RESUMO
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.
Assuntos
Humanos , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regulação para Cima , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Metiltransferases/metabolismo , Proteínas Culina/genéticaRESUMO
OBJECTIVE To investigate the inhibitory effect of berberine （BER） on the invasion and migration of human renal carcinoma cells and its potential mechanism. METHODS Using human renal carcinoma OSRC-2 cell as object， alamarBlue assay was adopted to detect the inhibitory effects of 0 （control group）， 25， 50， 75， 100， 125， 150， 175 and 200 μmol/L BER on the proliferation of OSRC-2 cell after treatment for 24 h and 48 h. After treated with 0（control group）， 50， 100 μmol/L BER for 48 h， the effect of BER on cell cycle was analyzed by flow cytometry. The migration of OSRC-2 cells in 24 h and 36 h was observed by cell scratch test， and the invasion ability of OSRC-2 cells in 24 h was detected by Transwell assay. The protein expression of methyltransferase-like 3 （METTL3） was detected by Western blot after treatment for 48 h， and RNA methylation quantification kit was used to detect the levels of N6-methyladenosine （m6A） in OSRC-2 cells. RESULTS Compared with control group， BER at different concentrations could significantly decrease the survival rate of OSRC-2 cells （P＜0.01）， and showed a dose-dependent and time-dependent manner. After 48 h of BER treatment at 50， 100 μmol/L， the cell was arrested in G0/G1 phase （P＜0.01）. Compared with control group， the migration and invasion abilities of cells in 50， 100 μmol/L BER group were significantly decreased （P＜0.05 or P＜0.01）； the protein expression of METTL3 and the level of m6A in RNA were significantly decreased （P＜0.01）. CONCLUSIONS BER can inhibit level of m6A by down-regulating the expression of METTL3， thereby inhibiting the invasion and metastasis of human renal carcinoma cells.
RESUMO
AIM: To investigate the role and mechanism of methyltransferase-like 3(METTL3)-mediated N6-methyladenosine(m6A)methylation modification in regulating biological activity of vascular endothelial cells in the pathogenesis of choroidal neovascularization.METHODS: Human umbilical vein endothelial cells(HUVEC)cultured in vitro were divided into the following groups: control group(normal culture), low density lipoprotein(LDL)group, fluorescence-labelled LDL(Dil-LDL)group, 12.5μg/mL and 25μg/mL oxidized LDL(ox-LDL)groups, 12.5μg/mL and 25μg/mL fluorescence-labelled ox-LDL(Dil-ox-LDL)groups, DMSO group, STM2457(METTL3 inhibitor)group, DAPT group; and monkey retina-choroidal endothelial cells(RF/6A)cultured in vitro were divided into control group, DMSO group, 12.5 μg/mL ox-LDL group, and DAPT group. Endocytosed lipoprotein level was examined through fluorescence microscopy. RNA m6A methylation level was detected through a dot blot assay. Protein and RNA levels of METTL3 or angiogenesis-related markers were measured through Western blot assays and real-time quantitative polymerase chain reaction(RT-qPCR), respectively. METTL3 expression and localization were investigated through immunofluorescence. Cell migratory and tube formation capacities were assessed through transwell migration and tube formation assays, respectively.RESULTS: Endocytosed lipoprotein levels in HUVECs exposed to Dil-LDL, 12.5μg/mL and 25μg/mL Dil-ox-LDL groups were significantly higher than those in the control group. 12.5μg/mL and 25μg/mL ox-LDL groups significantly increased m6A methylation(all P<0.05), METTL3 protein expression(all P<0.01), and cell migration and angiogenesis capacities(all P<0.01). METTL3 mRNA level was significantly unregulated in the 12.5μg/mL ox-LDL group(P<0.05). In comparison to the DMSO group, the addition of STM2457 caused significant decrease in m6A methylation level(P<0.05), expression of VEGF and other angiogenesis-related markers(all P<0.05), cell migration and angiogenesis capacities(all P<0.01)and the expression of NICD(P<0.05). However, there were no significant differences in METTL3 protein and mRNA levels(all P>0.05). The expression of VEGF and NICD(all P<0.05), as well as the ability of cell migration and angiogenesis of RF/6A, was all significantly decreased in the DAPT group compared to the DMSO group(all P<0.01).CONCLUSION: METTL3-mediated m6A methylation modification promotes angiogenesis in vascular endothelial cells via the Notch signaling pathway in the pathogenesis of choroidal neovascularization.
