ABSTRACT
Background: Increasing evidence has indicated an association between differentially expressed genes (DEGs) in tumor-infiltrating immune cells (TIICs) and clinical outcome. The aim of this research is to investigate the influence of tumor microenvironment on the gene expression profile of TIICs and to identify their potential markers for modulating immune cell function in prostate cancer. Methods: In our research, CIBERSORT algorithm was utilized to calculate the proportion of the TIICs in 164 tumor and 18 control samples from The Cancer Genome Atlas cohort. The differential expression analysis was conducted using R, and then the functional and the pathway enrichments of the DEGs were analyzed using Database for Annotation, Visualization, and Integrated Discovery, followed by integrated regulatory network analysis. Results: As a result, nTreg, B cells, Th1, and DC cells were significantly increased, accompanied by largely decreased NK and NKT cells. The expressed immune-related gene correlation analysis showed that the signature gene expression extent of CD8 T cells was positively associated with CD4 memory activated T cells but negatively correlated with that of CD4 memory resting T cells. In addition, a total of 128 differentially expressed genes were identified. CytoHubba analysis obtained six hub genes, of which three prognostic-associated potential key molecules including CAV1, FLNA, and VCL were mainly involved in biological processes associated with the regulation of organic substance and synaptic connections. Conclusions: This study provides a comprehensive understanding of the landscape of TIICs and the roles of the hub genes which may be valuable markers in prostate cancer diagnosis and immunotherapy.
ABSTRACT
Docetaxel is a first-line anticancer drug widely used in the treatment of advanced prostate cancer. However, its therapeutic efficacy is limited by its side effects and the development of chemoresistance by the tumor. Using a gene differential expression microarray, we identified 449 genes differentially expressed in docetaxel-resistant DU145 and PC3 cell lines as compared to docetaxel-sensitive controls. Moreover, western blotting and immunohistochemistry revealed altered expression of S100A4, ACKR3 and CDH1in clinical tumor samples. Cytoscape software was used to investigate the relationship between critical proteins and their signaling transduction networks. Functional and pathway enrichment analyses revealed that these signaling pathways were closely related to cellular proliferation, cell adhesion, cell migration and metastasis. In addition, ACKR3 knockout using the crispr/cas9 method andS100A4knockdownusing targeted shRNA exerted additive effects suppressing cancer cell proliferation and migration. This exploratory analysis provides information about potential candidate genes. It also provides new insight into the molecular mechanism underlying docetaxel-resistance in androgen-independent prostate cancer and highlights potential targets to improve therapeutic outcomes.
Subject(s)
Antigens, CD/genetics , Cadherins/genetics , Docetaxel/pharmacology , Receptors, CXCR/genetics , S100 Calcium-Binding Protein A4/genetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival , Drug Resistance, Neoplasm/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Male , Prostatic Neoplasms/genetics , TranscriptomeABSTRACT
BACKGROUND: Stathmin as a critical protein involved in microtubule polymerization, is necessary for survival of cancer cells. However, extremely little is known about Stathmin in glioblastoma. So, this study was designed to elucidate the function of Stathmin gene in the tumorigenesis and progression of glioblastoma cells. METHOD: The lentiviral interference vector pLV3-si-Stathmin targeting Stathmin gene and the control vector pLV3-NC were established for the co-transfection of 293T cells together with the helper plasmids. Viral titer was determined via limiting dilution assay. Then pLV3-si-Stathmin and pLV3-NC were stably co-transfected into U373 and U87-MG glioblastoma cells. Expression levels of Stathmin protein in each group were determined by using Western Blot, and the proliferation and migration ability of the cells with downregulated Stathmin were evaluated through CCK8 assay and transwell invasion assay, respectively. Cell cycles and cell apoptosis were detected with flow cytometry. Finally, the effect of Stathmin in tumor formation was determined in nude mice. RESULT: DNA sequencing and viral titer assay indicated that the lentiviral interference vector was successfully established with a viral titer of 4 × 108 TU/ml. According to the results from Western Blotting, Stathmin protein expression level decreased significantly in the U373 and U87-MG cells after transfected with pLV3-si-Stathmin, respectively, compared with those transfected with pLV3-NC. In glioblastoma cells, the cell proliferation and migration were greatly inhibited after the downregulation of Stathmin protein. Flow cytometry showed that much more cells were arrested in G2/M phasein Stathmin downregulated group, compared with the non-transfection group and NC group. But Stathmin downregulation did not induce significant cell apoptosis. Tumor formation assay in nude mice showed that tumor formation was delayed after Stathmin downregulation, with a reduction in both tumor formation rate and tumor growth velocity. CONCLUSION: Stathmin downregulation affected the biological behaviors of U373 and U87-MG glioblastoma cells, inhibiting the proliferation and migration of tumor cells. Stathmin gene may serve as a potential target in gene therapy for glioblastoma.
