ARHGAP42 promotes cell migration and invasion involving PI3K/Akt signaling pathway in nasopharyngeal carcinoma.
ABSTRACT: Rho GTPase-activating protein 42 was identified as an inhibitor of RhoA to maintain normal blood pressure homeostasis. However, the effect of ARHGAP42 in promoting cell malignancy in nasopharyngeal carcinoma is demonstrated in this study. Microarray and real-time quantitative PCR were used for a mRNA profiling of ARHGAP42 in nasopharyngeal primary and metastatic carcinoma tissues. Western blot and immunohistochemical staining were used for detecting the expression of ARHGAP42 protein in nasopharyngeal carcinoma tissues and cell lines. The overexpression and silence experiments of ARHGAP42 were performed in NPC cell lines using siRNA and expressive plasmid for evaluating cancer cell migration and invasion in vitro. Real-time quantitative PCR, western blot, and transwell test were employed for with the function of ARHGAP42 and its antisense lncRNA uc010rul. We confirmed the elevated expression of ARHGAP42 in metastatic NPC tissues of mRNA and protein for the first time. Immunohistochemical analysis indicated that NPC patients with highly ARHGAP42 expression were significantly associated with shorter metastasis-free survival. Knockdown of ARHGAP42 resulted in significant inhibition of nasopharyngeal cancer cell migration and invasion in vitro, and the overexpression of ARHGAP42 showed the opposite effects. In addition, the silence of uc010rul resulted in ARHGAP42 expression decrease and significant inhibition of nasopharyngeal cancer cell migration and invasion. High expression of ARHGAP42 is associated with poor metastasis-free survival of nasopharyngeal carcinoma patients. ARHGAP42 promotes migration and invasion of nasopharyngeal carcinoma cells in vitro; the antisense lncRNA may be involved in this effect.
Project description:Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors. Studies have indicated that long noncoding RNAs (lncRNAs) function as important regulators in progression of tumorigenesis. In this study, lncRNA small nucleolar RNA host gene 7 (SNHG7) was selected to identify how it functioned in the development of NPC. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect SNHG7 expression in paired NPC patient tissue samples and cell lines. The role of SNHG7 in the metastasis of NPC was detected through scratch wound assay and Transwell assay. RT-qPCR and western blot assay were used to discover the function of SNHG7 in epithelial-to-mesenchymal transition (EMT) process. Tumor metastasis assay was also performed in vivo. In this study, RT-qPCR results showed that SNHG7 expression in NPC samples was remarkably higher when compared with that in adjacent ones. Cell invasion and cell migration of NPC were inhibited due to silence of SNHG7 and were promoted due to overexpression of SNHG7. Moreover, results of further experiments revealed that the EMT-related proteins were regulated via knockdown or overexpression of SNHG7 in NPC. Furthermore, tumor metastasis of NPC was inhibited via knockdown of SNHG7 and was enhanced via overexpression of SNHG7 in nude mice. These results indicate that SNHG7 enhances NPC cell invasion and cell migration by eliciting the EMT process.
Project description:LncRNAs are reported to be involved in tumor proliferation, invasion and metastasis, and are considered as potential biomarkers and therapeutic targets for human cancer, including head and neck cancer. In this study, we screened the differentially low-expressed linc01513 by bioinformatic to detect its expression and biological effect on nasopharyngeal carcinoma (NPC). MTT was used to evaluate the effect of linc01513 on the proliferation of NPC cells. Wound healing assay was used to determine the cells migration ability. The matrix transwell was used to further detect the role of linc01513 in cell invasion. Western blot was used to detect the expression of epithelial-mesenchymal transformation (EMT)-induced transcription factors E-cadherin, vimentin and Slug. The results showed that silence of linc01513 could promoted the proliferation, migration and invasion of NPC cells. The <i>in vivo</i> experiment showed that overexpression of linc01513 could inhibit the volume and weight of xenograft tumors. Database prediction, RNA pull-down and RIP experiments suggested that linc01513 may play an anti-tumor effect by inhibiting PTBP1 protein level. It is suggested that linc01513 directly binds to PTBP1 protein and mediates the EMT process and malignant biological behavior of NPC cells, which provides a new molecular marker for the prognosis and treatment of NPC.
