A unique circ_0067716/EIF4A3 double-negative feedback loop impacts malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene.

Benzo(a)pyrene as a pervasive environmental contaminant is characterized by its substantial genotoxicity, and epidemiological investigations have established a correlation between benzo(a)pyrene exposure and the susceptibility to human lung cancer. Notably, much research has focused on the link between epigenetic alterations and lung cancer induced by chemicals, although circRNAs are also emerging as relevant contributors to the carcinogenic process of benzo(a)pyrene. In this study, we identified circ_0067716 as being significantly upregulated in response to stress injury and downregulated during malignant transformation induced by benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in human bronchial epithelial cells. The observed differential expression of circ_0067716 in cells treated with BPDE for varying durations suggests a strong correlation between this circRNA and BPDE exposure. The tissue samples of lung cancer patients also suggest that a lower circ_0067716 expression is associated with BPDE-DNA adduct levels. Remarkably, we demonstrate that EIF4A3, located in the nucleus, interacts with the flanking sequences of circ_0067716 and inhibits its biogenesis. Conversely, circ_0067716 is capable of sequestering EIF4A3 in the cytoplasm, thereby preventing its translocation into the nucleus. EIF4A3 and circ_0067716 can form a double-negative feedback loop that could be affected by BPDE. During the initial phase of BPDE exposure, the expression of circ_0067716 was increased in response to stress injury, resulting in cell apoptosis through the involvement of miR-324-5p/DRAM1/BAX axis. Subsequently, as cellular adaptation progressed, long-term induction due to BPDE exposure led to an elevated EIF4A3 and a reduced circ_0067716 expression, which facilitated the proliferation of cells by stabilizing the PI3K/AKT pathway. Thus, our current study describes the effects of circ_0067716 on the genotoxicity and carcinogenesis induced by benzo(a)pyrene and puts forwards to the possible regulatory mechanism on the occurrence of smoking-related lung cancer, providing a unique insight based on epigenetics.

Transcription factor SP1 and oncoprotein PPP1R13L regulate nicotine-induced epithelial-mesenchymal transition in lung adenocarcinoma via a feedback loop.

Tobacco remains the most common environmental carcinogen leading to the occurrence and development of lung cancer. Nicotine, a tumor promoter in cigarette smoke, has been shown to induce epithelial-mesenchymal transition (EMT), a cellular program required for the invasion and metastasis in tumor cells. Specificity Protein 1 (SP1) is a well-characterized transcription factor that can regulate the EMT process via transcriptionally activating E-cadherin expression. Protein Phosphatase 1 Regulatory Subunit 13 Like (PPP1R13L) is a newly identified oncoprotein previously reported to inhibit the transcriptional activity of SP1 via a direct protein-protein interaction. To reveal the underlying implication of the interconnections between PPP1R13L and SP1 in the nicotine-induced EMT process, the present study established an EMT cell model of lung cancer using 1 µM of nicotine, a dose close to human exposure, in which an alternate fluctuation in the expression of PPP1R13L and SP1 was captured. Subsequently, the direct inhibition of SP1 by PPP1R13L was demonstrated to be a critical mechanism underlying the involvement of PPP1R13L in the nicotine-induced EMT process. More interestingly, SP1 was further shown to transcriptionally activate PPP1R13L expression in a feedback manner. In addition, PPP1R13L and SP1 expression was found to be closely associated with the clinicopathological characteristics of lung cancer patients. Here we proposed a novel feedback regulation mechanism, in which SP1 may transcriptionally activate the PPP1R13L gene expression in the early stage of lung cancer to promote tumor growth, while the accumulation of PPP1R13L drives tumor invasion and metastasis by direct repression of SP1. Thus, this unique feedback loop between PPP1R13L and SP1 may play a vital role in chemical carcinogenesis and serve as a potential intervention target for lung cancer progression attributable to cigarette smoking.

A miR-15a related polymorphism affects NSCLC prognosis via altering ERCC1 repair to platinum-based chemotherapy.

