以部分磷酸根甲基化改質去氧核醣核酸之寡核苷酸透過中孔洞二氧化矽奈米粒子增強細胞內miR-21抑制之研究;Enhanced Intracellular miR-21 Suppression via Partially Phosphate-methylated DNA Oligonucleic Acid Using Mesoporous Silica Nanoparticles

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题名:	以部分磷酸根甲基化改質去氧核醣核酸之寡核苷酸透過中孔洞二氧化矽奈米粒子增強細胞內miR-21抑制之研究;Enhanced Intracellular miR-21 Suppression via Partially Phosphate-methylated DNA Oligonucleic Acid Using Mesoporous Silica Nanoparticles
作者:	蔡威霆;Cai, Wei-Ting
贡献者:	化學工程與材料工程學系
关键词:	反義寡核苷酸;寡核苷酸修飾;基因沉默;antisense oligonucleotides;oligonucleotide modifications;gene silencing
日期:	2024-07-04
上传时间:	2024-10-09 15:20:28 (UTC+8)
出版者:	國立中央大學
摘要:	微小RNA（microRNA, miRNA）是一種短小的非編碼RNA分子，其主要功能是在細胞內調控基因的表達。其中，miR-21在癌細胞中的高表達與促進細胞增殖、抑制細胞凋亡、促進侵襲和轉移等癌症相關特徵有很大的關聯性。因此針對miR-21的治療策略在癌症治療中具有重要意義。為了有效抑制miR-21在癌細胞中的表達，本研究使用反義寡核苷酸（Antisense oligonucleotides, ASOs）作為癌細胞治療手段。反義寡核苷酸是一種透過靶向特定RNA序列來調控RNA表達的分子，然而，反義寡核苷酸存在的限制包括與其靶向RNA的結合力、在體內的分布和代謝以及易被酶分解等問題。因此，我們開發一種ASOs的類型，稱為中性化DNA（Neutralized DNA, nDNA），其磷酸骨架上含有位點特異性甲基磷酸三酯（Methyl phosphotriester, MPTE）鍵，將帶負電荷的DNA磷酸二酯轉變為中性化的MPTE，並且可以設計在任意位置。nDNA減少了磷酸骨架上的負電荷，從而減少了ASO與RNA雙鏈之間的靜電排斥力，這提高了雜交親和力和雙鏈穩定性。雖然nDNA以前已被作為靈敏核苷酸測序探針(Probe)應用於聚合酶連鎖反應(Polymerase chain reaction, PCR)中，可在DNA或RNA樣本中辨別和定量特定的核苷酸序列，但nDNA在細胞內對RNA的結合與調控和基因治療中的潛力仍然未被充分探索。本研究主要在評估nDNA作為ASO 在抑制細胞內miR-21基因表達及下游基因之調控能力。在更具體的實驗設計中，本研究對nDNA進行了多種甲基化修飾，並評估了不同修飾位置對nDNA-ASO結合親和力和穩定性的影響。研究發現，當MPTE修飾位於N4-mid（修飾四個甲基在中間）位置時，nDNA對miR-21的抑制效果最佳，抑制持續時間也長達72小時。這表明精確定位的甲基化修飾不僅能提高nDNA的靶向能力，還能增強其在細胞內的穩定性。本研究證明透過調整MPTE的位置，部分甲基化的nDNA裝載到中孔洞二氧化矽奈米粒子（Mesoporous Silica Nanoparticle, MSN）上，可以有效地抑制細胞內的miRNA表達，更可以進一步在結腸癌細胞HCT116中調控下游的信使核糖核酸（messenger RNA, mRNA）表達。此外，在72小時內裝載nDNA ASO的MSN在降低miR-21水平方面，比裝載未修飾的DNA ASO的MSN表現出更高的效力。相較於未修飾的DNA ASO，nDNA ASO也導致了下游腫瘤抑制基因PTEN和PDCD4的mRNA水平增加。本研究發現，抑制miR-21後，下游的腫瘤抑制基因PDCD4和PTEN的mRNA表達顯著增強，這不僅抑制了腫瘤細胞的增殖，還促進了細胞的凋亡。本研究結果強調了nDNA在基因治療中的潛力，特別是透過精準調整甲基化位置來進行的癌症治療。這些結果顯示，nDNA不僅可以用於RNA調控，還能在癌症治療中發揮重要作用，提供了一種新的治療策略。整體來說，本研究顯示了中性化DNA在細胞內RNA調控和基因治療中的巨大潛力，尤其在癌症治療中，透過精準調整甲基化位置，可以顯著提高治療效果。本研究為未來的體內實驗提供了強而有力的基礎，並為開發新型癌症治療方法提供了重要的理論支持。;MicroRNAs (miRNAs) are short non-coding RNA molecules that play a crucial role in regulating gene expression within cells. miR-21 is highly expressed in cancer cells and is significantly associated with cancer-related characteristics such as promoting cell proliferation, inhibiting apoptosis, and enhancing invasion and metastasis. Therefore, therapeutic strategies targeting miR-21 hold great promise for cancer treatment. To effectively suppress the expression of miR-21 in cancer cells, this study employs antisense oligonucleotides (ASOs) as a therapeutic approach. ASOs are molecules that regulate RNA expression by targeting specific RNA sequences. However, limitations of ASOs include their binding affinity to target RNA, distribution and metabolism within