Tissue Slides were deparaffinized with subsequent washes in Xylene, 100% Ethanol, 90% Ethanol, 70% Ethanol, PBS, 3% Hydrogen peroxidase in methanol and dH2O. Pre-Hybridization was conducted by slides incubation with Hybridization Solution (Exiqon). 100 nM of custom LNA™ detection probe for MALAT1 (/5BiosG/ATGCTACTCTTCTAAGTCTTCA, Exiqon) was added to fresh Hybridization buffer 1X, heated at 95 °C for 4 min, and incubated on the slides at 54 °C overnight (in humidifying chamber). Subsequently, the tissue slides were washed with 5x, 1x and 0.02X SSC (saline-sodium citrate buffer) at 54 °C and after quickly with PBS. 2.5% horse serum was used for blocking, then slides were washed with Avidin D solution and then Biotin. The primary antibody was then added (Anti-Biotin Mouse for Malat1, Exiqon) and slides incubated at 4 °C overnight. Pan Specific antibody was used as secondary antibody while ABC Vectastain kit and DAB kit were used to visible stain the RNAs. Mayer’s Haematoxylin was used to visible stain the cell nuclei. The slides were then paraffinized, scanned and sent to the histopathologist for expression scoring.
Recombinant pmirGLO Dual Luciferase Plasmids production
To clone our target sequence Binding site A and B into a plasmid (pmirGLO Dual-Luciferase miRNA Target Expression Vector - Promega E1330), we synthesized two partially complementary oligoes for each with 4 nt overhangs compatible for cloning into the vector plasmid paying careful attention to the 5′ > 3′ orientations. When annealed, oligonucleotides form double stranded DNA with overhangs for cloning into Xbal site in pmirGLO vector. We used this protocol for obtain 3 different recombinant plasmids: pmirGLO-MALAT1 plasmid for binding site A, pmirGLO-MALAT1 plasmid for binding site B and also pmirGLO-MALAT1 plasmid with mutated binding sequence (negative control). The steps are explained below in detail.
Annealing and cloning procedure:
Anneal each pair of oligonucleotides: 1 μl oligo Fw (100 μM) + 1 μl oligo Rev. (100 μM) + 8 μL H2O for 10 μL total. Anneal in a thermocycler at 95 °C 5 min and then leave on the bench at RT to cool down for 1 h. Dilute the annealed oligonucleotides 1:200. The oligo sequences used are shown in Table 1.
Digestion vector reaction: linearize the pmirGLO vector with the appropriate restriction enzymes to generate overhangs that are complementary to the annealed oligonucleotide overhangs. 1 μg pmirGLO vector + 2 μl 10X RE Buffer + 1 μl Xbal (Promega- R6181) + Nuclease Free water to final volume of 20 μL. Incubate the digestion reaction at 37oC for at least 1 h. Run ~ 200 ng on agarose gel to ensure complete digestion. When digest is complete, heat inactivate at 65 °C for 20 min.
Ligation reaction: 100 ng pmirGLO Xbal digested vector + 2 μl annealed oligonucleotides duplex from step 1 (1:200 dilution) + 2 μl 10x DNA ligase buffer + 1 μl T4 ligase (T4 DNA Ligase- New England Biolabs) + Nuclease Free water to 20 μl final volume. Incubate the ligation reaction at 16 °C for 30 min and then at 65 °C for 10 min.
The pmirGLO-MALAT1 Dual Luciferase Plasmids obtained in the previous step were amplified individually in competent cells. First, the cells were thawed on ice for 5 min and mixed before transferring 50 μL to a chilled sterile polypropylene culture tube. Twenty-five nanograms of pmirGLO-MALAT1 plasmid was added to the competent cells and placed on ice for 10 min. Cells were heat-shocked at 42 °C for 45 s, placed on ice for 2 min, added to 450 μL of lysogeny broth (LB; Sigma-Aldrich) containing 100 μg/mL ampicillin (Sigma-Aldrich) and were shaken at 37 C° for 60 min. All 500 μL was plated on ampicillin containing LB-Agar plates overnight, and afterwards clones were isolated and shaken overnight at 37 °C in 5 mL of LB broth containing ampicillin. Plasmid was extracted using the QIAprep® Miniprep (QIAGEN). Cells were pelleted in a centrifuge and resuspended in 250 μL of Buffer P1. Two hundred fifty microliters of Buffer P2 was added and mixed by inverting the tube, and then 350 μL of neutralization solution (N3) was added and mixed by inverting the tube. Cells were centrifuged for 10 min at 14,000 x g. The supernatant was transferred to a QIAprep 2.0 spin column and centrifuged for 1 min at 12,000 x g. The flow through was discarded and two washes of 500 μL wash solution was added and the column centrifuged for 1 min at 12,000 x g each time. The plasmid was eluted from the column by adding 50 μL of elution buffer, waiting 2 min and centrifuging the columns at t 12,000 x g for 2 min. The concentration of the plasmid was then measured using a NanoDrop ND1000.
