miR-197在人口腔鳞状细胞癌中的表达及对细胞增殖的作用机制

Expression of miR-197 in human oral squamous cell carcinoma and its mechanism of action on cell proliferation

目的探讨miR-197在人口腔鳞状细胞癌(OSCC)中的表达及对细胞增殖的作用机制。方法选取2015年3月—2019年3月在海南医学院第一附属医院选择手术切除病灶的100例OSCC患者的肿瘤组织及癌旁组织(距离病灶边缘超过2.5 cm),qRT-PCR检测miR-197在OSCC组织及细胞系CAL27中的表达,Western blotting检测JAK2在OSCC组织及细胞系中的表达。构建沉默miR-197表达的病毒载体(miR-197-siRNA)及miR-197-siRNA阴性对照(miR-197-siRNA-NC),转染CAL27细胞,以未处理的CAL27细胞作为空白对照;倒置显微镜观察转染miRNA-197后的细胞形态;流式细胞术、EdU标记实验、Transwell小室实验分别检测miR-197对CAL27细胞凋亡率、DNA合成能力、侵袭能力的影响;荧光素酶实验基因报告分析miR-197与JAK2的作用关系,Western blotting检测各组细胞中JAK2的表达。结果qRT-PCR结果显示,与癌旁组织相比,miR-197在OSCC组织中的表达明显升高,JAK2在OSCC组织中的表达明显降低(P<0.05)。与人正常口腔黏膜角化细胞株HOKs相比,miR-197在人OSCC细胞株CAL27中的表达明显上升,JAK2在CAL27中的表达明显下降(P<0.05);与miR-197-siRNA-NC组相比,miR-197-siRNA组贴壁细胞数量明显减少,胞质空泡样,细胞堆积明显增多,染色质固缩,核膜破损严重,线粒体空泡。流式细胞术、EdU标记实验、平板集落克隆实验、软琼脂集落形成实验结果表明,与miR-197-siRNA-NC组相比,miR-197-siRNA组细胞凋亡率明显增加,细胞内DNA合成明显受阻,细胞增殖和侵袭明显受到抑制,差异均具有统计学意义(P<0.05);荧光素酶实验基因报告分析结果证明,miR-197与JAK2具有靶向调控关系;Western blotting结果表明,与miR-197-siRNA-NC组相比,miR-197-siRNA组细胞中JAK2的表达明显升高(P<0.05)。结论miR-197在OSCC中呈高表达,下调miR-197可抑制OSCC细胞增殖,这可能与JAK信号通路有关。

Objective To explore the expression of miR-197 in oral squamous cell cancer(OSCC)and its mechanism of action on cell proliferation.Methods The tumor tissue and paracancerous tissue(more than 2.5 cm away from the edge of the lesion)were collected from 100 OSCC patients who were selected for surgical resection of the lesion in the First Affiliated Hospital of Hainan Medical University between March 2015 and March 2019.qRT-PCR was used to detect the expression of miR-197 in OSCC tissue specimens and CAL27 cells.Western blotting was used to detect the expression of JAK2 miR-197,and the negative control miR-197-siRNA-NC were constructed,and then were transfected into CAL27 cells.The CAL27 cells untreated were set as the blank control group.The inverted microscope was used to observe the changes of cell morphology after transfection.Flow cytometry,EdU mark,and transwell chamber experiment were respectively used to detect the effects of miR-197 on the apoptosis rate,DNA synthesis,and invasive ability of CAL27 cells.The luciferase assay gene was used to analyze the relationship between miR-197 and JAK2,and Western blotting was used to detect the expression of JAK2 in each group.Results qRT-PCR results showed that the expression level of miR-197 in OSCC tissues was higher than that in the adjacent normal tissues,while the expression level of JAK2 in OSCC tissues was lower than that in the adjacent normal tissues(P<0.05).Compared with the normal human oral keratinocytes(HOKs),the expression of miR-197 in human OSCC cell line CAL27 was significantly increased,and the expression of JAK2 in human OSCC cell line CAL27 was significantly decreased(P<0.05).Compared with the miR-197-siRNA-NC group,the number of adherent cells in the miR-197-siRNA group was significantly reduced,and cytoplasmic vacuoles were observed,as well as cell accumulation,chromatin pyknosis,severe nuclear membrane damage and mitochondrial vacuoles.Compared with the miR-197-siRNA-NC group,the cell apoptosis rate of the miR-197-siRNA group was significantly increased,the intracellular DNA synthesis was significantly blocked,and the cell proliferation and invasion were significantly inhibited(P<0.05).And the luciferase assay gene report analysis showed that miR-197 had a targeted regulatory relationship with JAK2.Western blotting results showed that the expression level of JAK2 was significantly increased in miR-197-siRNA group as compared with the miR-197-siRNA-NC group(P<0.05).Conclusion miR-197 is highly expressed in OSCC,and down-regulation of miR-197 expression can inhibit the proliferation of OSCC cells.This may be related to the JAK signaling pathway.