Project description:Our previous study demonstrated that the NGX6b gene acts as a suppressor in the invasion and migration of nasopharyngeal carcinoma (NPC). Recently, we identified the novel isoform NGX6a, which is longer than NGX6b. In this study, we first found that NGX6a was degraded in NPC cells and that this degradation was mediated by ezrin, a linker between membrane proteins and the cytoskeleton. Specific siRNAs against ezrin increase the protein level of NGX6a in these cells. During degradation, NGX6a is not ubiquitinated but is degraded through a proteasome-dependent pathway. The distribution pattern of ezrin was negatively associated with NGX6a in an immunochemistry analysis of a nasopharyngeal carcinoma tissue microarray and fetus multiple organ tissues and Western blot analysis in nasopharyngeal and NPC cell lines, suggesting that ezrin and NGX6a are associated and are involved in the progression and invasion of NPC. By mapping the interacting binding sites, the seven-transmembrane domain of NGX6a was found to be the critical region for the degradation of NGX6a, and the amino terminus of ezrin is required for the induction of NGX6a degradation. The knockdown of ezrin or transfection of the NGX6a mutant CO, which has an EGF-like domain and a transmembrane 1 domain, resulted in no degradation, significantly reducing the ability of invasion and migration of NPC cells. This study provides a novel molecular mechanism for the low expression of NGX6a in NPC cells and an important molecular event in the process of invasion and metastasis of nasopharyngeal carcinoma cells.
Project description:BACKGROUND:The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN:CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored. RESULTS:NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC. CONCLUSION:Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC.
Project description:Epigenetic regulation plays an important role in the development and progression of nasopharyngeal carcinoma (NPC), but the epigenetic mechanisms underlying NPC metastasis remain poorly understood. Here, we demonstrate that hypermethylation of the UCHL1 promoter leads to its downregulation in NPC. Restoration of UCHL1 inhibited the migration and invasion of NPC cells in vitro and in vivo, and knockdown of UCHL1 promoted NPC cell migration and invasion in vitro and in vivo. Importantly, we found that UCHL1 interacts with CTTN, and may function as a ligase promoting CTTN degradation by increasing K48-linked ubiquitination of CTTN. Additionally, restoration of CTTN in NPC cells that overexpressed UCHL1 rescued UCHL1 suppressive effects on NPC cell migration and invasion, which indicated that CTTN is a functional target of UCHL1 in NPC. Our findings revealed that UCHL1 acts as a tumor suppressor gene in NPC and thus provided a novel therapeutic target for NPC treatment.
Project description:Accumulating evidences highlight the critical roles of long noncoding RNAs (lncRNAs) in a variety of cancers. LncRNA PXN-AS1-L was previously shown to exert oncogenic roles in hepatocellular carcinoma. However, the expression, role, and molecular mechanism of PXN-AS1-L in nasopharyngeal carcinoma (NPC) malignancy remain unknown. Here, we determined that PXN-AS1-L is upregulated in NPC tissues and cell lines. Increased expression of PXN-AS1-L predicts worse prognosis of NPC patients. PXN-AS1-L overexpression promotes NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. PXN-AS1-L silencing suppresses NPC cell proliferation, migration, and invasion in vitro. Mechanistically, PXN-AS1-L directly interacts with SAPCD2 mRNA 3'-untranslated region, prevents the binding of microRNAs-AGO silencing complex to SAPCD2 mRNA, and upregulates the mRNA and protein level of SAPCD2. SAPCD2 is also increased in NPC tissues. The expression of SAPCD2 is significantly positively associated with that of PXN-AS1-L in NPC tissues. Gain-of-function and loss-of-function experiments demonstrated that SAPCD2 also promotes NPC cell proliferation, migration, and invasion. Furthermore, depletion of SAPCD2 significantly reverses the roles of PXN-AS1-L in promoting NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. In conclusion, lncRNA PXN-AS1-L is upregulated in NPC and promoted NPC malignancy by upregulating SAPCD2 via direct RNA-RNA interaction.