Platinum-based chemotherapy is regarded as a preferential curative-intent option for non-small cell lung cancer (NSCLC), while the acquired drug resistance has become a major obstacle that limits its clinical application. Since the repair efficiency of tumour cells to platinum-DNA adducts plays a crucial role in chemotherapy resistance, we aimed to explore whether several meaningful polymorphisms of DNA repair genes were associated with the benefits of platinum-based chemotherapy in NSCLC patients. Firstly, six single nucleotide polymorphisms (SNPs) located in the 3'untranslated region (3'UTR) of three DNA repair genes were detected in 246 NSCLC patients receiving platinum-based chemotherapy and analysed the correlation of these candidate SNPs with the overall survival. Cox proportional hazard model showed that NSCLC patients carrying ERCC1 rs3212986 AA genotype had a shorter overall survival compared to those with CC. Mechanistically, we performed tumour chemosensitivity assay to observe the convincing linkage of rs3212986 polymorphism with ERCC1 expression and cisplatin sensitivity. The subsequent in vitro experiments identified that rs3212986 polymorphism altered the post-transcriptional regulation of ERCC1 via affecting the binding of miR-15a, and further changed the sensitivity to platinum analogue. It reminded that patients carrying ERCC1 rs3212986 CC homozygote were expected to respond better to platinum-based chemotherapy due to a lower expression of ERCC1. Compared with previous studies, our current comprehensive study suggested that rs3212986, a 3'UTR polymorphism in ERCC1, might have clinical relevance in predicting the prognosis of NSCLC patients receiving platinum-based chemotherapy.

Malignant transformation of human bronchial epithelial cells induced by benzo [a] pyrene suggests a negative feedback of TP53 to PPP1R13L via binding a possible enhancer element.

Previous studies have shown that PPP1R13L as an inhibitor of apoptosis protease TP53 can lead to abnormal cell proliferation and carcinogenesis, however, the function of PPP1R13L was complicated and the interaction between TP53 and PPP1R13L needs to be further explored. In the present study, a malignant transformation model of human bronchial epithelial cells induced by benzo (a) pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was established to observe the regulatory patterns between TP53 and PPP1R13L during carcinogenesis. In vitro experiments including CRISPR-Cas9 editing, RNA silence, Co-Immunoprecipitation and Chromatin Immunoprecipitation were applied to discuss their interactive effects. Additionally, TCGA data profile and our clinical samples of lung cancer were also used to analyze their relationship at the transcriptome level. Interestingly, we found that the mRNA and protein level of TP53 and PPP1R13L fluctuated as a wave in BPDE-induced malignant transformation under wild-type TP53 genetic background. Our results have also demonstrated that PPP1R13L acts as an inhibitor of TP53, while TP53 can regulate PPP1R13L via binding a possible enhancer of the first intron of PPP1R13L gene. Likewise, TCGA data and clinical samples have identified that in the case of TP53 mutation, TP53 expression was negatively correlated with PPP1R13L, while in the case of TP53 wild-type, TP53 expression was not correlated with PPP1R13L. It suggested that there existed a negative feedback of wild-type TP53 to PPP1R13L, which reminded a unique implication during chemical carcinogenesis.

Benzo[a]pyrene diol epoxide-induced transformed cells identify the significance of hsa_circ_0051488, a ERCC1-derived circular RNA in pulmonary squamous cell carcinoma.

