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Figure 3. MicroRNA analysis of EBC collected from unrestrained animals. (A) Timeline of the weekly EBC collections from animal pairs, separated by sex (circles for females, triangles for males), between healthy control mice (blue) and lung tumor-bearing mice (red), for a period of 16 weeks. Discovery analyses were performed using total small-RNA extracted from EBC collected at weeks 0, 5, 9, and 13 using Next-Generation Sequencing (NGS). Validation analyses were conducted using total small-RNA extracted from EBC at weeks 1, 8, and 15 using quantitative reverse transcription PCR (RT-qPCR). Proteomic analyses were conducted on EBC collected and pooled for weeks 12, 14, and 16 from control and tumor-bearing mice groups; (B) PCA plots for miRNA expression of healthy control mice measured at weeks 0, 5, 9, and 13 for both females and males (top), and lung tumor-bearing mice at the same time points for both females and males (bottom); (C) Heatmap classification of the top 233 miRNAs detected by NGS using small RNAs extracted from EBC of healthy control (blue) and lung tumor-bearing (red) mice at weeks 0 (yellow), 5 (green), 9 (pink), and 13 (purple) for both females (grey) and males (black). The purple box highlights miRNAs commonly identified between condensates from control and lung tumor-bearing animals. The red box highlights miRNAs predominantly identified in condensates obtained from lung tumor-bearing animals. Two miRNAs, namely miR-374a and miR-584, which are bolded in orange text, are identified to be predominantly upregulated in condensates of lung tumor-bearing animals and are also identified to be upregulated in EBC obtained from lung tumor-bearing animals (system v2.0) displayed in Figure 7C and D. Taqman^© qPCR analyses of hsa-miR-222 and has-miR-210 using total small-RNA purified from EBC collected at weeks 1 (blue), 8 (light purple), and 15 (dark purple), separately for females and males, with data calculated using the 2^ΔΔCt formula between healthy mice (i.e., using week 1 Ct values as the reference), and lung tumor-bearing mice, both normalized to exogenous ath-miR-159a (100 pg) as an internal “housekeeping” control that was spiked in EBC before RNA extractions and qPCR analyses.