange factors (RhoGEFs), 10 of 18 RhoGTPases (RhoA, RhoB, cdc42v1, cdc42v2, RhoU/Wrch1, RhoF/Rif, Rac2, RhoG, Rnd1 and RhoBTB1), ROCK1 and ROCK2. In conclusion, podosome distribution was affected by the OPG-induced inhibition of the expression of genes in the RhoGTPase signaling pathway. This resulted in damage to or destruction of the sealing zone, thus inhibiting osteoclast-mediated bone resorption activity.

Health risk assessment of heavy metal exposure to street dust in the zinc smelting district, Northeast of China.

Heavy metal contamination in the street dust due to metal smelting in the industrial district of Huludao city was investigated. Spatial distribution of Hg, Pb, Cd, Zn and Cu in the street dust was elucidated. Meanwhile, noncancer effect and cancer effect of children and adults due to exposure to the street dust were estimated. The maximum Hg, Pb, Cd, Zn and Cu contents in the street dust are 5.212, 3903, 726.2, 79,869, and 1532 mg kg(-1), and respectively 141, 181, 6724, 1257 and 77.4 times as high as the background values in soil. The trends for Hg, Pb, Cd, Zn and Cu are similar with higher concentrations trending Huludao zinc plant (HZP). The exponential equation fits quite well for the variations of Pb, Cd, Zn and Cu contents with distance from the pollution sources, but not for Hg. The biggest contribution to street dust is atmospheric deposition due to metal smelting, but traffic density makes slight contribution to heavy metal contamination. According to the calculation on Hazard Index (HI), in the case of noncancer effect, the ingestion of dust particles of children and adults in Huludao city appears to be the route of exposure to street dust that results in a higher risk for heavy metals, followed by dermal contact. The inhalation of resuspended particles through the mouth and nose is almost negligible. The inhalation of Hg vapour as the fourth exposure pathway to street dust is accounting for the main exposure. Children are experiencing the potential health risk due to HI for Pb larger than safe level (1) and Cd close to 1. Besides, cancer risk of Cd due to inhalation exposure is low.

[Heavy metal contents in insects collected from the Huludao City suffering pollution by zinc smelting and cholor-alkai production].

14 insect species, which were classified to three groups: the herbivorous, the polyphagous and the carnivorous, and earthworms were collected from the grasslands in Huludao City, Liaoning Province, China. Mercury, cadmium and lead contents in biota were determined to discuss the heavy metal pollution in organisms. Mercury, cadmium and lead contents were 0.168, 9.19 and 12.58 mg x kg(-1) in the herbivorous insects, respectively; 0.375, 24.43 and 17.71 mg x kg(-1) in the polyphagous insects, respectively; 0.928, 29.78 and 18.39 mg x kg(-1) in the carnivorous insects, respectively. It showed that heavy metal pollution in biota in Huludao City was heavy. Bioaccumulation abilities to heavy metals significantly differed with insect species. Snails and dragonflies could accumulate more mercury than the other insects and spiders could accumulate the most cadmium and lead in all insect species. These three metals investigated in insects were all sorted as the herbivorous < the polyphagous < the carnivorous. Cadmium and lead contents between the polyphagous and the carnivorous varied slightly. Correlation analysis showed that cadmium and lead contents were significantly related, but mercury and cadmium or mercury and lead were not. It indicated that cadmium and lead in insects were from the same pollution sources while mercury was more complex.

Mercury, cadmium and lead biogeochemistry in the soil-plant-insect system in Huludao City.

Mercury, cadmium, and lead concentrations of ashed plants and insects samples were investigated and compared with those of soil to reveal their biogeochemical processes along food chains in Huludao City, Liaoning Province, China. Concentration factors of each fragments of the soil-plant-the herbivorous insect-the carnivorous insect food chain were 0.18, 6.57, and 7.88 for mercury; 6.82, 2.01, and 0.48 for cadmium; 1.47, 2.24, and 0.57 for lead, respectively. On the whole, mercury was the most largely biomagnified, but cadmium and lead were not greatly accumulated in the carnivorous insects as expected when the food chain extended to the secondary consumers. Results indicated that concentration factors depended on metals and insects species of food chains.

Population health risk due to dietary intake of heavy metals in the industrial area of Huludao City, China.

For most people, diet is the main route of exposure to trace metals, so the assessment risks of these elements to human via dietary intake is important. The non-carcinogenic health risk of Hg, Pb, Cd, Zn and Cu to the adults and children via dietary intake in the industrial area of Huludao city, northeast of China was estimated. The industrial area of Huludao has been contaminated seriously by heavy metals due to heavy metals smelting. The target hazard quotients (THQs) and hazard index (HI) were calculated to evaluate the non-carcinogenic health risk from individual heavy metal and combined heavy metals due to dietary intake. Target hazard quotients for individual heavy metal from consuming individual foodstuff in the industrial area of Huludao were all less than one, indicating that health risk associated with the intake of a single heavy metal through consumption of only one kind of foodstuffs (e.g. vegetable) was the relative absent. However, consumption of the entire foodstuff would lead to potential health risks for children and adults, since hazard indexes (HIs) for heavy metals due dietary intake were higher than one. The relative contributions of Hg, Pb, Cd, Zn and Cu to the HIs were 1.7%, 11.7%, 24.0%, 23.4%, and 39.6% for adults, and 1.5%, 11.7%, 21.8%, 26.1%, and 38.8% for children. Cereal, sea product, and vegetable were the main sources of heavy metal intake from foodstuff for adults and children, but fruit, milk, bean, and egg were secondary contributors.

[Transfer characteristics of mercury, lead, cadmium, zinc and cuprum from soil to vegetable around zinc smelting plant].

The transfer characteristics of Hg, Pb, Cd, Zn and Cu from soil to vegetables near zinc smelting plant in Huludao City, China were investigated, and the sources of heavy metals in the soil and vegetable were also analyzed. The results indicate that the Hg, Pb, Cd, Zn and Cu contents of vegetables are 0.013, 5.476, 2.852, 41.16 and 1.515 mg/kg (fresh weight), respectively, and the environment around Huludao Zinc Plant are contaminated seriously. The transfer factors (TF) of heavy metals decrease in the order of Cd > Zn > Cu > Pb > Hg. The transfer factors of heavy metals from soil to leaves are higher than from soil to other tissues. The heavy metals in soil derive from atmosphere, and the parts of Pb in the leaves of vegetable derive from atmosphere. Uptake of gaseous mercury is the predominant pathway by which mercury accumulates in the vegetable.