let-7b and let-7c are determinants of intrinsic chemoresistance in renal cell carcinoma

Abstract

Background: Renal cell carcinoma (RCC) is characterized by inherent resistance to chemotherapy. Earlier studies demonstrated that microRNAs (miRNAs) might be involved in the chemosensitivity of cancers. MicroRNA let-7, a putative tumor suppressor, is dysregulated in many cancers. Our study aims to investigate the exact role of let-7 in chemotherapy sensitivity of 5-fluorouracil (5-FU) in RCC.

Methods: The clinical significance of let-7b and let-7c expression in surgically resected specimens was assessed by qRT-PCR. Cell proliferation assay and colony formation assay were used to assess the survival of 786-O cells treated with let-7b or let-7c combined with 5-FU. Western blot was used to detect the expression of Akt2 and caspase-7. Luciferase assay was used to detect the direct binding of let-7b and let-7c to the 3'-untranslated region (UTR) of Akt2.

Results: Expression of let-7b and let-7c was significantly decreased in 32 paired clear cell renal cell carcinoma tissue specimens and the dysregulation of let-7b was associated with pathological grade. Transfection of let-7b or let-7c combined with 5-FU inhibited proliferation and potentiated the antitumor efficacies of 5-FU at tolerated concentration. let-7b and let-7c suppressed the luciferase activity of reporter plasmid containing the 3'-UTR of Akt2. Overexpression of let-7b and let-7c reduced Akt2 expression, and Akt2 inhibition enhanced the sensitivity to 5-FU by affecting apoptotic pathway.

Conclusions: Expression of let-7b and let-7c was frequently decreased in clear cell renal cell carcinoma tissues. The dysregulation of let-7b and let-7c may be involved in chemoresistance of RCC cells to 5-FU by down-regulating Akt2.

Figure 1

Expression of let-7b and let-7c in the 32 paired clinical specimens. (A) Expression of let-7b and let-7c was lower in 32 RCC clinical specimens than in 32 normal kidney specimens (P < 0.05). (B) let-7b and let-7c expression correlated positively (r = 0.810, P < 0.05).

Figure 2

CCK-8 assay showed that let-7b and let-7c enhanced chemosensitivity of 786-O cells to 5-FU. (A) Relative cell survival of 786-O cells at certain time points (24, 48, 60, and 72 h) after transfected with let-7b or let-7c mimics and treated with 5-FU at progressive concentrations (1, 10, 10², 10³, and 10⁴ μmol/l). (B) Relative cell survival of different groups with previous treatment for 24, 48, 60, and 72 h, in which 5-FU is at concentrations of 10 and 10² μmol/l. Cell viability assay showed the inhibition of 786-O cells proliferation by transfection of let-7b and let-7c (*P < 0.05).

Figure 3

Colony formation assay was used to detect the cell growth activity. (A) Clones of transfected cells were less than control cells. The antitumor effects of 5-FU in transfected cells were more profound than those in the control cells. (B) Relative colony numbers of transfected cells and control cells. Less growth activity was found in transfected cells compared to control cells (*P < 0.05).

Figure 4

Akt2 is a functional target of let-7b and let-7c. (A) Schematic representation of Akt2 3′-UTRs as the putative target of let-7b and let-7c. (B) Western blot analysis demonstrated significant decreased expression of Akt2 in transfected cells. (C) Luciferase assay showed decreased reporter activity after co-transfection of Akt2 3′-UTR plasmid with let-7b or let-7c in 786-O cells (*P < 0.05).

Figure 5

Knockdown of Akt2 enhanced the chemosensitivity through caspase-7. (A) Relative cell survival of 786-O cells 48 h after transfected with Akt2 siRNA and treated with 5-FU. Proliferation of 786-O cells after transfection of Akt2 siRNA was significantly reduced compared with negative control (*P < 0.05). (B) Akt2 and caspase-7 protein levels were assessed by Western blot in 786-O cells transfected with si-Akt2 (#1, #2) and negative control. Akt2 knockdown showed the increased expression of caspase-7.