MicroRNA-302b Enhances the Sensitivity of Hepatocellular Carcinoma Cell Lines to 5-FU via Targeting Mcl-1 and DPYD

Abstract

MiR-302b is a member of miR-302-367 cluster. The miR-302-367 cluster played important roles in maintaining pluripotency in human embryonic stem cells (hESCs) and has been proved to be capable of suppressing cell growth in several types of cancer cell lines including Hepatocellular Carcinoma (HCC) Cell lines. However, the role that miR-302b plays in the 5-Fluorouracil (5-FU) sensitivity of HCC has not been known. This study showed that miR-302b could enhance the sensitivity to 5-FU in HCC cell lines and verified its two putative targeted genes responsible for its 5-FU sensitivity.

Keywords: 5-Fluorouracil (5-FU); hepatocellular carcinoma cell lines; miR-302b; sensitivity.

Figure 1

MiR-302b represses cell proliferation, cell cycle progression in liver cancer cell lines. (A) qRT-PCR showed that a significant increase after infection of miR-302b vector into HepG2/SMMC-7721, miR-ctrl as negative control; (B) MiR-302b suppressed the cell proliferation of HepG2/SMMC-7721, which was determined by MTT assay; (C) Overexpression of miR-302b in HepG2/SMMC-7721 blocked the cell cycle transition from G0/G1 to S phase; (D) Overexpression of miR-302b in HepG2/SMMC-7721 did not show obvious difference in apoptosis compared with the control cells. Error bars represent the S.D; * p < 0.05; ** p < 0.01, vs. the corresponding controls.

Figure 2

MiR-302b enhances the sensitivity to 5-FU in HepG2 and SMMC-7721 cells in a time- and dose- dependent manner. (A,B) 5-FU chemosensitivity was measured by MTT assay in miR-302b, miR-ctrl transfected or untransfected HepG2/SMMC-7721 cells treated with 1.6 μM 5-FU (A) or 3.2 μM 5-FU (B) for 24, 48 and 72 h. MiR-ctrl as the control; (C) Effect of miR-302b on chemosensitivity to 5-FU over 3.2 μM was measured at 72 h by MTT assay in HepG2/SMMC-7721 cells. MiR-ctrl as the control; (D) HepG2/SMMC-7721 cells were treated with miR-ctrl, miR-302b, with or without a combination of 3.2 μM 5-FU in the colony formation assays; (E) PI/Annexin V staining FACS analyses measured apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with miR-302b or miR-ctrl; (F) Western blot of caspase3 and cleaved PARP detected apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with miR-302b and miR-ctrl. Error bars represent the S.D.; * p < 0.05; ** p < 0.01, vs. the corresponding controls.

Figure 3

MiR-302b directly targets Mcl-1 by binding to the 3′ UTR and DPYD coding region. (A,D) schematic representation of miR-302b seed sequence within the 3′ UTR of Mcl-1 (A) and coding region of DPYD (D). Mutations in the seed region of miR-302b were respectively generated in the 3′ UTR of Mcl-1 and in the CDS of DPYD, as indicated by underline. The target genes sequences and their mutated ones were respectively cloned into the pmirGLO Dual-Luciferase miRNA Target Expression vector; (B,E) Analysis of luciferase activity; (C,F) Western blot measured the expression level of Mcl-1 (C) and DPYD (F) 48 h post transfection of miR-302b. Error bars represent the S.D; * p < 0.05; ** p < 0.01, vs. the corresponding controls.

Figure 4

Down-regulation of Mcl-1 or DPYD enhances the sensitivity to 5-FU in HepG2 and SMMC-7721 cells in a time- and dose- dependent manner. (A,B) qRT-PCR and Western blot analyses the expression level of Mcl-1 gene (A) and DPYD gene (B) after transfection of si-Mcl-1 and si-DPYD, respectively into SMMC-7721 cells; (C,D) 5-FU chemosensitivity was measured by MTT assay in si-Control, si-Mcl-1 transfected or untransfected HepG2/SMMC-7721 cells treated with 1.6 μM 5-FU (C) or 3.2 μM 5-FU (D) for 24, 48 and 72 h. Si-control as the control; (E,F) Same MTT assays were performed in si-DPYD, si-Control transfected or untransfected HepG2/SMMC-7721 cells treated with 0.8 μM 5-FU (E) or 1.6 μM 5-FU (F). Si-control as the control; (G) Effect of si-Mcl-1 and si-DPYD on Chemosensitivity to 5-FU over 3.2 μM was measured at 72 h by MTT assay in HepG2/SMMC-7721 cells; (H) PI/Annexin V staining FACS analyses measured apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with si-Control, si-Mcl-1 or si-DPYD; (I) Western blot of caspase3 and cleaved PARP detected apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with si-Control, si-Mcl-1 or si-DPYD. Error bars represent the S.D.; * p < 0.05; ** p < 0.01, vs. the corresponding controls.

Figure 4

Down-regulation of Mcl-1 or DPYD enhances the sensitivity to 5-FU in HepG2 and SMMC-7721 cells in a time- and dose- dependent manner. (A,B) qRT-PCR and Western blot analyses the expression level of Mcl-1 gene (A) and DPYD gene (B) after transfection of si-Mcl-1 and si-DPYD, respectively into SMMC-7721 cells; (C,D) 5-FU chemosensitivity was measured by MTT assay in si-Control, si-Mcl-1 transfected or untransfected HepG2/SMMC-7721 cells treated with 1.6 μM 5-FU (C) or 3.2 μM 5-FU (D) for 24, 48 and 72 h. Si-control as the control; (E,F) Same MTT assays were performed in si-DPYD, si-Control transfected or untransfected HepG2/SMMC-7721 cells treated with 0.8 μM 5-FU (E) or 1.6 μM 5-FU (F). Si-control as the control; (G) Effect of si-Mcl-1 and si-DPYD on Chemosensitivity to 5-FU over 3.2 μM was measured at 72 h by MTT assay in HepG2/SMMC-7721 cells; (H) PI/Annexin V staining FACS analyses measured apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with si-Control, si-Mcl-1 or si-DPYD; (I) Western blot of caspase3 and cleaved PARP detected apoptosis induced or not by 5-FU in SMMC-7721 cells transfected with si-Control, si-Mcl-1 or si-DPYD. Error bars represent the S.D.; * p < 0.05; ** p < 0.01, vs. the corresponding controls.