MicroRNA-101-3p inhibits nasopharyngeal carcinoma cell proliferation and cisplatin resistance through ZIC5 down-regulation by targeting SOX2.

This study aims to explore the mechanism of microRNA (miR)-101-3p-mediated SOX2/ZIC5 axis in the progression of cisplatin resistance of nasopharyngeal carcinoma (NPC). ZIC5 expression was analyzed with a bioinformatics database and detected in NPC cell lines. Cisplatin-resistant cells (HNE-1/DDP and C666-1/DDP) were transfected with sh-ZIC5, sh-SOX2, sh-SOX2 + pcDNA3.1-ZIC5, or miR-101-3p Agomir + pcDNA3.1-SOX2. MiR-101-3p, SOX2, and ZIC5 expression was assessed after transfection, and cancer associated phenotypes were evaluated after cisplatin treatment. The potential relationships among miR-101-3p, SOX2, and ZIC5 were analyzed. A xenograft mouse model of NPC was established with HNE-1 cells stably transfected or not transfected with oe-ZIC5 and subjected to tail vein injection of miR-101-3p Agomir and intraperitoneal injection of cisplatin. Overexpression of ZIC5 was found in cisplatin-resistant NPC cells. Downregulating ZIC5 in NPC cells decreased cell viability, promoted apoptosis, and reduced cisplatin resistance. SOX2 had a binding site on ZIC5, and SOX2 promoted proliferation, migration, and cisplatin resistance and inhibited cell apoptosis by up-regulating ZIC5. Mechanistically, miR-101-3p was decreased in cisplatin-resistant NPC cells and negatively targeted SOX2. Overexpression of miR-101-3p inhibited tumor growth and cisplatin resistance in xenograft mouse model, which was reversed by ZIC5 overexpression. In conclusion, the miR-101-3p/SOX2/ZIC5 axis was implicated in cancer associated phenotypes and cisplatin resistance in NPC.

Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced stemness and cisplatin resistance in nasopharyngeal carcinoma side population cells through the miR-579-3p/SIRT1/SSRP1 axis.

OBJECTIVE: To explore the effects of exosomes loaded with circular RNA PARD3 on EBV-miR-BART4-induced stemness and resistance of cisplatin in nasopharyngeal carcinoma side population (NPC-SP) cells through the miR-579-3p/SIRT1/SSRP1 axis. METHODS: Sixty-five cancer tissues and 65 noncancerous tissues were collected from NPC patients or patients with rhinitis. The expressions of circPARD3, miR-579-3p, SIRT1, and SSRP1 were detected by qRT-PCR, western blot, or immunohistochemistry. In vivo tumor formation assay was performed in nude mice. Immunofluorescence and qRT-PCR were conducted for the determination of CD44 and CD133 expressions, and flow cytometry combined with Hoechst 33,342 dye efflux for identifying SP cells, CCK-8 and EdU assays for cell proliferation, and Transwell assay for migration and invasion. RESULTS: CircPARD3, SIRT1, and SSRP1 were upregulated while miR-579-3p was downregulated in NPC tissues and cells. CircPARD3 was positively correlated with the expressions of SIRT1 and SSRP1, and miR-579-3p was negatively correlated with circPARD3, SIRT1, and SSRP1. Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells, while miR-579-3p reversed the effect of exosomal circPARD3 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. Additionally, miR-579-3p suppressed EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells by regulating SIRT1. SIRT1 upregulated SSRP1 expression by catalyzing H3K4 methylation and down-regulation of SSRP1 reversed the effect of SIRT1 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. CONCLUSION: Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells through the miR-579-3p/SIRT1/SSRP1 axis. Graphical Headlights • EBV-miR-BART4 induces the stemness and resistance of NPC-SP cells. • CircPARD3 regulates SIRT1 by miR-579-3p. • SIRT1 regulates SSRP1 expression by histone methylation. • Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced NPC-SP cell stemness and resistance by the miR-579-3p/SIRT1/SSRP1 axis.

LncRNA NR120519 Blocks KRT17 to Promote Cell Proliferation and Migration in Hypopharyngeal Squamous Carcinoma.

BACKGROUND: Hypopharyngeal carcinoma is the worst type of head and neck squamous cell carcinoma. It is necessary to identify the key molecular targets related to the carcinogenesis and development of hypopharyngeal carcinoma. METHODS: Differentially expressed lncRNAs in hypopharyngeal carcinoma were selected by microarray, and lncRNA-associated proteins were found by RIP assay. Colony formation, CCK-8, wound healing and Transwell assays were performed to detect the effects of lncRNA and its associated protein on cell proliferation and migration in vitro. Downstream pathways of lncRNA and its associated protein were detected by WB. Through a subcutaneous tumor model, the effects of lncRNA and its associated protein on cell proliferation were detected. The expressions of lncRNA and its associated protein in hypopharyngeal cancer tissues were detected by qRT-PCR and immunohistochemistry assays, respectively, and survival analyses were performed by Kaplan-Meier curve. RESULTS: A total of 542 and 265 lncRNAs were upregulated and downregulated in microarrays, respectively. LncRNA NR120519 was upregulated and promoted cell proliferation and migration of hypopharyngeal carcinoma in vitro and cell proliferation in vivo. RIP and WB assays showed that KRT17 was associated with and blocked by NR120519.The silencing of KRT17 promoted cell proliferation and the migration of hypopharyngeal carcinoma in vitro and cell proliferation in vivo by activating the AKT/mTOR pathway and epithelial-mesenchymal transformation (EMT). Finally, the NR120519 high expression and KRT17 low expression groups showed shorter overall survival. CONCLUSION: NR120519 activated the AKT/mTOR pathway and EMT by blocking KRT17 to promote cell proliferation and the migration of hypopharyngeal carcinoma.

