MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

Viviana Costa, Alessia Lo Dico, Aroldo Rizzo, Francesca Rajata, Marco Tripodi, Riccardo Alessandro, Alice Conigliaro, Viviana Costa, Alessia Lo Dico, Aroldo Rizzo, Francesca Rajata, Marco Tripodi, Riccardo Alessandro, Alice Conigliaro

Abstract

The survival rates in colon cancer patients are inversely proportional to the number of lymph node metastases. The hypoxia-induced Epithelial to Mesenchymal Transition (EMT), driven by HIF1α, is known to be involved in cancer progression and metastasis. Recently, we have reported that miR-675-5p promotes glioma growth by stabilizing HIF1α; here, by use of the syngeneic cell lines we investigated the role of the miR-675-5p in colon cancer metastasis.Our results show that miR-675-5p, over expressed in metastatic colon cancer cells, participates to tumour progression by regulating HIF1α induced EMT. MiR-675-5p increases Snail transcription by a dual strategy: i) stabilizing the activity of the transcription factor HIF1α and ii) and inhibiting Snail's repressor DDB2 (Damage specific DNA Binding protein 2).Moreover, transcriptional analyses on specimens from colon cancer patients confirmed, in vivo, the correlation between miR-675-5p over-expression and metastasis, thus identifying miR-675-5p as a new marker for colon cancer progression and therefore a putative target for therapeutic strategies.

Keywords: CRC; EMT; hypoxia; metastasis; miRNA675.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Figures

Figure 1. miR-675-5p inhibitor reduces metastatic features…
Figure 1. miR-675-5p inhibitor reduces metastatic features and promotes epithelial phenotype
(A) Real time-PCR for miR-675-5p in SW480 and SW620 cells. All data were normalized for U6 and ΔΔct was expressed as relative amount of miRNAs. Values are presented as the mean ± SD (of three independent experiments). SW620 vs SW480 **p < 0.001. (B) Real time-PCR for miR-675-5p and EMT master genes: Snail, Slug and E-cadherin in SW480 and SW620 cells. Data were normalized for β-actin, while U6 was used for miRNA normalization. ΔΔct is expressed as Fold of induction (FOI) with respect to expression in control samples. Data are the mean ± SD of three independent experiments. SW480 miRNA inhibitor vs SW480 scramble control: *p < 0.05; ***p < 0.0001; SW620 miRNA inhibitor vs SW620 scramble control *p < 0.05; **p < 0.001; ***p < 0.0001. (C) Immunofluorescences and median focal plane in confocal analysis for E-CADHERIN, ZO-1and SNAIL in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control, in blue the nuclear staining with DAPI. (D) Migration assay: Phase contrast micrographs (10×) showing the migration of SW480 and SW620 cells pre-treated with miR-675-5p inhibitor or scramble control. Right panel: Quantification of migration by counting the number of migrated cells (violet) per field (n = 6); *p < 0.05.
Figure 2. miR-675-5p activates EMT genes by…
Figure 2. miR-675-5p activates EMT genes by promoting HIF1α pathway
Real time-PCR for HIF1α (A) and VEGF-A (B) in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW480 miRNA inhibitor vs SW480 control *p < 0.05; SW620 miRNA inhibitor vs SW620 control *p < 0.05 (C) Real time-PCR for HIF1α in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in normoxia. Values are presented as the mean ± SD. SW620 inhibitor vs SW620 ctr **p < 0.001. ELISA assay for HIF-1α (D) and VEGFa (E) performed in SW480 and SW620 cells, transfected with miRNA inhibitor or scramble control, after 6 hours of hypoxia. Data are expressed as Absorbance (ABS) values at 450 nm. SW620 miRNA inhibitor vs SW620 scramble control **p < 0.001. Real time-PCR for hypoxia targets genes and EMT regulator genes (VEGF, SNAIL, SLUG) in SW480 cells (F) and SW620 cells (G) treated with miR-675-5p inhibitor or scramble control, after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 inhibitor vs SW620 control **p < 0.001. (H) Real time-PCR for lncH19 and HIF1α in SW620 cells treated with siRNA H19 or scramble control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 siH19 vs SW620 control *p < 0.05;. (I) Real time-PCR for Snail and Slug in SW620 cells transfected with siRNA H19 or scrambled control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. **p < 0.001. Data are the mean ± SD of three independent experiments.
Figure 3. Gain of function suggests a…
Figure 3. Gain of function suggests a critical role of miR-675-5p on colon cancer aggressiveness
(A) ELISA assay for HIF-1α performed in SW620 cells nuclear extracts after mimic or scramble-control transfection. Data are expressed as Absorbance (ABS) values at 450nm. SW620 mimic vs SW620 scramble control **p < 0.001. Data are the mean ± SD of three independent experiments. (B) Immunofluorescences and median focal plane of confocal analysis for HIF1α (red) in SW620 cells treated with miR-675-5p mimic or scrambled control, in blue the nuclear staining with DAPI. (C) Real-time PCR for HIF1α targets gene (Snail, Slug, VEGF, VEGFR-2) from SW620 cells after miR-675-5p mimic or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI of indicated genes after mimic transfection with respect to scramble control. (D) ELISA assay for VEGF levels in supernatants from SW620 cell lines 18 hours after mimic and scramble control transfection. Data are expressed as pg/ml of soluble VEGF. SW620 mimic vs SW620 scramble control ***p < 0,0001. Data are the mean ± SD of three independent experiments. (E) Migration assay: Phase contrast micrographs (10×) showing the migration of SW620 cells pre-treated with mimic miR-675-5p and scramble control. Right panel: Quantification of motility established by counting the number of migrated cells (violet) per field; SW620 mimic vs SW620 scramble control *p < 0.05.
Figure 4. miR-675-5p down regulates the repressor…
Figure 4. miR-675-5p down regulates the repressor DDB2
(A) Left: predicted seed sequence for miR-675-5p on DDB2 gene. Right: Real-time PCR for DDB-2 gene from SW620 cells after 18 hours of miR-675-5p mimic or miR-675-5p inhibitor or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to scramble control. SW620 mimic and inhibitor vs SW620 **p < 0,001. Data are the mean ± SD of three independent experiments. (B) Western blot for DDB2, HIF1α and β-actin in SW620 cells after 18 hours of miR-675-5p scramble control or inhibitor transfection.
Figure 5. miR -675-5p expression in human…
Figure 5. miR -675-5p expression in human colon carcinoma progression
(A) Real time-PCR for miR-675-5p in specimens obtained from colon carcinoma patients with or without metastasis. All data were normalized for U6 and ΔΔct was expressed as FOI of analysed genes in N > 0 vs N = 0. Values are presented as the mean ± SD. ***p < 0.001 gene expression in tumour N > 0 vs tumour N = 0. For statistical analysis t-test or one- or two-way analysis of variance (ANOVA), followed by Dunnett's or Bonferroni's multiple comparison test, were performed using Prism 4(GraphPad SoftwareInc., CA, USA). (B) Real-time PCR for lncH19 in specimens obtained from colon carcinoma patients with or without metastasis. Data were normalized for β-actin and ΔΔct was expressed as FOI of analysed genes in N > 0 vs N = 0.

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