Maintenance of cancer stemness by miR-196b-5p contributes to chemoresistance of colorectal cancer cells via activating STAT3 signaling pathway

Abstract

Emerging studies indicated that cancer stem cells represent a subpopulation of cells within the tumor that is responsible for chemotherapeutic resistance. However, the underlying mechanism is still not clarified yet. Here we report that miR-196b-5p is dramatically upregulated in CRC tissues and high expression of miR-196b-5p correlates with poor survival in CRC patients. Moreover, recurrent gains (amplification) contribute to the miR-196b-5p overexpression in CRC tissues. Silencing miR-196b-5p suppresses spheroids formation ability, the fraction of SP cells, expression of stem cell factors and the mitochondrial potential, and enhances the apoptosis induced by 5-fluorouracil in CRC cells; while ectopic expression of miR-196b-5p yields an opposite effect. In addition, downregulation of miR-196b-5p resensitizes CRC cells to 5-fluorouracil in vivo. Our results further demonstrate that miR-196b-5p promotes stemness and chemoresistance of CRC cells to 5-fluorouracil via targeting negative regulators SOCS1 and SOCS3 of STAT3 signaling pathway, giving rise to activation of STAT3 signaling. Interestingly, miR-196b-5p is highly enriched in the serum exosomes of patients with CRC compared to the healthy control subjects. Thus, our results unravel a novel mechanism of miR-196b-5p implicating in the maintenance of stem cell property and chemotherapeutic resistance in CRC, offering a potential rational registry of anti-miR-196b-5p combining with conventional chemotherapy against CRC.

Keywords: CRC; STAT3 signaling pathway; cancer stem cell; chemotherapeutic resistance; miR-196b-5p.

Figure 1. miR-196b-5p is upregulated in CRC and correlated with poor prognosis

(A–C) miR-196b-5p expression levels was markedly upregulated in CRC tissues as assessed by analyzing the E-GEOD-10259, E-GEOD-41655 and TCGA of CRC miRNA sequencing datasets. (D) Real-time PCR analysis of miR-196b-5p in 11 primary CRC tissues compared with the matched adjacent normal tissues (ANT). (E) Real-time PCR analysis of miR-196b-5p expression in 20 paired collected CRC tissue samples. Transcript levels were normalized to U6 expression. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (F) miR-196b-5p expression levels was markedly upregulated in CRC tissues compared with the matched adjacent normal tissues (ANT). (ANT, n = 20; CRC, n = 90). P < 0.001. (G) Kaplan–Meier analysis of overall survival curves of patients with CRC with high miR-196b-5p expression (> median, n = 45) versus low miR-196b-5p expression (< median, n = 45). P < 0.001, log-rank test. (H and I) Kaplan–Meier analysis of overall survival curves of CRC patients datasets from TCGA and E-GEOD-29623.

Figure 2. miR-196b-5p activates STAT3 signaling via targeting multiple negative regulators of STAT3 signaling

(A) Real-time PCR analysis of miR-196b-5p expression in the indicated cells. Transcript levels were normalized by U6 expression. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (B) Western blotting of SOCS1, SOCS2, SOCS3, SOCS4 and SOCS5 expression in the indicated cells. α-Tubulin served as the loading control. (C and D) Luciferase assay of cells transfected with pmirGLO-3′UTR reporter of SOCS1 and SOCS3 in miR-196b-5p overexpressing and silencing HCT116 and SW480 cells, respectively. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (E and F) MiRNP IP assay showing the association between miR-196b-5p and SOCS1,SOCS3 transcripts in HCT116 and SW480 cells. Pulldown of IgGantibody served as the negative control. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (G) STAT3 transcriptional activity was assessed by luciferase reporter constructs in the indicated cells. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (H) Western blotting of nuclear STAT3 expression. The nuclear protein p84 was used as the nuclear protein marker.

