Radioresistance Of Nasopharyngeal Carcinoma Cells Research Articles

Genome-Scale CRISPR-Cas9 Knockout Screening in Nasopharyngeal Carcinoma for Radiosensitive and Radioresistant Genes

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), and resistance of cancer cells to radiation is one of the major causes of treatment failure. In this study, the genome-wide CRISPR/Cas9-sgRNA library virus was used for the first time to screen for the functional genes associated with radiosensitivity and radioresistance in nasopharyngeal carcinoma, which lays the foundation for new research aiming to explain the mechanisms behind the radiosensitivity of nasopharyngeal carcinoma and makes some contribution to the discovery of new therapeutic targets and the development of new drugs to increase the sensitivity of NPC cells to radiotherapy.CRISPR/Cas9 library lentivirus screening in radiation-treated nasopharyngeal carcinoma cells was combined with second-generation sequencing technology to identify specific sgRNAs. A set of samples as contrast, and early library infection was the starting control for genetic change trend analysis. Two groups were exposed to doses of 0 and 2 Gy, and cell samples were collected 7 and 14 days after irradiation. The genomic DNA of living cells was used for PCR amplification in the coding region of sgRNA, and then high-throughput sequencing analysis was performed. The radiosensitivity or radioresistance of the screened functional genes in nasopharyngeal carcinoma were further verified by colony formation and cell proliferation experiments using a cell counting kit in a stable cell line with single gene knockout and qPCR analysis in radioresistant nasopharyngeal carcinoma cells.Eleven radiosensitive and radioresistant function genes were selected based on high-throughput sequencing and bioinformatics analysis. Compared with the control group, after the single gene FBLN5, FAM3C, MUS81, or DNAJC17 knockout, the colony formation and proliferation ability of nasopharyngeal carcinoma cells C666-1 and CNE1 were significantly promoted, while after the single gene CDKN2AIP and SP1 knockout, the colony formation and proliferation ability were significantly lower (P < 0.05). Compared to the negative control cells, the expression levels of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower in the C666-1-Rs and 5-8F-Rs cell lines (P < 0.05), while the expression levels of TOMM20, CDKN2AIP, SNX22, and SP1 were significantly higher (P < 0.05). KEGG enrichment analysis showed that the potential signaling pathway contributed to radiosensitivity or radioresistance in NPC, including the Fanconi anemia pathway and the TGF-beta signaling pathway.We found nine genes through CRISPR/Cas9-mediated genome-wide knockout involved in the radiosensitivity or radioresistance of NPC cells: four genes for radiosensitivity, FBLN5, FAM3C, MUS81, and DNAJC17; two genes for radioresistance: CDKN2AIP, SP1, while TOMM20 and SNX22 are potential radioresistant genes, and CALD1 is a potential radiosensitive gene.

Mechanistic Study of Irradiated Dying NPC Cells Promoting the Metastatic Ability of Surviving NPC Cells After Radiotherapy

<h3>Purpose/Objective(s)</h3> Distant metastasis remained the major cause of mortality in nasopharyngeal carcinoma (NPC) receiving radiotherapy despite favorable locoregional control in the past decades. Tumor microenvironment plays a critical role in tumor progression by secreting factors that regulate cancer cell metastasis. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of necrotic or apoptotic NPC cells killed by radiotherapy in distant metastasis. We hypothesize that necrotic or apoptotic NPC cells killed by radiotherapy could release metastasis-stimulating signals to the microenvironment and promote the metastatic ability of the surviving cancer cells. <h3>Materials/Methods</h3> Based on the co-culture system, cell migration assay and cell invasion assay were used to analyze the effect of lethally irradiated NPC CNE1 cell line on migration and invasion abilities of radioresistant NPC cell line CNE1-IR. Subsequently, ITRAQ was used to screen the key factors in this process, and ELISA, qRT-PCR and Western blot were used to detect the expression of slit2 in lethally irradiated CNE1 cells. CNE1-IR cells were infected with Slit2 shRNA lentivirus (LV3-shSlit2) or LV3-NC. Cell migration and invasion assay as well as tumor metastasis experiment in vivo were used to examine the role of Slit2 in promoting metastasis of NPC cell lines. Lastly, Cell migration, invasion assay and Co-IP assay were used to explore which Robo receptors was involved in Slit2 mediated metastasis of NPC cell lines. <h3>Results</h3> The migration and invasion abilities of CNE1-IR cells were significantly promoted (<i>P</i> < 0.01) when CNE1-IR was co-cultured with lethally irradiated CNE1 cells. ITRAQ found that the expression of Slit2 was upregulated in the supernatant of lethally irradiated CNE1 cells. ELISA assay confirmed that Slit2 in the supernatant of lethally irradiated CNE1 cells was significantly higher than control cells. qRT-PCR analysis and Western blot analysis also showed that the expressions of Slit2 was significantly overexpressed in irradiated NPC cells compared with unirradiated control CNE1 cells. Knockdown of Slit2 significantly suppressed lung and liver metastasis in vivo. The expressions of both Robo1 and Robo2 could be detected in CNE1 and CNE1-IR. Subsequently, addition of rSlit2 could enhance cell migration ability. In contrast, Robo1-depleted cells showed decreased migration ability and did not respond to rSlit2. When rSlit2 was added to CNE1-IR/shRobo2 cells, the number of trans-membrance CNE-IR cells was increased by about 26% compared with CNE1-IR/NC cells. Co-IP assay with anti-Robo1 antibody indicated that Robo1 was immunoprecipitated with Slit2, indicating the interaction of Robo1 and Slit2. <h3>Conclusion</h3> Lethally irradiated NPC cells could promote migration and invasion of radioresistant NPC cell lines. Slit2, a secreted factor released from irradiated dying NPC cells could promote metastasis of living/resistant NPC cells via Robo1 receptor.