the body, and enzymatic degradation. To address these issues, we developed a type of ASO called neutralized DNA (nDNA), which incorporates site-specific methyl phosphotriester (MPTE) linkages on its phosphate backbone, converting the negatively charged DNA phosphodiester into a neutral MPTE at designated positions. The nDNA reduces the negative charge on the phosphate backbone, thereby decreasing electrostatic repulsion between the ASO and RNA duplex, enhancing hybridization affinity and duplex stability. Although nDNA has previously been used as a sensitive nucleotide sequencing probe in polymerase chain reaction (PCR) to identify and quantify specific nucleotide sequences in DNA or RNA samples, its potential for binding and regulating RNA within cells and its role in gene therapy remain underexplored. This study focuses on evaluating the ability of nDNA as an ASO to inhibit miR-21 gene expression and regulate downstream genes within cells. In more specific experimental designs, this study introduced various methylation modifications and assessed the impact of different modification sites on the binding affinity and stability of nDNA-ASO. The study found that when MPTE modification was in the middle position, nDNA exhibited optimal miR-21 inhibition, with the effect lasting up to 72 hours. This suggests that precise localization of methylation not only enhances the targeting capability of nDNA but also improves its stability within cells. This study demonstrated that by adjusting the position of MPTE, partially and intermediately methylated nDNA loaded onto mesoporous silica nanoparticles (MSNs) effectively inhibited miRNA expression within cells and further regulated downstream messenger RNA (mRNA) expression in colon cancer cells (HCT116). Additionally, over 72 hours, nDNA ASO-loaded MSNs showed a greater reduction in miR-21 levels compared to unmodified DNA ASO-loaded MSNs. The miR-21 knockdown by nDNA ASO also resulted in increased mRNA levels of downstream tumor suppressor genes PTEN and PDCD4 compared to unmodified DNA ASO. This study found that inhibiting miR-21 levels with nDNA ASO significantly enhanced the mRNA expression of downstream tumor suppressor genes PDCD4 and PTEN, indirectly suppressing cancer cell growth. These results highlight the potential of nDNA in cancer gene therapy, through precise methylation, indicating its role in RNA regulation, offering a new therapeutic strategy. Overall, this study demonstrates the significant potential of nDNA in cancer gene therapy. Precise methylation can significantly improve RNA knockdown efficacy. This research provides a strong foundation for future in vivo experiments and offers an alternative strategy for gene cancer treatments.
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