Multi-platform analysis of methylation-regulated genes in human lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most frequent pathological type of lung cancer that has a poor prognosis and high mortality rate. DNA methylation plays a critical role in various biological processes during development, while dysregulation results in pathological consequences. Thus, this study aimed to identify DNA methylation-regulated genes involved in LUAD occurrence. Initially, 300 downregulated and 168 upregulated mRNA expression levels were identified in two databases: Gene Expression Omnibus (GEO) and The Cancer Genome Atlas. In addition, GEO was utilized to detect 243 DNA hyper-methylated sites. Based on our observations, it was possible to correlate downregulation of mRNA expression and DNA hyper-methylation of six genes (ABCA3, COX7A1, HOXA5, SLIT3, SOX17, and SPARCL1). Functional analysis of the six genes indicated that these genes are predominantly enriched in cancer-related pathways and may promote carcinogenesis by regulating epithelialmesenchymal transition processes. In conclusion, our study identified a panel of DNA methylation-regulated genes involved in LUAD and may serve as potential epigenetic markers for this type of carcinoma.

Radon-induced alterations in micro-RNA expression profiles in transformed BEAS2B cells.

Radon and its progeny are confirmed to be type I carcinogenic agents accounting for increased risks in 10% of observed lung cancers globally. However, the underlying carcinogenic mechanisms are largely unknown. In the present study, BEAS2B cells were directly exposed twice to 20,000 Bq/m(3) radon gas for 20 min once (first passage) and subsequently 10 times (fifth passage). The fifth-passage cells were then subcultured for 1 and 20 generations (named Rn5-1 and Rn5-20, respectively). Molecular mechanisms indicative of malignant transformation were assessed by determination of apoptosis, seroresistance, and microRNA (miRNA) expression profiles. The microRNA profiles were used to assess the functional annotations of the target genes. Data indicated an increased seroresistance and colony efficiency on soft agar, and enhanced apoptosis resistance in the Rn5-20 cells with significant differential expressions in some miRNA, including hsa-miR-483-3p, hsa-miR-494, hsa-miR-2115*, hsa-miR-33b, hsa-miR-1246, hsa-miR-3202, hsa-miR-18a, hsa-miR-125b, hsa-miR-17*, and hsa-miR-886-3p. Functional annotation demonstrated that these miRNA target genes were predominantly involved in the regulation of cell proliferation, differentiation, and adhesion during the process of malignant transformation, which is associated with signal pathways such as mitogen-activated protein kinase (MAPK), Int and Wg (Wnt), reactive oxygen species (ROS), nuclear factor κB (NF-κB), and other genes regulating cell cycles.

Effects of 1800-MHz radiofrequency fields on circadian rhythm of plasma melatonin and testosterone in male rats.

Radiofrequency fields (RF) at 1800 MHz are known to affect melatonin (MEL) and testosterone in male rats, but it remains to be determined whether RF affected circadian rhythm of these plasma hormones. Male Sprague-Dawley rats were exposed to 1800-MHz RF at 208 µw/cm² power density (SAR: 0.5762 W/kg) at different zeitgeber (ZT) periods of the day, including 0 (ZT0), 4 (ZT4), 8 (ZT8), 12 (ZT12), 16 (ZT16), and 20 (ZT20) h. RF exposure was 2 h/d for 32 d. From each rat, the concentrations of plasma MEL and testosterone were determined in plasma after RF exposure and compared with controls. The results confirmed the existence of circadian rhythms in the synthesis of MEL and testosterone, but revealed an inverse relationship in peak phase of these rhythms. These rhythms were disturbed after exposure to RF, with the effect being more pronounced on MEL than testosterone. The most pronounced effect of RF exposure on MEL and testosterone appears to be in rats exposed to RF at ZT 16 and ZT0 h, respectively. Data suggest that regulation of testosterone is controlled by MEL and that MEL is more sensitive to RF exposure.

Daytime sleepiness and its determining factors in Chinese obstructive sleep apnea patients.

