We previously reported the roles of microRNA (miR)-145 as a tumor-suppressor in human bladder cancer (BC) cells. On the other hand, the Warburg effect is a well-known feature in cancer specific metabolism. The functions of miR-145 in the Warburg effect are still largely unknown. Kruppel-like factor 4 (KLF4) is a transcription factors and is well known as a one of the essential factors of induced pluripotent stem cell. Also, it is possible that KLF4 also regulates the Warburg effect. In this study, we revealed that miR-145 affects the regulation of the Warburg effect through targeting KLF4 in BC cells.

METHODS

The expression levels of miR-145 between normal human urothelial cells (HUC), BC cells, and clinical BC samples were examined by a real-time PCR. Human BC T24 and 253 J-BV cells were transfected with miR-145, and the effects were examined by various experiments such as Western blotting analysis(WB), Hoechst33342 staining and immunostaining. The evaluation of targeting KLF4 by miR-145 was performed by luciferase assay. Moreover, the effects of knockdown of KLF4 on the various phenotypes of BC cells were also examined. Furthermore, the networks involving KLF4/PTBP1/PKMs in the Warburg effect related- molecules were examined by WB even in clinical BC samples. Finally, we examined immunohistochemical staining to evaluate KLF4 expression.

RESULTS

The expression levels of miR-145 were significantly down-regulated in clinical BC samples and BC cells compared with those in normal tissues and HUC. Luciferase assay showed that miR-145 directly targeted KLF4. Also, the Warburg related affect-genes were modulated by the transfection of miR-145 or siR-KLF4 in BC cells. Moreover, the expression levels of KLF4, PTBP1, and PKM2 is up-regulated in all clinical samples by WB. Finally, we observed KLF4-positive staining in the tumor by immunohistochemical staining.

CONCLUSIONS

In this study, we indicated that miR-145 affect the Warburg effect through targeting KLF4/PTBP1/PKMs axis in BC cells, which exhibited a significant cell growth inhibition.