研究报告: 基于AS1411适配体的DNA-RNA杂交载体的抗肿瘤研究

目的 研究连接适配体的DNA-RNA分子作为杂交载体靶向肿瘤细胞并导入功能性RNA分子进入细胞的有效性，以及对肿瘤细胞的影响。方法 设计合成短的互补DNA、RNA分子，组装成DNA-RNA杂合链；连接AS1411适配体为靶向分子，再分别连接p21 saRNA和TIGIT siRNA作为药物分子，记为P21 saRNA和TIGIT siRNA，构成杂交载体，通用结构式为AS1411-DNA/RNA-sxRNA；检测AS1411-DNA/RNA-sxRNA能否靶向结合并进入肿瘤细胞及其对瘤细胞生存、迁移、侵袭和凋亡的影响。结果 将设计的杂交载体各部分等摩尔加入杂交缓冲体系并于特定温度条件下孵育，TBM聚丙烯酰胺凝胶电泳检测到AS1411-DNA/RNA-sxRNA成功组装；AS1411-DNA/RNA-sxRNA杂交载体在10%血清条件下也显示出良好的抗降解稳定性；荧光显微镜和激光共聚焦显微镜下观察，SKOV3细胞表面及胞内存在绿色大量荧光信号，杂交载体成功进入肿瘤细胞。杂交载体孵育后：在mRNA水平上，p21基因表达（2.14±0.25）是对照组（1.02±0.10）2倍以上，P<0.05；TIGIT基因表达（0.63±0.09）低于对照组（1.09±0.15），P<0.05；在蛋白质水平上，p21基因表达（1.57±0.16）是对照组（1.10±0.09）1.5倍以上，P<0.05；TIGIT基因表达（0.61±0.12）低于对照组（1.01±0.07），P<0.05。CCK-8实验显示，P21 saRNA（3.10±0.13）和TIGIT siRNA（2.91±0.13）杂交载体孵育组与空白对照组（3.67±0.15）相比，卵巢癌细胞增殖能力显著下降（P<0.05）；划痕实验结果显示，P21 saRNA孵育组愈合率（42.53±2.90）%、TIGIT siRNA孵育组愈合率（36.23±3.43）%，明显低于空白对照组（76.47±3.64）%，P<0.05；Transwell检测迁移能力发现：P21 saRNA孵育组（128.25±5.36）、TIGIT siRNA孵育组（119.50±8.79）低于对照组（186.5±8.56）；侵袭能力：P21 saRNA孵育组（145.5±9.45）、TIGIT siRNA孵育组（112.25±5.63）也显著低于对照组（202.50±10.12），P<0.05；细胞凋亡率：P21 saRNA孵育组（11.74%±2.47%）、TIGIT siRNA孵育组（17.12%±2.04%）明显高于对照组（5.66%±1.44%），P<0.05。结论 所制备的AS1411-DNA/RNA-sxRNA杂交载体能够有效靶向肿瘤细胞，携带功能性小RNA靶向导入肿瘤细胞并调控目的基因表达，使肿瘤细胞的增殖、侵袭和迁移能力受到抑制；该结果为利用DNA-RNA偶联AS1411适配体作为靶向工具的杂交载体，靶向杀伤表面表达NCL蛋白的肿瘤细胞提供了实验基础。

Review: Anti-tumor Study of DNA-RNA Nanocarriers Linked to AS1411 Aptamer

Objective To study the effectiveness of DNA-RNA molecules linked with aptamers as hybrid vectors to target tumor cells and introduce functional RNA molecules into cells, as well as their effects on tumor cells.Methods Design and synthesize short complementary DNA and RNA molecules, and assemble them into DNA-RNA hybrid chains; connect AS1411 aptamer as targeting molecule, and then connect p21 saRNA and TIGIT siRNA as drug molecules, denoted as P21 saRNA and TIGIT siRNA , constitute a hybrid vector, the general structural formula is AS1411-DNA/RNA-sxRNA; detect whether AS1411-DNA/RNA-sxRNA can target and enter tumor cells and its effect on the survival, migration, invasion and apoptosis of tumor cells.Results Equimolar parts of the designed hybrid vector were added to the hybridization buffer system and incubated at a specific temperature. TBM polyacrylamide gel electrophoresis detected the successful assembly of AS1411-DNA/RNA-sxRNA; AS1411-DNA/RNA-sxRNA hybridized carriers are also showed good anti-degradation stability under the condition of 10% serum; observed under fluorescence microscope and laser confocal microscope, there were a large number of green fluorescent signals on the surface and in the cell of SKOV3 cells, and the hybrid carrier successfully entered the tumor cells. After the hybrid carrier incubated, at the mRNA level, the expression of p21 gene (2.14±0.25) was nearly 2 times that of the control group (1.02±0.10), P<0.05, the expression of TIGIT gene (0.63±0.09) was lower than that of the control group (1.09±0.15), P<0.05, at the protein level, the expression of p21 gene (1.57±0.16) was more than 1.5 times that of the control group (1.10±0.09), P<0.05, the expression of TIGIT gene (0.61±0.12) was lower than that of the control group (1.01±0.07) , P<0.05. The CCK-8 experiment showed that the proliferation ability of ovarian cancer cells decreased significantly (P<0.05) in the P21 saRNA (3.10±0.13) and TIGIT siRNA (2.91±0.13) groups, compared with the blank control group (3.67±0.15); the experimental results showed that the healing rate of P21 saRNA group ((42.53±2.90)%) and TIGIT siRNA group ((36.23±3.43)%) were significantly lower than those of the blank control group ((76.47±3.64)%), P<0.05; Transwell assay showed that: P21 saRNA group (128.25±5.36), TIGIT siRNA group (119.50±8.79) were lower than the control group (186.5±8.56), P<0.05. And the invasion ability: P21 saRNA group (145.5±9.45), TIGIT siRNA group (112.25±5.63) were also significantly lower than the control group (202.50±10.12), P<0.05; apoptosis rate: P21 saRNA group ((11.74±2.47)%) and TIGIT siRNA group ((17.12±2.04)%) were significantly higher than the control group ((5.66±1.44)%), P<0.05.Conclusion The prepared AS1411-DNA/RNA-sxRNA hybrid vector can effectively target tumor cells, carry functional small RNAs into tumor cells, regulate the expression of target genes, and inhibit the proliferation, invasion and migration of tumor cells. The results provide an experimental basis for using DNA-RNA conjugated AS1411 aptamer as a hybrid carrier as a targeting tool to target and kill tumor cells expressing NCL protein on the surface.