Identity of MMP1 and its effects on tumor progression in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant tumor worldwide with high morbidity and mortality. However, the diagnosis and molecular mechanisms of HNSCC remains poor. METHODS: Robust rank aggregation method was performed to excavate the differentially expressed genes (DEGs) in five datasets (GSE6631, GSE13601, GSE23036, GSE30784, GSE107591) from the Gene Expression Omnibus. Search Tool for the Retrieval of Interacting Genes (STRING) database extracted hub genes from the protein-protein interaction network. The expression of the hub genes was validated using expression profile from The Cancer Genome Atlas and Oncomine database. The module analysis and disease-free survival analysis of the hub genes were analyzed by Cytoscape and the Kaplan-Meier curve, respectively. The expression of hub genes was verified in clinical specimens. The functions of MMP1 which is most important in hub genes were explored in vitro and in vivo. RESULTS: Totally, 235 DEGs were identified in the present study which consists of 103 up-regulated and 132 down-regulated genes which were significantly enriched in the molecular function of calcium ion binding followed in the biological process of skin development. The mainly enriched pathways were ECM (extracellular matrix)-receptor interaction (hsa04512) and protein digestion and absorption (hsa04974). Six hub genes were screened out which showed dramatically increased expression in HNSCC samples compared with normal samples, including COL4A1, MMP1, PLAU, RBP1, SEMA3C, and COL4A2. These hub genes all showed worse disease-free survival with higher expression and were up-regulated in HNSCC clinical samples. MMP1 was proved to promote cell growth, migration, and phosphorylation of AKT in vitro and to promote liver metastasis in vivo. CONCLUSION: Bioinformatics analysis identified six key genes in HNSCC. Of these, MMP1 is the most likely biomarker. It activates the AKT pathway and promotes tumor progression.

Downregulation of S100A9 Reverses Cisplatin-Resistance and Inhibits Proliferation and Migration in Hypopharyngeal Carcinoma.

Purpose: Patients with hypopharyngeal carcinoma (HPC) often progress to an advanced clinical stage at diagnosis. Cisplatin has been widely used in first-line chemotherapy for advanced HPC. However, acquired chemotherapeutic resistance leads to recurrence, metastasis, and a poor survival rate. Therefore, identifying new drug targets to improve treatment effects is still in need. Methods: To screen the differential expression genes (DEGs) and proteins (DEPs), we conducted transcriptomic and proteomic analysis on cisplatin-sensitive cell lines (FaDu) and cisplatin-resistant cell lines (FaDu/DDP) of hypopharyngeal carcinoma. DEGs and DEPs, possibly the most associated with cisplatin-resistance, were verified by real-time polymerase chain reaction (RT-PCR) and western blot (WB), respectively, and the biological function of the screened S100A9 was further tested by CCK8, wound healing, and transwell assays. Results: We identified S100A9 as a target for resensitizing the response to cisplatin in an acquired resistance model. S100A9 overexpression was significantly related to cisplatin resistance. Functional studies in vitro models demonstrated that downregulation of S100A9 overcame cisplatin-resistance and inhibited proliferation and migration. Later, we verified that downregulation of S100A9 suppressed the interleukin-6 (IL6) expression and epithelial-mesenchymal transition (EMT) pathway. Conclusion: In all, S100A9 plays a crucial role in cisplatin-resistance, proliferation, and migration of HPC. Targeting S100A9 may become a novel strategy for the treatment of HPC.

Analysis of ceRNA network of differentially expressed genes in FaDu cell line and a cisplatin-resistant line derived from it.

BACKGROUND: Hypopharyngeal cancer accounts for 2% in head and neck cancers and has a poor prognosis. Cisplatin is a widely used chemotherapeutic drug in kinds of carcinomas, concluding hypopharyngeal cancer. However, the resistance of cisplatin appeared in recent years. Cisplatin-resistance has been partly explored before, but rarely in hypopharyngeal cancer. METHODS: We cultured the hypopharyngeal cancer cell (FaDu) and induced its cisplatin-resistant cell (FaDu/DDP4). Then we tested the differentially expressed genes (DEGs) between FaDu and FaDu/DDP4. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted on the DEGs, and we drew the ceRNA networks of DEGs. Finally, we chose eight miRNAs and six mRNAs for qRT-PCR to verify our microarray. RESULTS: We induced cisplatin-resistant FaDu/DDP4 and proved its chemoresistance. The resistance index (RI) of FaDu/DDP4 was 2.828. DEGs contain 2,388 lncRNAs, 1,932 circRNAs, 745 mRNAs and 202 miRNAs. These 745 mRNAs were classified into three domains and 47 secondary GO terms. In KEGG pathway enrichment, the "TNF signaling pathway", "IL-17 signaling pathway" and "JAK-STAT signaling pathway" were potentially significant signaling pathways. Then, 52 lncRNAs, 148 circRNAs, 155 mRNAs and 18 miRNAs were selected to draw the network. We noticed several potential targets (as miR-197-5p, miR-6808-5p, APOE, MMP1, S100A9 and CYP24A1). At last, the eight miRNAs and six mRNAs that are critical RNAs in ceRNA network were verified by qRT-PCR. CONCLUSION: The microarray helped to find DEGs in cisplatin-resistant hypopharyngeal cancer. TNF, IL-17 and JAK-STAT signaling pathways might be more significant for cisplatin-resistance. MiR-197-5p, miR-6808-5p, APOE, MMP1, S100A9 and CYP24A1 might be potential genes inducing resistance.