HOXA5 gain-of-function validation as miR-130a target

PRIMARY OUTCOME

validation of miR-130a-HOXA5 interaction

EXPECTED OUTCOMES

## Primary Outcomes **Direct Target Validation**: miR-130a inhibitor increases Firefly luciferase activity from WT HOXA5 3' UTR reporter by ≥1.8-fold vs. control inhibitor (p < 0.01). Mutation of miR-130a binding site abolishes this derepression (>85% reduction in derepression magnitude). **Endogenous HOXA5 Protein**: miR-130a inhibitor treatment elevates endogenous HOXA5 protein by ≥2.2-fold vs. control (p < 0.01, n≥4 biological replicates). HOXA5 mRNA shows modest or no change (±20%), indicating post-transcriptional regulation. ## Secondary Outcomes **Functional Consequences**: HOXA5 gain-of-function (via miR-130a inhibitor or FLAG-HOXA5 overexpression) produces: (a) reduced cell proliferation (≥25% decrease vs. control, p < 0.05), (b) increased apoptosis (≥1.5-fold caspase activation, p < 0.05), and (c) reduced cell migration (≥30% wound closure inhibition, p < 0.01), consistent with HOXA5 tumor suppressor role in cardiovascular relevant cell types.

SUCCESS CRITERIA

## Primary Success Criteria **Target Directness**: miR-130a inhibitor must produce ≥1.5-fold derepression of WT HOXA5 3' UTR reporter and simultaneous ≥2.0-fold upregulation of endogenous HOXA5 protein. The mut/miR-130a site reporter must show <30% derepression compared to WT, confirming direct miRNA:target pairing. **Specificity**: Non-target mRNAs with confirmed miR-130a regulation must also show derepression with miR-130a inhibitor (positive control). HOXA5 mRNAs without miR-130a sites must NOT respond to miR-130a inhibitor (negative control). ## Secondary Success Criteria **Concentration Response**: Dose-response curve for miR-130a inhibitor (1 nM to 100 nM) shows concentration-dependent HOXA5 protein upregulation with EC₅₀ in 5-20 nM range, consistent with LNA inhibitor pharmacology. **Rescue Experiment**: Co-transfection of miR-130a inhibitor + HOXA5 siRNA must abrogate the proliferative phenotype observed with miR-130a inhibitor alone, confirming HOXA5 as the critical mediator.

PROTOCOL

# 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).

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[HOXA5 gain-of-function validation as miR-130a target](http://scidex.ai/artifact/exp-e5b0f2a4-1681-4c91-986f-abe280cbd386)

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http://scidex.ai/artifact/exp-e5b0f2a4-1681-4c91-986f-abe280cbd386

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