MicroRNAs (miRNAs or miRs) play important roles in numerous cellular processes, including development, proliferation, tumorigenesis and apoptosis. It has been reported that miRNA expression is induced by ionizing radiation (IR) in cancer cells. However, the underlying molecular mechanisms are not yet fully understood. In this study, endogenous miR‑320a and its primary precursor (pri‑miR‑320a) were assayed by reverse transcription‑quantitative PCR (RT‑qPCR). Luciferase activities were measured using a dual‑luciferase reporter assay system. Western blot analysis was used to determine the protein expressions of upstream and downstream genes of miR‑320a. Cell apoptosis was evaluated by Annexin V apoptosis assay and cell proliferation was measured using the trypan blue exclusion method. The results revealed that miR‑320a expression increased linearly with the IR dose and treatment duration. Three transcription factors, activating transcription factor 2 (ATF2), ETS transcription factor (ELK1) and YY1 transcription factor (YY1), were activated by p38 mitogen‑activated protein kinase (MAPK) and mitogen‑activated protein kinase 8 (JNK) and by upregulated miR‑320a expression under IR conditions. In addition, it was identified that X‑linked inhibitor of apoptosis (XIAP) was an miR‑320a target gene during the IR response. By targeting XIAP, miR‑320a induced apoptosis and inhibited the proliferation of the cancer cells. On the whole, the results of this study demonstrated that miRNA‑320a, regulated by the p38 MAPK/JNK pathway, enhanced the radiosensitivity of cancer cells by inhibiting XIAP and this may thus prove to be a potential therapeutic approach with which to overcome radioresistance in cancer treatment.
Radiotherapy is necessary for 70% of malignant tumor patients. Local recurrence and metastasis are primary failure models, where radioresistance is one of important factors. It is critical to establish radioresistant tumor cell lines for understanding the mechanism of radioresistance. According to single radiation, fractioned radiation and the compound radiation method, four representative radiation models are classified: conventional radiation, repeated radiation, gradient radiation and other radiation. These different radiation models have difference in total dose and radiation model as well as the biological characteristics. Superior to other three models, the gradient fractioned irradiation model increase fractioned doses gradually along with the enhancement of radioresistance, which favorably balances the fractioned doses and the time of irradiated cells approaching to exponential growth phase. Clinically relevant radioresistant cell line (CRR) with a genotype in consistent with its parental cells is an important research direction on tumor radioresistance.
Key words:
Radiotherapy; Radiosensitivity; Radioresistance; Gradient irradiation
Introduction: Colorectal Cancer (CRC) accounts for 9% of cancer deaths globally. Hormonal pathways play important roles in some cancers. This study investigated the association of CRC expression of neurotensin (NTS), NTS receptors 1 and 3 (NTSR1 and NTSR3) and clinical outcomes. Methods: A prospective cohort study which quantifies the protein expression of NTS, NTSR1 and NTSR3 in human CRCs using immunohistochemistry. Expression levels were then compared with clinico-pathological outcome including histological grade, overall survival (OS) and disease-free survival (DFS). Results: Sixty-four patients were enrolled with median follow-up of 44.0 months. There was significantly higher expression of NTS in cancer tissue in CRC with higher T stages (p < 0.01), N stages (p = 0.03), and AJCC clinical stages (p = 0.04). There was significantly higher expression of NTS, NTSR1 and NTSR3 in cancer tissue compared to surrounding normal epithelium (median H-score 163.5 vs 97.3, p < 0.01). There was significantly shorter DFS in individuals with CRC with high levels of NTS compared to lower levels of NTS (35.8 months 95% CI 28.7–42.8 months vs 46.4 months 95% CI 42.2–50.5 months, respectively, p = 0.02). Above median NTS expression in cancer tissue was a significant risk factor for disease recurrence (HR 4.10, 95% CI 1.14–14.7, p = 0.03). Discussion: The expression of NTS and its receptors has the potential to be utilised as a predictive and prognostic marker in colorectal cancer for postoperative selection for adjuvant therapy and identify individuals for novel therapies targeting the neurotensinergic pathways. Conclusions: High NTS expression appears to be associated with more advanced CRC and worse DFS.