Figure 3. miR-196b-5p promotes stem cell properties in CRC cells

(A) Representative images of spheroids formed at 200-fold magnification were counted. Histograms showed the mean number of spheroids formed. Scale bars, 50 mm. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (B) Hoechst 33342 dye exclusion assay showed that overexpressing miR-196b-5p promoted the fraction of side population, whereas silencing miR-196b-5p decreased the fraction. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (C and D) Real-time PCR analysis of OCT4A, SOX2, NANOG and BMI-1 expression in the indicated cells. GAPDH was used as the loading control. Error bars represent the mean ± SD of three independent experiments. *P < 0.05.

Figure 4. miR-196b-5p promotes chemoresistance in CRC cells in vitro

(A) Annexin V-FITC/PI staining of the indicated cells under treatment of 5-FU. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (B) The JC-1 staining the indicated cells under treatment of 5-FU. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (C) Western blotting analysis of Bcl-2 and Bcl-xL in the indicated cells under treatment of 5-FU. (D and E) Analysis of the activities of caspase-3 (D) and caspase-9 (E) were detected by the cleaved forms of these two proteins. Error bars represent the mean ± SD of three independent experiments. *P < 0.05.

Figure 5. Inhibition of miR-196b-5p sensitizes CRC cells to 5-FU in vivo

(A) Representative images of tumor-bearing mice. The representative images were captured on day 14 and day 38 after inoculating HCT116 cells respectively. The intraperitoneal injection of 5-FU (50 mg/kg.d) started from two weeks after inoculating HCT116 cells and lasted for 24 days, namely 38 day after inoculating HCT116 cells. (B) Tumor volumes in the miR-196b-5p–overexpressing, miR-196b-5p–silenced, and control groups were measured on the indicated days. Data presented are the mean ± SD (C) Mice were euthanized, and tumors from each experimental group were excised. (D) Tumor weights of each group.

Figure 6. miR-196b-5p is detected in the serum and serum exosomes of CRC patients

(A) miR-196b-5p expression was elevated in the serum of CRC patients compared with the healthy controls from E-GEOD-25609 dataset (left panel) and ROC curve for miR-196b-5p in the serum of the healthy and CRC patients (right panel). (B) miR-196b-5p expression was elevated in the serum exosomes of CRC patients compared with the healthy controls from E-GEOD-39833 dataset (left panel) and ROC curve for miR-196b-5p in the serum exosomes of the healthy and CRC patients (right panel). (C) Correlation between mRNA expression levels of miR-196b-5p in 6 different CRC cells and the concentration of miR-196b-5p in their respective exosomes. (D) Endogenous expression of miR-196b-5p in diffetent CRC cell lines and miR-196b-5p expression in the exosomes from the supernatant of diffetent CRC cell lines. (E) Correlation between mRNA expression levels of miR-196b-5p in different CRC cells and the concentration of miR-196b-5p in their respective exosomes. (F) miR-196b-5p expression was elevated in the serum of CRC patients compared with the healthy controls (Health, n = 90; CRC, n = 150). P < 0.001. (G) ROC curve for miR-196b-5p in the serum of the healthy and CRC patients. (H) miR-196b-5p expression was elevated in the serum exosomes of CRC patients compared with the healthy controls (Health, n = 90; CRC, n = 150). P < 0.001. (I) ROC curve for miR-196b-5p in the serum exosomes of the healthy and CRC patients.

Figure 7. Clinical relevance of miR-196b-5p with SOCS1, SOCS3 and STAT3 signaling activity in human CRC tissues

(A) Analysis of miR-196b-5p expression with protein expression of SOCS1, SOCS3 and nuclear pSTAT3 in 8 CRC tissues. U6 was used as the control for RNA loading. miR-196b-5p expression levels were normalized to that miR-196b-5p expression of sample one. Loading controls were α-tubulin and p84 for the cytoplasmic and nuclear fractions. Each bar represents the mean ± SD of three independent experiments. *P < 0.05. (B) Hypothetical model illustrating that constitutive activation of the STAT3 signaling pathway by miR-196b-5p epigenetic disruption of multiple negative feedback loops contributes to the maintenance of stemness and chemoresistance in CRC cells.