Knocking down RAD51AP1 enhances chemosensitivity by inhibiting the self-renewal of CD133 positive ovarian cancer stem-like cells.

PURPOSE: This study was designed to investigate the function of RAD51AP1 in the self-renewal and chemosensitivity of CD133 positive (CD133+) ovarian cancer (OC) stem-like cells. METHODS: CD133+ (CD133 positive) OVCAR4 and CD133 negative (CD133-) OVCAR4 cells were separated from OVCAR4 by flow cytometry. Then, the separated CD133+OVCAR4 cells were divided into the following groups: Vector group; RAD51AP1 group; siNC group; si-RAD51AP1 group. Next, sphere-formation assay and colony forming assay were used to evaluate the self-renewal and proliferation ability of cells; western blot to detect the expression of RAD51AP1, transforming growth factor beta 1 (TGF-ß1) and SMAD4 proteins in tissues and cells; qRT-PCR to assess the mRNA levels of sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), NANOG and Kruppel-like factor 4 (KLF4). RESULTS: The performance of CD133+OVCAR4 cells was much better than that of CD133-OVCAR4 cells in sphere-formation assay and colony forming assay. Besides, compared with adjacent group and CD133-OVCAR4 cells, the expression level of RAD51AP1 increased significantly in OC group and CD133+OVCAR4 cells. Moreover, the over-expression of RAD51AP1 promoted the self-renewal and proliferation of CD133+OVCAR4 cells. On the contrary, knocking down the expression level of RAD51AP1 could inhibit the self-renewal and proliferation of CD133+OVCAR4 cells and improve the sensitivity of cells to chemotherapy drugs. CONCLUSION: The findings of this study showed that RAD51AP1 was highly expressed in OC tissue and CD133+OVCAR4 cells, and regulated the self-renewal and chemosensitivity of tumor cells through the TGF-ß1/SMAD4 signaling pathway.

Chimeric antigen receptor-T cells targeting epithelial cell adhesion molecule antigens are effective in the treatment of colorectal cancer.

OBJECTIVE: To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T). METHODS: A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts. RESULTS: It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05). CONCLUSION: This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.

The potential value of microRNA-145 for predicting prognosis in patients with ovarian cancer: A protocol for systematic review and meta-analysis.

BACKGROUND: As an anticancer gene, microRNA-145 (miRNA-145) inhibits the growth, migration, and invasion of cancer cells, and inhibits tumorigenesis by targeting various genes that are abnormally expressed in tumors. However, whether miRNA-145 can be applied as a biomarker for potential prognosis of ovarian cancer still remains controversial. Therefore, this study further explored the prognostic value and mechanism of miRNA-145 in ovarian cancer through meta-analysis and bioinformatics analysis. METHODS: Eligible studies were identified by searching the China National Knowledge Infrastructure, Chinese Biomedical literature Database, Chinese Scientific and Journal Database, Wan Fang database, PubMed, EMBASE, and Web of Science up to July 2021. Pooled hazard ratios with 95% confidence intervals for patient survival were calculated to investigate the effects of miRNA-145 on the prognosis of ovarian cancer. Survival curves of differential expression of miRNA-145 were analyzed by Oncomir. The target genes of miRNA-145 were predicted by miRTARbase and Diana-Tarbase V7.0 database. Enrichr database was applied to analyze the target genes by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Protein-protein interaction network of target genes was constructed from STRING database. Cytoscape software was used to screen the hub genes to meet the requirements. The Gene Expression Profiling Interactive Analysis database was applied to analyze the survival outcomes of hub genes. RESULTS: The results of this meta-analysis would be submitted to peer-reviewed journals for publication. CONCLUSION: This study provides high-quality evidence to support the relationship between miRNA-145 expression and ovarian cancer prognosis. Through bioinformatics analysis, we further explored the mechanism of miRNA-145 in ovarian cancer and related pathways.