[Effects of lncRNA RP11-770J1.3 and TMEM25 expression on paclitaxel resistance in human breast cancer cells].

OBJECTIVE: To investigate the effects of long non-coding RNA(lncRNA) RP11-770J1.3 and transmembrane protein 25 (TMEM25) on paclitaxel resistance in human breast cancer MCF-7/PR cell line. METHODS: The expression of lncRNA RP11-770J1.3 and TMEM25 in human breast cancer MCF-7(paclitaxel sensitive) and MCF-7/PR(paclitaxel resistant) cells were detected by quantitative RT-PCR. The synthetic interfering fragments of lncRNA RP11-770J1.3 and TMEM25 were transfected into MCF-7/PR cells. Sulforhodamine B assay was used to detect the sensitivity of MCF-7/PR cells to paclitaxel after interference of lncRNA RP11-770J1.3 and TMEM25. The expression of multidrug-resistance genes and proteins were detected by qRT-PCR and Western blot, respectively. RESULTS: lncRNA RP11-770J1.3 and TMEM25 were highly expressed in MCF-7/PR cells, and were significantly down-regulated after transfection of synthetic interfering fragments. Down-regulation of lncRNA RP11-770J1.3 and TMEM25 enhanced the sensitivity of MCF-7/PR cells to paclitaxel, and inhibited the expression of MRP, BCRP and MDR1/P-gp (all P<0.05). Such effects were more significant when lncRNA RP11-770J1.3 and TMEM25 were both down-regulated (all P<0.05). CONCLUSIONS: lncRNA RP11-770J1.3 and TMEM25 are highly expressed in MCF-7/PR cells, and the down-regulation of lncRNA RP11-770J1.3 and TMEM25 can enhance paclitaxel sensitivity in MCF-7/PR cells.

[CXC chemokine receptor 4 regulates breast cancer cell cycle through S phase kinase associated protein 2].

OBJECTIVE: To investigate the effect of CXC chemokine receptor 4 (CXCR4) on cell cycle of breast cancer and its molecular mechanisms. METHODS: The expression of CXCR4 and S phase kinase associated protein 2 (Skp2) was detected by real-time fluorescence quantitative PCR (fqRT-PCR) and Western blot in breast cancer cells. The expression of signal proteins and the downstream genes of Skp2 was detected by Western blot. The effect of CXCR4, PI3K/Akt pathway inhibitor LY294002 and ERK pathway inhibitor U0126 on cell cycle of breast cancer was detected by propidium iodide staining. RESULTS: Skp2 was significantly down-regulated in CXCR4-downregulated cells and up-regulated in CXCR4-upregulated cells. CXCR4 also regulated the expression of Skp2 and other downstream genes by signaling protein. The proportion of cells in G0/G1 phase increased and that in S phase declined in CXCR4-downregulated cell, and the effect was more significant when combined with the use of LY294002 or U0126. CONCLUSIONS: CXCR4 can affect cell cycle and inhibit the proliferation of breast cancer cells by regulating Skp2 gene expression through PI3K/Akt and ERK signaling pathway.

Colorectal cancer cell-derived exosomal miRNA-372-5p induces immune escape from colorectal cancer via PTEN/AKT/NF-κB/PD-L1 pathway.

