The Effect Of Let-7b And Mir-33a On A375Melanoma Cell Line Proliferation Aerobic Glycolysis And Genome Expression Profile

Objective:Determine the effect of let-7b and miR-33a on melanoma cell proliferation and aerobic glycolysis, screen genome expression profile and analyse the possible mechanism of these effect of let-7b and miR-33a.Method’.Analyse the expression level of let-7b and miR-33a in A375melanoma cell line and melanocytic nevus through real-time PCR; Interfere the expreesion level of let-7b and miR-33a in melanoma cell line A375by transfecting cell with microRNA mimics, the final expression is determined by real-time PCR.24h and48h after transinfection, determine the cell proliferation through MTT test, evaluate glycolytic rate through glucose comsumption and lactate production test. Extract RNA at24h after transfection and screen the genome expression profile by Affymetrix Human Genome U133plus2.0Array and analyse data by bioinformation method.Result:1.A375cell line has low expression of let-7b or miR-33a compared to melanocytic nevus.(p<0.05)2. At24h and48h after transfection, MTT test shows absorbance decreased in let-7b or miR-33a overexpression group (p<0.05), the result indicate that cell proliferation was inhibited.3. Glucose consumption and lactate production decreased after let-7b overexpressed within24h(p<0.05), but the ratio difference was not observed. Glucose consumption and lactate production and the ratio decreased within24h after miR-33a overexpressed (p<0.05). Glucose and lactate tested again at48h after transfection, Glucose consumption and lactate production and the ratio notablely decreased in miR-33a overexpression group(p<0.05), miR-33a overexpression inhibit glycolytic rate4. Genome array screening shows that let-7b overexpression result in255gene down regulated and72gene upregulated, miR-33a overexpression result in84gene down regulated and 126gene upregulated. Bioinformation analysis indicated the differentially expressed gene involve in a quality of signal transduction pathway and gene transcription, cell proliferation, apoptosis.Conclusion:1.Let-7b and miR-33a are low expressed in melanoma cell.2. Let-7b or miR-33a overexpression could inhibit cell proliferation3. MiR-33a overexpression inhibit aerobic glycolysis of melanoma cell line A375.4.let-7b/miR-33a overexpression result in genome express profile shift, bioinformation analyse shows gene transcription, apotosis, cell cycle arrest were mostly impact, while MAPK et al. were most involed signal transduction pathway.