ciRS-7可调控宫颈癌干细胞的干性及机制

ciRS-7 regulates the stemness of cervical cancer stem cells:effects and mechanisms

背景:研究表明ciRS-7参与多种肿瘤的发生发展,而其在宫颈癌干细胞领域的研究较少。目的:观察ciRS-7在宫颈癌组织及宫颈癌干细胞中的表达水平,并探讨其对宫颈癌干细胞干性的影响及作用机制。方法:①qRT-PCR检测ciRS-7在宫颈癌组织和正常宫颈组织中表达,并分析其与临床参数的关系;②分离培养原代宫颈癌细胞,流式细胞仪筛选出CD44+宫颈癌干细胞。qRT-PCR检测ciRS-7在CD44+宫颈癌干细胞中的表达;③经脂质体分别转染ciRS-7 siRNA(si-ciRS-7)和阴性对照siRNA (si-NC) 48 h后,MTS实验、软琼脂克隆形成实验、Transwell实验检测宫颈癌干细胞增殖能力、肿瘤球形成能力、转移能力,qRT-PCR检测各组宫颈癌干细胞中miR-7表达,Western blot检测各组宫颈癌干细胞中PTEN、p-PI3k和p-Akt蛋白表达;④将敲减ci RS-7的宫颈癌干细胞注射到BALB/c裸鼠皮下,观察抑制ciRS-7表达对宫颈癌干细胞体内成瘤能力的影响。结果与结论:①ciRS-7在宫颈癌组织中的表达显著高于正常宫颈组织,且与肿瘤大小及淋巴结转移相关(P <0.05),同时ciRS-7在宫颈癌干细胞中的表达显著升高(P <0.05);②与阴性对照组比较,si-ciRS-7组宫颈癌干细胞增殖减慢、肿瘤球形成能力下降、转移能力减弱、miR-7表达升高、PTEN抑癌蛋白表达升高、p-PI3k和p-Akt促癌蛋白表达降低,差异均有显著性意义(P <0.05);③si-ciRS-7组裸鼠成瘤体积显著小于阴性对照组(P <0.05);④结果表明,ciRS-7在宫颈癌组织和宫颈癌干细胞中高表达,ciRS-7可能通过上调miR-7的表达,调控PTEN/PI3K/AKT信号通路实现对宫颈癌干细胞干性的抑制作用。

BACKGROUND: Studies have shown that ciRS-7 is involved in the development of a variety of tumors, but there are few studies concerning the effect of ciRS-7 in cervical cancer stem cells.OBJECTIVE: To study the expression levels of ciRS-7 in cervical cancer tissues and cervical cancer stem cells, and to explore its effect on the stemness of cervical cancer stem cells and the relevant mechanism.METHODS:(1) qRT-PCR was used to detect the expression of ciRS-7 in cervical cancer tissues and normal cervical tissues, and its relationship with clinical parameters was analyzed.(2) Primary cervical cancer cells were isolated and cultured, and CD44+ phenotype of cervical cancer stem cells was screened by flow cytometry. q RT-PCR was then used to detect the expression of ciRS-7 in the cervical cancer stem cells.(3) After transfection of ci RS-7 siRNA(si-ciRS-7) and control(si-NC) for 48 hours, MTS was used to detect the proliferation of cervical cancer stem cells in each group. Soft agar cloning assay was used to detect the tumor formation ability of each group. Transwell assay was used to detect the metastatic ability of cervical cancer stem cells. The expression of miR-7 in cervical cancer stem cells was detected by qRT-PCR. The expressions of PTEN, p-Pi3k and p-Akt in cervical cancer stem cells were detected by western blot.(4) Cervical cancer stem cells with ci RS-7 knockdown were subcutaneously injected into BALB/c nude mice, to observe the effect of ci RS-7 on the tumorigenic ability of cervical cancer stem cells.RESULTS AND CONCLUSION:(1) The expression of ci RS-7 in cervical cancer tissues was significantly higher than that in normal cervical tissues, and it was correlated with tumor size and lymph node metastasis(P < 0.05). Meanwhile, the expression of ciRS-7 significantly elevated in cervical cancer stem cells(P < 0.05).(2) In the si-ciRS-7 cell group, the cell proliferation was slowed down, the tumor-forming ability was decreased, the ability to metastasis was weakened, the expression of miR-7 was increased, the expression of PTEN was increased, and the expression of p-Pi3k and p-Akt was decreased as compared with the negative control group(P < 0.05). The tumor-forming volume in the si-ciRS-7 cell group was significantly smaller than that in the negative control group(P < 0.05). In conclusion, ciRS-7 is highly expressed in cervical cancer tissues and cervical cancer stem cells. ciRS-7 may promote the stemness of cervical cancer stem cells by inhibiting the expression of miR-7 and regulating PTEN/PI3K/AKT signaling pathway.