Radiosensitive Nasopharyngeal Carcinoma Research Articles

USP5 Binds and Stabilizes EphA2 to Increase Nasopharyngeal Carcinoma Radioresistance.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment. However, the underlying mechanism of NPC radioresistance remains poorly understood, and the promising radiosensitizer for NPC radiotherapy is also lacked. Overexpression of USP5 and EphA2 has been linked to various cancers, and both the proteins have attracted considerable attention for the development of new anti-cancer drugs. Here, we report that USP5 interacts with EphA2, and increases EphA2 protein stability and expression by ubiquitin proteasome pathway in the NPC cells. Mebendazole (MBZ), a broad-spectrum anthelmintic drug, transcriptionally inhibits USP5 expression, and then promotes EphA2 ubiquitination degradation in the NPC cells. Functionally, USP5 enhances in vitro and in vivo NPC cell radioresistance via stabilizing EphA2, and MBZ decreases in vitro and in vivo NPC cell radioresistance via targeting USP5/EphA2 axis. Moreover, the levels of USP5 and EphA2 are significantly higher in the radioresistant NPCs than those in the radiosensitive NPCs, and both proteins for predicting patient prognosis are superior to individual protein. These findings suggest that USP5 binds and stabilizes EphA2 by ubiquitin proteasome pathway to promote NPC radioresistance, and MBZ increases NPC radiosensitivity by targeting USP5/EphA2 axis, and is a potential radiosensitizer in NPC and perhaps in other cancers.

Homeobox B2 promotes malignant behavior and contributes to the radioresistance of nasopharyngeal carcinoma by regulating forkhead box protein O1.

Background: Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the head and neck with heterogeneous racial and geographical distributions. Homeobox B2 (HOXB2) is a tumor promoter in many cancers. However, the biological role of HOXB2 in NPC has not been elucidated. Methods: Bioinformatics analysis was performed to identify the differentially expressed genes (DEGs) between samples of patients with radiosensitive and radioresistant NPC. qRT-PCR, western blotting and immunohistochemistry were used to detect the expression levels of the corresponding mRNA and proteins. Cell viability was detected by CCK-8 assay and colony-forming capability was evaluated using colony formation assays. Further, migration and invasion abilities were examined using wound-healing and transwell chamber assays, respectively. Cellular apoptosis after irradiation was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Results: HOXB2 was identified as a potential regulator of radioresistance in NPC. Our in vitro results indicate that HOXB2 overexpression (HOXB2-OE) promoted malignant behaviors including invasion, migration, proliferation, and inhibited the irradiation-induced apoptosis of NPC cells. Consistent with these results, HOXB2 knockdown (HOXB2-sh) exhibited the opposite trends in these biological activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were enriched in the FOXO signaling pathway. Mechanistically, western blotting showed that HOXB2-OE inhibited forkhead box protein O1 (FOXO1) expression in NPC cells. Thereafter, we transferred the FOXO1-OE plasmid to HOXB2-OE NPC cells and found that overexpression of FOXO1 reversed cell proliferation, migration, invasion, and radioresistance profiles promoted by HOXB2 overexpression. Conclusion: Our findings showed that HOXB2 acts as a tumor promoter in NPC, activating malignant behaviors and radioresistance of tumors via FOXO1 regulation. Moreover, the inactivation of HOXB2 or activation of FOXO1 are potential strategies to inhibit tumor progression and overcome radioresistance in NPC.

Enhancing nasopharyngeal carcinoma cell radiosensitivity by suppressing AKT/mTOR via CENP-N knockdown