OBJECTIVE: The aim of this study was to characterize excessive daytime sleepiness (EDS) in a large cohort of Chinese patients with various severity of obstructive sleep apnea-hypopnea syndrome (OSAHS), and investigate its correlations with clinical/polysomnographic variables. MATERIALS AND METHODS: A total of 1,035 consecutive Chinese patients with snoring (mean age ± SD 45 ± 15 years, BMI 26.6 ± 4.3 kg/m(2)) were examined by overnight polysomnography, and subjective EDS was assessed using the Epworth Sleepiness Scale (ESS). RESULTS: The 1,035 patients were compared according to severity of sleep-disordered breathing: AHI <5 (primary snoring group or normal overall AHI) (24.1%), AHI 5-20 (mild OSAHS, 21.7%), AHI >20-40 (moderate OSAHS 16.5%), and AHI >40 (severe OSAHS 37.7%). ESS score progressively increased as the severity of OSAHS aggravated among these patients. More severe OSAHS patients were characterized by EDS, nocturnal hypoxemia, and disruption of sleep structure. Progressive worsening of nocturnal hypoxemia was observed from mild to severe OSAHS patients with a strong correlation with ESS score. The stepwise multiple regression analysis performed to evaluate the correlations of individual clinical and polysomnographic variables with the ESS score revealed that the ESS score significantly correlated with the oxygen desaturation index (ODI), apnea-hypopnea index (AHI), and body mass index (BMI), and ODI was the strongest determinant of ESS score. CONCLUSION: EDS is correlated with the severity of OSAHS. More severe patients are characterized by higher ESS score, higher BMI, and progressive worsening of nocturnal hypoxemia. Nocturnal hypoxemia is a major determinant of EDS in Chinese OSAHS patients.

Screening of differential expressive genes in murine cells following radon exposure.

This study was designed to construct and identify the subtracted cDNA library in peripheral blood cells of BALB/c mice and tracheal-bronchial epithelial cells of Wistar rats following exposure to radon inhalation. Two groups of the animals were exposed in a radon chamber at an accumulative dose of 100 WLM, while control animals were housed in a room at a background dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and suppression subtractive hybridization (SSH) assay were performed. The obtained forward and reverse cDNA fragments were directly inserted into a pMD-18 vector and transformed into Escherichia coli JM109. In total, 593 white bacterial clones were selected from both forward- and reverse-subtracted libraries. Among them, 81 clones were chosen for their differential expressions based on reverse Northern blot. Portions of these cDNA clones were also verified by a quantitative real-time polymerase chain reaction (PCR). The screening resulted in 14 upregulative and 8 downregulative known function/annotation genes, which were revealed to be functionally related to cell proliferation, cell oxidative and DNA damage, apoptosis, and tumor promotion. Access numbers were obtained from the GenBank for 11 unknown expressed sequence tags (EST). Analysis of biological roles of these cDNA fragments may provide further insight into mechanisms underlying adverse molecular events induced by high-dose radon exposure.

[Antagonistic effect of microwave on hematopoietic damage of mice induced by gamma-ray irradiation].

OBJECTIVE: To investigate antagonistic effect of microwave on hematopoietic damage of mice induced by gamma-ray irradiation. METHODS: Male healthy Kunning mice were treated with low dose microwave radiation before exposure to (60)Co gamma-ray irradiation of 8.0 Gy. The 30-day survival rate and average survival time of the mice after the treatment were examined. Peripheral blood parameters and the organ indexes of thymus and spleen were also observed in the irradiated mice. After exposure to 5.0 Gy gamma irradiation, indexes of hematopoietic foci formation of bone marrow cells (CFU-GM) and the proliferation activity of BMNCs were examined. The serum concentration of hemopoietic factors (GM-CSF and IL-3) were detected by ELISA kits. RESULTS: Pre-exposure with 120 microW/cm(2) 900 MHz microwave increased the 30-day survival rate (P < 0.05) and the number of white blood cells of gamma-ray treated mice. The increases of the organ indexes of thymus and spleen, proliferation activity of BMNCs and CFU-GM hematopoietic foci numbers, as well as the higher serum concentration of GM-CSF and IL-3 were observed in the microwave pre-exposure group. CONCLUSION: Low dose microwave radiation may exert potential antagonistic effects on hematopoietic injuries induced by ionizing radiation. The underlying mechanisms might be related with stimulation of hematopoietic growth factors expression, promotion of HSCs/HPCs proliferation, suppression on the reduction of HSCs/HPCs caused by (60)Co gamma-ray, and enhanced construction of the hematopoietic system.

