miR-375 Mediated Acquired Chemo-Resistance in Cervical Cancer by Facilitating EMT

<div><p>Acquired chemo-resistance is one of the key causal factors in cancer death. Emerging evidences suggest that miRNA and epithelial–mesenchymal transition play critical roles in the chemo-resistance in cancers. Here, we showed the association of paclitaxel-resistance with miR-375 over-expression and epithelial–mesenchymal transition inducement in cervical cancer. Using different cervical cancer cell models, we found that paclitaxel transiently induced up-regulation of miR-375 expression, proliferation inhibition, transition from epithelial to mesenchymal phenotype, and consequently impaired paclitaxel sensitivity. Forced over-expression of miR-375 may suppress Ecadherin expression by a directly targeting pathway, which led to paclitaxel resistance. Contrarily, re-expression of Ecadherin partly reversed epithelial–mesenchymal transition phenotype and miR-375 induced paclitaxel-resistance. Our findings suggest that paclitaxel-induced miR-375 over-expression facilitates epithelial–mesenchymal transition process via directly targeting Ecadherin, proliferation inhibition, and consequently results in chemo-resistance in cervical cancer cells. A reversion of miR-375 or Ecadherin expression may be a novel therapeutic approach for overcoming chemo-resistance in cervical cancer.</p></div

Forced expression of Ecadherin attenuates the ability miR-375 to mediate EMT and paclitaxel-resistance.

<p>Immunoblotting (a) and MTS (b) analysis of Ecadherin and other EMT related proteins in SiHa with and without miR-375 overexpression after the Ecadherin-expressing or negative lentivirus vector transfection. Data in (a) and (b) were presented as the mean of triplets samples from independent experiments. All error bars indicated s.e.m. (**p≤0.01, *p≤0∶05). (c) Morphological changes in SiHa with and without miR-375 over-expression after the Ecadherin-expressing or negative lentivirus vector transfection. The representative cells with morphological changes had been labeled.</p

TGF-β1 or EGF-β induces EMT and paclitaxel resistance in cervical cells.

<p>(a) Visible morphological changes in SiHa and CaSki under TGF-β1 or EGF-β inducement. (b) MTS analysis of paclitaxel-sensitivity in cervical cancer cells. (c) Immunoblotting analysis of Ecadherin and other EMT related proteins after TGF-β1 or EGF-β induction. (d) miR-375 expression in SiHa and CaSki under TGF-β1 or EGF-β inducement. Data in b–d were presented as mean of triplets samples from independent experiments. All error bars indicated s.e.m. (**p≤0.01, *p≤0.05).</p

Sequences of primers for miRNAs, RNU6, and EEF1A1.

<p>Sequences of primers for miRNAs, RNU6, and EEF1A1.</p

Conditionally over-expressed miR-375 induces EMT via targeting Ecadherin in cervical cancer cells.

<p>(a) Phase contrast images of cervical cancer cells infected with the pre-miR-375 or negative lentivirus vector and blank control. (b) Immunoblotting measurement of Ecadherin and EMT related proteins in cervical cancer tercells infected with the pre-miR-375 or negative lentivirus vector. (c) A predicted duplex formation between human CDH1 (Ecadherin) 3′-UTR and miR-375. A reduced luciferase activity was observed after cotransfection of Ecadherin 3′-UTR wild type vector with miR-375, but not mutated or empty vector (p = 0.014). Data in a–c were presented as the mean of triplets samples from independent experiments. All error bars indicated s.e.m. (**p≤0.01, *p≤0.05).</p

miR-375 over-expression correlates with Ecadherin expression in post-chemotherapy human cervical carcinoma tissues.

<p>(a) Ecadherin and miR-375 expression were reversely correlated in all tissues including pre- and post-chemotherapy (r = −0.905, P = 1.81×10⁻³). (b) Staining of human-specific Ecadherin expression in three representatives (1 PD and 2 PR) (×200). A decreased Ecadherin expression was shown in the tissues after chemotherapy (p = 0.0023).</p

Paclitaxel inhibits proliferation, induces EMT, and up-regulates miR-375 expression simultaneously in cervical cancer cells.

<p>(<b>a</b>) Visible morphological changes from “cobblestone”-like to “fibroblast”-like cells was observed in SiHa after 72 h paclitaxel treatment. The representative cells with morphological changes were labeled (<b>b</b>) Immunoblotting measured the expression of Ecadherin and other EMT related proteins (vimentin, Ncadherin, and fibronectin) in cervical cancer cells (SiHa and CaSki) under different amounts (0, 5, 10, 20 nM) of paclitaxel treatment. The expression of Ecadherin protein was decreased and the expressions of vimentin, Ncadherin, and fibronectin protein were increased in cells treated with paclitaxel for 72 hours in a dose-responsive manner. All the expressions of the proteins were normalized to ß-actin and the data in bar graphs are presented as mean of triple samples from three independent experiments. All error bars indicated s.e.m. (**p≤0.01, *p≤0.05). (<b>c,</b><b>d</b>) The transient cell proliferation inhibition and morphological changes induced by paclitaxel were observed. The proliferation changes in SiHa and CaSki cells after paclitaxel administration were analyzed by CellTrace CFSE cell proliferation (<b>c</b>) and MTT assay (<b>d</b>) at 24 h, 72 h, 96 h, day 5, day 7, day 14, and day 21, respectively. The proliferation rate of paclitaxel treated SiHa and CaSki cells was significantly reduced at day 7 and restored at day 21 after paclitaxel administration. The survived CaSki cells were restored into normal morphology at day 21 after paclitaxel administration.</p