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Title: [Lentivirus media miR-1246 knockdown inhibits tumor growth and promotes apoptosis of SiHa cells].
Author: Du P, Lai YH, Yao DS, Lu Y, Chen JY, Ding N.
Journal: Zhonghua Fu Chan Ke Za Zhi; 2018 Jul 25; 53(7):481-486. PubMed ID: 30078258.
Abstract:
Objective: To study the effect of lentivirus-mediated microRNA (miR) -1246 RNA interference (RNAi) on biological characteristics and behaviors in cervical cancer cells as well as to identify the downstream signaling pathways affected. Methods: MiR-1246 specific cDNA was synthesized and cloned into the recombinant lentiviral vector (LV-miR-1246-inhibitor) . The SiHa cells were devided into three groups: no viral infection (negative control, NC) , infection with control virus (LV-NC) , and infection with miR-1246-inhibitor virus (LV-miR-1246-inhibitor) . The expression of the miR-1246 was detected by reverse transcription (RT) -PCR. Cell growth was analyzed by cell counting kit 8 (CCK-8) assay. The invasion was dectected by transwell matrige gel. Cell apoptosis was detected by flow cytometer. The growth of xenograft tumors was also investigated. Expression of thrombospondin-2 (THBS2) , matrix metalloproteinase (MMP) 2, 9 were also evaluated in the cells. Results: (1) The expression level of miR-1246 in SiHa cells (0.11±0.13) was significantly lower in group LV-miR-1246-inhibitor than those in the group LV-NC and the group NC (1.14±0.86 and 1.30±0.73, respectively; P<0.01) . (2) The proliferation of SiHa was also markedly suppressed in CCK-8 at 96 hours (P<0.01) . (3) The number of group LV-miR-1246-inhibitor was significantly less than those in the LV-NC and NC groups in transwell invasion assay (71±4, 162±5 and 188±5, respectively; P<0.01) . (4) The apoptosis rate of SiHa cells in the group LV-miR-1246-inhibitor [ (16.10±3.37) %] was significantly lower than those of group LV-NC and group NC [ (5.67±0.89) % and (1.78±0.08) %,P<0.01]. (5) The tumor volume in the nude mice group LV-miR-1246-inhibitor [ (287±59) mm(3)] was significantly lower than those in the LV-NC and NC groups [ (571±137) and (657±144) mm(3), respectively; P<0.01]. (6) Compared with the LV-NC group and the NC group, THBS2 protein expression in the tumor tissue of the nude mice in the group LV-miR-1246-inhibitor was significantly increased (P<0.05) , while the expression levels of MMP-2 and MMP-9 protein were significantly decreased (P<0.01) . Conclusion: These results suggest that miR-1246 functions during cervical cancer pathogenesis and tumor formation via the THBS2, MMP signaling pathway. 目的： 探讨慢病毒介导的微小RNA（miR）-1246表达下调后，对子宫颈癌细胞系SiHa细胞生物学行为的影响，以及对其下游蛋白表达的调控作用。 方法： 针对人miR-1246的靶序列合成含有反向互补序列的寡核苷酸片段并克隆至慢病毒载体，构建重组慢病毒LV-miR-1246-抑制物（inhibitor）。实验分为3组，即抑制组（感染重组慢病毒LV-miR-1246-inhibitor）、阴性对照组（LV-NC组，感染空载体病毒颗粒）和空白对照组（NC组，无干预措施仅加培养基）。采用逆转录（RT）-PCR技术检测3组SiHa细胞中miR-1246的表达水平，活细胞计数（CCK-8）法、体外侵袭实验、流式细胞仪分别检测3组SiHa细胞的增殖、侵袭、凋亡情况。建立子宫颈癌裸鼠移植瘤模型，观察miR-1246表达下调对移植瘤生长的影响，蛋白印迹（western blot）法检测3组裸鼠移植瘤组织中血小板反应蛋白2（THBS2）及基质金属蛋白酶（MMP）2、MMP-9蛋白的表达。 结果： （1）miR-1246表达：抑制组SiHa细胞中miR-1246表达水平（0.11±0.13）明显低于LV-NC组、NC组（分别为1.14±0.86、1.30±0.73，P<0.01）。（2）增殖能力：培养不同时间（分别为24、48、72、96 h）后，抑制组SiHa细胞的A值均明显低于LV-NC组、NC组，分别比较，差异均有统计学意义（P<0.01）。（3）侵袭能力：抑制组的穿膜细胞数[（71±4）个]明显少于LV-NC组、NC组[分别为（162±5）、（188±5）个，P<0.01]。（4）细胞凋亡率：抑制组SiHa细胞凋亡率[（16.10±3.37）%]明显高于LV-NC组、NC组[分别为（5.67±0.89）%、（1.78±0.08）%，P<0.01]。（5）移植瘤体积：抑制组裸鼠移植瘤体积[（287±59）mm(3)]明显小于LV-NC组、NC组[分别为（571±137）、（657±144）mm(3)，P<0.01]。（6）与LV-NC组、NC组比较，抑制组裸鼠移植瘤组织中THBS2蛋白的表达水平明显升高（P<0.05），而MMP-2、MMP-9蛋白的表达水平明显下降（P<0.01）。 结论： 子宫颈癌SiHa细胞中miR-1246的表达下调后，抑制细胞增殖、促进细胞凋亡、降低细胞侵袭能力；其机制可能是通过上调THBS2蛋白的表达，进一步影响MMP的表达而实现。.

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