Hypoxia-associated circPRDM4 promotes immune escape via HIF-1α regulation of PD-L1 in hepatocellular carcinoma.

BACKGROUND: Hypoxia is a hallmark of cancer, and is closely intertwined with tumor immune evasion. Circular RNAs (circRNAs) have been implicated in tumor response to immune checkpoint blockades. However, hypoxia-associated circRNAs that orchestrate the association between hypoxia and response to immunotherapy remain poorly understood. Here, we aimed to determine the roles of hypoxia-associated circRNAs in immune escape of hepatocellular carcinoma (HCC) cells. METHODS: Differentially expressed hypoxia-associated circRNAs were determined using high-throughput sequencing technology. HCC patients treated with PD-1 blockade were enrolled to assess the clinical significance of circPRDM4. RT-qPCR, western blotting, flow cytometry, T cell-mediated tumor cell killing assay, and enzyme linked immunosorbent assay were used to investigate the roles of circPRDM4 in immune escape of HCC cells in vitro. Patient-derived xenograft mouse models and adoptive human tumor infiltrating lymphocyte-CD8+ T cell transfer were adopted to evaluate the effects of circPRDM4 in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification, dual-luciferase reporter assays, dot blotting, DNA in situ hybridization, and immunoprecipitation were utilized to examine the interaction between circPRDM4, HIF-1α, and CD274 promoter. RESULTS: We identified circPRDM4 as a hypoxia-associated circRNA in HCC. circPRDM4 was upregulated in responders to PD-1 blockade and associated with therapeutic efficacy. In vitro and in vivo experiments showed that circPRDM4 induced PD-L1 expression and promoted CD8+ T cell-mediated immune escape under hypoxic conditions. Mechanistically, circPRDM4 acted as a scaffold to recruit HIF-1α onto CD274 promoter, and cemented their interaction, ultimately promoting the HIF-1α-mediated transactivation of PD-L1. CONCLUSIONS: These findings illustrated that circPRDM4 promoted immune escape of HCC cells by facilitating the recruitment of HIF-1α onto the promoter of CD274 under hypoxia, thereby inhibiting CD8+ T cell infiltration in the tumor microenvironment. This work may provide a novel prognostic biomarker and therapeutic candidate for HCC immunotherapy.

SGK1 inhibits cellular apoptosis and promotes proliferation via the MEK/ERK/p53 pathway in colitis.

AIM: To investigate the role of serum-and-glucocorticoid-inducible-kinase-1 (SGK1) in colitis and its potential pathological mechanisms. METHODS: SGK1 expression in mucosal biopsies from patients with active Crohn's disease (CD) and normal controls was detected by immunohistochemistry. We established an acute colitis model in mice induced by 2,4,6-trinitrobenzene sulfonicacid, and demonstrated the presence of colitis using the disease activity index, the histologic activity index and hematoxylin and eosin staining. The cellular events and potential mechanisms were implemented with small interference RNA and an inhibitor of signaling molecule (i.e., U0126) in intestinal epithelial cells (IECs). The interaction between SGK1 and the signaling molecule was assessed by co-immunoprecipitation. RESULTS: SGK1 expression was significantly increased in the inflamed epithelia of patients with active CD and TNBS-induced colitis model (0.58 ± 0.055 vs 0.85 ± 0.06, P < 0.01). At the cellular level, silencing of SGK1 by small interference RNA (siSGK1) significantly inhibited the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1) and the downstream molecule extracellular signal regulated protein kinase (ERK) 1/2, which induced the upregulation of p53 and Bcl-2-associated X protein, mediating the subsequent cellular apoptosis and proliferation in IECs. Cells treated with MEK1 inhibitor (i.e., U0126) before siSGK1 transfection showed a reversal of the siSGK1-induced cellular apoptosis. CONCLUSION: Our data suggested that SGK1 may protect IECs in colitis from tumor necrosis factor-α-induced apoptosis partly by triggering MEK/ERK activation.

Clinicopathological significance and prognostic value of the expression of the cancer stem cell marker CD133 in hepatocellular carcinoma: a meta-analysis.