ObjectiveInvestigating the impact of centromere protein N (CENP-N) on radiosensitivity of nasopharyngeal carcinoma (NPC) cells.MethodsUsing immunohistochemistry and immunofluorescence to detect CENP-N expression in tissues from 35 patients with radiosensitive or radioresistant NPC. Assessing the effect of combined CENP-N knockdown and radiotherapy on various cellular processes by CCK-8, colony formation, flow cytometry, and Western blotting. Establishing a NPC xenograft model. When the tumor volume reached 100 mm3, a irradiation dose of 6 Gy was given, and the effects of the combined treatment were evaluated in vivo using immunofluorescence and Western blotting techniques.ResultsThe level of CENP-N was significantly reduced in radiosensitive tissues of NPC (p < 0.05). Knockdown of CENP-N enhanced NPC radiosensitivity, resulting in sensitizing enhancement ratios (SER) of 1.44 (5-8 F) and 1.16 (CNE-2Z). The combined treatment showed significantly higher levels of proliferation suppression, apoptosis, and G2/M phase arrest (p < 0.01) compared to either CENP-N knockdown alone or radiotherapy alone. The combined treatment group showed the highest increase in Bax and γH2AX protein levels, whereas the protein Cyclin D1 exhibited the greatest decrease (p < 0.01). However, the above changes were reversed after treatment with AKT activator SC79. In vivo, the mean volume and weight of tumors in the radiotherapy group were 182 ± 54 mm3 and 0.16 ± 0.03 g. The mean tumor volume and weight in the combined treatment group were 84 ± 42 mm3 and 0.04 ± 0.01 g.ConclusionKnockdown of CENP-N can enhance NPC radiosensitivity by inhibiting AKT/mTOR.

Inhibition of MNK pathway sensitizes nasopharyngeal carcinoma to radiotherapy.

Improving the clinical management of nasopharyngeal carcinoma (NPC) is an unmet need owing to the high incidence of treatment failure caused by radioresistance. In our study, we observed increased phosphorylation of translation initiation factor 4E (eIF4E), regulated by MAP kinase-interacting kinase (MNK), in NPC cells following irradiation treatment. Using siRNA to deplete MNK, we found that radiation-induced eIF4E phosphorylation was eliminated, NPC cell sensitivity to radiation was enhanced, and radioresistant NPC cell viability was reduced. Furthermore, we tested three pharmacological MNK inhibitors (eFT508, CGP57380, and cercosporamide) and found that they were effective against radioresistant NPC cells and synergized with irradiation. In-vivo experiments confirmed that eFT508, at a tolerable dose, inhibited the growth of radioresistant NPC and synergized with radiation in a radiosensitive NPC xenograft model. Our research highlights the activation of MNK-mediated survival mechanisms in NPC in response to radiotherapy and the potential of combining radiation with MNK inhibitors as a sensitizing strategy. Notably, eFT508 is currently being investigated in clinical trials for cancer treatment, and our findings may prompt the initiation of clinical trials using eFT508 in radioresistant NPC patients.

M6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis

Radiotherapy is a valid treatment for nasopharyngeal carcinoma (NPC), and radioresistance is the main cause of local NPC treatment failure. However, the underlying mechanisms and valuable markers of radioresistance for NPC remain have not been established. In this study, we observed that the m6A mRNA demethylase fat mass and obesity-associated protein (FTO) was significantly upregulated in radioresistant NPC tissues and cells relative to parental radiosensitive NPC tissues and cells. FTO enhances radioresistance by repressing radiation-induced ferroptosis in NPC. Mechanistically, FTO acts as an m6A demethylase to erase the m6A modification of the OTUB1 transcript and promote the expression of OTUB1, thereby inhibiting the ferroptosis of cells induced by radiation and finally triggering the radiotherapy resistance of NPC. Furthermore, our in vivo experiment results showed that the FTO inhibitor, FB23-2, and the ferroptosis activator, erastin, altered tumor responsiveness to radiotherapy in NPC cell lines and patient-derived xenografts. Our findings reveal, for the first time, that FTO enhances NPC radiotherapy resistance by withstanding radiation-induced ferroptosis, suggesting that FTO may serve as a potential therapeutic target and valuable prognostic biomarker in patients with NPC.

