ABSTRACT: Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been analysed. Herein, analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by approximately 80% in a murine liver metastasis model. Animals: Balb/c mice (6- to 8-week-old males) were obtained from Charles River Laboratories Spain SA (Barcelona, Spain). All the procedures were approved by the Ethical Committee for Animal Experimentation (CEEA) of the University of the Basque Country (EHU/UPV) in accordance with institutional, national and international guidelines regarding the protection and care of animal use for scientific purposes. Mice were kept in the animal facility of EHU/UPV and had access to standard chow and water ad libitum. Colorectal cancer cells: Murine colorectal cancer C26 cells (ATCC, Manassas, VA) syngeneic with Balb/c mice were used. The cells were grown under standard conditions in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 microg/ml) and amphotericin B (0.25 microg/ml), all purchased from Life technologies, Carlsbad, CA. Control and tumor-activated LSECs culture: Control and tumour activated primary cultures of LSECs were isolated form livers with colorectal cancer metastasis or from healthy ones. To do so, Balb/c mice were anesthetized with isofluorano and small cut done in their left broadside. Then, 50 microl for each mice of C26 colon carcinoma cells were inoculated in the spleen at 2 x106 cells/ml concentration and the peritoneum and skin were sutured. The control mice were inoculated with PBS 1x buffer. Fourteen days later, all mice were sacrificed and the tumour activated LSECs and control LSECs were isolated by differential centrifugation. To do so, mice were perfused with collagenase P from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) through cava vein. The obtained cell suspension was twice centrifuged resulting in a parenchymal (PC)-enriched pellet and a non PC-enriched supernatant. Then, the non PC-enriched supernatant was layered on Percoll gradients (25% on top of 50%) to obtain LSEC. After a centrifugation the interphase between the two density cushions was recollected with purified non-PC enriched LSECs and Kupffer cells (KC). Then, the KCs were separated by adherence and isolated LSECs were washed and cultured with RPMI 1640 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% FBS and used in different experiments 24 hours after the isolation. (Life technologies, Carlsbad, CA). miRNA microarray analysis : The purified total RNA was analysed using Agilent mouse miRNA microarrays. Release 18.0, 8x60K (v18) microarray slides (Agilent Technologies, USA), with 1,200 mouse miRNAs represented, were used. Briefly, 100 ng of total RNA was labelled and hybridized following the standard Agilent Protocol for the miRNA Microarray System with a miRNA Complete Labelling and Hybridization Kit, including Agilent miRNA Spike-ins, and the results were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies, USA). The scanned TIFF image files were processed using Agilent Feature Extraction Software vs 10.7.3.1 to extract raw data and obtain QC reports. The microarray raw data were normalized using the quantile method. To find the statistically significant differentially expressed miRNAs (DEMs) between two groups of samples, we calculated the mean of each probe across all the samples of the group. Next, we filtered out all the probes whose absolute value of the difference of mean values between the two groups was less than a selection threshold thetaDEM = 4 (that corresponds to a fold change of 2 in log2 scale), and we applied a Student's t-test with a significance threshold alphaDEM = 0.05. The PCA and the hierarchical clustering of genes and samples were performed with one minus the correlation metric and the unweighted average distance (UPGMA) linkage method. All the analysis algorithms and graphics were implemented functions in Matlab.
