Apatite nanoparticle(sCA)-siTIMP1 delivery in NIH3T3
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PROVIDER: PRJNA526507 | ENA |
REPOSITORIES: ENA
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Project description:Expression profiling of sCA-siRNA delivery in NIH3T3 cells (siControl vs. siTIMP1) Goal was to determine the off target effects of sCA-siTIMP1 in NIH3T3 cells.
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Project description:Purpose: A super carbonate apatite (sCA) nanoparticle is an in vivo pH-sensitive delivery system for siRNA and microRNA. These carriers accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Systemic administration of sCA incorporating siRNA and microRNA has demonstrated superb tumor suppressive effects in vivo. We recently observed that sCA could deliver abundant nucleic acids to the inflammatory sites in rheumatoid arthritis mouse model. Based on the success, we tried to examine whether sCA could deliver sufficient amounts of miRNA into the colorectum inflamed by dextran sodium sulfate (DSS) treatment. Methods: We performed a RNA sequencing analysis of the DSS-treated colon walls. DSS was administered for 4 days and sCA-miR-29a, sCA-miR-29b, sCA-NC-miR was injected on days 1, 2, 3. On day 4, colorectum was removed and the mRNA samples were subject to the RNA sequencing analysis. Results: RNA sequencing of the rectum samples showed a number of enhanced or reduced gene expression in DSS treated NC-miR group on day 4 compared to normal mice. Such tendency of upregulation or downregulation was also noted in DSS-treated NC-miR group on day 2. Comparison of DSS treated samples on day 4 among NC-miR, miR-29a and miR-29b groups, revealed that several gene expression related to the interferon pathway was reversed by miR-29a or miR-29b towards the normal controls. These include Stat1, Stat2, IRF7, IRF9, and IFIT1. Conclusions: Many molecules in the interferon signaling pathway were activated in DSS-induced colitis on day 4 and Stat1, Stat2, IRF7, IRF9, and IFIT1 were key molecules in the interferon related pathways. These findings suggest that sCA-miR-29a or sCA-miR-29b may inhibit type 1 IFN and type 2 IFN pathways which are otherwise activated by DSS treatment.
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Project description:RNA interference (RNAi) has immense potential to modulate cell functions. However, effective delivery of siRNA while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next-generation RNA sequencing (RNA-seq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a 3-fold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ~14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 h after treatment, while 11% of the treated population was annexin V+-siRNA-. RNA-seq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor (TLR), retinoic acid-inducible gene 1 (RIG-I)-like receptor, and nuclear factor-κB (NF-κB) signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the MSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.
2016-09-30 | GSE87497 | GEO
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