ERCC1 is a gene for repairing DNA damage whose function is related to carcinogenic-induced tumorigenesis and the effectiveness of platinum therapies. Circular RNAs (circRNAs) are products of posttranscriptional regulation with pleiotropic effects on the pathogenesis of lung cancer. We aim to identify that specific circRNAs derived from ERCC1 can regulate key biological processes involved in the development of lung cancer. We performed bioinformatics analysis, in vitro experiments, and analyzed clinical samples, to determine the biological features of a certain ERCC1-derived circRNA termed as hsa_circ_0051488 in benzo[a]pyrene diol epoxide-induced malignant transformed cell and lung cancer cell. The well-established model of transformed cells provided an ideal platform for analyzing the molecular characteristics of this circRNA in the malignant transformation of lung epithelial cell, which supports that hsa_circ_0051488 functions in the onset and growth of lung squamous cell carcinoma (LUSC). Further analysis indicates that the absence of hsa_circ_0051488 promoted the proliferation of cells with the malignant phenotype. Extensive experiments confirm that hsa_circ_0051488 is present in the cytoplasm and functioned as a competing endogenous RNA. In particular, hsa_circ_0051488 binds to mir-6717-5p, thereby modulating the expression of SATB2 gene, a lung cancer suppressor. Furthermore, our in silico experiments indicate that SATB2 can inhibit multiple tumor pathways and its expression positively correlated with the tumor suppressor gene CRMP1. These findings suggest a possible regulatory mechanism of hsa_circ_0051488 in LUSC, and that the newly discovered hsa_circ_0051488/miR-6717-5p/SATB2 axis may be a potential route for therapeutic intervention of LUSC.

LncRNA EWSAT1 Regulates the Tumorigenesis of NSCLC as a ceRNA by Modulating miR-330-5p/ITGA5 Axis.

The aim of the study is to investigate how lncRNA EWSAT1 regulates the tumorigenesis of non-small cell lung cancer (NSCLC) as a ceRNA by modulating miR-330-5p/ITGA5 axis. qRT-PCR was conducted to evaluate the expression of EWSAT1 in NSCLC tissue. Then, A549 cells were selected and divided into Blank shScramble, shEWSAT1, miR-330-5p inhibitor, shEWSAT1 + miR-330-5p inhibitor, and siITGA5 and miR-330-5p inhibitor + siITGA5 groups. Besides, a series of in-vitro experiments were carried out to determine the changes in cell proliferation, apoptosis, invasion, and migration in each group. In addition, xenograft models were also constructed on nude mice to detect the tumor volume and weight, and the expression of Ki67 and apoptosis in xenograft tumor were evaluated. In NSCLC tissue and cell, EWSAT1 was upregulated significantly, demonstrating a correlation with tumor diameter, differentiation, lymph node metastasis, and TNM stage. Dual luciferase reporter gene assay confirmed targeting relationships among miR-330-5p, EWSAT1, and ITGA5. In comparison with the Blank group, the number of cell clones in the shEWSAT1 group and siITGA5 decreased, with declined invasion and migration but increased apoptotic rate. Meanwhile, ITGA5, MMP-2, and MMP-9 were downregulated with upregulated cleaved caspase-3. However, the changes above were totally reversed in the miR-330-5p inhibitor group, and miR-330-5p inhibitor transfection abolished the effect of shEWSAT1. In addition, subcutaneous xenotransplantation showed that the tumor growth in shEWSAT1 group retarded significantly, with downregulation of Ki67 and increase apoptotic rate. Silencing EWSAT1 could inhibit the expression of ITGA5 via upregulating miR-330-5p, thus, resulting in the inhibition of NSCLC cell growth.

Mitochondrial Ca2+ regulation in the etiology of heart failure: physiological and pathophysiological implications.

Heart failure (HF) represents one of the leading causes of cardiovascular diseases with high rates of hospitalization, morbidity and mortality worldwide. Ample evidence has consolidated a crucial role for mitochondrial injury in the progression of HF. It is well established that mitochondrial Ca2+ participates in the regulation of a wide variety of biological processes, including oxidative phosphorylation, ATP synthesis, reactive oxygen species (ROS) generation, mitochondrial dynamics and mitophagy. Nonetheless, mitochondrial Ca2+ overload stimulates mitochondrial permeability transition pore (mPTP) opening and mitochondrial swelling, resulting in mitochondrial injury, apoptosis, cardiac remodeling, and ultimately development of HF. Moreover, mitochondria possess a series of Ca2+ transport influx and efflux channels, to buffer Ca2+ in the cytoplasm. Interaction at mitochondria-associated endoplasmic reticulum membranes (MAMs) may also participate in the regulation of mitochondrial Ca2+ homeostasis and plays an essential role in the progression of HF. Here, we provide an overview of regulation of mitochondrial Ca2+ homeostasis in maintenance of cardiac function, in an effort to identify novel therapeutic strategies for the management of HF.

