人肾癌干细胞的分离、鉴定及其特异性microRNA的筛选和功能研究
发布时间：2018-08-15 12:57
【摘要】：目的 鉴定和分离以CD105+为标志的人肾癌干细胞,并通过比较CD105-shRNA和不同microRNA在肾癌干细胞和普通肾癌细胞中的功能,探讨CD105和干细胞相关microRNA通过杀灭肾癌干细胞来治疗肾癌的可行性和有效性。 方法 采取ACHN肾癌细胞系在免疫缺陷小鼠BALB/c-nu皮下成瘤的方式获得肾癌组织细胞,并通过外源性anti-hCD105-PE抗体标记和流式细胞术分离CD105阳性的人肾癌干细胞。分别采用成球实验、成上皮诱导分化实验鉴定CD105(+)细胞的肿瘤干细胞特性,并通过MTS和EdU细胞增殖实验、周期测定、集落形成和细胞衰老程度测定等手段鉴定以CD105(+)为标志的肾癌干细胞的增殖特性和细胞衰老程度。在此基础上,采用microRNA数据库检索、文献检索和qRT-PCR验证的方式筛选在干细胞和非干细胞中差异性表达的microRNA,并采用双荧光素酶的方式确定microRNA和其靶蛋白mRNA3'端非编码区之间的结合程度,最终将筛选得到的microRNA采用mimics或者慢病毒转染的方式探讨其在肾癌干细胞的体外成球能力、顺铂耐药能力、迁移能力中的作用。 结果 CD105(+)肾癌细胞的成球能力明显强于CD105(-)细胞系ACHN,且在RPMI-1640培养基中培养两周后,干细胞标志CXCR4、OCT-4、NANOG、HIF1A、DLK-1和EZH2逐渐降低；在DMEM-HG+10%FBS培养基中培养两周后,其干细胞标志CXCRH-4、NANOG、 DLK-1、OCT-4和KLF4表达明显下降。在其他鉴定实验中,虽然ACHN-CD105(+)细胞的增殖速度下降且存在G0期阻滞,但其形成集落和成球能力均强于ACHN-CD105(-)细胞,衰老程度也显著低于ACHN-CD105(-)细胞。在此基础上,我们鉴定出27个在CD105(+)肾癌干细胞中差异性表达的microRNA,并证实miR-34a、miR-155、miR-200a可不同程度结合CD105的mRNA从而下调其表达,使CD105(+)肾癌干细胞对顺铂耐药性下降,而采用shRNA敲降CD105的方式,我们也发现CD105具有维持ACHN-CD105(+)细胞耐药性并防止其衰老的作用；同时我们也证明miR-335可结合OCT-4蛋白的mRNA从而降低其表达,并产生抗迁移、降低成球率和抑制SOX-2、OCT-4、NANOG、KLF-4等干细胞因子表达的效应。 结论 经ACHN细胞系于BALB/c-nu裸鼠皮下移植瘤细胞分选获得的CD105(+)细胞具有肿瘤干细胞特性,可初步认定为肾癌干细胞；miR-34a、miR-155、miR-200a可通过下调CD105降低CD105(+)肾癌干细胞对顺铂的耐药性；miR-335可通过下调OCT-4抑制CD105(+)肾癌干细胞的迁移能力、成球能力。 第一部分人肾癌干细胞的分离、培养和鉴定 目的 分离和鉴定以CD105(+)为标志的人肾癌干细胞 方法 将肾癌细胞系ACHN接种于BALB/c-nu裸鼠的腋窝皮肤下,一月后将形成的肿瘤消化成单细胞悬液并采用anti-hCD105-PE流式抗体4-C孵育半小时,于流式分选仪上分离得到ACHN-CD105(+)细胞并采用肾癌干细胞培养基扩增和冻存细胞。采用体外成球实验、RPMI-1640+10%FBS和DMEM-HG+10%FBS培养基诱导分化实验、顺铂耐药性实验、MTS和EdU细胞增殖实验、周期测定、集落形成实验和细胞衰老程度测定的方式鉴定ACHN-CD105(+)细胞的增殖、分化和成瘤特性。 结果 CD105(+)细胞仅占ACHN细胞系皮下移植瘤细胞的3.05%,符合肿瘤干细胞理论中“极低比例的细胞驱动肿瘤生长”的观点。ACHN-CDl05(+)可在肾癌干细胞培养基中持续扩增并维持CD105和干细胞相关因子的表达水平。在后续功能鉴定中,ACHN-CD105(+)细胞的成球率明显高于ACHN-CD105(-)细胞,且在RPMI-1640+10%FBS和DMEM-HG+10%FBS培养基中培养两周后干细胞相关因子CXCR-4、OCT-4、NANOG表达下降,顺铂耐药实验显示ACHN-CD105(+)细胞的耐药性明显强于ACHN-CD105(-)细胞,细胞增殖实验和集落形成实验显示ACHN-CD105(+)细胞增殖速度低于ACHN-CD105(-)细胞且有GO期阻滞现象,但其集落形成能力显著优于后者。细胞衰老程度测定显示,ACHN-CD105(+)细胞衰老程度低于ACHN-CD105(-)细胞。 结论 经ACHN细胞系于BALB/c-nu裸鼠皮下移植瘤细胞分选获得的CD105(+)细胞具有肿瘤干细胞特性,可初步认定为肾癌干细胞。 第二部分miRNA在人肾癌干细胞中的功能和机制研究 目的 筛选可抑制ACHN-CD105(+)干细胞特性的miRNA并探讨其作用机制 方法 采用miRNA数据库筛选、文献检索和qRT-PCR验证的方式筛选具有抑制ACHN-CD105(+)干细胞特性的miRNA,以双荧光素酶实验确定miRNA和其靶蛋白mRNA之间的结合程度并采取顺铂耐药性实验、细胞衰老程度测定、细胞迁移实验和成球实验检测经过筛选获得的miRNA的有效性和用于在体实验的可行性。 结果 我们筛选得到27个在ACHN-CD105(+)和ACHN-CD105(-)细胞间差异性表达的miRNA,后期又确定了miR-34a、miR-155、miR-200a的靶标为CD105, miR-335的靶标为OCT-4。在转染了miR-34a、miR-155、miR-200a的化学合成mimics之后,ACHN-CD105(+)细胞对顺铂的耐药性显著下降；在转染miR-335的过表达慢病毒后,ACHN-CD105(+)细胞的迁移迁移能力和成球能力下降,包括OCT-4、NANOG、SOX-2、KLF-4在内的多种干细胞相关因子表达降低。 结论 miR-34a、miR-155、miR-200a可通过下调CD105降低CD105(+)肾癌干细胞对顺铂的耐药性;miR-335可通过下调OCT-4抑制CD105(+)肾癌干细胞的迁移能力、成球能力。
[Abstract]:objective
