BackgroundTriple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required.ResultsHere, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils.ConclusionsUsing a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0228-7) contains supplementary material, which is available to authorized users.
Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E–3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E–3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.
Background Over 96% of high‐grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63 , which shares many of the downstream targets and functions of p53 , is rarely mutated in cancer. Methods A novel strategy is presented for circumventing alterations in p53 by inducing the tumor‐suppressor isoform TAp63 (transactivation domain of tumor protein p63 ) through its direct downstream target, microRNA‐130b (miR‐130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. Results Treatment with miR‐130b resulted in: 1) decreased migration/invasion in HEYA8 cells ( p53 wild‐type) and disruption of multicellular spheroids in OVCAR8 cells ( p53 ‐mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B‐cell lymphoma ( Bcl )‐inhibitor B‐cell lymphoma 2‐like protein 11 ( BIM ). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR‐130b. In vivo, combined miR‐130b plus CDDP exhibited greater therapeutic efficacy than miR‐130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR‐130b in 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP‐treated mice, and 20% of DOPC–miR‐130b plus CDDP‐treated mice were living tumor free. Systemic injections of scL–miR‐130b plus CDDP in a clinically tested, tumor‐targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. Conclusions The miR‐130b/ TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad‐spectrum therapeutic options for the majority of p53 ‐altered cancers.
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