AC138128.1 an Intronic lncRNA originating from ERCC1 Implies a Potential Application in Lung Cancer Treatment.

Lung cancer is one of the most devastating tumors with a high incidence and mortality worldwide. Polymorphisms and expression of ERCC1 commonly predicted the occurrence and prognosis of lung cancer. However, few studies have focused on long non-coding RNAs related to ERCC1 though some studies reminded the importance of its post-transcriptional regulation. In the present study, an intronic lncRNA AC138128.1 originated from ERCC1 was firstly identified in microarray chip and database, and its possibility as a novel biomarker to predict lung cancer treatment was further discussed. Firstly, the qRT-PCR data showed that AC138128.1 expression was much lower in lung cancer comparing with its para-cancer tissues, which further analyzed by ROC curve. Similarly, the difference was also verified in 16HBE, A549 and LK2 cells. Then AC138128.1 expression was found to have an increasing trend in a dose or time-dependent manner after cisplatin treatment. Finally, the subcellular distribution of AC138128.1 reminded that AC138128.1 was mainly expressed in the nucleus. Interestingly a positive relationship between AC138128.1 and ERCC1 expression was only found in cancer tissues, which reminded AC138128.1 may be involved in the regulation of ERCC1. Therefore, as a preliminary exploration of the lncRNA originated from ERCC1, the present study suggested AC138128.1 is of potential value in predicting platinum analogue benefit in lung cancer.

Rs3212986 polymorphism, a possible biomarker to predict smoking-related lung cancer, alters DNA repair capacity via regulating ERCC1 expression.

Single nucleotide polymorphisms (SNPs) in 3'UTR of key DNA repair enzyme genes are associated with inter-individual differences of DNA repair capacity (DRC) and susceptibility to a variety of human malignancies such as lung cancer. In this study, seven candidate SNPs in 3'UTR of DRC-related genes including ERCC1 (rs3212986, rs2336219, and rs735482), OGG1 (rs1052133), MLH3 (rs108621), CD3EAP (rs1007616), and PPP1R13L (rs6966) were analyzed in 300 lung cancer patients and controls from the northeast of China. Furthermore, we introduced ERCC1 (CDS+3'UTR) or CD3EAP (CDS) cDNA clone to transfect HEK293T and 16HBE cells. Cell viability between different genotypes of transfected cells exposed to BPDE was detected by CCK-8 assay, while DNA damage was visualized using γH2AX immunofluorescence and the modified comet assay. We found that minor A-allele of rs3212986 could reflect a linkage with increasing risk of NSCLC. Compared with CC genotype, AA genotype of ERCC1 rs3212986 was a high-risk factor for NSCLC (OR = 3.246; 95%CI: 1.375-7.663). Particularly stratified by smoking status in cases and controls, A allele of ERCC1 rs3212986 also exhibited an enhanced risk to develop lung cancer in smokers only (P < 0.05). Interestingly, reduced repair efficiency of DNA damage was observed in 293T ERCC1(AA) and 16HBE ERCC1(AA), while no significant difference was appeared in two genotypes of CD3EAP (3' adjacent gene of ERCC1) overexpressed cells. Our findings suggest that rs3212986 polymorphism in 3'UTR of ERCC1 overlapped with CD3EAP may affect the repair of the damage induced by BPDE mainly via regulating ERCC1 expression and become a potential biomarker to predict smoking-related lung cancer.

Intracerebroventricular streptozotocin-induced Alzheimer's disease-like sleep disorders in rats: Role of the GABAergic system in the parabrachial complex.

