ABSTRACT
Lung cancer is the main cause of cancer-related deaths, and its morbidity and mortality is the highest, which seriously affects patients' survival time and quality of life. To develop new targets of tumor cells and expand the beneficiary population of lung cancer patients have become a hot and difficult research topic in recent years. RNA binding protein human antigen R (HuR) plays an important role in the development and progression of lung cancer. It can affect the transcription, translation and synthesis of multiple genes, proteins and molecules of lung cancer cells by binding to specific mRNA in lung cancer cells, and promote proliferation, differentiation and apoptosis of lung cancer cells. HuR is of great significance in molecular diagnosis, treatment and prognosis of lung cancer. This article will review the biological characteristics of HuR and research progress of its interaction with HuR-related regulatory factors in lung cancer based on relevant domestic and foreign literatures.
ABSTRACT
Objective@#To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro.@*Methods@#The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction.@*Results@#(1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05).@*Conclusions@#After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.
ABSTRACT
Objective To investigate the expressions of RNA-binding protein human antigen R(HuR), fatty acid binding protein type 4(FABP4),fatty acid synthetase(FASN),and lipoprotein lipase(LPL)during the differentiation of human adipocytes, and to explore their possible roles. Methods Human adipose-derived mesenchymal stem cells were induced by adipogenic differentiation,and the adipogenesis of cells was observed by oil red O staining. The expressions of HuR,FABP4,FASN,and LPL mRNA and protein were detected by real-time PCR and Western blotting. After HuR was silenced by siRNA, the change of adipogenesis for human adipose-derived mesenchymal stem cells was observed and the expressions of adipogenic genes were detected. Results The expressions of HuR,FABP4,FASN,and LPL mRNA and protein were significantly increased after human adipose-derived mesenchymal stem cells were induced to differentiate into adipocytes(all P<0.01). After HuR expression was down-regulated by siRNA,the adipogenic level of human adipose-derived mesenchymal stem cells was reduced,with decreased protein levels of FABP4,FASN,and LPL(all P<0.05),which were without changes for their mRNA levels. Conclusion HuR promotes the differentiation of human adipocytes mainly via regulating the changes of FABP4,FASN,and LPL protein levels.
ABSTRACT
Objective Intercellular adhesion molecule-1 (ICAM-1) plays an important role in mediating pulmonary infiltration of neutrophils .The aim of the study was to observe the expression of ICAM-1 and its potential regulators MK 2/HuR in pulmonary micro-vascular endothelial cells ( PMVEC ) in mice with acute respiratory distress syndrome ( ARDS) induced by lipopolysaccharide ( LPS) . Methods Ten 6-8 weeks old healthy C57BL/6 mice were randomly divided into an LPS and a control group of equal number , the former injected intraperitoneally with LPS diluted in 100 μL PBS while the latter with PBS only , both at 5 mg per kg of the body weight .At 24 hours after injection , all the mice were sacrificed .Real-time PCR was used to determine the mRNA expressions of HuR and ICAM-1 in the PMVECs, Western bolt employed to detect the protein expressions of MK2, HuR and ICAM-1, and flow cytometry adopted to measure the ICAM-1 expression on the surface of the PMVECs and pulmonary infiltration of neutrophils . Results The W/D ratio in the lung tissue of the mice was significantly lower in the LPS than in the control group (3.61 ±0.28 vs 6.16 ±0.40, P<0.05), while the rate of neutrophil infiltration markedly higher in the former than in the latter ([13.92 ±3.23]%vs [3.24 ±1.24]%, P<0.05).The mRNA and protein expressions of ICAM-1 in the PMVECs were significantly elevated in the LPS group as compared with that in the control (P<0.05), and so was the mRNA expression of HuR (P<0.05).No remarkable changes were observed in the expressions of total MK 2 and HuR proteins, but phosphorylated MK2 (p-MK2) and cytoplasmic HuR were increased in the LPS-stimulated mice. Conclusion Specific blockage or reduction of the HuR expression in PMVECs may lower the expression of ICAM-1, reduce neutrophil infiltration , and lessen pathophysiological changes in mice with ARDS .
ABSTRACT
Objective: To study the effects of polyporus polysaccharide (PPS) on the proliferation of human lung cancer A549 cells and the corresponding molecular mechanism. Methods: The A549 cells in control group were normally treated and the cells in experimental groups were incubated with different doses of PPS. The MTT method and flow cytometry were used to analyze the influence of drugs on cell proliferation. The qRT-PCR was used to detect mRNA levels of A549 cells and Cyclin D1 mRNA stability. The levels of human antigen R (HuR) protein in plasma and nuclei and cyclin D1 protein were detected by Western blotting. Results: Compared with the control, the cell growth was obviously inhibited (P<0.05), the Cyclin D1 mRNA stability and Cyclin D1 mRNA were decreased in the mid-and high-dose PPS groups (P<0.05), the total protein of HuR was slightly changed, but the cytoplasm protein of HuR was down-regulated (P<0.05) and the nucleus protein of HuR was up-regulated (P<0.05). Conclusion: PPS can suppress the proliferation of A549 cells, which is possibly affected by down-regulating the cytoplasm protein of HuR, lowering the stability of Cyclin D1 mRNA, and thus decreasing the expression of Cyclin D1 protein.