BACKGROUND: Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer. METHODS: Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot. RESULTS: Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05). CONCLUSIONS LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.
Humans ; Lung Neoplasms/pathology* ; RNA, Long Noncoding/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Up-Regulation ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Methyltransferases/metabolism* ; Cullin Proteins/genetics*

OBJECTIVE: To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism. METHODS: GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway. RESULTS: The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01). CONCLUSION PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.
Humans ; Animals ; Mice ; Up-Regulation ; beta Catenin/metabolism* ; Mice, Nude ; Brain Neoplasms/pathology* ; Lactic Acid ; Cell Line, Tumor ; Glioma/pathology* ; Cell Proliferation ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/genetics* ; Gene Expression Regulation, Neoplastic

The thalamocortical (TC) circuit is closely associated with pain processing. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 channel is predominantly expressed in the ventral posterolateral thalamus (VPL) that has been shown to mediate neuropathic pain. However, the role of VPL HCN2 in modulating TC circuit activity is largely unknown. Here, by using optogenetics, neuronal tracing, electrophysiological recordings, and virus knockdown strategies, we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex (S1HL) as well as mechanical hypersensitivity following spared nerve injury (SNI)-induced neuropathic pain in mice. Either pharmacological blockade or virus knockdown of HCN2 (shRNA-Hcn2) in the VPL was sufficient to alleviate SNI-induced hyperalgesia. Moreover, shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit. Together, our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.
Animals ; Mice ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics* ; Neuralgia ; RNA, Small Interfering ; Thalamus/metabolism* ; Up-Regulation

OBJECTIVES: The pathogenesis of androgenetic alopecia (AGA) is related to the level of androgen and its metabolic pathways. The binding of androgen and androgen receptor (AR) depends on the assistance of heat shock protein 27 (HSP27). HSP27 combined with microRNAs (miR)-1 can regulate AR levels. However, it is not clear whether HSP27 and miR-1 jointly participate in the pathogenesis of AGA. This study aims to investigate the role of AR up-regulation in the pathogenesis of AGA and underlying mechanisms. METHODS: A total of 46 male AGA patients (AGA group), who admitted to the First Affiliated Hospital of Guangzhou Medical University from September 2019 to February 2020, and 52 healthy controls admitted to the same period were enrolled in this study. Serum levels of dihydrotestosterone (DHT) and HSP27 in patients and healthy controls were measured by ELISA. Western blotting was used to detect the protein expression of HSP27 and AR in scalp tissues of patients and the healthy controls. The levels of HSP27, AR, and miR-1 were analyzed using real-time PCR. Human dermal papilla cells were transfected with HSP27 siRNA to inhibit the expression of HSP27. MiR-1 and miR-1 inhibitors were transfected simultaneously or separately into cells and then the changes in AR protein expression were detected. RESULTS: The levels of DHT and HSP27 in the AGA group were (361.4±187.7) pg/mL and (89.4±21.8) ng/mL, respectively, which were higher than those in the control group [(281.8±176.6) pg/mL and (41.2±13.7) ng/mL, both P<0.05]. However, there was no significant difference in serum HSP27 and AR levels among AGA patients with different degrees of hair loss (P>0.05). Correlation analysis showed that there was a positive correlation between HSP27 level and DHT level in the AGA patients (P<0.05). The level of HSP27 mRNA in scalp tissue was negatively correlated with that of miR-1 mRNA (P<0.05). Compared with the control group, the levels of HSP27 protein, AR protein, HSP27 mRNA, and AR mRNA in scalp tissues of AGA group were significantly increased (P<0.05). The up-regulation of HSP27 in scalp tissues of AGA patients was closely related to the increased levels of AR. However, the level of miR-1 in scalp tissues of AGA patients was significantly down-regulated, contrary to the expression of AR (P<0.05). Further in cell studies showed that inhibition of HSP27 or miR-1 expression in human dermal papilla cells could inhibit the expression of AR, and inhibition of both HSP27 and miR-1 expression was found to have an accumulative effect on AR, with statistically significant differences (all P<0.05). CONCLUSIONS HSP27 could combine with miR-1 to up-regulate AR levels, which is closely related to the development of AGA.
Alopecia/pathology* ; HSP27 Heat-Shock Proteins/metabolism* ; Humans ; Male ; MicroRNAs/genetics* ; RNA, Messenger ; Receptors, Androgen/metabolism* ; Up-Regulation

