摘要
背景:研究表明,miRNA对肿瘤干细胞的更新分化有调节作用,有关miRNA-17-92在胃癌干细胞更新及增殖中的作用尚未完全阐明。目的:分析miRNA-17-92在胃癌干细胞自我更新及增殖中的作用。方法:1利用细胞培养使胃癌干细胞贴壁分化并送miRNA芯片检测,寻找并验证不断缺失的miRNA。2构建并转染mi RNA-17-92分子慢病毒稳定转染细胞。3通过tumorsphere实验研究miRNA-17-92与胃癌细胞更新的关系。4通过MTT、平板克隆试验检测miRNA-17-92与胃癌干细胞增殖的关系。结果与结论:1miR-19b/20a/92a在胃癌干细胞贴壁分化过程中表达逐渐减低。2慢病毒携带的miRNA-17-19基因在MKN28细胞和CD44^-/EpCAM^-细胞中的表达均明显增加;瞬时转染pre-miR-19b/20a/92a能使CD44^-/EpCAM^-和MKN28的miRNA表达增高,瞬时转染pre-miR-19b/20a/92a拮抗剂能使SGC7901和CD44^+/Ep CAM^+的miRNA表达降低;过表达lenti-miR-19b/20a/92a能显著增加胃癌细胞形成肿瘤球的能力;在化疗药的作用下,lenti-miR-19b/20a/92a感染细胞的生存时间延长;瞬时转染pre-miR-19b/20a/92a能够显著增加CD44^+/EpCAM^+细胞数,转染其拮抗剂可以降低CD44^+/EpCAM^+细胞数。3miR-19b/20a/92a稳定表达组的胃癌细胞增殖速度较对照组快。瞬间转染miR-19b/20a/92a前体组加快胃癌细胞的增殖速度,而瞬时转染其拮抗物组减慢胃癌细胞的增殖速度;瞬间转染miR-19b/20a/92a前体组的克隆数较对照组多,而瞬时转染miR-19b/20a/92a拮抗物组的克隆数较对照组少。结果表明miR-19b/20a/92a基因在胃癌干细胞分化过程中不断缺失,miR-17-92基因能够促进胃癌干细胞的更新和增殖。
BACKGROUND: Studies have shown that micro RNAs(miRNAs) have moderating effect on the renewal and differentiation of cancer stem cells. However, there is no complete understanding on the effect of microRNA-17-92 gene on gastric cancer stem cell renewal and proliferation. OBJECTIVE: To explore the effect of miRNA-17-92 in promoting self-renewal and proliferation of gastric cancer stem cells. METHODS:(1) The gradually reduced mi RNAs during gastric cancer stem cell self-renewal were investigated using mi RNA array based on RNAs from differentiated and adherent cells.(2) The miRNA-17-92 was constructed and transfected to gastric cancer stem cells.(3) The effects of miRNA-17-92 on the self-renewal of gastric cancer stem cells were studied by tumor sphere assay in vitro.(4) The effects of miRNA-17-92 on the proliferation of gastric cancer stem cells were investigated by MTT assay and colony formation assay.RESULTS AND CONCLUSION:(1) miR-19b/20a/92 a expression gradually reduced in the adhesion and differentiation of gastric cancer stem cells.(2) The expression of lentivirus carrying miRNA-17-19 gene in MKN28 cells and CD44^-/EpCAM^- cells were significantly increased; transient transfection of pre-miR-19b/20a/92 a increased the expression of CD44^-/EpCAM^- and MKN28 mi RNA, transient transfection of pre-mi R-19b/20a/92 a antagonists reduced the expression of SGC7901 and CD44^+/Ep CAM^+ miRNA; overexpression of lenti-miR-19b/20a/92 a significantly increased the ability of gastric cells to form tumor spheres; chemotherapy drugs prolonged the survival time of lenti-mi R-19b/20a/92a-infected cells; transient transfection of pre-mi R-19b/20a/92 a significantly increased the number of CD44^+/EpCAM^+ cells, but transfection of pre-miR-19b/20a/92 a antagonist reduced the number of CD44^+/EpCAM^+ cells.(3) MTT proliferation assay showed that gastric cancer cell proliferation rate in miR-19b/20a/92 a stably expressing group was faster than that in the control group. Transient transfection of miR-19b/20a/92 a precursor accelerated the growth rate of gastric cancer cells, and transient transfection of its antagonist slowed down the growth rate of gastric cancer cells. Colony formation assay showed that transient transfection of miR-19b/20a/92 a precursor significantly increased the colony formation number as compared with the control group; transient transfection of miR-19b/20a/92 a antagonist reduced the colony formation as compared with the control group. These findings indicate that miR-19b/20a/92 a gene presents with continuous deletion in gastric cancer stem cell differentiation process, and miRNA-17-92 gene can promote the renewal and proliferation of gastric cancer stem cells.
出处
《中国组织工程研究》
CAS
北大核心
2015年第50期8077-8083,共7页
Chinese Journal of Tissue Engineering Research