Dual luciferase assay
The Dual-Luciferase Reporter Assay System (Promega) was used to detect firefly and renilla luciferase from the pmirGLO-MALAT1 A/B plasmids and pmirGLO-MALAT1 Mut (mutated site) plasmid in Mimic miR-423-5p LNCaP stable cell line or LNCaP pLKO.1-puro (empty vector). Twenty-four hours after transduction with recombinant plasmids (separately) using Lipofectamine 3000, the cells were washed with PBS in preparation for lysis. Cells were no more than 95% confluent at the time of lysis. One hundred microliters of Passive lysis Buffer was added to each well and the plate was rocked gently for 15 min. One hundred microliters of Luciferase Assay Reagent II was added in each well and after 15 min the measurement for firefly luciferase activity was done with Luminometer Infinite M200 Pro TECAN. Then, 100 μL of Stop & Glo reagent was added and renilla luciferase activity was measured after 15 min. Each sample was read twice, and luciferase activity (firefly luciferase / renilla luciferase) was normalized to the LNCaP pLKO.1-puro (empty vector).
Proximity ligation assay
PLA assay was performed using the Duolink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich, St Louis, MO, USA) and following the manufacturer recommendations.
Cells were washed once with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde (v/v) in PBS on ice for 30 min. The samples were permeabilized with 70% Ethanol (v/v) in Nuclease Free Water overnight at + 4 °C and washed three times with PBS. The samples were then pre-hybridized with Hybridization solution 2X (Exiqon) for 6 h at 54 °C (in humidify chamber). One hundred nanometers of specific oligonucleotide probes (Custom LNA™ detection probe for MALAT1 and miRCURY LNA miRNA Detection Probe, Exiqon) were added to fresh Hybridization buffer 1X, heated at 95 °C for 4 min, and incubated on fixed/permeabilized cells at 54 °C overnight (in humidify chamber). Subsequently, the cells were washed with 0.02X SSC (saline-sodium citrate buffer) for 1 h at 54 °C and after quickly with PBS. The samples were incubated with 2/3 drops of blocking solution (Duolink® PLA, Sigma-Alderich) for 1 h at 37 °C (in humidify-chamber) and then with the appropriate primary antibodies in Diluent Antibody solution (Duolink® PLA, Sigma-Alderich) at 4 °C overnight. The probe solution was prepared by diluting the corresponding species-specific minus PLA probe and plus PLA probe (Duolink® PLA, Sigma-Alderich) 1:5 into Duolink Antibody Diluent. After two washes with 1X Wash buffer A, the coverslips were transferred to a prewarmed humidified chamber and incubated with the probe solution for 1 h at 37 °C.
The subsequent PLA ligation and amplification steps were performed according to the manufacturer’s instructions (Duolink® PLA, Sigma-Alderich). Briefly, the PLA probe solution was aspirated from the cells and washed twice with wash buffer A for 5 min each under gentle agitation. Cells were incubated with freshly prepared ligation mix for 30 min at 37 °C. Samples were then washed twice with wash buffer A for 5 min each with gentle agitation and incubated with freshly prepared amplification mix for 100 min at 37 °C. To enhance the signal intensity, the amplification step can be extended to 2–4 h if needed. Cells were washed twice with wash buffer B for 10 min with gentle agitation and a final wash with 0.01× wash buffer B for 1 min. Coverslips were mounted onto glass slides using Duolink In Situ Mounting Medium with DAPI. Images were captured by Leica TCS SP5 fluorescent microsystems using appropriate filters. All images were taken with the same exposure time and the same threshold to allow subsequent quantitative analysis in the respective channel. At least 50 nuclei per sample were imaged and the images were processed with ImageJ. Experiments were repeated three times.
