A Preliminary Study Of MiR-378a-3p Affecting Energy Metabolism And Apoptosis Of Eca-109 Cells Through Intervention Of Key Enzymes Of Warburg Effect

Objective: To investigate the relationship between microRNA-378a-3p(miR-378a-3p)and key glycolytic enzymes,and the effect of miR-378a-3p on apoptosis and energy metabolism of esophageal squamous carcinoma cell Eca-109.Methods: Eca-109 esophageal squamous carcinoma cell line was used to divide into blank control group,negative control group and experimental group(24h and 48 h groups after miR-378a-3p gene transfection)The specific method is as follows:1)Using GV272 as a vector and using PCR technology to amplify PKM in vitro to construct a GV272-PKM overexpression vector;2)Detection of changes in protein expression levels of GAPDH,PGK-1,ENO1,PKM2,LDHA,GLUT1,HK2,PFKL,ALDO and other proteins after miR-378a-3p intervention by Western blotting;3)The effect of miR-378a-3p transfection group 24 h and 48 h on ALDOA,GLUT1,HK2,PFKL,GAPDH,PGK-1,ENO1,PKM2,LDHA and other key glycolytic enzyme activities was verified by ELISA;4)Verify the downstream target genes of miR-378a-3p by detecting luciferase activity;5)The effect of miR-378a-3p expression on energy metabolism(ATP production)was detected by ultraviolet spectrophotometry;6)The effect of miR-378a-3p expression on apoptosis of esophageal squamous carcinoma cells.Results: 1)The GV272-PKM overexpression vector was successfully constructed,and the sequencing results indicated that the sequence construction was consistent with the theoretical prediction;2)Western blot results showed that compared with the blank control group and the negative control group,the expression levels of HK2,ALDOA,PKM2 and LDHA protein decreased at 24 h and48h after transfection of miR-378a-3p(P <0.05)The expression level of GAPDH protein in the miR-378a-3p group at 24 h was significantly reduced(P <0.01);3)The results of enzyme-linked immunosorbent assay showed that compared with the blank control group,esophageal squamous cell carcinoma cells Eca-109 were inhibited against GLUT1,HK2,ENO1,PKM2,GAPDH,LDHA,PFKL and other protein activities at 24 h after transfection,The activity of HK2,GLUT1,ENO1 protein was obviously inhibited,and the enzyme activity decreased(P <0.001);4)When GLUT-1,PKM2,and ALDO gene wild-type were co-transfected with miR-378a-3p,dual luciferase reporter gene activity was significantly inhibited,suggesting that GLUT-1,PKM2,and ALDO may be miR-378a-3p Downstream target genes;5)After transfection of miR-378a-3p,the results of ultraviolet spectrophotometry showed that: ATP content was significantly reduced,and metabolism was reduced(P <0.01);6)Compared with the blank control group and the negative control group,the miR-378a-3p transfection group significantly increased the apoptosis rate of esophageal squamous cell carcinoma Eca-109 at 24 h and 48h(P <0.001).Conclusion:The GLUT-1,PKM2,and ALDO genes were identified as the target genes of miR-378a-3p through verification.miR-378a-3p blocks the energy metabolism of esophageal cancer cells by reducing the expression of key glycolytic enzymes,thereby increasing cell apoptosis.