Down-regulation of miR-21 Induces Differentiation of Chemoresistant Colon Cancer Cells and Enhances Susceptibility to Therapeutic Regimens

Abstract

MicroRNAs are endogenous posttranscriptional modulators that negatively control the expression of their target genes and play an important role in the development and progression of many malignancies, including colorectal carcinoma. In particular, expression of microRNA-21 (miR-21) is greatly increased in chemotherapy-resistant (CR) colon cancer cells that are enriched in undifferentiated cancer stem/stem-like cells (CSCs/CSLCs). We hypothesize that miR-21 plays a critical role in regulating differentiation of CR colon cancer cells. Indeed, we observed that downregulation of miR-21 in CR colon cancer cells (HCT-116 or HT-29) by antisense miR-21 induced differentiation, as evidenced by marked increases in cytokeratin-20 (CK-20) expression and alkaline phosphatase activity. These changes were accompanied by a significant reduction in the expression of colon CSC/CSLC marker CD44, colonosphere formation, and T-cell factor/lymphoid enhancer factor (TCF/LEF) activity but increased the expression of proapoptotic programmed cell death 4 gene. Induction of differentiation greatly increased sensitivity of CR colon cancer cells to the growth inhibitory properties of all three regimens tested: 5-fluorouracil + oxaliplatin (FUOX), difluorinated curcumin (CDF), and the combination of CDF and FUOX. However, the magnitude of inhibition of growth by either CDF (75%) alone or CDF + FUOX (80%) was much higher than that observed with only FUOX (40%). Growth inhibition by CDF and CDF + FUOX in differentiating CR colon cancer cells was associated with a 98% to 99% reduction in the expression of CD44 and epidermal growth factor receptor (EGFR). However, down-regulation of CK-20 in CR colon cancer cells produced no significant change in cellular growth in the absence or presence of FUOX, when compared with the corresponding controls. The current observation suggests that CDF and CDF + FUOX are highly effective in inhibiting growth and reducing colon CSCs/CSLCs in anti-miR-21-induced differentiating CR colon cancer cells and supports our contention that differentiation enhances susceptibility of CR cancer cells to conventional and nonconventional therapeutic regimen.

Figure 1

Down-regulation of miR-21 in CR colon cancer HCT-116 cells through transfection of anti-miR-21 (A) lowers the levels of both pri-miR-21 and mature miR-21, (B) decreases the expression of CD44 and EGFR, and (C) stimulates the expression of PDCD4 but reduces CD44. In this and all subsequent studies, the control cells were transfected with scrambled miRNAs. The data represent means ± SD of three independent experiments.

Figure 2

Down-regulation of miR-21 in CR HCT-116 cells by anti-miR-21 (A) decreases the ability of cells to form spheroids as shown by the representative photographs (magnification, x10) taken after 7 days of transfection with either anti-miR-21 or scrambled miRNA (control), (B) the number of spheres formed 7 days following transfection of anti-miR-21 or scrambled miRNA; nontransfected CR-HCT-116 cells were used as controls. Values are means ± SD. P < . 05, compared with scramble or untreated controls. (C) qRT-PCR (upper panel) showing up-regulation of CK-20 mRNA in CR HCT-116 cells 48 hours following transfection of anti-miR-21, when compared with corresponding scramble control (*P < .001). Western blot (lower panel) showing increased expression of CK-20 in CR HCT-116 cells following transfection of anti-miR-21 when compared with the corresponding control cells; β-actin was used as a loading control. (D) Induction of alkaline phosphatase activity in CR HCT-116 cells 48 hours following transfection of anti-miR-21 (n = 3). Values are means ± SD. *P < .05, compared with scramble transfected control.

Figure 3

Down-regulation of either miR-21 or β-catenin in CR colon cancer cells (HCT-116 or HT-29) leads to attenuation of transcriptional activation of TCF/LEF. (A) Relative transcriptional activities of TCF/LEF in CR colon cancer HCT-116 cells after down-regulation of either miR-21 by anti-miR-21 or β-catenin by the corresponding siRNA, when compared with the corresponding controls that were similarly treated with scramble miRNA or siRNA. *P < .001, compared with the corresponding scramble controls. (B and C) qRT-PCR showing down-regulation of β-catenin in CR colon cancer HT-29 or HCT-116 cells by the corresponding siRNA leads to reduction in CD44 expression and increase in CK20 expression in CR colon cancer HT-29 cells (B) and HCT-116 cells (C), compared with the corresponding controls. The data represent means ± SD of three independent experiments, *P < .001.

Figure 4

Down-regulation of miR-21 increases the CR colon cancer cell susceptibility to conventional therapeutic FUOX, nonconventional therapeutic CDF, and the combination of FUOX plus CDF. (A) MTT assay showing growth inhibition of anti-miR-21 or scramble miRNA-transfected CR HCT-116 cells in response to FUOX (100 µM 5-FU + 2.5 µM oxaliplatin), CDF (2.0 µM), or the combination of CDF+FUOX compared to the untreated cells (control). The cells were exposed to the therapeutic agents for 48 hours. Each value represents mean ± SD of six observations; *P < .001. (B) qRT-PCR showing anti-miR-21.transfected CR HCT-116 cells; expression of CD44 and EGFR is greatly inhibited in response to FUOX, CDF, or CDF + FUOX. Scramble miRNA-transfected CR cells were used as control; *P < .001. The CR cells were maintained in FUOX (50 µM 5-FU + 1.25 µM oxaliplatin) throughout as stated above in Materials and Methods section.

Figure 5

Down-regulation of CK-20 in CR colon cancer HT-29 cells causes no apparent change in proliferation and susceptibility to FUOX. (A) Real-time qRT-PCR showing down-regulation of CK-20 48 hours following transfection with CK-20 siRNA. Data represent means ± SD of three independent experiments; *P < .001, compared to the control. (B) Western blot analysis of CK-20 following downregulation of CK-20. (C) MTT assay showing the CR HT-29 cell proliferation and its susceptibility to FUOX after transfection with CK-20 siRNA, anti-miR-21, and the scramble transfected (control). Data represent means ± SD of three independent experiments.