| PuroseTo isolate and identify renal cancer stem cells signified with CD105(+), and screen potentially therapeutic miRNAs targeting ACHN-CD105(+) cells and explore the underlying mechanismsMethodsA xenograft model was established subcutaneously in the axillary fossa of BALB/c-nu with well-acknowledged renal cancer cell line ACHN1month prior to FACS. ACHN-CD105(+) cells could be sorted with anti-hCD105-PE from ACHN xenograft tumor at a proportion of3.05%and proliferated and cryopreserved in renal cancer stem cell expansion medium. Sphere formation assay, RPMI-1640+10%FBS and DMEM-HG+10%FBS differentiation assay, cisplatin-based chemoresistance assay, MTS and EdU-based cell proliferation assay, cell cycle analyses, colony formation assay and β-galactosidase sensescence assay were utilized to assess the stemness-related property and determine the proliferation pattern. Based on the previous research on renal cancer stem cell, miRNAs targeting ACHN-CD105(+) were determined by the query of miRNA databases and literacies and verified by qRT-PCR. Dual Luciferase Assay was utilized to confirm the binding between miRNAs and corresponding target mRNAs. Cisplatin-based chemoresistance assay, β-galactosidase cell senescence assay, cell migration assay and sphere formation assay were adopted to assess the efficacy and efficiency of the screened miRNAs in the treatment of renal cancer.Results ACHN-CD105(+) only accounted for3.05%in the whole ACHN xenograft tumor cell population, which is in accordance with the canonical cancer stem cell theory that a small fraction of cells drive tumor growth. Also, ACHN-CD105(+) cells could be sorted, cryopreserved and proliferated without differentiation or sternness genotypic change within at least50passages. In subsequent identification experiements, ACHN-CD105(+) beared an obviously elevated capability to form spheres than ACHN-CD105(-) cells and could be induced to differentiate and lose sternness genotypes as CXCR4, OCT-4and NANOG after2weeks’ culture in RPMI-1640+10%FBS and DMEM-HG+10%FBS. Moreover, ACHN-CD105(+) manifested an improved cisplatin resistance compared to ACHN-CD105(-). In cell proliferation-related assays, ACHN-CD105(+)’s proliferation seemed to be retardated and shows GO arrest as seen in cell cycle analyses, but its ability to form colonies was better than ACHN-CD105(-) cells, which is also complies with the theory that cancer stem cells proliferate slowly with a paradoxically enhanced capability to maintain the tumor growth. Additionally, ACHN-CD105(+) cells were younger than ACHN-CD105(-) cells in our β-galactosidase cell senescence assay. Based on findings above, We identified27differently expressed miRNAs in ACHN-CD105(+) and ACHN-CD105(-) cells, of which we pinpointed miR-34a, miR-155, miR-200a targeting CD105while miR-335pairs OCT-4. ACHN-CD105(+) cells transfected with miR-34a, miR-155and miR-200a showed remarkably decreased cisplatin-resistance while ACHN-CD105(+)-LV_miR-335experienced diminished sphere forming and migration ability. Also, miR-335could decrease various stemness genes expression including OCT-4, NANOG, SOX-2and KLF-4.ConclusionACHN-CD105(+) cells sorted from ACHN xenograft tumor sample beared the majority property of cancer stem cells, thus it can be preliminarily seen as renal cancer stem cells. miR-34a, miR-155and miR-200a could diminish the cisplatin resistance of ACHN-CD105(+) cells by downregulating CD105while miR-335could inhibit ACHN-CD105(+) cells’ sphere formation and migration capability by pulling down OCT-4. Part ⅠIsolationl, Identifictaion and Culture of Human Renal Cancer Stem CellsPurposeTo isolate and identify renal cancer stem cells signified with CD105(+)MethodsA xenograft model was established subcutaneously in axillary fossa of BALB/c-nu with well-acknowledged renal cancer cell line ACHN1month prior to FACS. ACHN-CD105(+) cells could be sorted with anti-hCD105-PE from ACHN xenograft tumor at a proportion of3.05%and proliferated and cryopreserved in renal cancer stem cell medium. Sphere Formation Assay, RPMI-1640+10%FBS and DMEM-HG+10%FBS differentiation assay, cisplatin-based drug-resistance assay, MTS and EdU-based cell proliferation assay, cell cycle analyses, colony formation assay and β-galactosidase sensescence assay were utilized to assess the sternness-related property and determine the proliferation pattern.ResultsACHN-CD105(+) only accounted for3.05%in the whole ACHN xenograft tumor cell population, which is accordance with the canonical cancer stem cell theory that a small fraction of cells drive tumor growth. Also, ACHN-CD105(+) cells could be sorted, cryopreserved and proliferated without differentiation or stemness genotypic change within at least50passages. In the subsequent identification experiements, ACHN-CD105(+) beared an obviously elevated capability to form spheres than ACHN-CD105(-) cells and could be induced to differentiate to lose stemness genotypes as CXCR4, OCT-4and NANOG after2weeks’ culture in RPMI-1640+10%FBS and DMEM-HG+10%FBS. Moreover, ACHN-CD105(+) manifested an improved cisplatin resistance compared to ACHN-CD105(-). In cell proliferation-related assays, ACHN-CD105(+)’s proliferation seemed to be retardated and shows GO arrest as seen in cell cycle analyses, but its ability to form colonies was better than ACHN-CD105(-) cells, which is also complies with the theory that cancer stem cells proliferate slowly with a paradoxically enhanced capability to restart the tumor growth. Additionally, ACHN-CD105(+) cells were younger than ACHN-CD105(-) cells in our P-galactosidase cell senescence assay.ConclusionACHN-CD105(+) cells sorted from ACHN xenograft tumor sample beared the majority property of cancer stem cells, thus it can be preliminarily seen as renal cancer stem cell. Part ⅡFunction of microRNAs in Human Renal Cancer Stem CellsPurposeTo screen potentially therapeutic miRNAs targeting ACHN-CD105(+) cells and explore the underlying mechanismsMethodsmiRNAs targeting ACHN-CD105(+) were determined by the query of miRNA databases and literacies and verified by qRT-PCR. Dual Luciferase Assay was utilized to confirm the binding between miRNAs and their corresponding target mRNAs. Cisplatin-based chemoresistance assay, β-galactosidase cell senescence assay, cell migration assay and sphere formation assay were adopted to assess the efficacy and efficiency of the screened miRNAs in the treatment of renal cancer.ResultsWe identified27differently expressed miRNAs in ACHN-CD105(+) and ACHN-CD105(-) cells, of which we pinpointed miR-34a, miR-155, miR-200a targeting CD105while miR-335pairs OCT-4. ACHN-CD105(+) cells transfected with miR-34a, miR-155and miR-200a showed remarkably decreased cisplatin-resistance while ACHN-CD105(+)-LV_miR-335experienced diminished sphere forming and migration ability. Also, miR-335could decrease various stemness genes expression including OCT-4, NANOG, SOX-2and KLF-4.ConclusionmiR-34a, miR-155and miR-200a could diminish the cisplatin resistance of ACHN-CD105(+) cells by downregulating CD105while miR-335could inhibit ACHN-CD105(+) cells’ sphere formation and migration capability by pulling down OCT-4. |
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