Tumor cells can escape immune surveillance by changing their own escape or expressing abnormal genes and proteins, resulting in unlimited proliferation and invasive growth of cells. These changes are related to microRNAs (miRNAs), which reduce the killing effect of immune cells, devastate the immune response, and interfere with apoptosis through the aberrant expression of relevant miRNAs. In the preliminary phase of this study, miRNAs in clinical plasma exosomes of colorectal cancer patients were differentially analyzed by RNA sequencing technology, and miR-372-5p derived from extracellular vesicles (sEVs) was found to be a key signaling molecule mediating the regulation of macrophages by colorectal cancer (CRC). miRNA-372-5p is upregulated in colorectal cancer patient tissues and serum, as well as colorectal cancer cell lines and their exosomes. Subsequently, we found that macrophages could take up sEV secreted by colorectal cancer cells HCT116, affecting the expression of the immune checkpoint PD-L1, resulting in the generation of a tumor-immunosuppressive microenvironment and suppression of T cell activation in CRC. Gene enrichment mapping and database revealed that miR-372-5p regulates PD-L1 expression in colorectal cancer through the homologous phosphatase-tensin (PTEN)-phosphatidylinositol 3-kinase-protein kinase B (AKT)-nuclear factor-κB (NF-κB) pathway. Further studies confirmed that miRNA-372-5p-treated macrophages co-cultured with T cells affected the regulation of PD-L1 expression through the PTEN/AKT/NF-κB signaling pathway, resulting in decreased CD3+CD8+ T cell activity, decreased cytokine IL-2 and increased IFN-γ. And miRNA-372-5p could down-regulate the expression of PD-L1 in HCT116 through the PTEN/AKT/NF-κB pathway, inhibit tumor cell proliferation and promote apoptosis. Conclusion: Colorectal cancer cell-derived exosome miR-372-5p can be phagocytosed by colorectal cancer and macrophage cells, regulate the expression of PD-L1 in colorectal cancer cells and macrophages by targeting the PTEN/AKT/NF-κB pathway, and induce the immunosuppressive microenvironment of CRC to promote CRC development. This suggests that inhibiting the secretion of HCT116-specific sEV-miR-372-5p or targeting PD-L1 in tumor-associated macrophages could be a novel approach for CRC treatment and possibly a sensitizing approach for CRC anti-PD-L1 therapy.

[Construction of pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and their interaction in living cells].

OBJECTIVE: To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells. METHODS: DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay. RESULTS: The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm. CONCLUSION: The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.

[Low expression of lncRNA-GAS5 promotes epithelial-mesenchymal transition of breast cancer cells in vitro].

OBJECTIVE: To investigate the role of long non-coding RNA growth arrest-specific transcript 5 (lncRNA-GAS5) in breast cancer progression and epithelial-mesenchymal transition (EMT) of the cancer cells. METHODS: Real-time quantitative PCR (qRT-PCR) was used to detect the expression of lncRNA-GAS5 in 37 pairs of breast cancer and adjacent non-tumor tissues and in parental MCF-7 cells and paclitaxel-resistant MCF-7 (MCF-7/PR) cells, and the correlation of lncRNA-GAS5 expression with the clinical stage and lymph node metastasis of breast cancer was investigated. The expressions of the genes related with cell cycle and EMT at both the mRNA and protein levels were detected using qRT-PCR, Western blotting and immunohistochemistry. The changes in the biological behaviors and morphology of breast cancer cells with either lncRNA-GAS5 knockdown or overexpression were observed. Nude mouse models were established bearing breast cancer xenografts derived from MCF-7/PR cells or MCF-7/PR cells over-expressing lncRNA-GAS5, and the inhibitory effect of paclitaxel on tumor growth was evaluated. RESULTS: The transcriptional levels of lncRNA-GAS5 were significantly lower in breast cancer tissues than in the adjacent non-tumor tissues (P<0.05), and decreased lncRNA-GAS5 expression was significantly correlated with TNM stage and lymph node metastasis of breast cancer (P<0.05). lncRNA-GAS5 expression was also significantly lowered in paclitaxel-resistant breast cancer cells and showed a positive correlation with P21 expression and a negative correlation with CDK6. MCF-7 cells during EMT presented with a lowered expression of lncRNA-GAS5, whereas lncRNA-GAS5 over-expression strongly suppressed MCF-7/PR cell migration and invasion, and increased the susceptibility of the cells to paclitaxel. In the tumor-bearing nude mouse models, lncRNA-GAS5 overexpression in the tumor cells obviously enhanced the inhibitory effect of paclitaxel on tumor growth and lung metastasis by reversing the EMT marker proteins. CONCLUSION: A decreased expression of lncRNA-GAS5 promotes lung metastasis of breast cancer by inducing EMT, suggesting the potential of lncRNA-GAS5 as a therapeutic target in breast cancer.

Involvement of miR-451 in resistance to paclitaxel by regulating YWHAZ in breast cancer.