NanoString nCounter gene expression analysis
A total of 730 cancer-related human genes and 40 internal reference genes were contained on cancer pathway panel reporter and capture probe. For analysis, LNCaP pLKO.1-puro and LNCaP Mimic miR-423-5p cells were used. One hundred nanograms of total RNA was used for hybridization, and analyzed on an nCounter Digital Analyzer (NanoString Technologies, Inc., Seattle, WA) according to the manufacturer’s instructions. Briefly, a hybridization reaction was set up at room temperature for each sample with the following components: 3 μL of Reporter CodeSet, 5 μL of hybridization buffer, up to 5 μL (100 ng) of sample RNA, and 2 μL of Capture ProbeSet. Briefly the tubes were spined down and immediately placed in the pre-heated 65 °C thermal cycler for at least 16 h. When the hybridization time is up, two of the 96 well plates were centrifuged at 670 g for 2 min brake off and the Prep Station machine was initiated. Data quality control was implied using nSolver analysis software (NanoString Technologies). Reporter counts were normalized to each sample using positive control and housekeeping genes. The mean values were shown to fold change or log2 transformation.
Gene ontology (GO) analysis
Metascape.org was used to enrich genes for GO biological processes and pathways.
The software identified all statistically enriched terms (GO Biological Processes, GO Cellular Components and GO Molecular Functions), accumulative hypergeometric p-values and enrichment factors were calculated and used for filtering. Remaining significant terms were then hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. Then 0.3 kappa score was applied as the threshold to cast the tree into term clusters.
Tumour transplantation assay
Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were acquired (Charles River, U.K.) and kept in accordance with the Animals (Scientific Procedures) Act 1986. The mice were used for tumour transplantation Assay (Xenotransplantation assay) by subcutaneous injection of PC3 control (PC3 Control/Luc2) or PC3 miR-423-5p-mimic (PC3 miR-423-5p-mimic/Luc2 (3 × 106 suspended in 100ul PBS). Tumours’ growth and metastases’ formation were monitored using a Perkin Elmer™ bioluminescence-based in vivo imager.
Bioinformatic and statistical analyses
In silico validation was performed by analysing the gene expression of MALAT1 with clinical parameters using the following datasets: Lapointe (n = 26) , Tomlins (n = 75) , Varambally (n = 19)  and The Cancer Genome Atlas (TCGA) (n = 281) . The gene expression profiles were generated using gene expression microarrays (Lapointe, Tomlins and Varambally) and RNA-sequencing (TCGA). The gene expression of MALAT1 was selected and min-max normalised across the complete patient population within each dataset. Gene expression of MALAT1 across healthy, primary, and metastatic prostate cancer was compared using datasets of Lapointe, Tomlins and Varambally. A comparison of MALAT1 expression wit Gleason scoring (GS6, GS7, GS8 and GS9 + 10) and disease-recurrence was performed using the TCGA dataset. Unpaired t-test was used to analyse differences between the sample groups, comparing disease status and Gleason score. Kaplan-Meier curves were generated presenting disease-free survival (DFS) in relation to MALAT1 expression. Patient groups were separated according to median expression and survival curves were analysed using Mantel-Cox test. P-values below 0.05 were considered to be significant (p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = *** and p ≤ 0.0001 = ****). Analysis was performed using GraphPad Prism 8. The in silico identification miR-423-5p binding sites on MALAT-1 was analysed using TargetScan (http://www.targetscan.org/vert_71/).
For TMA analysis all data are presented as the mean ± SEM for at least three independent experiments. For each experiment, the statistical tests are indicated in the Results section. The student’s t-test was conducted using Prism 8 (Graphpad Software, La Jolla, CA, USA). Significant (‘*’/p = < 0.05), very significant (‘**’/p = < 0.01), highly significant (‘***’/p = < 0.001) or very highly significant (‘****’/p = < 0.0001). Spearman’s Rho bivariate analysis and X squared test of contingency were performed using IBM SPSS Ver. 25, p-value < 0.05 is considered statistically significant.
Other statistical analyses were performed using the GraphPad Prism 8 student t-test and Anova.