To conduct a meta-analysis to assess the association between CD133 expression and clinicopathological significance and prognostic value in hepatocellular carcinoma patients. Studies were identified via an electronic comprehensive literature search through the Pubmed, Chinese CNKI, and Wanfang databases. This meta-analysis was performed using Stata statistical software version 12.0. The outcomes included various clinicopathological and survival parameters (P < 0.05 was consider to indicate a statistical significance). A total of 21 studies comprising 2592 patients were included in this meta-analysis. CD133 overexpression was significantly associated with a series of clinicopathological parameters, such as low tumor differentiation (pooled odds ratio (OR) = 2.26, 95% CI: 1.59-3.21, P < 0.00001), advanced tumor stage (pooled OR = 2.17, 95% CI: 1.70-2.77, P < 0.00001), vascular invasion (pooled OR = 2.06, 95% CI: 1.25-3.39, P = 0.005), and vascular thrombosis (pooled OR = 1.47, 95% CI: 1.08-1.99, P = 0.015). However, CD133 expression was not correlated with hepatitis, cirrhosis, α-fetoprotein level, tumor number, tumor size, encapsulation, or metastasis. Regarding survival outcome, CD133 overexpression was significantly correlated with poor overall survival (pooled hazard ratio (HR) = 2.01, 95% CI: 1.45-2.80, P = 0.00002) and poor disease-free survival (pooled HR = 1.82, 95% CI: 1.45-2.29, P < 0.00001). This meta-analysis indicated that CD133 overexpression is significantly associated with clinicopathological factors and poorer survival outcome.

Multi-target lentivirus specific to hepatocellular carcinoma: in vitro and in vivo studies.

BACKGROUND & AIMS: We aimed at investigating the effects of the targeted transduction of the Wtp53-pPRIME-miR30-shRNA gene into liver cancer cells, under the mediation of anti-alpha fetoprotein scFv-directed lentivirus, and the inhibitory effect of this system on liver cancer cells. METHODS: The result of infection was observed by fluorescence microscopy. Polymerase chain reaction and Western blotting were used to demonstrate the successful transduction and transcription of the Wtp53-pPRIME-miR30-shRNA-IGF1R gene. Cell growth was observed via the Cell-Counting Kit-8 Method, and cell apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. To observe further the effects of AFP-Wtp53-pPRIME-miR30-shRNA-IGF1R therapy in animals, models of BALB-C nude mice bearing subcutaneous human hepatocellular carcinoma were established. The influence of the growth of subcutaneously transplanted tumor, expression of Wtp53 protein, apoptosis, and microvessel formation on the overall level of AFP-Wtp53 pPRIME-miR30-shRNA-IGF1R were also evaluated. RESULTS: Recombinant lentivirus was successfully constructed, and its functional plaque-forming unit titer was determined as 4.58 × 10(9)plaque-forming units/ml. A positive strand was detected by polymerase chain reaction and Western blotting. Lentiviral construction worked effectively in AFP-positive liver cancer cells. In vitro and in vivo experiments showed that the recombinant lentivirus was more efficacious in inhibiting the proliferation of Hep3B cells. CONCLUSIONS: The Wtp53-pPRIME-miR30-shRNA gene can be subjected to targeted transduction into liver cancer cells under the mediation of anti-alpha fetoprotein scFv-directed lentivirus. The Wtp53-pPRIME-miR30-shRNA system has targeting ability and lethal effects on liver cancer cells.

Surgical outcomes for gastric cancer of a single institute in southeast China.

BACKGROUND: In recent years, with social and economic development and lifestyle changes, the incidence of gastric cancer as well as the surgical results and prognoses of patients with gastric cancer have changed significantly in southeast China. METHODS: A total of 1,451 patients were divided into 2 groups according to admission time periods. Trends in clinicopathologic characteristics and operative outcomes of these patients were analyzed retrospectively. RESULTS: The numbers of old and young patients were significantly increased in period 2 compared with period 1. Tumors located in the proximal stomach increased from 20.26% to 36.83%. The incidence of early gastric cancer was significantly increased from period 1 to period 2. Lymph node metastasis was seen more prevalently in period 2 than in period 1. The rate of operation-related major complications decreased from 5.23% to 1.43%. Operative mortality was .49% in period 1 and .24% in period 2. The 5-year survival rate increased from 38.40% to 53.99%. CONCLUSIONS: Early diagnosis, standardized surgical treatment including pertinent lymph node dissection, and better perioperative care notably improve the outcomes of patients with gastric cancer.

Comparative proteome analysis of neural retinas from type 2 diabetic rats by two-dimensional electrophoresis.

PURPOSE: The purpose of this study was to isolate, identify, and analyze diabetes-related protein changes that occur in neural retinas in vivo. METHODS: Total proteins were extracted from neural retinas of normal and 8-weeks diabetic Sprague-Dawley (SD) rats and separated by two-dimensional gel electrophoresis (2-DE). Some protein spots exhibiting statistically significant variations (p < 0.05) were selected randomly and identified by mass spectrometry (MS or MS/MS). The protein alphaA-crystallin was chosen as a target for specific immunodetection using Western blot to corroborate the variation found by 2-DE. RESULTS: Twenty protein spots identified included alphaA-crystallin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine (Gln) synthetase, and so forth. Western blotting analyses confirmed that alphaA-crystallin protein expression was upregulated in diabetic retina. CONCLUSIONS: In this study, we isolate, identify, and analyze diabetes-related protein changes that occur in neural retinas in vivo. Further investigation of candidate proteins may identify novel pharmacological targets for diabetic retinopathy.