AIM: Sleep disorders are common in Alzheimer's disease (AD) and assumed to directly influence cognitive function and disease progression. This study evaluated sleep characteristics in a rat model of AD that was induced by intracerebroventricular streptozotocin (STZ) administration and assessed the possible underlying mechanisms. METHODS: Cognition ability was assessed in the Morris water maze in rats. Sleep parameters were analyzed by electroencephalographic and electromyographic recordings. Neuronal activity in brain areas that regulate sleep-wake states was evaluated by double-staining immunohistochemistry. High-performance liquid chromatography with electrochemical detection was used to detect neurotransmitter levels. RESULTS: Fourteen days after the STZ injection, the rats exhibited sleep disorders that were similar to those in AD patients, reflected by a significant increase in wakefulness and decreases in nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. The c-Fos expression analysis indicated that neuronal activity and the number of neurons in the dorsal raphe nucleus and locus coeruleus decreased in STZ-injected rats. In the ventrolateral preoptic nucleus (VLPO), the activity of γ-aminobutyric acid (GABA) neurons was suppressed. In the arousal-driving parabrachial nucleus (PBN), GABAergic activity was suppressed, whereas glutamatergic activity was promoted. The neurotransmitter analysis revealed a reduction in GABA in the VLPO and PBN and elevation of glutamate in the PBN. A direct injection of the GABAA receptor antagonist bicuculline in the PBN in normal rats induced a similar pattern of sleep disorder as in STZ-injected rats. A microinjection of GABA in the PBN improved sleep disorders that were induced by STZ. CONCLUSION: These results suggest that the reduction in GABAergic inhibition in the PBN and VLPO may be involved in sleep disorders that are induced by STZ. Our novel findings encourage further studies that investigate mechanisms of sleep regulation in sporadic AD.

The lncRNA RHPN1-AS1 downregulation promotes gefitinib resistance by targeting miR-299-3p/TNFSF12 pathway in NSCLC.

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib has exhibited notable clinical efficacy in non-small cell lung cancer (NSCLC) patients. However, its therapeutic efficacy is ultimately limited by the development of gefitinib resistance. The present study aimed to investigate the effects of the long non-coding RNA, RHPN1-AS1 on gefitinib resistance in NSCLC and explore the underlying mechanisms. In this study, RHPN1-AS1 was observed to be downregulated in gefitinib resistant patients and NSCLC cell lines. Besides, decreased expression of RHPN1-AS1 was found to be associated with poor prognosis of NSCLC patients. RHPN1-AS1 knockdown conferred gefitinib resistance to gefitinib sensitive NSCLC cells, whereas the overexpression of RHPN1-AS1 sensitized gefitinib resistant NSCLC cells to gefitinib treatment. Mechanistically, RHPN1-AS1 was found to positively regulate the expression of TNFSF12 by directly interacting with miR-299-3p. Collectively, RHPN1-AS1 modulates gefitinib resistance through miR-299-3p/TNFSF12 pathway in NSCLC. Our findings indicate that RHPN1-AS1 may serve as not only a prognostic biomarker for gefitinib resistance but also as a promising therapeutic biomarker and target for the treatment of NSCLC patients.

Different splicing isoforms of ERCC1 affect the expression of its overlapping genes CD3EAP and PPP1R13L, and indicate a potential application in non-small cell lung cancer treatment.

Numerous genes are arranged in complex overlapping and interlaced patterns, and such arrangements potentially contribute to the regulation of gene expression. Previous studies have demonstrated that a region in chromosome 19q13.2-3 encompassing the overlapping genes excision repair cross-complementation group 1 (ERCC1), CD3e molecule associated protein (CD3EAP) and protein phosphatase 1 regulatory subunit 13 like (PPP1R13L) was found to be associated with the risk and prognosis of non-small cell lung cancer (NSCLC). The present study confirmed the hypothesis that there are co-expression patterns among these overlapping genes. The suggestive bioinformatic evidence of The Cancer Genome Atlas was verified by quantitative polymerase chain reaction (qPCR) analysis of NSCLC tissue samples. In addition, a cisplatin-induced DNA damage cell model was assessed by microarray analysis, qPCR and 3' rapid amplification of cDNA ends (3'RACE) to verify and quantify the expression levels of co-expressed alternative splicing isoforms in the NSCLC tissues, as well as in cancer A549 and normal 16HBE cells. The expression of CD3EAP exon 1 was demonstrated to be significantly associated with PPP1R13L exon 1, while CD3EAP exon 3 was significantly associated with ERCC1 exon 11 in normal and NSCLC tissues. It was observed that short transcripts of ERCC1, CD3EAP and PPP1R13L are co-expressed in A549 cells and full-length transcripts are co-expressed in 16HBE cells. Furthermore, a novel transcriptional regulation pattern was described based on the positional associations of overlapping genes. The region encompassing the overlapping genes ERCC1, CD3EAP and PPP1R13L may be involved in linking the upstream and downstream genes, while the different splicing isoforms of ERCC1 affect the expression of its overlapping genes, suggesting potential application in cisplatin resistance in NSCLC treatment.