Key radioresistance regulation models and marker genes identified by integrated transcriptome analysis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a malignancy that is endemic to China and Southeast Asia. Radiotherapy is the usual treatment, however, radioresistance remains a major reason for failure. This study aimed to find key radioresistance regulation models and marker genes of NPC and clarify the mechanism of NPC radioresistance by RNA sequencing and bioinformatics analysis of the differences in gene expression profiles between radioresistant and radiosensitive NPC tissues. A total of 21 NPC biopsy specimens with different radiosensitivity were analyzed by RNA sequencing. Differentially expressed genes in RNA sequencing data were identified using R software. The differentially expressed gene data derived from RNA sequencing as well as prior knowledge in the form of pathway databases were integrated to find sub‐networks of related genes. The data of RNA sequencing with the GSE48501 data from the GEO database were combined to further search for more reliable genes associated with radioresistance of NPC. Survival analyses using the Kaplan–Meier method based on the expression of the genes were conducted to facilitate the understanding of the clinical significance of the differentially expressed genes. RT‐qPCR was performed to validate the expression levels of the differentially expressed genes. We identified 1182 differentially expressed genes between radioresistant and radiosensitive NPC tissue samples. Compared to the radiosensitive group, 22 genes were significantly upregulated and 1160 genes were downregulated in the radioresistant group. In addition, 10 major NPC radiation resistance network models were identified through integration analysis with known NPC radiation resistance‐associated genes and mechanisms. Furthermore, we identified three core genes, DOCK4, MCM9, and POPDC3 among 12 common downregulated genes in the two datasets, which were validated by RT‐qPCR. The findings of this study provide new clues for clarifying the mechanism of NPC radioresistance, and further experimental studies of these core genes are warranted.

Effect of GADD45G on the radioresistance of nasopharyngeal carcinoma cells.

The development of radioresistance by nasopharyngeal carcinoma (NPC) cells almost always results in tumor recurrence and metastasis, making clinical treatment of the disease difficult. In this study, the mechanism of radioresistance in NPC cells was investigated. First, a gene array and quantitative reverse-transcription-PCR assays were used to screen for genes exhibiting significantly altered expression in the DNA damage signaling pathway. Based on those results, GADD45G was further studied in the context of radioresistance. A GADD45G-knockout NPC cell line (CNE-2R-KO) was constructed using CRISPR-Cas9 technology and used for a comparison of differences in radioresistance with other radiosensitive and radioresistant NPC cells, as evaluated using colony formation assays. Cell cycle changes were observed using flow cytometry. Cell proliferation and migration were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and wound healing assays, respectively. The sequencing results revealed the successful construction of the CNE-2R-KO cell line, the radiosensitivity of which was higher than that of its parent radioresistant cell line owing to the GADD45G knockout. This was likely related to the increase in the number of cells in the G1 phase and decrease in those in the S1 phase as well as the increased cell proliferation rate and decreased migratory ability. GADD45G is associated with radioresistance in NPC cells and likely has a role in the occurrence and metastasis of NPC.

Cancer-associated fibroblasts promote the survival of irradiated nasopharyngeal carcinoma cells via the NF-\u03baB pathway

BackgroundIrradiation has emerged as a valid tool for nasopharyngeal carcinoma (NPC) in situ treatment; however, NPC derived from tissues treated with irradiation is a main cause cancer-related death. The purpose of this study is to uncover the underlying mechanism regarding tumor growth after irradiation and provided potential therapeutic strategy.MethodsFibroblasts were extracted from fresh NPC tissue and normal nasopharyngeal mucosa. Immunohistochemistry was conducted to measure the expression of α-SMA and FAP. Cytokines were detected by protein array chip and identified by real-time PCR. CCK-8 assay was used to detect cell proliferation. Radiation-resistant (IRR) 5-8F cell line was established and colony assay was performed to evaluate tumor cell growth after irradiation. Signaling pathways were acquired via gene set enrichment analysis (GSEA). Comet assay and γ-H2AX foci assay were used to measure DNA damage level. Protein expression was detected by western blot assay. In vivo experiment was performed subcutaneously.ResultsWe found that radiation-resistant NPC tissues were constantly infiltrated with a greater number of cancer-associated fibroblasts (CAFs) compared to radiosensitive NPC tissues. Further research revealed that CAFs induced the formation of radioresistance and promoted NPC cell survival following irradiation via the IL-8/NF-κB pathway to reduce irradiation-induced DNA damage. Treatment with Tranilast, a CAF inhibitor, restricted the survival of CAF-induced NPC cells and attenuated the of radioresistance properties.ConclusionsTogether, these data demonstrate that CAFs can promote the survival of irradiated NPC cells via the NF-κB pathway and induce radioresistance that can be interrupted by Tranilast, suggesting the potential value of Tranilast in sensitizing NPC cells to irradiation.

