Fig 2: The expression of IGF1R and mTOR in HCC cell lines and tissues were inversely correlated with the levels of miR-99a and miR-497(A) The protein levels of IGF1R and mTOR in HepG2 cell line were higher than them in liver normal cell lines (L-02). (B) Representative immunohistochemical staining for IGF1R and mTOR from HCC tissues and non-tumourous liver tissues. Upper: IGF1R immunostaining; Lower: mTOR immunostaining (×200). (C) The immunohistochemistry(IHC) scores of IGF1R and mTOR in HCC tissues were significantly higher than them of matched non-cancerous liver tissues. *P < 0.05, **P<0.005, compared to control. And correlation analyses were performed between IHC scores of IGF1R/mTOR and the mRNA levels of miR-99a/miR-497 expression in HCC tissues (n = 30).

Fig 3: Up-regulation of miR-99a and miR-497 inhibited liver cancer cell proliferation, delayed cell cycle progression, and promoted apoptosis in liver cancer cell, which were reversed by up-regulating IGF1R and mTOR(A) Ectopic expression of miR-99a and miR-497 suppressed HegG2 and Hep3B cell lines proliferation at 48h and 72h. And these anti-growth effects would be attenuated after co-transfection with antisense miR-99a and miR-497 (miR-99a-in and miR-497-in). (B) After transfection of miR-99a and miR-497 mimics or co-transfection with miR-99a-in and miR-497-in to HCC cells, the DNA content of PI-stained cells were analyzed by flow-cytometry. Cell-cycle distribution of G0/G1 phase presented here was in HepG2 cells. (C) The stained cell number ratios of HepG2 and Hep3B cell lines are presented in bar graph. (D) Evaluation of apoptosis by annexin V-PITC (AV) and propidium iodide (PI) staining and analysis by flow-cytometry in HepG2 and Hep3B cells after transfection. The apoptotic cell number are counted in bar graph. (E) Validation of drug-mediated IGF1R and mTOR activating in HepG2 cells. The relative IFG1R and mTOR mRNA levels, as determined by real-time qPCR, are expressed as fold changes after normalization to the internal control (β-actin). (F) Cell growth was monitored in HepG2 cells at 48h using the CCK-8 assay. NC: negative control microRNA. *P<0.05, **P<0.005, ***P<0.001, compared to NC. #P<0.05, compared to transfection with miR-99a or miR-497.

Fig 4: Synergistic effects of miR-497 and miR-99a on retarding HCC cell proliferation, which related with synergistic co-targeting on IGF1R and mTOR(A) Co-expression of miR-99a and miR-497 exerted stronger suppression on HegG2 and Hep3B cell proliferation than that of miR-497 or miR-99a alone at 48h and/or 72h. (B) Evaluation of apoptosis by flow-cytometry in HepG2 and Hep3B cells transfected with of miR-99a and/or miR-497 (apoptotic cell distribution phase presented here was in HepG2 cells). The apoptotic cell number are counted in bar graph. (C) The levels of IGF1R and mTOR mRNAs analyzed by qRT-PCR, GAPDH was used as control. (D) The protein levels of IGF1R and mTOR were detected by Western blot assay,ß-actin was used as control. The results were reproducible in three independent experiments. NC: negative control microRNA. *P<0.05, **P<0.005, ***P<0.001, compared to NC. #P<0.05, ##P<0.005, compared to co-transfection with miR-99a and miR-497.

Fig 5: MiR-99a and miR-497 repressed the growth of HepG2-engrafted tumors and decreased the expression of IGF1R and mTOR in vivo(A) Engrafted tumors from scrambled and miR-mimics-treated mice groups were dissected and photographed at the day 25 after transplantation. (B) Tumor volumes at the indicated days during the experiment for the groups. (C) Tumor weights averages between scrambled and miR-mimics-treated mice groups at the day 25 after engraftment. (D) Western blotting analyses of the protein levels of IGF1R and mTOR in HepG2-engrafted tumors. ß-actin was used as control. The results were reproducible in three independent experiments. NC: negative control microRNA. **P<0.005, ***P<0.001, compared to NC. #P<0.05, ##P<0.005, ###P<0.001, compared to co-transfection with miR-99a and miR-497.