The Long Non-Coding RNA XIST Controls Non-Small Cell Lung Cancer Proliferation and Invasion by Modulating miR-186-5p.

BACKGROUND/AIMS: Long non-coding RNAs (lncRNAs) are key players in the development and progression of human cancers. The lncRNA XIST (X-inactive specific transcript) has been shown to be upregulated in human non-small cell lung cancer (NSCLC); however, its role and molecular mechanisms in NSCLC cell progression remain unclear. METHODS: qRT-PCR was conducted to assess the expression of XIST and miR-186. Cell proliferation was detected using MTT assay. Cell invasion and migration were evaluated using transwell assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Luciferase reporter assay was used to identify the direct regulation of XIST and miR-186. A RNA immunoprecipitation was used to analyze whether XIST was associated with the RNA-induced silencing complex (RISC). RESULTS: We confirmed that XIST was upregulated in NSCLC cell lines and tissues. Functionally, XIST knockdown inhibited cancer cell proliferation and invasion, and induced apoptosis in vitro, and suppressed subcutaneous tumor growth in vivo. Mechanistic investigations revealed a reciprocal repressive interaction between XIST and miR-186-5p. Furthermore, we showed that miR-186-5p has a binding site for XIST. Our data also indicated that XIST and miR-186-5p are likely in the same RNA induced silencing complex. CONCLUSION: Together, our data revealed that XIST knockdown confers suppressive function in NSCLC and XIST may be a novel therapeutic marker in this disease.

Tenuifolin, a saponin derived from Radix Polygalae, exhibits sleep-enhancing effects in mice.

BACKGROUND: Radix Polygalae, the dried root of Polygala tenuifolia, has been extensively used as a traditional Chinese medicine for promoting intelligence and tranquilization. Polygalasaponins extracted from the root of P. tenuifolia possess evident anxiolytic and sedative-hypnotic activities. Previous studies have reported that tenuifolin was a major constituent of polygalasaponins. PURPOSE: The currently study aims to investigate the hypnotic effect and possible mechanism of tenuifolin in freely moving mice. DESIGN/METHODS: The hypnotic effects of tenuifolin (20, 40 and 80mg/kg, p.o.) were assessed by electroencephalographic (EEG) and electromyographic (EMG) analysis. Double-staining immunohistochemistry test was performed to evaluate the neuronal activity of sleep-wake regulating brain areas. High performance liquid chromatograph- electrochemical detection (HPLC-ECD) and ultrafast liquid chromatography-mass spectrometry (UFLC-MS) were used for the detection of neurotransmitters. Locomotor activity was measured by Open-field Test. RESULTS: Tenuifolin at doses of 40 and 80mg/kg (p.o.) significantly prolonged the total sleep time by increasing the amount of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep, associated with the significant increase in the bouts of episodes respectively. After administration of tenuifolin, the cortical EEG power spectral densities during NREM and REM sleep were similar to that of natural sleep (vehicle) and thus compatible with physiological sleep. Double-immunohistochemistry staining test showed that tenuifolin increased the c-Fos positive ratios of GABAergic NREM sleep-promoting neurons in ventrolateral preoptic area (VLPO), cholinergic REM sleep-promoting neurons in laterodorsal tegmental area (LDT) and pontomesencephalic tegmental area (PPT) and decreased the c-Fos positive ratios in wake-promoting neurons (locus coeruleus (LC) and perifornical area (Pef)). Neurotransmitter detections revealed that tenuifolin significantly reduced the noradrenaline (NA) levels in LC, VLPO, PPT and LDT, elevated the GABA levels in VLPO, LC and Pef and increased the acetylcholine (Ach) levels in LDT and PPT. In addition, tenuifolin did not cause any change to locomotor activity. CONCLUSION: Taken together, these results provide the first experimental evidence of the significant sleep-enhancing effect of tenuifolin in mice. This effect appears to be mediated, at least in part, by the activation of GABAergic systems and/or by the inhibition of noradrenergic systems. Moreover, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant P. tenuifolia in the development of phytomedicines with hypnotic properties.