Carbon ion triggered immunogenic necroptosis of nasopharyngeal carcinoma cells involving necroptotic inhibitor BCL-x.

To explore the potential and mechanisms of necroptosis, a form of immunogenic cell death, induced by carbon ion as compared to photon beams in established photon resistant- (PR-) and sensitive nasopharyngeal carcinoma (NPC) cells. MLKL is considered a central executor of necroptosis and phosphorylation of MLKL (p-MLKL) was a critical event of necroptosis. The clonogenic survival and DNA microarray demonstrated that after repeated photon irradiation, radiosensitive NPC cells became apoptosis-resistant but could be effectively inhibited by carbon ion irradiation. The relative biologic effectiveness (RBE) at D10 and D37 were 2.15 and 2.78 for PR-NPC cells. Carbon ion induced delayed DNA damage repair, cell cycle arrest, cytogenetic damage, morphological change and cell necrosis, indicating the possibility of necroptosis in both PR- and sensitive NPC cell types. The lower expression of necroptotic inhibitors (caspase-8 and Bcl-x) and higher level of MLKL in PR-NPC cells showed it was relatively more predisposed to necroptosis compared to the sensitive cells. Subsequent experiments demonstrated the significant upregulation of p-MLKL in the PR-NPC cells treated by carbon ion (4 Gy) compared with photon irradiation at both physical (4 Gy) and RBE (10 Gy) doses (P≤0.0001). Moreover, carbon ion induced a robust (up to 28 folds) p-MLKL in the PR-NPC cells as well as sensitive cells (up to 6-fold) coupled with a lower level of BCL-x expression and increased GM-CSF implicated in resculputure of immune system. These results suggested that carbon ion could induce necroptosis of NPC cells, especially in PR-NPC cells, and its mechanisms involve BCL-x.

Down regulation of lactotransferrin enhanced radio-sensitivity of nasopharyngeal carcinoma

IntroductionIt is reported that LTF had a radiation resistance effect, and its expression in nasopharyngeal carcinoma (NPC) was significantly down-regulated. However, the mechanism of down-regulated LTF affecting the sensitivity of radiotherapy has remained elusive. MethodsWe re-analyzed the microarray data GSE36972 and GSE48503 to find differentially expressed genes (DEGs) in NPC cell line 5−8 F transfected with LTF or vector control, and the DEGs between radio-resistant and radio-sensitive NPC cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein-protein interaction network (PPI) analysis of DEGs were performed to obtain the node genes. The target genes of miR-214 were also predicted to complement the mechanism associated with radiotherapy resistance because it could directly target LTF. ResultsThis study identified 1190 and 1279 DEGs, respectively. GO and KEGG analysis showed that apoptotic process and proliferation, PI3K-Akt signaling pathway were significantly enriched pathways. Four nodes (DUSP1, PPARGC1A, FOS and SMARCA1) associated with LTF were screened. And 42 target genes of miR-214 were cross-linked to radiotherapy sensitivity. ConclusionsThe present study demonstrates the possible molecular mechanism that the down-regulated LTF enhances the radiosensitivity of NPC cells through interaction with DUSP1, PPARGC1A, FOS and SMARCA1, and miR-214 as its superior negative regulator may play a role in regulating the radiotherapy effect.

Construction and analysis of miRNA-mRNA regulatory networks in the radioresistance of nasopharyngeal carcinoma.

Radiotherapy has been the major treatment strategy for nasopharyngeal carcinoma (NPC), while the occurrence of radioresistance may lead to cancer recurrence or progression. This study aimed to identify the key microRNAs (miRNAs) and their target genes in the development of NPC radioresistance. Public microarray data were searched and analyzed to screen the differentially expressed miRNAs (DEMs) and genes (DEGs) between radioresistant and radiosensitive NPC samples. MiRNA-mRNA networks were constructed. As a result, 5 DEMs and 195 DEGs were screened out. The DEGs were enriched in various signaling pathways, such as Cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, and Toll-like receptor signaling pathway. Several hub genes, such as IGF2, OLA1, BBS10, MMP9, and BBS7 were identified. A regulatory miRNA-mRNA network containing 87 miRNA-mRNA pairs was constructed. Then, 14 key miRNA-mRNA pairs that contained the hub genes were further filtered out. In the networks, miR-203a-3p had the largest number of target genes. Afterwards, the candidate pairs (miR-203a-3p/BTK and miR-484/OLA1) have been verified by a qRT-PCR assay. In summary, we identified several miRNAs and hub genes via big data screening. A total of 87 miRNA-mRNA pairs (including 14 key pairs) were predicted to play a crucial role in the development of NPC radioresistance. These data provide a bioinformatics basis for further exploring the molecular mechanism of radiotherapy resistance in NPC. Future studies are needed to validate the results.