MicroRNA and mRNA Interaction Network Regulates the Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cigarette Smoke.

This study analyzes the correlation and interaction of miRNAs and mRNAs and their biological function in the malignant transformation of BEAS-2B cells induced by cigarette smoke (CS). Normal human bronchial epithelial cells (BEAS-2B) were continuously exposed to CS for 30 passages (S30) to establish an in vitro cell model of malignant transformation. The transformed cells were validated by scratch wound healing assay, transwell migration assay, colony formation and tumorigenicity assay. The miRNA and mRNA sequencing analysis were performed to identify differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between normal BEAS-2B and S30 cells. The miRNA-seq data of lung cancer with corresponding clinical data obtained from TCGA was used to further identify lung cancer-related DEMs and their correlations with smoking history. The target genes of these DEMs were predicted using the miRDB database, and their functions were analyzed using the online tool "Metascape." It was found that the migration ability, colony formation rate and tumorigenicity of S30 cells enhanced. A total of 42 miRNAs and 753 mRNAs were dysregulated in S30 cells. The change of expression of top five DEGs and DEMs were consistent with our sequencing results. Among these DEMs, eight miRNAs were found dysregulated in lung cancer tissues based on TCGA data. In these eight miRNAs, six of them including miR-96-5p, miR-93-5p, miR-106-5p, miR-190a-5p, miR-195-5p, and miR-1-3p, were found to be associated with smoking history. Several DEGs, including THBS1, FN1, PIK3R1, CSF1, CORO2B, and PREX1, were involved in many biological processes by enrichment analysis of miRNA and mRNA interaction. We identified the negatively regulated miRNA-mRNA pairs in the CS-induced lung cancer, which were implicated in several cancer-related (especially EMT-related) biological process and KEGG pathways in the malignant transformation progress of lung cells induced by CS. Our result demonstrated the dysregulation of miRNA-mRNA profiles in cigarette smoke-induced malignant transformed cells, suggesting that these miRNAs might contribute to cigarette smoke-induced lung cancer. These genes may serve as biomarkers for predicting lung cancer pathogenesis and progression. They can also be targets of novel anticancer drug development.

Radon-induced proteomic profile of lung tissue in rats.

The aim of this study was to investigate the differential expression of proteins in lung of rats following long-term exposure to radon. The total proteins of lung tissue from Wistar rats exposed to radon for cumulative doses up to 100, 200, or 400 WLM (working level months) were isolated by two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. Comparison of the 2-DE images between the control and radon-exposed groups resulted in 14 upregulated and 9 downregulated protein spots, of which 15 were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). The simultaneous up-expressions of RAGE and S100A6 indicated that both proteins might be applied as biomarkers for lung injury induced by long-term radon exposure.

Screening of differential expression genes in bone marrow cells of radon-exposed mice.

This study was designed to screen for differential expression genes in bone marrow cells of mice exposed to radon inhalation. Based upon established pathological findings in mouse, differential screening of gene expressions was conducted by using the SSH method. Among 285 cDNA clones selected from both forward- and reverse subtracted libraries, 45 were chosen for their differential expressions based on reverse Northern blot and quantitative real-time PCR analysis. Of these, up-regulation of the mRNA levels of E-cadherin and down-regulation of the replication protein A1 (RPA1) and casein kinase 1 delta (CKI delta) were also verified by a quantitative real-time PCR. Biological roles of these obtained cDNAs are described and the results of the screening may provide important clues for further investigations of the adverse molecular events induced by radon exposure.

Circadian rhythms and different photoresponses of Clock gene transcription in the rat suprachiasmatic nucleus and pineal gland.

The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light: 12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6 rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (<0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P<0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P>0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG) and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P<0.05), and the rhythmic profile was anti-phasic to that under DD condition (P<0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P<0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P<0.05). (3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P<0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.