The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model.

Nucleotide excision repair (NER) is an important defense mechanism of the body to exogenous carcinogens and mutagens, such as benzo[a]pyrene (B[a]P). Genetic polymorphisms in ERCC2/XPD, a critical element in NER, are thought to be associated with individual's cancer susceptibility. Although ERCC2/XPD Lys751Gln (rs13181) is the most studied polymorphism, the impact of this polymorphism on DNA repair capacity to carcinogen remains unclear. In the present study, cDNA clones carrying different genotypes of ERCC2/XPD (Lys751Gln) were introduced into an ERCC2/XPD deficient cell line (UV5) in a well-controlled biological system. After B[a]P treatment, cell growth inhibition rates and DNA damage levels in all cells were detected respectively. As expected, we found that the DNA repair capacity in UV5 cells was restored to levels similar to wildtype parent AA8 cells upon introduction of the cDNA clone of ERCC2/XPD (Lys751). Interestingly, after B[a]P treatment, transfected cells expressing variant ERCC2/XPD (751Gln) showed an enhanced cellular sensitivity and a diminished DNA repair capacity. The wildtype genotype AA (Lys) was found to be associated with a higher DNA repair capacity as compared to its polymorphic genotype CC (Gln). These data indicate that ERCC2/XPD Lys751Gln polymorphism affects DNA repair capacity after exposure to environmental carcinogens such as B[a]P in this well-controlled in vitro system and could act as a biomarker to increase the predictive value to develop cancer.

Different neural circuitry is involved in physiological and psychological stress-induced PTSD-like "nightmares" in rats.

Posttraumatic nightmares are a core component of posttraumatic stress disorder (PTSD) and mechanistically linked to the development and maintenance of this disorder, but little is known about their mechanism. We utilized a communication box to establish an animal model of physiological stress (foot-shock [FS]) and psychological stress (PS) to mimic the direct suffering and witnessing of traumatic events. Twenty-one days after traumatic stress, some of the experimental animals presented startled awakening (i.e., were startled awake by a supposed "nightmare") with different electroencephalographic spectra features. Our neuroanatomical results showed that the secondary somatosensory cortex and primary auditory cortex may play an important role in remote traumatic memory retrieval in FS "nightmare" (FSN) rats, whereas the temporal association cortex may play an important role in PS "nightmare" (PSN) rats. The FSN and PSN groups possessed common emotion evocation circuits, including activation of the amygdala and inactivation of the infralimbic prefrontal cortex and ventral anterior cingulate cortex. The decreased activity of the granular and dysgranular insular cortex was only observed in PSN rats. The present results imply that different types of stress may cause PTSD-like "nightmares" in rodents and identified the possible neurocircuitry of memory retrieval and emotion evocation.

Girdin correlated with autophagy in invasive ductal breast carcinomas.

AIMS AND BACKGROUND: Increasing evidence claims that autophagy is essential for breast cancer progression. Girdin was found highly expressed in breast cancers. It has been reported that Girdin attenuates autophagy in HeLa cells. We explored the relationship between Girdin expression and autophagic patterns in breast cancer. METHODS AND STUDY DESIGN: In the study, Girdin expression and autophagic activity were investigated in a series of 99 invasive ductal breast carcinomas after immunohistochemical staining for the autophagy-associated proteins LC3-II and Girdin. RESULTS: The level of Girdin expression negatively correlated with LC3-II level, which represents autophagic activity (r = -0.289), and positively correlated with lymph node metastasis (r = 0.472). Girdin level was found no different in the "diffuse cytoplasmic" and "stone-like" patterns of LC3-II. CONCLUSIONS: Up-regulated autophagy was negatively associated with Girdin level. There was a significant correlation between Girdin expression and lymph nodes metastasis in invasive ductal breast carcinoma.

