Quantitative Analysis of HOTAIR and miR-193a Expression

ISH and IHC were performed and subsequent results were analyzed as previously described [29 (link)]. In brief, the triple digoxigenin-labeled antisense locked nucleic acid (LNA)-modified probes for HOTAIR and miR-193a were synthesized by Boster Biotech (Wuhan, China). ISH was conducted according to the manufacturer’s instruction of the HOTAIR and microRNA ISH Optimization Kits (Boster, Wuhan, China). IHC was performed using anti-EZH2 antibody (1:250, Abcam, Cambrige, MA, USA) according to the manufacturer’s instructions. The original magnification: ×200.The specific evaluation of gene expression via ISH or IHC was calculated as previously described [30 (link)]. In brief, sections with no labeling or less than 5% labeled cells were scored as 0, 5%–30% of cells as 1, 31%–70% of cells as 2, and ≥71% as 3. The staining intensity was scored similarly using 0 for negative staining, 1 for weakly positive, 2 for moderately positive, and 3 for strongly positive. The scores of percentage of positive tumor cells and staining grade were calculated to generate the immune-reactive score for each specimen. A combined score of 0–1 indicates negative expression (−), 2–3 indicates weak expression (+), 4–5 indicated moderate expression (++), and 6 indicates strong expression (+++). Each sample was examined separately and scored by two pathologists [30 (link)].