[Comments on the Confucian physician].

Confucianism gradually permeated and influenced the development of TCM from the Song dynasty, and the term "Confucian physician" is still in use today. With the impact of Confucianism, whether in the compilation of the medical classics or the explanation and conclusion of the medical theories as well as in medical education and ethics, all developed dramatically. But the Confucianism had also a negative effect on the development of medicine. For example, SU Dong-po cured the epidemics with "Sheng san zi", but he exaggerated its action and recorded it. The later intellectuals learnt from him without differentiation and many people suffered. Another example is, with the influence of ideas of "serve the parents" and "help the public", adult children treated their parents by cutting their own thigh. Even some wealthy and intelligent people blindly applied the prescription without differentiation.

[The effect of entecavir treatment on HBV-specific immunity in patient with chronic hepatitis B and its relationship to HBeAg sero-conversion].

OBJECTIVE: To investigate the effect of Entecavir treatment on HBV-specific immunity in patient with chronic hepatitis B and its relationship to HBeAg sero-conversion. METHODS: Serum aminotransferase (ALT), HBV DNA and HBeAg were monitored before the after entecavir treatment. At the same time point, HBV-specific T cell proliferation was determined by [3H] T-dR incorporation assay, while HBV-specific IFN-alpha secretion was measured by ELISA. RESULTS: The level of HBV DNA and ALT was significantly decreased after entecavir treatment. Same is the titer of HBeAg. In addition, HBeAg sero-conversion was observed in some of them, in whom the HBV-specific T cell proliferation and IFN-alpha production were significantly increased. CONCLUSION: Entecavir treatment resulted in increased HBV-specific immunity along with the inhibition of HBV replication.

[Roles of the histaminergic receptors in the locus ceruleus in stress-induced carotid baroreflex resetting in rats].

AIM: To explore the roles of H1 and H2 receptors in the locus ceruleus (LC) in the carotid baroreflex (CBR) resetting resulted from foot-shock stress. METHODS: Male SD rats were divided into two groups (n=18) at random: unstressed and stressed group. The latter were subjected to unavoidable electric foot-shock twice daily for a week and each session of foot-shock lasted 2 hours. The left and right carotid sinus regions were isolated from the systemic circulation in all animals anesthetized with pentobarbital sodium. The intracarotid sinus pressure (ISP) was altered in a stepwise manner in vivo. ISP-mean arterial pressure (MAP), ISP-Gain relationship curves and reflex characteristic parameters were constructed by fitting to the logistic function with five parameters. The changes in CBR performance induced by stress and the effects of microinjection with histaminergic receptors antagonists into the LC on the responses of CBR to stress were examined. RESULTS: Stress significantly shifted the ISP-MAP relationship curve upwards (P < 0.05) and obviously moved the middle part of ISP-Gain relationship curve downwards (P < 0.05), and decreased the value of the MAP range and maximum gain (P < 0.05), but increased the threshold pressure, saturation pressure, set point and ISP at maximum gain (P < 0.05). Microinjection of selective H1 or H2 receptor antagonist, chlorpheniramine (CHL, 0.5 microg/microl) or cimetidine (CIM, 1.5 microg/microl) into the LC, significantly attenuated the above-mentioned changes in CBR performance induced by stress and the alleviate effect of CIM was less remarkable than that of CHL (P < 0.05). The responses of CBR under stress to H1 or H2 receptor antagonist generally occurred 20 min after the administration and lasted approximately for 16 min. Microinjection with the same dose of CHL or CIM into the LC in the unstressed group did not change CBR performance significantly (P > 0.05). However, microinjection of CHL or CIM into the LC could not completely abolish the stress-induced changes in CBR. CONCLUSION: The stress results in a resetting of CBR and a decrease in reflex sensitivity. The stress-induced changes in CBR may be mediated, at least in part, by activating the brain histaminergic system. The H1 and H2 receptors in the LC, especially, Hi receptors may play an important role in the resetting of CBR under stress. The descending histaminergic pathway from the hypothalamus to LC may be involved in these effects. Moreover, the effects of stress on CBR also have other mechanisms.