We transfected miR- 148a��Cexpressing HepG2 cells with HPIP or HPIP siRNA. As anticipated, HPIP reexpression in miR-148a-HepG2 cells reversed the inhibition of AKT and ERK mediated by miR- 148a , and HPIP knockdown abolished the ability of miR-148a to repress AKT and ERK . The knockdown results might be rescued by siRNA-resistant HPIP expression. Moreover, HPIP knockdown had related results to miR-148a overexpression on regulation of AKT and ERK . These information recommend that miR-148a represses AKT and ERK with the inhibition of HPIP. miR-148a suppresses the mTOR pathway by inhibition of HPIP/ AKT and HPIP/ERK pathways. Given that AKT and ERK can activate the mTOR pathway and miR-148a represses activation of AKT and ERK, we made a decision to investigate whether miR-148a represses the mTOR pathway. Western blot analysis showed that, consistent using the benefits of miR-148a inhibition of AKT and ERK phosphorylation, miR-148a overexpression in HepG2 cells decreased the ranges of total mTOR and phosphorylation of mTOR and phosphorylation of S6K1 and 4E-BP1, 2 mTOR kinase targets, also since the mTOR downstream effectors c-myc and cyclin D1, whereas knockdown of miR-148a with miR-148a inhibitor had opposite effects .
Upcoming, we determined if miR-148a inhibition within the mTOR pathway was as a result of the OSI-930 inhibition of HPIP. We transfected miR- 148a-HepG2 cells with HPIP or HPIP siRNA. Certainly, HPIP reexpression in miR-148a-HepG2 cells reversed the inhibition from the mTOR pathway mediated by miR-148a , and HPIP knockdown abolished the means of miR-148a to suppress the mTOR pathway . The knockdown results may very well be rescued by siRNA-resistant HPIP expression. Additionally, HPIP knockdown had very similar results to miR-148a overexpression about the regulation with the mTOR pathway . These outcomes indicate that miR-148a suppresses the mTOR pathway with the inhibition of HPIP.
To more identify if miR-148a represses the mTOR pathway as a result of inhibition of HPIP-mediated activation of ERK, AKT, and mTOR, we taken care of HPIP-transfected continue reading this} HepG2 cells with PD98059, LY294002, and rapamycin, which are MAPK/ ERK1/2, PI3K/AKT, and mTOR pathway inhibitors, respectively. Intriguingly, inhibition of ERK1/2, AKT, and mTOR by PD98059, LY294002, and rapamycin, respectively, abolished the skill of HPIP to activate ERK, AKT, and mTOR also as mTOR targets . Furthermore, AKT or ERK reexpression in miR-148a-HepG2 cells reversed the inhibition within the mTOR pathway mediated by miR-148a , plus the inhibition of AKT and ERK by LY294002 and PD98059 abolished the ability of miR-148a to repress mTOR signaling .
It has to be mentioned that PD98059, LY294002, and rapamycin at rather high concentrations inhibited the expression of complete mTOR, but very low concentrations of PD98059, LY294002, and rapamycin didn’t . Taken with each other, our information suggest that miR- 148a represses the mTOR pathway by way of inhibition of HPIPmediated activation of ERK and AKT. mTOR exists in two distinct complexes: mTORC1 and mTORC2 .