MicroRNAs (miRNAs) have been identified as major post-transcriptional regulators of the initiation and progression of human cancers, including breast cancer. However, the detail role of miR-451 has not been fully elucidated in breast cancer. In this study, we aimed to investigate the biological role and molecular mechanisms of miR-451 in drug resistance in breast cancer cell lines and in xenograft model. We show that miR-451 is decreased in human breast cancer specimens and in paclitaxel-resistant (PR) cells. Ectopic expression of miR-451 could inhibit the cell migration and invasion, promoted apoptosis, induced cell-cycle arrest Furthermore, tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ) was identified as a direct target of miR-451. Remarkably, the expression of YWHAZ is inversely correlated with the level of miR-451 in human breast cancer samples. Co-treatment with miR-451 mimics and YWHAZ-siRNA significantly enhanced YWHAZ knockdown in both SKBR3/PR and MCF-7/PR cells Moreover, miR-451 markedly inhibited expression of ß-catenin via YWHAZ and subsequently inhibited downstream gene cyclin D1, c-Myc expression. The results of xenograft model in vivo showed that intratumor injection of miR-451 agomir induced a tumor-suppressive effect in SKBR3/PR drug-resistant xenograft model. Taken together, our findings suggested that miR-451 might be considered as important and potential target in paclitaxel-resistant breast cancer treatment.

N-peptide of vMIP-â ¡ reverses paclitaxel-resistance by regulating miRNA-335 in breast cancer.

Acquisition of resistance to paclitaxel is one of the most important problems in treatment of breast cancer patients, but the molecular mechanisms underlying sensitivity to paclitaxel remains elusive. Emerging evidence has demonstrated that microRNAs (miRNAs) play important roles in regulation of cell growth, migration and invasion through inhibiting the expression of its target genes. In our previous studies, we have shown that microRNA-335 (miR­335) decreased obviously between paclitaxel-resistant (PR) and parental breast cancer cells through miRNA microarray. However, the roles of miR­335 in breast cancer progression and metastasis are still largely unknown. NT21MP was designed and synthesized as an antagonist with CXCR4 to inhibit cellular proliferation and induce apoptosis. Therefore, the aim of this study was to explore the underlying mechanism of miR­335 and NT21MP in reverse PR in breast cancer cells. In this study, we found that miR­335 expression is significantly lower in PR MCF­7 and SKBR-3 cells (MCF­7/PR and SKBR-3/PR) compared with their parental MCF­7 and SKBR-3 cells. Functional experiments showed that overexpression of miR­335 and NT21MP increased the number of apoptosis cells, arrested cells in G0/G1 phase transition, and suppressed cell migration and invasion in vitro. Dual luciferase assays revealed that SETD8 is a direct target gene of miR­335. Furthermore, miR­335 markedly inhibited expression of SETD8 via Wnt/ß­catenin signaling and subsequently inhibited the expression of its downstream genes cyclin D1, and c­Myc. Additionally, ectopic expression of miR­335 or depletion of its target gene SETD8 could enhance the sensitivity of PR cells to paclitaxel. Taken together, these date elucidated that NT21MP and miR­335 mediated PR of breast cancer cells partly through regulation of Wnt/ß­catenin signaling pathway. Activation of miR­335 or inactivation of SETD8 could be a novel approach for the treatment of breast cancer.

[The mechanism of polypeptide derived from viral macrophage inflammatory protein II modulates SDF-1α/CXCR4-induced migration].

AIM: To assess whether NT21MP, the synthetic antagonist 21-mer peptide derived from viral macrophage inflammatory protein II inhibits human SKBR3 cells migration by interfering with SDF-1α/CXCR4 signaling. METHODS: The levels of CXCR4 were detected in breast cancer cells SKBR3 and MCF-7 by RT-PCR and immunohistochemistry. The effect of SDF-1α-induced SKBR3 migration (chemotaxis) in the presence and absence of NT21MP was determined using the Boyden chamber migration assay. Intracellular Ca(2+); concentration was measured by fluorometric analysis. Western blot analyses were performed to quantify phosphorylated ERK1/2 and FAK expression levels. RESULTS: The expression of CXCR4 was higher in SKBR3 than MCF-7 cells; SKBR3 migration increased in SDF-1α-treated cells. In contrast, AMD3100, an inhibitor of CXCR4 effectively inhibited SKBR3 migration. SKBR3 migration was decreased when the cells were exposed to NT21MPdose dependently(P<0.05). NT21MP also blocked Ca(2+); influx(P<0.05), an important signal for SKBR3 migration. In addition, NT21MP significantly decreased SDF-1α-induced SKBR3 migration and downregulated SDF-1α-induced express of phospho-ERK1/2 and phospho-FAK(P<0.05). CONCLUSION: The results showed that NT21MP has an inhibitory effect on SDF-1α-induced SKBR3 migration. The plausible mechanism of action could be upstream blockage of Ca(2+); influx and the downstream reduction of ERK1/2 and FAK signals.