To identify and isolate human renal cell carcinoma stem cells marked by CD105+ and to compare the functions of CD105-shRNA and different microRNAs in renal cell carcinoma stem cells and normal renal cell carcinoma cells, to explore the feasibility and effectiveness of CD105 and stem cell-related microRNAs in the treatment of renal cell carcinoma by killing renal cell carcinoma stem cells.
Method
ACHN renal carcinoma cell line was subcutaneously tumorigenic in BALB/c-nu immunodeficient mice. CD105-positive human renal carcinoma stem cells were isolated by anti-hCD105-PE antibody labeling and flow cytometry. The proliferation characteristics and senescence degree of renal cell carcinoma stem cells marked by CD105 (+) were identified by MTS and EdU cell proliferation test, cell cycle test, colony formation and senescence degree test. Differentially expressed microRNA was used to determine the binding degree between microRNA and its target protein mRNA 3'-terminal non-coding region by dual luciferase method. Finally, the screened microRNA was transfected with mimics or lentiviruses to explore its role in globular formation, cisplatin resistance and migration of renal cancer stem cells in vitro.
Result
The globular ability of CD105 (+) renal carcinoma cells was significantly stronger than that of CD105 (-) cell line ACHN, and the expression of stem cell markers CXCR4, OCT-4, NANOG, HIF1A, DLK-1 and EZH2 decreased after two weeks in RPMI-1640 medium. The expression of stem cell markers CXCRH-4, NANOG, DLK-1, T-4 and KLF4 decreased significantly after two weeks in DMEM-HG+10% FBS medium. He identified 27 differentially expressed microRNAs in CD105 (+) renal cancer stem cells and confirmed the presence of microRNAs in these cells. Mi-34a, Mi-155, and Mi-200a can down-regulate the expression of CD105 mRNA to some extent, thus reducing the resistance of CD105 (+) renal cancer stem cells to cisplatin. By knocking down CD105 with shRNA, we also found that CD105 can maintain the drug resistance of ACHN-CD105 (+) cells and prevent their aging. We also proved that Mi-335 can bind to OCT-4 eggs. White mRNA reduces its expression and produces anti-migration, decreases the rate of globulation and inhibits the expression of stem cell factors such as SOX-2, OCT-4, NANOG and KLF-4.
conclusion
CD105 (+) cells derived from ACHN cell line subcutaneously transplanted in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be initially identified as renal cancer stem cells; microRNAs-34a, microRNAs-155, microRNAs-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105 (+); microRNAs-335 can inhibit CD105 (+) renal cancer by down-regulating OCT-4. Cell migration ability and ball forming ability.