T63 induces apoptosis in nasopharyngeal carcinoma cells through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway.

BackgroundNasopharyngeal carcinoma (NPC) is a common malignant tumor in china. T63 is one of novel curcumin analogues, which reduces the tumorigenic potential effectively.MethodsWe investigated the mechanism of anti-cancer activity on NPC cells treatment with T63 for the first time. Cell viability was monitored using the method of MTT and colony formation assay. Morphological changes observed by fluorescence microscope with Hoechst33342 staining. The cell cycle, the rates of cell apoptotic and mitochondrial membrane potential were identified by flow cytometry, and apoptotic proteins were identified by western blot analysis.ResultsIn our study, we found a significant decrease in the number of viable cells and in colony formation abilities of NPC cells (CNE2 and CNE2R) after 24, 48 and 72 h treatment with T63. T63 promoted morphologic changes of apoptosis and increased apoptotic rate. T63 promoted also cell cycle arrest of G2/M phases followed by decreased cell viability in CNE2 and CNE2R after 48 h. Then the loss of the mitochondrial membrane potential and release of cytochrome-c were demonstrated. Western blotting analysis found a decrease in Bcl-2/Bax ratio and activation of caspase-8, -9, -3 and PARP. And the changes of PTEN and p-Aktp473 suggested PTEN/PI3K/Akt might be an important signaling pathway in the anticancer role of T63.ConclusionsT63 definitely induced apoptosis in radiosensitive and radioresistant NPC cell lines. But the apoptotic effect was stronger on radioresistant cells than that on radiosensitive cells through activation of mitochondria-mediated apoptotic pathway.

M6A Reader YTHDC2 Promotes Radiotherapy Resistance of Nasopharyngeal Carcinoma via Activating IGF1R/AKT/S6 Signaling Axis.

N6-methyladenosine (m6A) modification has been reported as a critical regulator of gene transcript expression. Although m6A modification plays important roles in tumor development, its role in therapeutic resistance remains unknown. In this study, we aimed to examine the expression level of m6A-modification related proteins and elucidate the effect of m6A-related proteins on radiation response in nasopharyngeal carcinoma (NPC). Among the genes that participated in m6A modification, YTHDC2, a m6A reader, was found to be consistently highly expressed in radioresistant NPC cells. Knocking down of YTHDC2 expression in radioresistant NPC cells improved the therapeutic effect of radiotherapy in vitro and in vivo, whereas overexpression of YTHDC2 in radiosensitive NPC cells exerted an opposite effect. Bioinformatics and mechanistic studies revealed that YTHDC2 could physically bound to insulin-like growth factor 1 receptor (IGF1R) messenger RNA and promoted translation initiation of IGF1R mRNA, which in turn activated the IGF1R-AKT/S6 signaling pathway. Thus, the present study suggests that YTHDC2 promotes radiotherapy resistance of NPC cells by activating the IGF1R/ATK/S6 signaling axis and may serve as a potential therapeutic target in radiosensitization of NPC cells.

RETRACTED ARTICLE: Downregulation of RKIP promotes radioresistance of nasopharyngeal carcinoma by activating NRF2/NQO1 axis via downregulating miR-450b-5p

