MicroRNA-143 inhibits tumor growth and angiogenesis and sensitizes chemosensitivity to oxaliplatin in colorectal cancers

Abstract

Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Recently, downregulation of microRNA-143 (miR-143) has been observed in CRC tissues. Here in this study, we found that miR-143 expression was downregulated both in CRC patients' blood samples and tumor specimens. MiR-143 expression levels were strongly correlated with clinical stages and lymph node metastasis. Furthermore, insulin-like growth factor-I receptor (IGF-IR), a known oncogene, was a novel direct target of miR-143, whose expression levels were inversely correlated with miR-143 expression in human CRC specimens. Overexpression of miR-143 inhibited cell proliferation, migration, tumor growth and angiogenesis and increased chemosensitivity to oxaliplatin treatment in an IGF-IR-dependent manner. Taken together, these results revealed that miR-143 levels in human blood and tumor tissues are associated with CRC cancer occurrence, metastasis and drug resistance, and miR-143 levels may be used as a new diagnostic marker and therapeutic target for CRC in the future.

Keywords: IGF-IR; angiogenesis; chemotherapy; microRNA-143; tumorigenesis.

Figure 1. MiR-143 expression levels are downregulated in blood samples and cancer tissues and correlated with CRC clinical stages. (A) The relative expression levels of miR-143 in plasma of CRC patients were detected and normalized to those of the spiked-in control cel-miR-39. **p < 0.001 indicates significant difference comparing miR-143 expression levels in the plasma from CRC patients and from healthy subjects. (B) Expression levels of miR-143 in 62 pairs of CRC tumor tissues and adjacent normal specimens were analyzed by stem-loop qRT-PCR and normalized to the levels of U6. The fold changes were obtained by the ratio of miR-143 abundance in cancer tissues to that in the adjacent normal tissues. *p < 0.05 indicates significant difference comparing miR-143 expression in tumor tissues with adjacent normal tissues. (C) Relative expression levels of miR-143 in different stages of cancer tissues. *p < 0.05 indicates significant difference comparing miR-143 expression in Duke’s stage C+D with stage A, while ^#p < 0.05 comparing miR-143 expression in Duke’s stage C+D with stage B. (D) Relative expression levels of miR-143 in different types of lymph node metastasis. *p < 0.05 comparing miR-143 expression in positive lymph node metastasis with negative one.

Figure 2. MiR-143 overexpression suppresses proliferation and migration of SW1116 cells. (A) Cell viability was evaluated. Results were means ± SE from three independent experiments performed in sextuple. (B) SW1116 cells stably overexpressing miR-143 or miR-control were cultured to 90% confluence. A sterile 200 μl pipette tip was used to scratch the cells to form a wound. The wound gaps were photographed (top) and measured (bottom).

Figure 3. IGF-IR is a direct target of miR-143. (A) Putative seed-matching sites (in bold and italic) or mutant sites (underlined) between miR-143 and 3′-UTR of IGF-IR. (B) Luciferase activities of reporter constructs containing wild-type (WT) or mutant (MT) 3′-UTR of IGF-IR were assayed and normalized to those of renilla activities (internal control). Data were presented as means ± SE from three independent experiments with triple replicates per experiment. (C and D) Total proteins were subjected to western blotting and detected for IGF-IR expression levels. Relative densities of protein expression signals were calculated and normalized to GAPDH protein levels from three separate experiments. Data were means ± SE (E) The correlation analysis was performed between IGF-IR protein levels and miR-143 expression levels in CRC tissues. The IHC scores were used to describe protein levels of IGF-IR in tumor tissues, which were evaluated by experienced pathologists in a blind manner. (F) Linear regression curves of miR-143 expression and IGF-IR protein levels. *p < 0.05 comparing miR-143 with scrambled miRNA control (miR-NC).

Figure 4. MiR-143 inhibits angiogenesis and tumorigenesis in vivo. (A) Angiogenesis assay by chorioallantoic membrane (CAM) model as described in Methods. Top: Representative CAM plugs. Bar = 2 mm. Bottom: The branches of blood vessels, which were counted as the index of angiogenesis was obtained from the CAMs of 8–10 embryos per treatment 96 h after implantation. The data represented as mean ± SE of blood vessel numbers were normalized to those of the control. (B) Tumor growth assay in nude mice (n = 6). Representative pictures of xenograft tumors are shown. Bar = 2 mm. (C) Tumor growth curve upon implantation. (D) The average weights of xenograft tumors. Data were means ± SE (E) Protein levels of IGF-IR in xenograft tumors. (F) qRT-PCR analysis of VEGF mRNA levels in xenograft tumors. Data were presented as means ± SE *p < 0.05 indicates significant difference comparing miR-143 treatment and miR-NC (control).

Figure 5. MiR-143 increases chemosensitivity of CRC cells to oxaliplatin treatment through IGF-IR. (A and B) Cell viability was evaluated in cells stably expressing scrambled miRNA control (miR-NC), miR-143 or miR-143 + IGF-IR, respectively, with (A) or without (B) the oxaliplatin treatments at different doses. Data were means ± SE. (C and D) Cells stably expressing miR-NC, miR-143 or miR-143 + IGF-IR were treated with 4 µM of oxaliplatin at indicated time points and subjected to apoptosis analysis by flow cytometry (C) and western blotting (D). Data were means ± SE **p < 0.001 indicated significance between group of miR-143 and control group miR-NC, while ^#p < 0.05 represented significance between group of miR-143 and group of miR-143 + IGF-IR.