RESUMO
ObjectiveTo explore the effect and mechanism of Sishenwan-containing serum on aerobic glycolysis in human colon cancer HCT116 cells. MethodCell counting kit-8 （CCK-8） was used to detect the cell viability of colon cancer HCT116 cells after treatment with Sishenwan-containing serum （2.5%， 5%， and 10%） for 24， 48， 72 h. The concentration of lactic acid， the content of intracellular glucose， and the activity of hexokinase （HK） and fructose-6-phosphate kinase （PFK） in the cell culture medium were detected by the micro-method. The content of glucose transporter 1 （GluT1） mRNA was detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of GluT1 and methyltransferase-like 3 （MettL3） was detected by Western blot. The expression of GluT1 in cells was detected by immunofluorescence and the level of N6-methyladenosine （m6A） RNA methylation was detected by colorimetry. ResultCompared with the normal serum， 2.5%， 5%， and 10% Sishenwan-containing serum had no significant effect on the viability of HCT116 cells at 24 h， while 10% Sishenwan-containing serum showed a significant inhibitory effect on the viability of HCT116 cells at 48 h （P<0.05）. Hence， 10% Sishenwan-containing serum was used in subsequent experiments， and the intervention time was 48 h. Compared with the normal serum， 10% Sishenwan-containing serum could reduce lactate production （P<0.05）， down-regulate glucose uptake （P<0.05）， and blunt the activities of HK and PFK， the key rate-limiting enzymes of glycolysis （P<0.05）. Meanwhile， 10% Sishenwan-containing serum could decrease the expression of GluT1 protein （P<0.01） and mRNA （P<0.05） and reduce the proportion of cells expressing GluT1 （P<0.01）. Compared with the normal serum， Sishenwan-containing serum also decreased the protein content of MettL3 （P<0.05） and the methylation level of m6A RNA （P<0.01）. ConclusionSishenwan can inhibit glycolysis in colon cancer cells， and its inhibitory mechanism may be related to reducing MettL3 overexpression， inhibiting m6A RNA methylation， and down-regulating GluT1 and the activities of intracellular aerobic glycolysis-related enzymes such as HK and PFK.
RESUMO
Objective:To investigate the expression and prognostic value of glycolytic metabolism related methyltransferase-like 3 (METTL3) in resectable primary liver cancer.Methods:The METTL3 mRNA expression data, clinicopathological data and prognostic information of 364 patients with primary liver cancer and 50 healthy normal liver tissues were downloaded from the Cancer Genome Atlas (TCGA) database, and gene set enrichment analysis was performed. Then the tissue samples of 239 patients with primary liver cancer who underwent radical hepatectomy in Chongqing Armed Police Corps Hospital from January 2016 to may 2019 were selected. Among them, 110 cases contained matched normal liver tissue adjacent to the cancer. The expression of METTL3 protein was detected by immunohistochemical staining. Kaplan-Meier survival curve was drawn, and log-rank test was used for comparison; Cox proportional hazard model was used to evaluate the risk factors affecting the prognosis in patients with primary liver cancer.Results:TCGA database analysis result showed that the expression level of METTL3 mRNA in liver cancer tissue was significantly higher than that in normal liver tissue: 3.72 (3.19, 4.03) vs. 2.45 (2.11, 2.68), and there was statistical difference ( P = 0.007); Kaplan-Meier survival curve analysis result showed that the median overall survival time and disease-free survival time in patients with high expression of METTL3 mRNA were significantly shorter than those in patients with low expression of METTL3 mRNA (43.0 months vs. 72.0 months and 22.0 months vs. 