Dysregulation of RKIP and NRF2 has been widely involved in the therapy resistance of multiple malignances, however, their relation and the corresponding mechanisms, especially in radiation response, have not been elucidated. In this study, we revealed that RKIP could negatively regulate the expression of NRF2 in nasopharyngeal carcinoma (NPC) cells. Depletion or ectopic expression of NRF2 countered the pro- or anti- radioresistant effects of RKIP knockdown or overexpression on NPC cells, respectively, both in vitro and in vivo. Furthermore, our results indicated that NQO1 was positively regulated by NRF2 and served as the downstream effector of RKIP/NRF2 axis in regulation of NPC radioresistance. Mechanistically, miR-450b-5p, being positively regulated by RKIP in NPC cells, could sensitize NPC cells to irradiation by directly targeting and suppressing the level of NRF2. Besides, we analyzed the level of aforementioned molecules in NPC tissues. The results indicated that RKIP was significantly downregulated, NRF2 and NQO1 were notably upregulated in NPC tissues compared with in normal nasopharyngeal mucosa (NNM) tissues. Furthermore, RKIP and miR-450b-5p were remarkably lower, yet NRF2 and NQO1 were notably higher, in radioresistant NPC tissues relative to in radiosensitive NPC tissues. Consistent with the pattern in NPC cells, the RKIP/miR-450b-5p/NRF2/NQO1 axis was significantly correlated in NPC tissues. Downregulation of RKIP and miR-450b-5p, and upregulation of NRF2 and NQO1, positively correlated to malignant pathological parameters such as primary T stage, Lymph node (N) metastasis, and TNM stage. Finally, RKIP and miR-450b-5p served as favorable prognostic indicators, and NRF2 and NQO1 acted as unfavorable prognostic biomarkers in patients with NPC. Collectively, our outcomes reveal that RKIP downregulation promotes radioresistance of NPC by downregulating miR-450b-5p and subsequently upregulating and activating NRF2 and NQO1, highlighting RKIP/miR-450b-5p/NRF2/NQO1 axis as a potential therapeutic target for improving the radiosensitivity of NPC.

Diffusion kurtosis imaging and tumour microstructure for monitoring response to radiotherapy in human nasopharyngeal carcinoma xenografts.

To investigate the correlations and feasibility of diffusion kurtosis imaging (DKI) parameters and tumour histopathology after radiotherapy in human nasopharyngeal carcinoma (NPC) xenografts on nude mice. Seventy-two nude mice were used for the construction of CNE-1 (radio-insensitive) and CNE-2 (radio-sensitive) NPC xenograft models, followed by fraction irradiation at different doses of X-ray. The nude mice were randomly divided into six groups in each cell line models according to the dose of X-ray they have received and with six mice in each group. DKI scan was performed after radiation. DKI parameters, tumour histopathology and AQP-1 biomarkers were detected. One-way ANOVA and Pearson's correlation analysis were used in statistical analysis. In CNE-1 and CNE-2 NPC xenografts, D values were increased (P<0.01 and P<0.001), while K values (P<0.01 and P<0.001) and tumour size (P<0.001 and P<0.001) were reduced during fraction irradiation. Additionally, cell density (CD) and AQP-1 expressions were decreased, and necrosis ratio (NR) was increased in CNE-2 xenografts after fraction irradiation (P<0.001). The changes in D values were negatively correlated with tumour size (r=-0.856, P<0.001), CD (r=-0.918, P<0.001), AQP-1 mRNA (r=-0.856, P<0.001) and protein (r=-0.381, P=0.022) expressions while positively correlated with NR (r=0.908, P<0.001) in CNE-2 xenografts. The changes in K values were positively correlated with tumour size (r=0.964, P<0.001), CD (r=0.888, P<0.001), AQP-1 mRNA (r=0.955, P<0.001) and protein (r=0.330, P=0.049) expression levels while negatively correlated with NR (r=-0.930, P<0.001). However, in CNE-1 xenografts, there were no correlation between DKI parameters and the expression of AQP-1. Changes in D and K parameters after fractional irradiation are closely related with NPC cellular and pathological characteristics, especially size reduction and necrosis induction. These parameters exhibit potential abilities of monitoring the response to fractional irradiation in radio-sensitive NPC xenografts.

MiR-181a Upregulation Promotes Radioresistance of Nasopharyngeal Carcinoma by Targeting RKIP.

