Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells

Abstract

In this study, high-throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G(1) and G(2) phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133(+hi)CD44(+hi) colon cancer stem-like cells that exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid-modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation through G(1) and G(2) phase arrest mediated in part through the suppression of HDAC4. miR-140 may be a candidate target to develop novel therapeutic strategy to overcome drug resistance.

Figure 1

Venn diagram analysis of overlapping miRNAs based on chemosensitivity to doxorubicin, cisplatin and ifosfamide in human osteosarcoma xenografts.

Figure 2

Chemosensitivity analysis in HCT 116 (wt-p53) cells. Cells were transfected with 100nM miR-140, miR control, or siHDAC4, then treated with MTX (a) or 5-FU (b) for 72 h, and cell viability was determined by the WST-1 assay. miR control was used as the negative control. Numbers are indicated as mean ± SD.

Figure 3

Impact of miR-140 on cell proliferation in U-2 OS cells (wt-p53) (a), HCT 116 (wt-p53) cells (b), MG63 cells (mut-p53) (c) and HCT 116 (null-p53) cells (d). Each cell group was transfected with 100 nM miR control or miR-140, cell numbers were determined by the WST-1 assay. All the data represent mean ± SD.

Figure 4

Impact of miR-140 on cell cycle. (a) Cell cycle analysis by flow cytometry in U-2 OS and MG63 cells or HCT 116 (wt-p53) and HCT 116 (null-p53) cells transfected with 100 nM miR control or miR-140. The values of G1/S and G2/S ratio in the miR control were set as 1, the bar graphs showed the relative quantity of G1/S and G2/S ratio in the miR-140 transfected cells compared to the miR control as mean ± SD. This experiment was repeated two separate times, and similar results were obtained. The representative flow cytometry pattern was shown. (b) Western immunoblot analysis of p53 and p21 expression in HCT 116 (wt-p53) (left panel) and HCT 116 (null-p53) (right panel) cells, α-tubulin was used as a protein loading control.

Figure 5

HDAC 4 is the target of miR-140. (a) Sequence comparison analysis of 3′-UTRs of mouse and human HDAC4 mRNAs with miR-140 interaction site. (b) Expression of HDAC4 mRNA in U-2 OS and HCT 116 (wt-p53) cells analyzed by real time qRT-PCR. (*P<0.05, Student's t test, n=3). (c) Protein expression analysis of HDAC4 in HCT 116 (wt-p53) and HCT 116 (null-p53) cells transfected with miR-140, oligofectamine alone (vehicle control) and miR control were used as the negative controls. (d) HCT 116 (wt-p53) and HCT 116 (null-p53) cells were transfected with LNA anti-miR140 and scramble-miR (LNA-control), HDAC4 protein was quantified by Western immunoblot.

Figure 6

Expression of miR-140 in human colon cancer stem-like cells is elevated. (a) Flow cytometry analysis of sorted CD133^+hi/CD44^+hi HCT 116 (wt-p53) colon cancer stem-like cells using labeled CD133-PE and CD44-FITC antibodies. (b) Expression levels of miR-140 in colon cancer stem-like cells and colon cancer cells were analyzed by real time qRT-PCR (*P<0.05, Student's t test, n=3). (c) CD133^+hi/CD44^+hi colon cancer stem-like cells were more resistant to 5-FU treatment. CD133^+hi/CD44^+hi and control HCT 116 (wt-p53) cells were incubated with lethal dose 5-FU (100 μM) for 48 h, the dead cells were determined by the FITC Annexin V and PI detection kit (top panel, **P<0.01, Student's t test, n=3). CD133^+hi/CD44^+hi HCT 116 (wt-p53) colon cancer stem-like cells transfected with LNA anti-miR140 became sensitive to 5-FU treatment. CD133^+hi/CD44^+hi cells were transfected with 100 nM of LNA anti-miR140, 24 h later, cells were incubated with 100 μM of 5-FU for 48 h. The dead cells were determined by the FITC Annexin V and PI detection kit (lower panel, *P<0.05, Student's t test, n=3).