OBJECTIVETo investigate the effects of hypoxia on lipid metabolism in the normal human hepatic cell line L02 and to investigate the underlying molecular mechanisms.

METHODSL02 cells were exposed to hypoxic conditions (experimental groups: at 1% O2, 5% CO2, 94% N2 for 3, 6, 12, 24, or 48 hours) or normoxic conditions (control group: at 21% O2). Lipid droplet accumulation and triglyceride content were measured in each group by oil red O staining and biochemical assay, respectively. The mRNA expression levels of hypoxia-inducible factor (HIF)-2a and sterol regulatory element binding protein (SREBP)-1c were detected by reverse transcription-polymerase chain reaction. Protein expression levels of HIF-2a, adipose differentiation-related protein (ADRP), and Fas were tested by Western blot analysis.

RESULTSLipid droplet accumulation and the triglyceride content were significantly higher in the hypoxia group than the normoxia group. In addition, the hypoxia groups had significantly down-regulated mRNA expression of SREBP-1c (12h: 0.236+/-0.043, 24 h: 0.287+/-0.044, 48 h: 0.342+/-0.049 vs. normoxia: 0.503+/-0.037; F = 28.37, P less than 0.01) and FAS protein (12 h: 0.562+/-0.054, 24 h: 0.674+/-0.062, 48 h: 0.682+/-0.057 vs normoxia: 0.857+/-0.069; F = 16.08, P less than 0.01). In normoxic cells, little or no expression of HIF-2a protein was detected by Western blot. In hypoxic cells, HIF-2a protein expression peaked at 6h (0.973+/-0.067). ADRP protein expression was significantly higher in hypoxia groups than in the normoxia group (12 h: 0.319+/-0.043, 24 h: 0.732+/-0.056 and 48 h: 0.873+/-0.066 vs. 0.211+/-0.019; all, P less than 0.05.

CONCLUSIONExposure to hypoxic conditions might induce lipidosis in normal human hepatic cells by stimulating HIF-2a and ADRP expression.

Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Hypoxia ; Cell Line ; Down-Regulation ; Hepatocytes ; metabolism ; Humans ; Lipid Metabolism ; Membrane Proteins ; metabolism ; Oxygen ; metabolism ; Perilipin-2 ; Sterol Regulatory Element Binding Protein 1 ; metabolism

Humans ; Hemangioendothelioma, Epithelioid/pathology* ; Trans-Activators ; Transcription Factors ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; Hemangioendothelioma

Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics* ; Biomarkers, Tumor ; Humans ; Perivascular Epithelioid Cell Neoplasms/surgery* ; Stomach

To clarify the function and molecular mechanism of miR-155 in myogenic differentiation of C2C12, we constructed adenovirus over-expression vector of miR-155, then C2C12 cells were infected by adenovirus and induced myogenic differentiation. First, we observed the morphology of C2C12 after differentiation. Then the mRNA and protein expressions of myogenic markers (MyoD, MyoG and MyHC) were detected by qPCR and western blotting. Subsequently, the dual luciferase reporter gene assay was carried out to validate putative target gene (TCF4) of miR-155. Meanwhile, mRNA level of TCF4 was analyzed after over-expressing miR-155. The results show that over-expressed miR-155 reduced myotubes formation. Moreover, the mRNA and protein expression of MyoG and MyHC decreased significantly (P < 0.01). Further research demonstrated miR-155 bound the one (4532-4538) of three putative sites (1487-1493,1516-1522, 4532-4583) of TCF4 by luciferase reporter gene assay and the mRNA level of TCF4 decreased notably (P < 0.05). The data suggest that miR-155 inhibited myogenic differentiation of C2C12 through targeted TCF4.
Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; Cell Differentiation ; Cell Line ; Genetic Vectors ; Mice ; MicroRNAs ; genetics ; Myoblasts ; cytology ; Myogenin ; genetics ; metabolism ; Myosin Heavy Chains ; genetics ; metabolism ; RNA, Messenger ; genetics ; Transcription Factor 4

