Screening and evaluation of Smo-siRNA targeted to inhibition of Molt-4 cell proliferation

doi:10.3969/j.issn.2095-4344.0483

Abstract

Abstract:

BACKGROUND: Studies have shown that the occurrence and development of T lymphocytic leukemia is related to the abnormality of Hedgehog pathway. The Smo gene is a key gene in this signaling pathway and controls the transmission of Hedgehog signaling into the cell membrane.
OBJECTIVE: To design and screen a highly efficient and specific Smo-siRNA which is able to downregulate the Smo gene expression in Molt-4 cells, thereby inhibiting the Molt-4 cells proliferation and inducing apoptosis.
METHODS: (1) Smo-siRNAs numbered 1, 2 or 3, and the scrambled non-siRNA control (SC) were obtained by chemosynthesis. Untreated and sc-treated cells were used as controls. (2) Smo expression levels in Molt-4 cells were analyzed using qRT-PCR at 24, 48, 72 hours after siRNAs delivered by NuclefectorTM. Cell proliferation in vitro was assayed by the cell counting kit-8. The morphology and percentage of apoptotic cells were revealed by Hoechst33258 staining and flow cytometry, respectively.
RESULTS AND CONCLUSION: (1) Smo-siRNAs were successfully transferred into Molt-4 cells, and exhibited best silencing results. After transfection with Smo-siRNA1, the mRNA level of Smo was significantly reduced (P < 0.05), and the lowest level was at 48 hours after transfection. (2) Cell proliferation of Molt-4 cells was significantly inhibited by Smo-siRNA at 24 hours after transfection. (3) Hoechst staining results showed morphological changes of Molt-4 were in accordance with those of apoptotic cells. (4) The apoptotic rate was significantly increased in the Smo-siRNA group compared with the control group (P < 0.05). Findings from this study showed that suppression of Smo by RNA interference could effectively inhibit proliferation and induce apoptosis in Molt-4 cells, indicating that Smo-siRNA as gene targeted therapy or synergistic treatment has therapeutic potential in T-cell malignancies.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: RNA, Small Interfering, Leukemia, T-Cell, Cell Proliferation, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Zhu Hua-min, Wang Xu, Xu Yan, Yang Li-jian, Chen Shao-hua, Wu Xiu-li, Li Yang-qiu. Screening and evaluation of Smo-siRNA targeted to inhibition of Molt-4 cell proliferation[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2104-2108.

Figures/Tables 1

2.1 Smo-siRNA特异性抑制Smo在Molt-4 T细胞中表达水平利用转染试剂将Alexa Red Oligo转染进入Molt-4细胞，转染后6 h在倒置荧光显微镜下可见部分细胞显示红色荧光。Smo-siRNA转染Molt-4细胞为C-005程序，通过荧光显微镜和流式细胞仪检测转染效率为(60.12±10.13)%。转染24，48，72 h，各组Molt-4细胞Smo mRNA相对表达量发生变化，见图1。其中，Smo-siRNA1，2转染组Molt-4细胞Smo mRNA 相对表达量均出现下降，并且分别与空白对照组、阴性对照组相比差异均有显著性意义(P < 0.05)，其中以48 h Smo mRNA相对表达量降低最明显；而Smo-siRNA3组Molt-4细胞Smo mRNA相对表达量与空白对照组、阴性对照组相比差异均无显著性意义(P > 0.05)。 2.2 Smo-siRNA抑制Molt-4 T细胞增殖转染后24，48，72 h，CCK-8法检测Smo-siRNA作用后Molt-4细胞的生长情况，见图2。结果显示：转染后24 h，Smo-siRNA1组细胞增殖受抑制，与空白对照组、阴性对照组相比差异均有显著性意义(P < 0.05)；Smo-siRNA2，3组细胞增殖受抑制，但是与空白对照组、阴性对照组相比差异无显著性意义(P > 0.05)；转染后48，72 h，Smo-siRNA1组细胞增殖受抑制，与空白对照组、阴性对照组相比差异均有显著性意义(P < 0.05)，其中以72 h抑制作用最明显；Smo- siRNA2，3组细胞增殖受抑制，但是与空白对照组、阴性对照组相比差异无显著性意义(P > 0.05)。 2.3 Hoechst染色检测细胞凋亡形态转染Smo-siRNA1 72 h后，收集各组Molt-4细胞，进行Hoechst33258染色，荧光显微镜下观察到均可见明显的凋亡细胞，细胞核膜完整，体积变小，核质固缩，部分细胞核发生边集、破碎、裂解。空白对照组和MOCK组细胞未见明显凋亡(图3)。 2.4 流式细胞仪检测细胞凋亡率转染Smo-siRNA1序列72 h后，收集各组细胞用流式细胞仪AnnexinV/PI双染法检测细胞凋亡率，见图4。结果显示：Smo-siRNA1处理组细胞的凋亡明显增加，与空白对照组、MOCK组相比差异有显著性意义(P < 0.05)，而空白对照组、MOCK组之间相比差异无显著性意义(P > 0.05)。"

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