Part 1 isolation, culture and identification of human renal cancer stem cells
objective
Isolation and identification of human renal cancer stem cells marked by CD105 (+)
Method
ACHN cell line was inoculated into the axillary skin of BALB/c-nu nude mice. After one month, the tumor was digested into a single cell suspension and incubated with anti-hCD105-PE flow cytometry antibody 4-C for half an hour. ACHN-CD105 (+) cells were isolated from the axillary skin of BALB/c-nu nude mice by flow sorter. The cells were amplified and cryopreserved in vitro. The proliferation, differentiation and tumorigenesis of ACHN-CD105(+) cells were identified by RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, cisplatin resistance, MTS and EdU cell proliferation, cell cycle, colony formation and cell senescence.
Result
CD105 (+) cells accounted for only 3.05% of ACHN subcutaneously transplanted tumor cells, which accorded with the view of "very low proportion of cells driving tumor growth" in cancer stem cell theory. The rate of cell globulation was significantly higher than that of ACHN-CD105 (-) cells. After two weeks of culture in RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, the expression of stem cell-related factors CXCR-4, OCT-4 and NANOG was decreased. Cisplatin resistance test showed that ACHN-CD105 (+) cells were more resistant than ACHN-CD105 (-) cells. Cell proliferation test and colony formation test were obvious. The results showed that the proliferation rate of ACHN-CD105 (+) cells was lower than that of ACHN-CD105 (-) cells and the colony forming ability of ACHN-CD105 (-) cells was significantly better than that of ACHN-CD105 (-) cells.
conclusion
CD105 (+) cells derived from ACHN cell line in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be identified as renal cancer stem cells.
The second part is about the function and mechanism of miRNA in human renal cancer stem cells.
objective
Screening of miRNA that inhibits ACHN-CD105 (+) stem cell characteristics and its mechanism
Method
MicroRNAs that inhibit ACHN-CD105 (+) stem cells were screened by microNA database screening, literature retrieval and qRT-PCR. The binding degree between microRNAs and their target protein mRNA was determined by double luciferase assay. Cisplatin resistance test, cell senescence test, cell migration test and globular test were carried out. The validity of the selected miRNA and its feasibility for in vivo experiments are obtained.
Result
We screened 27 differentially expressed microRNAs between ACHN-CD105 (+) and ACHN-CD105 (-) cells, and later identified that the targets of microRNAs-34a, microRNAs-155, and microRNAs-200a were CD105 and that of microRNAs-335 were OCT-4. After transfection of microRNAs-34a, microRNAs-155, and microRNAs-200a, the resistance of ACHN-CD105 (+) cells to cisplatin decreased significantly. Migration, migration and globulation ability of ACHN-CD105 (+) cells decreased after exposure to lentiviruses overexpressing microRNA-335, and expression of many stem cell-related factors, including OCT-4, NANOG, SOX-2 and KLF-4, decreased.
conclusion
Mi-34a, Mi-155, and Mi-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105; Mi-335 can inhibit the migration and globulation of CD105 (+) renal cancer stem cells by down-regulating OCT-4.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11
本文编号：2184272
[Abstract]:objective
To identify and isolate human renal cell carcinoma stem cells marked by CD105+ and to compare the functions of CD105-shRNA and different microRNAs in renal cell carcinoma stem cells and normal renal cell carcinoma cells, to explore the feasibility and effectiveness of CD105 and stem cell-related microRNAs in the treatment of renal cell carcinoma by killing renal cell carcinoma stem cells.
Method
ACHN renal carcinoma cell line was subcutaneously tumorigenic in BALB/c-nu immunodeficient mice. CD105-positive human renal carcinoma stem cells were isolated by anti-hCD105-PE antibody labeling and flow cytometry. The proliferation characteristics and senescence degree of renal cell carcinoma stem cells marked by CD105 (+) were identified by MTS and EdU cell proliferation test, cell cycle test, colony formation and senescence degree test. Differentially expressed microRNA was used to determine the binding degree between microRNA and its target protein mRNA 3'-terminal non-coding region by dual luciferase method. Finally, the screened microRNA was transfected with mimics or lentiviruses to explore its role in globular formation, cisplatin resistance and migration of renal cancer stem cells in vitro.