Urothelial carcinomas (UCs) are malignant tumors that arise from the lower and upper urinary tract and are characterized by multiple recurrences. Aristolochic acid (AA) is a potent nephrotoxin and human carcinogen associated with UC. East Asian populations with a high UC prevalence have an unusual genome-wide AA-induced mutational pattern. To address the genomic differences and clonal relatedness between primary and recurrent tumors in the UCs with AA pattern, we investigated the genomic differences and tumor microenvironment (TME) of AA and non-AA UCs. 17 UC patients were recruited, with nine documented AA exposure. Eleven of them showed recurrence. After-surgery tissues of primary and paired recurrent tumors were collected. Capture-based targeted deep sequencing was performed using a commercial panel consisting of 520 cancer-related genes. Tumor-infiltrating lymphocytes (TILs) were identified with an immunofluorescence-based microenvironment analysis panel (MAP). Hierarchical clustering based on the COSMIC signatures confirmed two significant subtypes: AA Sig and non-AA Sig. AA Sig was associated with AA-containing herbal drug intake, recurrence, and higher tumor mutation burden (TMB). The clonal architecture of UCs revealed three types of clonal evolution patterns. Non-AA Sig cohort showed shared clonal origin of primary and recurrent tumors. AA Sig showed heterogeneity and had multiple independent origins. Recurrent tumors as second primary tumors in AA Sig showed immunoreactive TME, indicating a better response with immune checkpoint inhibitor therapy. The AA mutational signature and unique immune profiles are helpful molecular markers to distinguish AA exposure from other carcinogens. These results also provide new insights into the origin of recurrent UCs that could affect treatment strategies.
Abstract Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild‐type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild‐type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.
We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.
Oxidative stress is a well-established event in the pathology of several neurobiological diseases. Sirt3 is a nicotinamide adenine nucleotide (NAD+)-dependent protein deacetylase that regulates mitochondrial function and metabolism in response to caloric restriction and stress. This study aims to investigate the role of Sirt3 in H2O2 induced oxidative neuronal injury in primary cultured rat cortical neurons. We found that H2O2 treatment significantly increased the expression of Sirt3 in a time-dependent manner at both mRNA and protein levels. Knockdown of Sirt3 with a specific small interfering RNA (siRNA) exacerbated H2O2-induced neuronal injury, whereas overexpression of Sirt3 by lentivirus transfection inhibited H2O2-induced neuronal damage reduced the generation of reactive oxygen species (ROS), and increased the activities of endogenous antioxidant enzymes. In addition, the intra-mitochondrial Ca2+ overload, but not cytosolic Ca2+ increase after H2O2 treatment, was strongly attenuated after Sirt3 overexpression. Overexpression of Sirt3 also increased the content of mitochondrial DNA (mtDNA) and the expression of mitochondrial biogenesis related transcription factors. All these results suggest that Sirt3 acts as a prosurvival factor playing an essential role to protect cortical neurons under H2O2 induced oxidative stress, possibly through regulating mitochondrial Ca2+ homeostasis and mitochondrial biogenesis.
Cigarette smoking worsens asthma and is associated with reduced response to corticosteroid therapy. As cigarette smoke is known to have immunomodulatory effects, we hypothesized that one mechanism by which smoking mediates its adverse effect is by reduction of the numbers of bronchial mucosal dendritic cells (DCs), which control B-cell growth and T-cell responses.We set out to sample the bronchial mucosa in smoking and never-smoking patients with asthma and to count DCs, B cells, and cells expressing genes for two key T-lymphocyte regulatory cytokines.Twenty-one never-smoker patients with asthma (6 steroid naive), 24 smoker patients with asthma (9 steroid naive), and 10 healthy never-smokers (control subjects) were recruited and their endobronchial biopsy samples were immunostained for detection of mature DCs (CD83(+)), Langerhans cells (CD1a(+)), B lymphocytes (CD20(+)), and helper T-cell type 1 (IFN-gamma) and helper T-cell type 2 (IL-4) cytokine-expressing cells.The number (per square millimeter) of CD83(+) mature DCs was significantly lower in smoker patients with asthma (median [range]: 37 [0, 131]) in comparison with never-smoker steroid-naive and steroid-treated patients with asthma (76 [24, 464]; p = 0.006) or control subjects (85 [40, 294]; p = 0.004). Moreover, B cells were fewer in smoker (26 [4, 234]) versus never-smoker steroid-naive and steroid-treated patients with asthma (45 [10, 447]; p = 0.01) and in smoker steroid-naive patients with asthma (23 [4, 111]) versus control subjects (34 [10, 130]; p = 0.05). The number of cells expressing IFN-gamma showed a trend toward fewer in smoker (70 [6, 24]) versus never-smoker steroid-naive patients with asthma (144 [44, 323]; p = 0.10).There are important and statistically significant differences in the number of CD83(+) mature DCs and B cells in the large airways of smokers with asthma. We speculate that their reductions may render patients with asthma less responsive to corticosteroids and more susceptible to infection.
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