The level of phosphorylated Akt predominantly reflects the expressive status of CerbB2 in invasive breast cancer.

Although some evidence has been documented on EGFR/PI3K mediation of Akt activation in breast cancers, ILK and DNA-PK have not been investigated so far. The aim of this study was to analyze the expression of phosphorylated Akt (pAkt) in breast cancer, with respect to its upstream regulators. The immunostaining of pAkt (Ser473) in 70 invasive breast cancers revealed that status of CerbB2 could play a major role in Akt phosphorylation, while ILK was also involved in the stimulated level of pAkt. The results would provide an important clue for the activation of Akt and potential targeted therapy in breast cancer.

[Aberrant expression and correlative analysis of P16 in breast cancers].

OBJECTIVE: To evaluate the relationship between the aberrant expression of P16 and the clinic-pathological features in breast cancers. METHODS: In our study, 72 cases of breast cancer were collected and the expressions of P16, HER-2 (human epidermal growth receptor-2), ER (estrogen receptor), PR (progesterone receptor), P53, Ki-67 were measured by immunohistochemistry. The correlations between the P16 and the clinic-pathological features (menstruation, tumor size, histological grade, lymph node metastasis were statistically analyzed. RESULTS: Aberrant expression of P16 was detected in 36.1%(26/72)of breast cancers. Not only was the staining of P16 increased,but also the subcellular localization was changed from nuclear to cytoplasm or whole cell staining. In general, the occasional cell staining (+) usually presented in nuclear, as expression increased, P16 showed mainly in cytoplasm or whole cells (+++) even diffusive staining in extracellular, and the moderate staining was multi-focal both in nuclear and cytoplasm. The expression of P16 was significantly increased in ER negative group compared with ER positive group (78.6% vs. 9.1%,P=0.000). Statistical analysis showed a significant correlation of high Ki-67 index with the group of P16 positive (Z =-0.263, P=0.003). In addition, significant difference was calculated between pre- and post-menopause (55.6% vs. 24.4%, P=0.008)and P16 expressions were also more credited to the poor-differentiated group in histological grading: 11.8% (2/17) for highly differentiated group, 27.6% (8/29) for moderately differentiated group and 61.5% (16/26) for poorly differentiated group, respectively (P=0.002). CONCLUSION: The aberrant expression of P16 in breast cancers correlated closely with loss of estrogen receptor, high proliferation index or high histological grade, predisposed to the patients of pre-menopause, implicating that the aberrant expression of P16 should be a predictor of poor response to endocrine therapy or more aggressive behavior.

Girdin locates in centrosome and midbody and plays an important role in cell division.

Girdin is a downstream effector of epidermal growth factor receptor (EGFR)-AKT and interacts with actin and microtubule. Increasing evidence confirmed that Girdin played an important role in cell migration. Here we report that Girdin also regulates cell division. Overexpression or suppression of Girdin leads to attenuated cell proliferation. Imaging of mitotic cells revealed that Girdin is located in the cell division apparatus such as centrosome and midbody. The sub-cellular localization of Girdin was dependent on the domains, which interacted with actin or microtubules. Overexpression of Girdin lead to increased centrosome splitting and amplification. In addition, data show that pAKT also locates in both the centrosome and midbody, indicating the regulating role of AKT in Girdin-mediated cell division. To elucidate the effect of Girdin on tumor growth in vivo, HeLa cells infected with retrovirus harboring either control or Girdin shRNAs were injected subcutaneously into the immunocompromised nude mice. Downregulation of Girdin by shRNA markedly inhibited the cell growth of subcutaneously transplanted tumors in nude mice. These data demonstrate that Girdin is important for efficient cell division. Taking our previous data into consideration, we speculate that Girdin regulates both cell division and cell migration through cytoskeletal molecules.