Result
The globular ability of CD105 (+) renal carcinoma cells was significantly stronger than that of CD105 (-) cell line ACHN, and the expression of stem cell markers CXCR4, OCT-4, NANOG, HIF1A, DLK-1 and EZH2 decreased after two weeks in RPMI-1640 medium. The expression of stem cell markers CXCRH-4, NANOG, DLK-1, T-4 and KLF4 decreased significantly after two weeks in DMEM-HG+10% FBS medium. He identified 27 differentially expressed microRNAs in CD105 (+) renal cancer stem cells and confirmed the presence of microRNAs in these cells. Mi-34a, Mi-155, and Mi-200a can down-regulate the expression of CD105 mRNA to some extent, thus reducing the resistance of CD105 (+) renal cancer stem cells to cisplatin. By knocking down CD105 with shRNA, we also found that CD105 can maintain the drug resistance of ACHN-CD105 (+) cells and prevent their aging. We also proved that Mi-335 can bind to OCT-4 eggs. White mRNA reduces its expression and produces anti-migration, decreases the rate of globulation and inhibits the expression of stem cell factors such as SOX-2, OCT-4, NANOG and KLF-4.
conclusion
CD105 (+) cells derived from ACHN cell line subcutaneously transplanted in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be initially identified as renal cancer stem cells; microRNAs-34a, microRNAs-155, microRNAs-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105 (+); microRNAs-335 can inhibit CD105 (+) renal cancer by down-regulating OCT-4. Cell migration ability and ball forming ability.
Part 1 isolation, culture and identification of human renal cancer stem cells
objective
Isolation and identification of human renal cancer stem cells marked by CD105 (+)
Method
ACHN cell line was inoculated into the axillary skin of BALB/c-nu nude mice. After one month, the tumor was digested into a single cell suspension and incubated with anti-hCD105-PE flow cytometry antibody 4-C for half an hour. ACHN-CD105 (+) cells were isolated from the axillary skin of BALB/c-nu nude mice by flow sorter. The cells were amplified and cryopreserved in vitro. The proliferation, differentiation and tumorigenesis of ACHN-CD105(+) cells were identified by RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, cisplatin resistance, MTS and EdU cell proliferation, cell cycle, colony formation and cell senescence.
Result
CD105 (+) cells accounted for only 3.05% of ACHN subcutaneously transplanted tumor cells, which accorded with the view of "very low proportion of cells driving tumor growth" in cancer stem cell theory. The rate of cell globulation was significantly higher than that of ACHN-CD105 (-) cells. After two weeks of culture in RPMI-1640+10% FBS and DMEM-HG+10% FBS medium, the expression of stem cell-related factors CXCR-4, OCT-4 and NANOG was decreased. Cisplatin resistance test showed that ACHN-CD105 (+) cells were more resistant than ACHN-CD105 (-) cells. Cell proliferation test and colony formation test were obvious. The results showed that the proliferation rate of ACHN-CD105 (+) cells was lower than that of ACHN-CD105 (-) cells and the colony forming ability of ACHN-CD105 (-) cells was significantly better than that of ACHN-CD105 (-) cells.
conclusion
CD105 (+) cells derived from ACHN cell line in BALB/c-nu nude mice have the characteristics of tumor stem cells and can be identified as renal cancer stem cells.
The second part is about the function and mechanism of miRNA in human renal cancer stem cells.
objective
Screening of miRNA that inhibits ACHN-CD105 (+) stem cell characteristics and its mechanism
Method
MicroRNAs that inhibit ACHN-CD105 (+) stem cells were screened by microNA database screening, literature retrieval and qRT-PCR. The binding degree between microRNAs and their target protein mRNA was determined by double luciferase assay. Cisplatin resistance test, cell senescence test, cell migration test and globular test were carried out. The validity of the selected miRNA and its feasibility for in vivo experiments are obtained.
Result
We screened 27 differentially expressed microRNAs between ACHN-CD105 (+) and ACHN-CD105 (-) cells, and later identified that the targets of microRNAs-34a, microRNAs-155, and microRNAs-200a were CD105 and that of microRNAs-335 were OCT-4. After transfection of microRNAs-34a, microRNAs-155, and microRNAs-200a, the resistance of ACHN-CD105 (+) cells to cisplatin decreased significantly. Migration, migration and globulation ability of ACHN-CD105 (+) cells decreased after exposure to lentiviruses overexpressing microRNA-335, and expression of many stem cell-related factors, including OCT-4, NANOG, SOX-2 and KLF-4, decreased.
conclusion
Mi-34a, Mi-155, and Mi-200a can reduce the resistance of CD105 (+) renal cancer stem cells to cisplatin by down-regulating CD105; Mi-335 can inhibit the migration and globulation of CD105 (+) renal cancer stem cells by down-regulating OCT-4.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11
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