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

OBJECTIVE: To analyze the changes in the gene expression profile of T cells in CML patients after TCRζ up-regulation expression, and to explore the molecular mechanism of T cell reactivation after transgenic up-regulation of TCRζ. METHODS: The peripheral blood mononuclear cells(PBMCs) from 3 newly untreated chronic-stage CML patients were collected, and the CD3 RESULTS: A total of 2248 differentially-expressed genes were obtained, including 553 up-regulated genes and 1695 down-regulated genes in experimental group as compared with those in control group (P<0.05) . The GO and KEGG enrichment analyses showed that differentially expressed genes involved in the biological processes related to T cell immune function, such as TCR signaling pathway, T cell proliferation and activation. Some of core genes involved in promoting the TCR signaling pathway, T cell proliferation, activation and apoptosis pathways were significantly up-regulated, while some core genes involved in inhibiting T cell activation were significantly down-regulated. CONCLUSION The molecular mechanism of the significantly improved T cell activation and proliferation ability in CML patients after TCRζ up-regulation may be related to the differential transcripts mediated signaling pathways of T cell activation, proliferation and apoptosis.
Humans ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Receptors, Antigen, T-Cell/genetics* ; T-Lymphocytes ; Up-Regulation

OBJECTIVES: To analyze the expressions and distributions of hypoxia-inducible factor-1α (HIF-1α), CD147, and glucose transporter 1 (GLUT1) in epidermis from psoriasis vulgaris and normal people, and to explore the associations among these proteins and their roles in hypoxic HaCaT cell line. METHODS: The expression levels of HIF-1α, CD147, and GLUT1 were determined by immunohistochemistry staining in skin biopsies from 48 psoriasis vularis patients and 33 healthy subjects. Cobalt chloride (CoCl RESULTS: HIF-1α, CD147, and GLUT1 were highly expressed and the glycolytic capacity was increased in lesions of psoriasis vulgaris; HIF-1α upregulated the expression of CD147 and GLUT1, increased the lactate production and decreased the ATP level in CoCl CONCLUSIONS Glycolytic capacity increases in the injured keratinocytes of psoriasis vulgaris, suggesting that HIF-1α, CD147, and GLUT1 are associated with glycolysis, which can be considered as the promising targets for psoriasis therapy.
Basigin ; Glucose Transporter Type 1 ; Glycolysis ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Psoriasis/genetics* ; Transcriptional Activation ; Up-Regulation

BACKGROUND: Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells. METHODS: In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance. RESULTS: MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells. CONCLUSIONS MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.
Adenocarcinoma of Lung/genetics* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics* ; MicroRNAs/genetics* ; Up-Regulation/genetics* ; rho-Associated Kinases/genetics* ; rhoA GTP-Binding Protein/genetics*

OBJECTIVE: To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats. METHODS: Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting. RESULTS: BYHWD treatment significantly promoted the recovery of neurological function ( CONCLUSIONS Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.
Animals ; Brain Ischemia/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Ischemic Stroke/drug therapy* ; Male ; MicroRNAs/genetics* ; Neurogenesis/drug effects* ; Rats ; Rats, Sprague-Dawley ; Up-Regulation/drug effects* ; Vascular Endothelial Growth Factor A/genetics*

OBJECTIVE: To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC，KLF4，LIN28，NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future. METHODS: Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4（hBMMSCs-OCT4）were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs. RESULTS: Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426). CONCLUSION Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2.
Bone Marrow ; Humans ; Induced Pluripotent Stem Cells ; Mesenchymal Stem Cells ; Nanog Homeobox Protein ; genetics ; Octamer Transcription Factor-3 ; genetics ; Transcription Factors ; Up-Regulation