Hypoxia-inducible factors (HIFs) are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. This study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer. The data showed that HIFs were over-expressed in gastric cancer and that activation of the target genes was observed mainly in the early stages. Moreover, the results of the present study revealed that only HIF-1alpha, but not HIF-2alpha dimerizes with HIF-1beta and then regulates expression of target genes in response to hypoxia. The results of the present study demonstrate that HIF-1alpha and HIF-1beta enhances expression of VEGF and glucose metabolism-related genes in response to hypoxia in gastric cancer. These data offer important information regarding HIF pathways in the development of gastric cancer.
Aryl Hydrocarbon Receptor Nuclear Translocator/*genetics/metabolism ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Glucose/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Neovascularization, Pathologic/genetics/metabolism ; Stomach Neoplasms/*genetics/metabolism ; Vascular Endothelial Growth Factor A/*genetics/metabolism

The expression levels of hypoxia-inducible factor 1alpha (HIF-1&alpha;) and HIF-2&alpha; in pancreatic cancer (PC) and their association with clinicopathologic characteristics were investigated in order to elucidate their roles in the development of PC. HIF-1&alpha; and HIF-2&alpha; mRNA levels in 20 patients with PC were detected by quantitative real-time polymerase chain reaction. The expression of HIF-1&alpha; and HIF-2&alpha; protein in samples from other 90 patients with PC was measured by immunohistochemistry. Correlations between the expression of HIF-1&alpha; or HIF-2&alpha; and clinicopathologica features and prognosis were analyzed. The expression of both HIF-1&alpha; and HIF-2&alpha; mRNA was up-regulated in most cancer tissues (P<0.05). HIF-1&alpha; staining was weakly positive in most cancer tissues and strongly positive in adjacent pancreas tissues (P<0.05). Clinicopathologic analysis revealed that relatively strong HIF-1&alpha; expression in cancer tissues was related to greater invasion (P<0.05), higher tumor pathologic stage (P<0.05), higher American Joint Committee on Cancer (AJCC) stage (P<0.05) and shorter overall survival time (P<0.05). Conversely, HIF-2&alpha; staining was strongly positive in most cancer tissues and weakly positive in adjacent pancreas tissues. Clinicopathologic analysis revealed that relatively strong HIF-2&alpha; expression in cancer tissues was related to less invasion (P<0.05), lower tumor pathologic stage (P<0.05), lower AJCC stage (P<0.05) and longer overall survival time (P<0.05). Moreover, the HIF-1&alpha;(high)/HIF-2&alpha;(low) group showed a shorter survival time than the HIF-1&alpha;(low)/HIF-2&alpha;(high) group. In conclusion, although HIF-1&alpha; and HIF-2&alpha; mRNA expression patterns are the same, their protein expression patterns are significantly different and they play different roles in PC. Combined analysis of HIF-1&alpha; and HIF-2&alpha; expression might be useful to predict the prognosis of patients with PC.
Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Prognosis ; RNA, Messenger ; genetics

Objective: To investigate the clinicopathological features of pulmonary granular cell tumors (pGCTs) and to improve the diagnostic accuracy of the tumor. Methods: A total of 5 pGCTs were diagnosed from February 2016 to January 2022 at Shanghai Pulmonary Hospital, Tongji University School of Medicine and Fudan University Shanghai Cancer Center, China. Immunohistochemical staining, and analysis of the clinicopathological characteristics were performed. Results: The average age of the pGCTs patients was 46 years (ranging from 24 to 54 years), with 3 females and 2 males. One case occurred in the bronchus with multiple nodules in the lung, 2 cases occurred in the bronchial opening, and 2 cases were solitary nodules in the lung. The maximum diameter of the tumors ranged from 12 to 15 mm (mean size 14 mm). Microscopically, the tumor showed infiltrative growth and consisted of round, oval or polygonal cells. Abundant eosinophilic cytoplasm was noted, and the nucleoli were prominent. None of the 5 cases showed any mitosis or necrosis. Immunohistochemical and histochemical study showed positive staining for S-100 (5/5), SOX10 (5/5), Vimentin (5/5), TFE3 (4/5), PAS (3/5), and amylase-digested-PAS (3/5), while 4 cases were negative for CD68. TFE3 FISH analyses on 2 cases showed that no signal abnormality was detected in these 2 cases. The average proliferation index of Ki-67 was 2.2% (range 0-5%). There was no recurrence in 4 cases of pGCTs with a follow-up time ranging from 2 months to 60 months. Conclusions: pGCTs are very rare tumors, most likely originating from Schwann cells. Immunohistochemical staining is the conventional diagnostic tool for pGCTs diagnosis. Recognition of this entity is essential for pathologists to avoid misdiagnosis and unnecessary treatments.
Female ; Humans ; Male ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; Biomarkers, Tumor ; Bronchi ; China ; Granular Cell Tumor/surgery* ; Lung ; S100 Proteins ; Adult ; Middle Aged

