HOXA5 Gain-of-Function Validation as miR-130a Target Protocol
Phase 1: Plasmid Construction and Virus Production (Days 1-14)
Expression Vector Construction: Clone full-length human HOXA5 (NM_001518) into AAV2/9 expression plasmid under CMV promoter (pAAV-CMV-HOXA5-IRES-eGFP). Verify insert via Sanger sequencing (bidirectional). Generate untagged HOXA5 and FLAG-HOXA5 fusion constructs for separate experimental arms.
viral Production: Co-transfect HEK293T cells (80% confluent, 10 cm dishes) with pAAV-CMV-HOXA5, pAAV2/9 helper, and pXR5 rep/cap plasmids using PEI (1:3 DNA:PEI ratio). Harvest viruses at 72 hours, concentrate via iodixanol gradient ultracentrifugation. Titer via qPCR (genome copies/mL, target ≥10¹² GC/mL).
...HOXA5 Gain-of-Function Validation as miR-130a Target Protocol
Phase 1: Plasmid Construction and Virus Production (Days 1-14)
Expression Vector Construction: Clone full-length human HOXA5 (NM_001518) into AAV2/9 expression plasmid under CMV promoter (pAAV-CMV-HOXA5-IRES-eGFP). Verify insert via Sanger sequencing (bidirectional). Generate untagged HOXA5 and FLAG-HOXA5 fusion constructs for separate experimental arms.
viral Production: Co-transfect HEK293T cells (80% confluent, 10 cm dishes) with pAAV-CMV-HOXA5, pAAV2/9 helper, and pXR5 rep/cap plasmids using PEI (1:3 DNA:PEI ratio). Harvest viruses at 72 hours, concentrate via iodixanol gradient ultracentrifugation. Titer via qPCR (genome copies/mL, target ≥10¹² GC/mL).
Target Validation Reporter Construction: Clone full-length 3' UTR of human HOXA5 (包含 miR-130a binding sites) into psiCHECK-2 reporter vector (Promega). Generate mutant reporter with 4 bp seed-region deletion in miR-130a binding site. Verify via sequencing.
Phase 2: miR-130a Target Inhibition (Days 15-21)
miR-130a Inhibition: Synthesize and transfect hsa-miR-130a-3p inhibitor (miRCURY LNA, Qiagen, 50 nM final concentration) into HEK293T or HeLa cells. Include negative control inhibitor (Qiagen #1027324). For rescue experiments, co-transfect miR-130a inhibitor + FLAG-HOXA5 expression plasmid (10:1 ratio).
Luciferase Reporter Assay: At 24 hours post-transfection, seed cells in 96-well plates (10,000 cells/well). At 48 hours, transfect with psiCHECK-2-WT or psiCHECK-2-MUT reporters (100 ng/well). At 72 hours, lyse cells and measure Firefly/Renilla luciferase (Dual-Glo kit, Promega). Calculate relative reporter activity (Firefly/Renilla normalized to empty vector baseline).
Phase 3: Endogenous HOXA5 Upregulation by miR-130a Inhibitor (Days 22-28)
miR-130a Inhibitor Treatment: Treat HeLa cells (6-well plates, 70% confluent) with miR-130a inhibitor (50 nM) or negative control for 48 hours. Collect cells for RNA and protein analysis.
HOXA5 Expression Analysis: Extract RNA (Trizol), synthesize cDNA (High-Capacity RT kit), and perform qRT-PCR for HOXA5 (TaqMan #Hs00394156_m1) and GAPDH endogenous control (ΔΔCt method). For protein, lyse cells in RIPA buffer and probe western blot with anti-HOXA5 (1:500, Abcam ab115328) and anti-β-actin (1:5000).
Functional Downstream Validation: Assay phenotypically relevant endpoints: cell proliferation (CellTiter-Glo, 72-hour timecourse), apoptosis (Caspase-Glo 3/7, n=4), and migration (wound closure assay, 24-hour timeframe).