BackgroundRadioresistance is the leading cause of treatment failure for nasopharyngeal carcinoma (NPC). Therefore, screening the critical regulators in radioresistance and revealing the underlying mechanisms is imperative for improvement of therapeutical efficacy in NPC.Materials and methodsOur previous study has proved that miR-181a may serve as a pro-radioresistant miRNA. In this study, we explored the expression of miR-181a in NPC, especially in radioresistant NPC samples, by qPCR. Moreover, the clinical significance of miR-181a level was also analyzed. Furthermore, the functions of miR-181a, both in vitro and in vivo, were detected via a serial of assays such as CCK-8, plate clone survival, apoptosis, and xenograft tumor model. The downstream target of miR-181a was also validated by dual luciferase reporter assay and the roles of miR-181a’s target in the regulation of NPC radioresistance were investigated.ResultsThe results revealed that miR-181a was significantly upregulated in NPC, especially in radioresistant NPC. MiR-181a level is positively correlated to lymph node metastasis and advanced TNM stages and negatively associated with overall survival rate in NPC. Ectopic expression of miR-181a in radiosensitive NPC cells, or overexpression of miR-181a inhibitor in radioresistant NPC cells, could enhance or impair the radioresistance of NPC cells supported by the results from both in vitro and in vivo, respectively. Mechanistically, dual luciferase report assay indicated that miR-181a could directly target RKIP. Moreover, both in vitro and in vivo experimental outcomes indicated that RKIP restoration and knockdown could antagonize the effects of miR-181a and miR-181a inhibitor in the regulation of NPC radioresistance.ConclusionCollectively, the findings of this study proved that miR-181a is upregulated and promotes radioresistance by targeting RKIP in NPC. Targeting miR-181a/RKIP axis may be a valid path for reinforcing radiosensitivity and eventually improving the outcomes of clinical treatment in NPC.

CircRNA_000543 knockdown sensitizes nasopharyngeal carcinoma to irradiation by targeting miR-9/platelet-derived growth factor receptor B axis

Radiation therapy is the mainstay of salvage therapy for nasopharyngeal carcinoma (NPC). However, radiation therapy efficacy is limited by radioresistance that can cause local recurrence and distant metastases. Circular RNAs (circRNAs) have been involved in radioresistant esophageal cancer cells. In this study, we aim to investigate the role of circRNAs in the radioresistant NPC. Human circRNA array was performed in radioresistant and radiosensitive NPC samples to screen the differentially expressed circRNAs. We found that circRNA_000543 expression was upregulated in NPC tissues compared adjacent tissues, and was higher in radioresistant samples than in radiosensitive NPC samples. NPC patients with high circRNA_000543 expression displayed poorer overall survival compared with the patients with low circRNA_000543. Further investigations demonstrated that circRNA_000543 knockdown sensitized NPC cells by targeting miR-9/platelet-derived growth factor receptor B (PDGFRB) axis. CircRNA_000543 may be a potential therapeutic target for radioresistant NPC.

SA17EXTRACTING STABILITY INCREASES THE SNP HERITABILITY OF EMOTIONAL PROBLEMS IN YOUNG PEOPLE

Radiation therapy is the mainstay of salvage therapy for nasopharyngeal carcinoma (NPC). However, radiation therapy efficacy is limited by radioresistance that can cause local recurrence and distant metastases. Circular RNAs (circRNAs) have been involved in radioresistant esophageal cancer cells. In this study, we aim to investigate the role of circRNAs in the radioresistant NPC. Human circRNA array was performed in radioresistant and radiosensitive NPC samples to screen the differentially expressed circRNAs. We found that circRNA_000543 expression was upregulated in NPC tissues compared adjacent tissues, and was higher in radioresistant samples than in radiosensitive NPC samples. NPC patients with high circRNA_000543 expression displayed poorer overall survival compared with the patients with low circRNA_000543. Further investigations demonstrated that circRNA_000543 knockdown sensitized NPC cells by targeting miR-9/platelet-derived growth factor receptor B (PDGFRB) axis. CircRNA_000543 may be a potential therapeutic target for radioresistant NPC.

