ABSTRACT
@#Objective To investigate the expression of methyltransferase like-3(METTL3)in esophageal cancer tissues and its effects on the proliferation,migration,apoptosis and glutamine metabolism of cells.Methods The expression of METTL3 in esophageal cancer and paraneoplastic tissues was detected by qRT-PCR,Western blot and Immunohistochemistry. The effects of METTL3 inhibitor STM2457 on cell proliferation and migration of human esophageal squamous carcinoma cell lines Eca109 and KYSE150 were detected by CCK8,cloning assay,scratch test and Transwell assay. The effects on cell cycle and cell apoptosis were measured by flow cytometry and TUNEL experiment. TMT/iTRAQ quantitative proteomics experiment was used to identify the effects on downstream related signaling pathways. Glutamine assay,glutamate assay and Western blot were used to analyze the effect on glutamine metabolism in esophageal cancer cells.Results METTL3 gene expression was significantly upregulated in esophageal cancer tissues(t = 5. 024,P < 0. 000 1). STM2457 inhibited METTL3 expression in Eca109 and KYSE150 cells,significantly inhibited the cell proliferation and migration,blocked the cell cycle in G0/G1 phase,increased the cell apoptosis,decreased the glutamine uptake and glutamate production,and down-regulated the expression of glutamine-related proteins alanine-serine-cysteine transporter 2(ASCT2),glutaminase(GLS)and glutamate dehydrogenase 1(GLUD1). The glutamine uptake and glutamate production in Eca109 and KYSE150 cells decreased significantly after METTL3 knockdown(glutamine uptake:t = 24. 52-41. 01,each P < 0. 01;glutamate production:t = 8. 431-11. 83,each P < 0. 01);After METTL3 overexpression,the glutamine uptake and glutamate production in Eca109 and KYSE150 cells increased significantly(glutamine uptake:t = 5. 803 and 56. 13,respectively,each P < 0. 01;glutamate production:t = 11. 06 and 4. 695,respectively,each P < 0. 01). After METTL3 knockdown,the expression levels of glutamine metabolism related proteins ASCT2,GLS and GLUD1 in Eca109 and KYSE150 cells were significantly down-regulated,while after METTL3 overexpression,the expression levels of ASCT2,GLS and GLUD1 were significantly up-regulated.Conclusion METTL3 is highly expressed in esophageal cancer and may promote cell proliferation by mediating glutamine metabolism in esophageal cancer cells.
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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.
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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.