miR-185-5p通过IGFBP2调节口腔鳞状细胞癌细胞的增殖、凋亡

甘志恒; 余子敬; 毛敏; 施俊; 蒙建超; 夏凌云

miR-185-5p通过IGFBP2调节口腔鳞状细胞癌细胞的增殖、凋亡

miR-185-5p regulates the proliferation and apoptosis of oral squamous cell carcinoma cells through IGFBP2

摘要

摘要:
目的： 探究miR-185-5p对口腔鳞状细胞癌（OSCC）细胞增殖、凋亡的作用及其机制。
方法： 运用实时定量荧光聚合酶链反应（qRT-PCR）试剂盒检测72例OSCC病人的癌组织及癌旁组织及正常人口腔角质形成细胞HOK、口腔鳞状细胞癌细胞SCC-15、HSC-3、Cal-27中 miR-185-5p、胰岛素样生长因子结合蛋白2（IGFBP2）的表达；脂质体法将NC组（不做任何处理）、agomiRNA组（转染agomiRNA）、agomiR-185-5p组（转染agomiR-185-5p）、si-NC组（转染si-NC）、si-IGFBP2组（转染si-IGFBP2）、agomiR-185-5p + pcDNA组(共转染agomiR-185-5p和pcDNA）、agomiR-185-5p + pcDNA-IGFBP2组（共转染agomiR-185-5p和pcDNA-IGFBP2）转染SCC-15细胞。细胞计数试剂盒（CCK8）实验、克隆形成实验、5-乙炔基-2′-脱氧尿苷（EdU）染色检测细胞的增殖情况；流式细胞术检测细胞的凋亡；蛋白印迹（Western Blotting）实验检测IGFBP2蛋白；双荧光素酶报告实验检测细胞miR-185-5p与IGFBP2的结合力。
结果： 与癌旁组织相比，癌组织（Ⅰ ~ Ⅱ期、Ⅲ期、Ⅳ期）中miR-185-5p表达降低，IGFBP2表达升高，且呈负相关（r = –0.412，P ＜ 0.05）；miR-185-5p高表达预示病人的3年总存活率升高。与HOK组相比，SCC-15、HSC-3、Cal-27组细胞中miR-185-5p表达降低，过表达miR-185-5p的SCC-15细胞活性、克隆形成率及EdU阳性率均降低，凋亡率升高（P ＜ 0.05）。敲减IGFBP2具有相似的作用。miR-185-5p靶向结合IGFBP2，并负向调控IGFBP2。过表达IGFBP2部分逆转miR-185-5p对SCC-15细胞的增殖抑制和凋亡促进。
结论： miR-185-5p在OSCC中低表达，过表达miR-185-5p可抑制OSCC细胞增殖，促进凋亡，预测病人预后，这与靶向IGFBP2相关。

Abstract:
Objective To investigate the effect and mechanism of miR-185-5p on proliferation and apoptosis of oral squamous cell carcinoma (OSCC) cells.
Methods qRT-PCR kit was used to detect the expression of miR-185-5p and insulin-like growth factor binding protein 2 (IGFBP2) in 72 patients with OSCC, as well as in normal human oral keratinocytes Hok, oral squamous cell carcinoma cells SCC-15, HSC-3 and Cal-27. NC group (without any treatment), agomiRNA group (transfected agomiRNA), agomiR-185-5p group (transfected with agomiR-185-5p), si-NC group (transfected with si-NC), si-IGFBP2 group (transfected si-IGFBP2), agomiR-185-5p pcDNA group (co-transfected agomiR-185-5p and pcDNA), agomiR-185-5p + pcdna-IGFBP2 group (co-transfected agomiR-185-5p and pcDNA-IGFBP2) were transfected into SCC-15 cells by liposome method. Cell counting Kit (CCK8), clone formation test and 5-ethynyl-2 ′- deoxyuridine (EDU) staining were used to detect the proliferation of cells. Apoptosis was detected by flow cytometry. IGFBP2 protein was detected by western blot (WB). The binding of miR-185-5p to IGFBP2 was detected by double luciferase report assay.
Results Compared with adjacent tissues, the expression of miR-185-5p decreased and IGFBP2 increased in cancer tissues (stage Ⅰ - Ⅱ, stage Ⅲ and stage Ⅳ) (r = –0.412, P ＜ 0.05). The high expression of miR-185-5p indicated an increase in the 3-year overall survival rate of patients. Compared with HOK group, the expression of miR-185-5p decreased in SCC-15, HSC-3 and Cal-27 groups, meanwhile the activity, clone formation rate and EDU positive rate of SCC-15 cells overexpressing miR-185-5p decreased, the apoptosis rate increased (P ＜ 0.05). Knockdown of IGFBP2 had a similar effect. miR-185-5p targeted IGFBP2 and negatively regulated IGFBP2. Overexpression of IGFBP2 partially reversed the proliferation inhibition and apoptosis promotion of SCC-15 cells by miR-185-5p.
Conclusion miR-185-5p is low expressed in OSCC. Overexpression of miR-185-5p can inhibit the proliferation of OSCC cells, promote apoptosis and predict the prognosis of patients, which is related to targeting IGFBP2.

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