| 摘要: | 微小RNA（microRNA，miRNA）是調控基因表達的重要分子。在癌症生物學中，miRNA的表達失調極為常見，並且對腫瘤的發展、轉移以及治療抗藥性具有重要影響。近年來，眾多臨床試驗已經展開，旨在評估基於miRNA調控治療方法的安全性與有效性。
在眾多miRNA中，miR-21和miR-155因其在多種癌症類型中的表達異常而引起了廣泛關注。這些miRNA參與調控細胞增殖、凋亡、遷移、侵襲及腫瘤微環境互動等多個生物學過程。因此，我們認為，若能同時針對miR-21和miR-155進行調控抑制，將能在癌症治療中展現出相當大的潛力。
本研究利用反義寡核苷酸（Antisense oligonucleotides, ASO）抑制癌細胞中miRNA的表達。ASO是經過化學修飾的小型寡核苷酸，能以序列特異性結合目標RNA，從而改變蛋白質表達。不同的修飾可提高ASO的穩定性、標靶親和力、生物利用度，並增強細胞攝取。我們開發的一種新型ASO，稱為中性化DNA（Neutralized DNA, nDNA）。此ASO在磷酸骨架上引入甲基磷酸三酯鍵（Methyl phosphotriester, MPTE），將負電荷的磷酸二酯結構轉化為中性結構，此修飾可減少雜交時的靜電排斥力，進而增強與目標RNA的雜交親和力及雙鏈穩定性，期待獲得更佳的調控行為。而此研究為了將nDNA送入細胞中，我們則是選用中孔洞二氧化矽奈米粒子（Mesoporous Silica Nanoparticle, MSN）作為載體。
本實驗室先前已評估過nDNA對miR-21及其下游基因的調控能力，接下來將進行miR-155的調控評估，更重要的是，本研究將進一步探討使用nDNA同時調控兩種miRNA的可行性。
本研究首先對miR-155反義股的nDNA進行不同數量和位置的甲基化修飾，評估其對雜交親和力及穩定性的影響。結果顯示，當甲基化修飾位於N2-2G（兩個甲基修飾位於序列中的鳥嘌呤上）時，對人體血漿培養基中的miR-155逆轉錄反應產生最佳抑制效果，且相比一般DNA，其抑制效果更為突出。隨後，我們證實將此nDNA裝載於MSN後，能順利進入細胞並有效抑制miR-155的表達。抑制miR-155後，下游腫瘤抑制基因TP53INP1和PTEN的信使RNA（mRNA）表達量顯著上升，進而抑制腫瘤生長，從而降低癌細胞的存活率。
接著，我們進一步使用nDNA進行miR-21和miR-155的共抑制實驗。結果顯示，細胞中兩種miRNA的表達量隨對應ASO的濃度而變化，而下游腫瘤抑制基因PTEN、RECK和TP53INP1的表達量也隨之上升。特別值得注意的是，PTEN是miR-21和miR-155的共同下游靶標，在共抑制實驗中其mRNA表達量的上升比起單獨抑制時更為顯著，證明我們成功實現了兩種miRNA的共同抑制。進一步的癌細胞存活率分析發現，相比於miR-21，miR-155對HCT116細胞的抑制效果更為明顯。我們推測，這一差異可能與下游蛋白標靶有關。miR-21主要影響細胞增殖、遷移和侵襲，而miR-155則直接靶向與細胞凋亡相關的TP53INP1，使得在短時間內抑制miR-155比miR-21更有效地減少細胞存活。在細胞計數的實驗中，這一推測也得到了初步驗證。
綜上所述，本研究展示了中性化DNA作為反義寡核苷酸（ASO）以及對多種miRNA共同調控的巨大潛力，為未來開發更高效、精準的癌症治療方法提供了實驗基礎和新的思路。
;MicroRNAs (miRNAs) are important molecules that regulate gene expression. In cancer biology, miRNA expression dysregulation is common and significantly impacts tumor development, metastasis, and therapeutic resistance. In recent years, numerous clinical trials have been conducted to evaluate the safety and efficacy of miRNA-based therapies.
Among various miRNAs, miR-21 and miR-155 have attracted widespread attention due to their abnormal expression in many cancer types. These miRNAs are involved in regulating several biological processes, including cell proliferation, apoptosis, migration, invasion, and interactions within the tumor microenvironment. Therefore, we believe that simultaneously targeting and inhibiting miR-21 and miR-155 has great potential in cancer therapy.
This study utilizes antisense oligonucleotides (ASOs) to inhibit miRNA expression in cancer cells. ASOs are small oligonucleotides that have been chemically modified to bind to target RNA sequences with high specificity, thereby altering protein expression. Different modifications can improve ASO stability, target affinity, bioavailability, and enhance cellular uptake. We developed a novel ASO called neutralized DNA (nDNA). This ASO introduces methyl phosphotriester (MPTE) linkages into the phosphate backbone, converting the negatively charged phosphodiester structure into a neutralized form. This modification reduces electrostatic repulsion during hybridization, enhancing hybridization affinity and duplex stability with the target RNA. To deliver nDNA into cells, we used mesoporous silica nanoparticles (MSNs) as the carrier.
Our lab previously evaluated the regulatory effects of nDNA on miR-21 and its downstream genes. We will next assess the regulation of miR-155 and further explore the feasibility of co-targeting both miRNAs with nDNA.
This study initially modified the antisense strand of miR-155 nDNA with different quantities and positions of methylation to assess its impact on hybridization affinity and stability. The results revealed that when methylation was introduced at N2-2G (two methyl modifications at guanine residues within the sequence), it produced the optimal inhibition effect in the reverse transcription reaction of miR-155 in human plasma culture medium, with a significantly stronger inhibitory effect compared to unmodified DNA. Subsequently, we confirmed that loading this nDNA onto MSN allowed it to successfully enter cells and effectively suppress miR-155 expression. After inhibiting miR-155, the mRNA expression levels of downstream tumor suppressor genes TP53INP1 and PTEN were significantly elevated, which in turn suppressed tumor growth and dramatically reduced cancer cell viability.
Next, we further investigated the co-suppression of miR-21 and miR-155 using nDNA. The results showed that the expression levels of both miRNAs decreased with increasing concentrations of the corresponding ASO, while the mRNA expression of downstream tumor suppressor genes PTEN, RECK, and TP53INP1 also increased accordingly. Notably, PTEN is a common downstream target of both miR-21 and miR-155. Upon dual inhibition, its mRNA expression increased more significantly than with the inhibition of either miRNA alone, demonstrating the successful simultaneous suppression of both miRNAs. In further cancer cell viability assays, we found that miR-155 was more effective than miR-21 in inhibiting the survival of HCT116 cells. We hypothesize that this difference may be related to their downstream protein targets. MiR-21 primarily affects cell proliferation, migration, and invasion, while miR-155 directly targets TP53INP1, a protein involved in apoptosis. This suggests that inhibiting miR-155 in the short term is more effective than miR-21 in reducing cell viability. This hypothesis was preliminarily validated through cell count assays.
In conclusion, this study demonstrates the immense potential of neutralized DNA as an antisense oligonucleotide (ASO) platform and its capability for the simultaneous regulation of multiple oncogenic miRNAs. These findings provide a strong experimental foundation and novel insights for the development of more efficient and precise miRNA-targeted cancer therapeutics. |
