Radiation Biology

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SU_45_2452 - miR-15a regulation of endothelial radiation-induced inflammatory cell death

Sunday, October 21
1:15 PM - 2:45 PM
Location: Innovation Hub, Exhibit Hall 3

miR-15a regulation of endothelial radiation-induced inflammatory cell death
S. Rana1, R. Ruhl1, C. Espinosa1, C. R. Thomas Jr2, and S. Anand1; 1Oregon Health & Science University, Portland, OR, 2Oregon Health and Science University, Portland, OR

Purpose/Objective(s): miR-15a targets a major determinant of endothelial cell (EC) radiosensitivity, acid sphingomyelinase (SMPD1), an enzyme known to facilitate rapid endothelial apoptosis via enhanced production of the pro-apoptotic sphingolipid ceramide, primarily after ablative doses of radiation. Apart from endothelial apoptosis regulation, recent insight in vascular miR-15a inhibition has defined its influence on immune function by increasing inflammatory cytokines and immune cell infiltrate. Within this context, we hypothesize that miR-15a inhibition will increase tumor cell death through preservation of SMPD1 and ceramide biosynthesis enhancing radiation-induced endothelial pyroptosis, or inflammatory cell death, and concomitant inflammatory cytokine upregulation.

Materials/Methods: Using TaqMan Human miRNA microarray panels, microRNAs (miRs) were profiled in human umbilical vein endothelial cells (HUVECs) treated with either single 2 Gy or 20 Gy dose via Cs-137. miR-target prediction bioinformatics were used to identified distinct miRs targeting SMPD1. In vitro gain and loss of studies were performed for miR transfections in HUVECs and CT26 colorectal carcinoma cells with RNAimax reagent and luminescence based assays for proliferation and apoptosis (Cell titer glo and Caspase Glo, Promega). CXCL10 and Fas expression was measured via qRT-PCR. Caspase 1 activation was measured using Caspase 1 Glo Inflammasome Assay. Gasdermin D (GSDMD) was measured via Western blot. A CT26 syngeneic colorectal carcinoma (CRC) flank xenograft model in BALB/c mice was used for in vivo assessment of miR-15a inhibitor, which was administered via tail vein unencapsulated or encapsulated in vascular targeted 7C1 nanoparticles. CD31 immunofluoresence staining was performed on tumor sections to measure endothelial cell area.

Results: Among miRs targeting SMPD1, miR-15a exhibited the greatest differential change in HUVECs at 6 hours post-IR between low and high dose radiation. Lower dose was associated with higher miR-15a and vice versa. Exogenous miR-15a mimic significantly decreased SMPD1 mRNA and protein expression. miR-15a levels were reciprocal in magnitude to SMPD1 mRNA levels. miR-15a inhibition decreased cell proliferation in both HUVECs and CT26 colorectal cells and increased apoptosis when combined with radiation. Associated with increased apoptosis, miR-15a inhibition significantly increased Fas expression in HUVECs compared to radiation alone. Further, miR-15a inhibition increased inflammasome-associated caspase 1, GSDMD expression, and inflammatory CXCL10 production. Both systemic and vascular targeted delivery of miR-15a inhibitor significantly diminished tumor growth and decreased endothelial cell density in vivo in our CRC flank tumor model.

Conclusion: Acting upon the vasculature, our data demonstrates miR-15a inhibition increases SMPD1 in ECs, decreases proliferation in vitro and in vivo, and stimulates pyroptosis to abrogate tumor growth.

Author Disclosure: S. Rana: Patent/License Fees/Copyright; MICRORNA INHIBITORS AS ANTI-CANCER THERAPEUTICS. R. Ruhl: None. C. Espinosa: None. S. Anand: Patent/License Fees/Copyright; MICRORNA INHIBITORS AS ANTI-CANCER THERAPEUTICS.

Shushan Rana, MD

Disclosure:
Employment
Oregon Health & Science University: Employee: Employee

Ownership
MICRORNA INHIBITORS AS ANTI-CANCER THERAPEUTICS: Patent/License Fees/Copyright

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