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

doi:10.3969/j.issn.2095-4344.1690

Abstract

Abstract:

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. qRT-PCR was then used to detect the expression of ciRS-7 in the cervical cancer stem cells. (3) After transfection of ciRS-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 ciRS-7 knockdown were subcutaneously injected into BALB/c nude mice, to observe the effect of ciRS-7 on the tumorigenic ability of cervical cancer stem cells.
RESULTS AND CONCLUSION: (1) The expression of ciRS-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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Uterine Cervical Neoplasms, Neoplastic Stem Cells, RNA Interference, Tissue Engineering

CLC Number:

Cheng Haiyan, Long Heming, Xie Xiaoying, Li Feng. ciRS-7 regulates the stemness of cervical cancer stem cells: effects and mechanisms[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2009-2015.

Figures/Tables 1

2.1 ciRS-7在宫颈癌组织和正常宫颈组织中的表达 qRT-PCR检测ciRS-7在40例宫颈癌组织中的表达及与临床参数的关系。结果显示：ciRS-7在宫颈癌组织中的表达高于正常宫颈组织，见图1A，且在肿瘤T>4 cm及淋巴结转移的组织中高表达(P < 0.05)，见图1B，C。 2.2 宫颈癌干细胞的分选利用流式细胞仪将新鲜肿瘤组织原代分离培养的肿瘤球进行分选，结果筛选出CD44+细胞约占全部细胞的3.01%。Western blot检测肿瘤干细胞标记基因Nanog、Sox2蛋白在CD44+细胞中的表达显著高于CD44-细胞，见图2。qRT-PCR检测结果显示ciRS-7在CD44+细胞中的表达为7.21±0.53，显著高于在CD44-细胞中的表达(1.02±0.07)，差异有显著性意义(P < 0.05)。 2.3 MTS检测抑制ciRS-7表达对宫颈癌干细胞增殖的影响与阴性对照组相比，si-ciRS-7组细胞增殖能力明显减慢，差异有显著性意义(P < 0.05)，见图3。 2.4 软琼脂克隆实验检测抑制ciRS-7表达对宫颈癌干细胞克隆形成能力的影响 si-ciRS-7组肿瘤克隆球形成明显小于阴性对照组，见图4。 2.5 Transwell小室检测抑制ciRS-7表达对宫颈癌干细胞转移能力的影响 si-ciRS-7组和阴性对照组细胞穿膜数分别为21.31±4.87，63.51±9.24。si-ciRS-7组细胞转移能力明显低于阴性对照组，差异有显著性意义(P < 0.05)，见图5。 2.6 抑制ciRS-7表达对宫颈癌干细胞体内成瘤能力的影响 si-ciRS-7组裸鼠瘤体体积显著低于阴性对照组，差异有显著性意义(P < 0.05)，见图6。 2.7 抑制ciRS-7表达对宫颈癌干细胞中miR-7表达的影响采用qRT-PCR测定si-ciRS-7组和阴性对照组宫颈癌干细胞中miR-7表达水平，分别为7.65±1.01，1.00±0.02，两组比较差异有显著性意义(P < 0.05)。 2.8 抑制ciRS-7表达对宫颈癌干细胞中PTEN、p-PI3k和p-Akt蛋白表达的影响与阴性对照组相比，si-ciRS-7组PTEN抑癌蛋白表达显著升高，p-PI3k和p-Akt促癌蛋白表达显著降低，见图7。"

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