Retraction: MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance By Targeting A20/NF-κB Signaling Pathway.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-125b in NPC radioresistance, one of upregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-125b was frequently upregulated in the radioresistant NPCs, and its increment was significantly correlated with NPC radioresistance, and was an independent predictor for poor patient survival. In vitro radioresponse assays showed that miR-125b inhibitor decreased, whereas miR-125b mimic increased NPC cell radioresistance. In a mouse model, therapeutic administration of miR-125b antagomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we confirmed that A20 was a direct target of miR-125b and found that miR-125b regulated NPC cell radioresponse by targeting A20/NF-κB signaling. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 overexpression decreased while A20 knockdown increased NPC cell radioresistance both in vitro and in vivo Moreover, A20 was significantly downregulated while p-p65 (RelA) significantly upregulated in the radioresistant NPCs relative to radiosensitive NPCs, and miR-125b expression level was negatively associated with A20 expression level, whereas positively associated with p-p65 (RelA) level. Our data demonstrate that miR-125b and A20 are critical regulators of NPC radioresponse, and high miR-125b expression enhances NPC radioresistance through targeting A20 and then activating the NF-κB signaling pathway, highlighting the therapeutic potential of the miR-125b/A20/NF-κB axis in clinical NPC radiosensitization. Mol Cancer Ther; 16(10); 2094-106. ©2017 AACR.

Downregulation of EB virus miR-BART4 inhibits proliferation and aggressiveness while promoting radiosensitivity of nasopharyngeal carcinoma.

This study aims to explore the role of Epstein-Barr virus (EBV) miR-BART4 in occurrence and progression of nasopharyngeal carcinoma (NPC) and its effect on radiosensitivity. The expressions of EBV and miR-BART4 in 108 cases of NPC tissues and 97 cases of chronic nasopharyngeal inflammation tissues were determined by real time quantitative polymerase chain reaction (PCR), and the relationship between the expression of miR-BART4 and the clinicopathological features of NPC was analyzed. Cell lines, HONEl, CNEl, CNE2, C666-1, 6-10B, and NP-69 were used to compare the expression of miR-BART4, in which the CNE2 cells were selected for further experiments. CNE2 cells were grouped into blank group, negative control (NC) group, miR-BART4 inhibitors group and miR-BART4 mimics group. Cells in above groups were under radiation of 6 Gy X ray for 12 h before grouped into control group, 6 Gy group, NC + 6 Gy group, miR-BART4 inhibitors + 6 Gy group and miR-BART4 mimics + 6 Gy group. Cell proliferation, clone formation ability, cell apoptosis, invasion and migration ability were measured by MTT assay, clone formation assay, flow cytometry (FCM), Transwell assay and scratch test, respectively. Western blot analysis was used to detect the expression of apoptosis-related proteins (cleaved caspase-3, Bax and Bcl-2) and epithelial-mesenchymal transition (EMT) marker protein E-cadherin and Vimentin. mRNA and protein expression of PTEN were detected by qRT-PCR and western blot. Bioinformatics software and luciferase activity experiments were used to verify the targeting relationship between miR-BART4 and PTEN. Positive rate of EBV in NPC tissues (93.5%) was remarkably higher than that in chronic nasopharyngeal inflammation tissues (21.6%). miR-BART4 was highly expressed and mRNA and protein expression of PTEN was lowly expressed in EBV positive NPC tissues compared with EBV negative NPC tissues and chronic nasopharyngeal inflammation tissues. The expression of miR-BART4 was related to the clinical stage, lymph node metastasis and differentiation degree of NPC. Expression of miR-BART4 in CNE2, CNEl, HONEl, C666-1, 6-10B, 5-8F cells was higher than that in NP-69 cells. In CNE2 and C666-1 cell experiments, compared with blank group and NC group, miR-BART4 inhibitors group had decreased miR-BART4 expression, increased mRNA and protein expression of PTEN, cell survival rate, invasion and migration ability and increased cell apoptosis rate, which is totally contrary to the observation in miR-BART4 mimics group. The radiosensitive NPC tissues had higher miR-BART4 expression than that in radio-resistance NPC tissues. In comparison to 6 Gy group and NC + 6 Gy group, cell survival rate and clone number was inhibited, but the cell apoptosis rate was increased in miR-BART4 inhibitors +6 G group, in contrary to the observation in miR-BART4 inhibitors + 6 Gy group. Bioinformatics software and luciferase activity experiments confirmed that miR-BART4 could inhibit the expression of PTEN. EBV may promote development and progression of NPC by up-regulating miR-BART4 expressions, consequently inhibiting its radiosensitivity, whose effect may be related to the targeting inhibition of PTEN expression.