Objective: To investigate the clinicopathological features of perivascular epithelioid cell tumor (PEComa) of the lung. Methods: Eight PEComa cases of the lung diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China from July 2008 to December 2021 were collected and subject to immunohistochemical staining, fluorescence in situ hybridization and next generation sequencing. The relevant literature was reviewed and the clinicopathological features were analyzed. Results: There were 5 males and 3 females, aged from 18 to 70 years (mean 39 years). There were 3 cases of the right upper lung, 3 cases of the left lower lung, 1 case of the left upper lung and 1 case of the right middle lung. Seven cases were solitary and 1 case was multifocal (4 lesions). Seven cases were benign while one was malignant. The tumors were all located in the peripheral part of the lung, with a maximum diameter of 0.2-4.0 cm. Grossly, they were oval and well circumscribed. Microscopically, the tumor cells were oval, short spindle-shaped, arranged in solid nests, acinar or hemangiopericytoma-like patterns, with clear or eosinophilic cytoplasm. The stroma was rich in blood vessels with hyalinization. Coagulated necrosis and high-grade nuclei were seen in the malignant case, and calcification was seen in 2 cases. Immunohistochemically, the tumor cells were positive for Melan A (8/8), HMB45 (7/8), CD34 (6/8), TFE3 (4/7), and SMA (3/8). All cases were negative for CKpan and S-100. TFE3 (Xp11.2) gene fusion was examined using the TFE3 break-apart fluorescence in situ hybridization in 5 cases, in which only the malignant case was positive. The next generation sequencing revealed the SFPQ-TFE3 [t(X;1)(p11.2;p34)] fusion. Follow-up of the patients ranged from 12 to 173 months while one patient was lost to the follow-up. The malignant case had tumor metastasis to the brain 4 years after the operation and then received radiotherapy. Other 6 cases had no recurrence and metastasis, and all the 7 patients survived. Conclusions: Most of the PEComas of the lung are benign. When there are malignant morphological features such as necrosis, high-grade nuclei or SFPQ-TFE3 gene fusion, close follow-up seems necessary.
Male ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Perivascular Epithelioid Cell Neoplasms/pathology* ; Lung/pathology* ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics* ; Necrosis ; Biomarkers, Tumor/analysis*

Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; metabolism ; Carcinoma, Renal Cell ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Humans ; Melanoma ; genetics ; metabolism ; Microphthalmia-Associated Transcription Factor ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Perivascular Epithelioid Cell Neoplasms ; genetics ; metabolism ; Sarcoma, Clear Cell ; genetics ; metabolism ; Translocation, Genetic

OBJECTIVETo investigate the expression and mutation of Mad1, Mxi1 and Rox genes in leukemia cells.

METHODSExpression and mutation of Mad1, Mxi1 and Rox genes in bone marrow mononuclear cells (BMMNC) from 26 de novo acute leukemia (AL) patients, and in peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, as well as in 7 human leukemic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.

RESULTSRT-PCR showed that all the above cells expressed Mad1, Mxi1 and Rox mRNA. SSCP revealed four polymorphisms: two in Mad1, one each in Mxi1 and Rox. DNA sequencing detected nine missense mutations: two in Mad1 in AL patients, four in Mxi1 (three in AL patients and one in KG-1 cell line), and three in Rox in AL patients. The mutations of Mad1, Mxi1 and Rox mRNA were detected in 2, 3 and 3 patients, respectively.

CONCLUSIONIt is for the first time to demonstrate the mutations of Mad1, Mxi1 and Rox genes in AL patients suggesting these mutated genes involve in the pathogenesis of leukemia.

Adolescent ; Adult ; Aged ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Female ; Humans ; Leukemia ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; metabolism ; Polymorphism, Single-Stranded Conformational ; Repressor Proteins ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism