A novel homeostatic loop of sorcin drives paclitaxel-resistance and malignant progression via Smad4/ZEB1/miR-142-5p in human ovarian cancer

The primary chemotherapy of ovarian cancer (OC) often acquires chemoresistance. Sorcin (SRI), a soluble resistance-related calcium-binding protein, has been reported to be an oncogenic protein in cancer. However, the molecular mechanisms of SRI regulation and the role and aberrant expression of SRI in chemoresistant OC remain unclear. Here, we identified SRI as a key driver of paclitaxel (PTX)-resistance and explored its regulatory mechanism. Using transcriptome profiles, qRT-PCR, proteomics, Western blot, immunohistochemistry, and bioinformatics analyses, we found that SRI was overexpressed in PTX-resistant OC cells and the overexpression of SRI was related to the poor prognosis of patients. SRI was a key molecule required for growth, migration, and PTX-resistance in vitro and in vivo and was involved in epithelial–mesenchymal transition (EMT) and stemness. Mechanistic studies showed that miR-142-5p directly bound to the 3ʹ-UTR of SRI to suppress its expression, whereas a transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) inhibited the transcription of miR-142-5p by directly binding to the E-box fragment in the miR-142 promoter region. Furthermore, ZEB1 was negatively regulated by SRI which physically interacted with Smad4 to block its translocation from the cytosol to the nucleus. Taken together, our findings unveil a novel homeostatic loop of SRI that drives the PTX-resistance and malignant progression via Smad4/ZEB1/miR-142-5p in human OC. Targeting this SRI/Smad4/ZEB1/miR-142-5p loop may reverse the PTX-resistance.

Total RNAs from OVCAR-3 and OV3R-PTX cells were extracted using TRIzol reagent (Invitrogen). RNA samples were randomly assigned numbers and carried out by CapitalBio Technology (Beijing, China). Agilent human gene expression microarray V4.0 was performed according to the manufacturer's instructions. The differentially expressed genes were screened with threshold criteria of adjusted P < 0.05 and fold change ≥ 2. For proteomics analysis, cell lysates in RIPA lysis buffer were applied for mass spectrometric detection carried out at Fudan University.

Transfection of siRNA, miRNA, and plasmid and infection of shRNA lentivirus
Small interfering RNAs (siRNAs) for human SRI and ZEB1 and corresponding negative controls (NC) were synthesized in GenePharma (Shanghai, China). The miR-142-5p and miR-147a mimics, inhibitors, and corresponding controls (Ctrl) were purchased from GenePharma. SRI and ZEB1 overexpressing plasmids were constructed in mammalian expression vectors pcDNA4.1/TO/myc-His (B) and pcDNA3.1 (Invitrogen), respectively. The cells were transfected with siRNAs, mimics, inhibitors, and plasmids or their NCs using X-tremeGENE Transfection Reagent

Quantitative RT-PCR
Total RNA was extracted from cultured cells using the RNA-Quick Purification Kit (Yishan Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. Total mRNA, primary miRNA, and mature miRNA were reversely transcribed and PCR was performed as described previously.

Extraction of the total, cytoplasmic, and nuclear proteins and western blot
Total protein was extracted with SDS lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with the proteinase inhibitors (Beyotime) and phosphatase inhibitor (Nanjing KeyGen Biotech Co., Ltd.). The cytoplasmic and nuclear proteins were extracted with an extraction kit (#SC-003; Invent Biotechnologies, Inc) according to the manufacturer's instructions. Western blot analysis was performed as described previously. Immunoblots were quantified using Tanon-4500 Gel Imaging System with GIS ID Analysis Software v4.1.5 (Tanon Science & Technology Co., Ltd., Shanghai, China).

Immunohistochemical staining (IHC)
Briefly, the 4% paraformaldehyde-fixed, paraffin-embedded tissue specimens were sectioned (4 µm thick), deparaffinized in xylene, and rehydrated in a descending alcohol series. Endogenous peroxide activity was quenched by 3% hydrogen peroxide in methanol for 15 min. After blocking with 10% normal goat serum for 40 min at room temperature, the section was incubated with an anti-SRI antibody (1:300 dilution, Abcam) at 4°C overnight, followed by incubation with biotinylated anti-rabbit secondary antibody at room temperature for 1 h. After washing, the signal was detected using a DAB kit. The immunoreactive staining of SRI was evaluated by the scores of the sum of the percentage of positive cells (0, <5%; 1, 5-25%; 2, 25-50%; 3, 50-75%; and 4, >75%) and the intensity score (0, no coloration; 1, pale brown; 2, brown; and 3, dark brown). The final scores of more than 2 were considered positive.

Immunofluorescence (IF) and confocal microscopy
After plating cells onto coverslips in a 24-well plate overnight, cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After blocking with goat serum, cells were incubated with primary antibodies dilution in 3% BSA at 4°C overnight, followed by secondary antibody incubation for 1 h at room temperature in the dark. DAPI (Beyotime) was added for nuclei staining. Images were captured by a fluorescence microscope (Olympus Corporation, Tokyo, Japan). For confocal microscopy assays, cells were grown in confocal dishes (Beyotime) overnight. Following the above procedures, images were captured by a confocal laser-scanning microscope (Leica SP5, Wetzlar, Germany).

Flow cytometry
The apoptotic cells were detected by Annexin V conjugated with fluorescein isothiocyanate (BD Pharmingen) and propidium iodide (PI) complied with the manufacturer's instructions and counted by flow cytometry (Beckman Coulter, Inc.).

Paclitaxel cytotoxicity assay
Cells were seeded at a 96-well plate at a density of 1×10 4 cells/well and then treated with different doses of PTX for 48 h. Cell growth was detected using a CCK-8 kit. The relative cell viability (%) was calculated as a percentage of viable cell proportion for PTX-treated cells vs. untreated control cells.
The luciferase activity was measured after 48 h transfection.