MicroRNA-330 inhibited cell proliferation and enhanced chemosensitivity to 5-fluorouracil in colorectal cancer by directly targeting thymidylate synthase

Abstract

Colorectal cancer (CRC) is the third most common cancer in males and the second most common in females, worldwide. Currently, 5-fluorouracil (5-FU)-mediated chemotherapy is the adjuvant treatment for patients with CRC following surgical resection. However, a number of patients with CRC develop 5-FU resistance, which is a major challenge for the effective treatment of cancer. Therefore, it is important to investigate the molecular mechanisms underlying chemoresistance and the therapeutic treatments that may improve the treatment of CRC. The present study demonstrated that microRNA (miR)-330 was significantly downregulated in CRC tissues and cell lines. Ectopic miR-330 expression decreased cell proliferation and enhanced cell chemosensitivity to 5-FU via the cell apoptosis pathway in CRC. In addition, thymidylate synthase (TYMS) was revealed to be a direct target gene of miR-330 in CRC. Knockdown of TYMS inhibited CRC cell proliferation, and enhanced cell chemosensitivity to 5-FU by promoting cell apoptosis. In conclusion, the results of the present study indicated that miR-330 targeted TYMS to inhibit the proliferation and enhance the chemosensitivity of CRC cells to 5-FU by promoting cell apoptosis. The present study provided important insight into the molecular mechanism underlying 5-FU-mediated chemoresistance and a novel therapeutic strategy for the enhancement of efficacy in CRC treatment.

Keywords: 5-fluorouracil; chemosensitivity; colorectal cancer; microRNA-330; proliferation; thymidylate synthase.

Figure 1.

The expression level of miR-330 in CRC tissues. (A) miR-330 was downregulated in CRC tissues compared with matched NATs. (B) The expression level of miR-330 was decreased in CRC cell lines compared with the FHC cell line. (C) miR-330 was significantly upregulated in HCT119 and HT29 cells following transfection with miR-330 mimics. *P<0.05 compared with their respective controls. miR-330, microRNA-330; CRC, colorectal cancer; NATs, normal adjacent tissues; NC, negative control.

Figure 2.

CCK8 assays revealed that the upregulation of miR-330 resulted in the growth inhibition of HCT116 and HT29 cells. *P<0.05 compared with the NC. CCK-8, Cell Counting Kit-8; miR-330, microRNA-330; NC, negative control.

Figure 3.

Chemosensitivity assays demonstrated that enforced miR-330 expression decreased the survival rate of HCT116 and HT29 cells compared with NC-transfected HCT116 and HT29 cells. *P<0.05 compared with the NC. miR-330, microRNA-330; NC, negative control.

Figure 4.

Overexpression of miR-330 significantly increased the rate of apoptosis of (A) HCT116 and (B) HT29 cells induced by 5-FU. *P<0.05 compared with the NC. miR-330, microRNA-330; 5-FU, 5-fluorouracil; FITC, fluorescein isothiocyanate; NC, negative control.

Figure 4.

Overexpression of miR-330 significantly increased the rate of apoptosis of (A) HCT116 and (B) HT29 cells induced by 5-FU. *P<0.05 compared with the NC. miR-330, microRNA-330; 5-FU, 5-fluorouracil; FITC, fluorescein isothiocyanate; NC, negative control.

Figure 5.

TYMS is a direct target gene of miR-330 in vitro. (A) The miR-330 binding site in the 3′UTR of TYMS and the TYMS 3′UTR mutant sequence. (B) miR-330 inhibited PGL3-TYMS-3′UTR Wt luciferase activity in HEK293T cells (P<0.05), whereas PGL3-TYMS-3′UTR Mut revealed no response to miR-330. (C) TYMS mRNA expression levels were not significantly altered following transfection with miR-330 mimics. (D) The TYMS protein was downregulated in miR-330 mimic-transfected HCT116 and HT29 cells compared with the NC groups. *P<0.05 compared with the NC. TYMS, thymidylate synthase; miR-330, microRNA-330; 3′UTR, 3′untraslated region; Wt, wild-type; Mut, mutant; NC, negative control.

Figure 6.

Effects of TYMS expression in HCT116 and HT29 cells. (A) TYMS siRNA decreased the expression level of TYMS in HCT116 and HT29 cells. (B) Cell growth was suppressed in TYMS siRNA transfected HCT116 and HT29 cells compared with the NC siRNA groups. (C) Chemosensitivity assays demonstrated that TYMS siRNA enhanced the chemosensitivity of HCT116 and HT29 cells to 5-FU. (D) Inhibition of TYMS increased apoptosis of HCT116 and HT29 cells induced by 5-FU. *P<0.05 compared with the NC. TYMS, thymidylate synthase; siRNA, short interfering RNA; NC, negative control; 5-FU, 5-fluorouracil.