Project description:Recent studies demonstrated that long non-coding RNAs (lncRNAs) deregulated in many cancer tissues including nasopharyngeal carcinoma (NPC) and had critical roles in cancer progression and metastasis. In this study, we aimed to assess a lncRNA LINC01420 expression in NPC and explore its role in NPC pathogenesis. Our research revealed that the expression level of LINC01420 in NPC tissues were higher than nasopharyngeal epithelial (NPE) tissues. Moreover, NPC patients with high LINC01420 expression level showed poor overall survival. Knockdown LINC01420 inhibited NPC cell migration and invasion in vitro. In summary, LINC01420 may play a critical role in NPC progression and may serve as a potential prognostic biomarker in NPC patients.
Project description:The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.
Project description:Background:Mounting evidence has reported that microRNA-154-5p (miR-154-5p) is involved in the development of multiple cancers, but its function in nasopharyngeal carcinoma (NPC) remains not well investigated. Methods:Real-time quantitative PCR (qRT-PCR) was used to detect miR-154-5p expression in NPC tissues and cells. CCK8, colony formation, wound healing and transwell assays were performed to assess cell proliferation, migration and invasion. Dual-luciferase reporter assays and Western blots were performed to confirm the target gene of miR-154-5p. Rescue experiments were conducted to explore the influence of target gene KIF14 on the functions of miR-154-5p. Xenograft tumor model was conducted to detect the effect of miR-154-5p in vivo. Results:qRT-PCR results revealed that the expression of miR-154-5p was down-regulated in NPC tissues and cell lines compared to normal nasopharyngeal tissues and cell line. Overexpression of miR-154-5p inhibited cell migration and invasion. However, miR-154-5p had no influence on the proliferation of NPC cells. MiR-154-5p overexpression suppressed xenograft tumor metastasis in vivo. Dual-luciferase reporter analysis identified KIF14 as a target gene of miR-154-5p. Rescue experiments showed that knockdown of KIF14 reversed the effect of inhibiting miR-154-5p expression on NPC cell migration and invasion. Conclusion:Taken together, miR-154-5p suppresses tumor migration and invasion by targeting KIF14 in NPC. The newly identified miR-154-5p/KIF14 interaction offers further insights into the progression of NPC, which may represent a novel target for NPC diagnosis and treatment.
Project description:Background:E2F transcription factor 3 (E2F3) is oncogenic and dysregulated in various malignancies. Complex networks involving microRNAs (miRNAs) and E2F3 regulate tumorigenesis and progression. However, the potential roles of E2F3 and its target miRNAs in nasopharyngeal carcinoma (NPC) are rarely reported. Methods:E2F3 expression was detected in human NPC tissues and cell lines through quantitative real-time PCR. NPC cell proliferation, migration, and invasion were evaluated in vitro by colony forming, cell counting kit-8, wound healing, and Transwell invasion assays. Publicly available database software was used to explore the target miRNAs of E2F3. Dual-luciferase reporter assay was performed to identify the direct relationship. The function of miRNAs in vivo was investigated by using a tumor xenograft model. Results:E2F3 was upregulated in NPC cell lines and tissues, and its exotic expression promoted NPC cell invasion and migration. E2F3 was identified as a target of miR-432, which restrained NPC cell invasion and migration in vitro and in vivo. Further experiments revealed that miR-432 repressed the invasion and migration potential of NPC cells by modulating E2F3 expression. Conclusion:miRNA-432 suppressed the malignant biological behavior of NPC cells by targeting E2F3. This study provided further insights into NPC prognosis and treatment.