Subject(s)
Cell Proliferation/genetics , Down-Regulation/genetics , Glioblastoma/metabolism , Stathmin/genetics , Animals , Cell Line, Tumor , Genetic Vectors , Glioblastoma/genetics , Glioblastoma/pathology , Mice , Stathmin/metabolism , TransfectionABSTRACT
BACKGROUND: Stathmin as a critical protein involved in microtubule polymerization, is necessary for survival of cancer cells. However, extremely little is known about Stathmin in glioblastoma. So, this study was designed to elucidate the function of Stathmin gene in the tumorigenesis and progression of glioblastoma cells. METHOD: The lentiviral interference vector pLV3-si-Stathmin targeting Stathmin gene and the control vector pLV3-NC were established for the co-transfection of 293T cells together with the helper plasmids. Viral titer was determined via limiting dilution assay. Then pLV3-si-Stathmin and pLV3-NC were stably co-transfected into U373 and U87-MG glioblastoma cells. Expression levels of Stathmin protein in each group were determined by using Western Blot, and the proliferation and migration ability of the cells with downregulated Stathmin were evaluated through CCK8 assay and transwell invasion assay, respectively. Cell cycles and cell apoptosis were detected with flow cytometry. Finally, the effect of Stathmin in tumor formation was determined in nude mice. RESULT: DNA sequencing and viral titer assay indicated that the lentiviral interference vector was successfully established with a viral titer of 4 × 108 TU/ml. According to the results from Western Blotting, Stathmin protein expression level decreased significantly in the U373 and U87-MG cells after transfected with pLV3-si-Stathmin, respectively, compared with those transfected with pLV3-NC. In glioblastoma cells, the cell proliferation and migration were greatly inhibited after the downregulation of Stathmin protein. Flow cytometry showed that much more cells were arrested in G2/M phasein Stathmin downregulated group, compared with the non-transfection group and NC group. But Stathmin downregulation did not induce significant cell apoptosis. Tumor formation assay in nude mice showed that tumor formation was delayed after Stathmin downregulation, with a reduction in both tumor formation rate and tumor growth velocity. CONCLUSION: Stathmin downregulation affected the biological behaviors of U373 and U87-MG glioblastoma cells, inhibiting the proliferation and migration of tumor cells. Stathmin gene may serve as a potential target in gene therapy for glioblastoma.
Subject(s)
Animals , Mice , Down-Regulation/genetics , Glioblastoma/metabolism , Cell Proliferation/genetics , Stathmin/genetics , Transfection , Glioblastoma/genetics , Glioblastoma/pathology , Cell Line, Tumor , Stathmin/metabolism , Genetic VectorsABSTRACT
We investigated the plant regeneration and production of flavonoids in three high-yield flavonoids transgenic Saussurea involucrata hairy roots C17, C27 and C46 by quantification of two phytohormones GA3 and IAA. The results showed that GA3 concentration at more than 1.0 mg/L could induce adventitious shoots in the hairy root lines. The highest shoot regeneration rate, about 82%, was obtained when the hairy roots C17 were cultured with 2.0 mg/L GA3. The results on HPLC and UV spectrophotometry showed that exogenous application of both GA3 and IAA increased the content of flavonoids in the hairy roots. The contents of flavonoids and apigenin in the hormone-treated hairy roots and regenerates were higher comparing with those in the untreated hairy roots and the regenerates. However, the content of flavonoids was not related to tissue weight, and was negatively related to the regeneration efficiency.