38.0 months, HR = 1.7 and 1.4, P = 0.002 and 0.024). Gene set enrichment analysis result showed that METTL3 mRNA was related to impaired glucose metabolism. In the clinical sample study, the positive expression rate of METTL3 protein in liver cancer tissue was significantly higher than that in normal liver tissue adjacent to cancer: 54.55% (60/110) vs. 14.55% (16/110), and there was statistical difference ( χ2 = 38.92, P<0.01). Among 239 patients with primary liver cancer, 132 cases (55.23%) had positive expression of METTL3 protein in liver cancer tissue, and 107 cases had negative expression. Compared with METTL3 protein negative patients, METTL3 protein positive patients had higher body mass index; higher TNM stage, BCLC stage and tissue grade; more lesions; larger tumor sizes; more common satellite nodules; higher leukocyte count, platelet count and neutrophil count, and there were statistical differences ( P<0.05 or <0.01). Kaplan-Meier survival curve analysis result showed that the median overall survival time and disease-free survival time in patients with METTL3 protein positive were significantly shorter than those in patients with METTL3 negative (18.0 months vs. 38.0 months and 13.0 months vs. 27.0 months, P<0.01). Univariate and multivariate Cox analysis result showed that METTL3 protein positive in liver cancer tissue was an independent risk factor affecting overall survival time and disease-free survival time ( HR = 1.840 and 2.096, 95% CI 1.298 to 2.608 and 1.469 to 2.991, P<0.01). Conclusions:METTL3 is involved in glycolytic metabolism of primary liver cancer, and can be used as a promising biomarker to evaluate the prognosis of patients with primary liver cancer.
RESUMO
Objective:To explore the changes of mRNA N6-methyladenosine methylation level and methyltransferase-like 3 (METTL3) and demethylase fat mass and obesity-associated (FTO) in the blood of patients with Alzheimer's disease (AD) compared with normal controls.Methods:From January 2020 to June 2021, totally 40 AD patients treated in the outpatient and inpatient department of Neurology of the Affiliated Hospital of Jining Medical University were selected as the patient group, and 40 healthy volunteers as the control group. The blood samples were collected to extract plasma and peripheral blood mononuclear cells for enzyme-linked immunosorbent assay (ELISA), Western blot (WB), quantitative real-time PCR (qPCR) and m6A methylation quantification experiments respectively to detect the methylation levels of METTL3, FTO and m6A. The data were analyzed by SPSS 23.0 statistical software for t-test. Results:The plasma concentrations of METTL3 and FTO protein in AD group were lower than those in control group (METTL3: (22.33±3.01)ng/mL, (25.63±1.70)ng/mL, t=6.055, P<0.01; FTO: (63.51±4.95)pg/mL, (69.60±4.60)pg/mL, ( t=5.704, P<0.01). The band gray values of METTL3 and FTO protein in blood cells in AD group were lower than those in control group (METTL3: 0.399 5±0.028 7, 0.676 6±0.053 3, t=7.935, P=0.001; FTO: 0.439 4±0.017 8, 0.782 6±0.087 6, t=6.652, P=0.003). The expression levels of METTL3 and FTO in blood cell RNA in AD group were lower than those in control group (METTL3: 0.387 8±0.020 3, 1.010 0±0.177 0, t=6.041, P=0.004; FTO: 0.442 8±0.037 1, 1.003 0±0.090 4, t=9.931, P=0.001). The levels of m6A in blood cell RNA in AD group were lower than those in control group((0.000 571±0.000 167)%, (0.002 514±0.001 284)%, t=6.041, P=0.004). Conclusion:The levels of METL3, FTO and m6A methylation are down-regulated in the plasma and peripheral blood mononuclear cells of patients with AD, indicating that there is a certain association between mRNA N6-methyladenosine methylation and AD.