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.

Radioresistant Nasopharyngeal Carcinoma Cells Exhibited Decreased Cisplatin Sensitivity by Inducing SLC1A6 Expression.

Cisplatin-based regimens are commonly used for the treatment of nasopharyngeal carcinoma (NPC) in patients who receive concurrent chemoradiotherapy. The sensitivity of NPC cells to cisplatin is closely associated with the efficacy of radiation therapy. In this study, we established two radioresistant NPC cell lines, HONE1-IR and CNE2-IR, and found that both cell lines showed reduced sensitivity to cisplatin. RNA-sequence analysis showed that SLC1A6 was upregulated in both HONE1-IR and CNE2-IR cell lines. Downregulation of SLC1A6 enhanced cisplatin sensitivity in these two radioresistant NPC cell lines. It was also found that the expression of SLC1A6 was induced during radiation treatment and correlated with poor prognosis of NPC patients. Notably, we observed that upregulation of SLC1A6 led to elevating level of glutamate and the expression of drug-resistant genes, resulted in reduced cisplatin sensitivity. Our findings provide a rationale for developing a novel therapeutic target for NPC patients with cisplatin resistance.

Epstein-Barr virus (EBV) encoded microRNA BART8-3p drives radioresistance-associated metastasis in nasopharyngeal carcinoma.

Radiotherapy plays an important role in the treatment of nasopharyngeal carcinoma (NPC), however, 20% of patients with NPC exhibit unusual radioresistance. Patients with radioresistance are at risk of recurrence, so it is imperative to explore the mechanism of resistance to radiotherapy. In the past, studies on the mechanism of radioresistance have been restricted to DNA damage and related cell cycle remodeling or apoptosis. So far, no studies have explored the relationship between radioresistance and metastasis. Through the analysis of clinical samples, we observed that the metastasis rate of recurrent NPC was much higher than that of primary patients. In vitro and in vivo experiments showed that NPC cells with acquired radioresistance exhibited a stronger ability for invasion and metastasis. Mechanistically, we found that the Epstein-Barr virus (EBV)-encoded miRNA BART8-3p was increased in patients with NPC, and its expression was positively correlated with adverse prognostic factors, such as radioresistance. Besides this, miR-BART8-3p promoted the epithelial-mesenchymal transition, invasion, and metastasis of radioresistant NPC cells by targeting and inhibiting their PAG1 host gene. These findings suggested a novel role for EBV-miR-BART8-3p in promoting NPC radioresistance-associated metastasis and highlighted its potential value as a prognostic indicator or therapeutic target.

MiR-138-1-3p alters the stemness and radiosensitivity of tumor cells by targeting CRIPTO and the JAK2/STAT3 pathway in nasopharyngeal carcinoma.

BackgroundTumor resistance to radiotherapy is one of the main obstacles to the clinical treatment of nasopharyngeal carcinoma (NPC). Improving the radiosensitivity of tumor cells has an important clinical significance in treatment of clinical NPC. This study aimed to identify that miR-138-1-3p as a novel therapeutic target in radioresistant NPC cells and found its targets, CRIPTO and the JAK2/STAT3 pathway.MethodsRadioresistant C666-IR and HK-1R cells were derived from the NPC cell lines C666-1 and HK-1. The different microRNAs (miRNAs) and their targeting genes were analyzed between C666-1 and C666-IR cells using microarray bioinformatics. Western blot, qRT-PCR, gene transfection, Luciferase reporter assay, and confocal laser scanning microscopy were applied for the analysis of the different genes.ResultsMiR-138-1-3p was found to target CRIPTO, which involved in the epithelial-mesenchymal transition (EMT) and JAK2/STAT3 signaling pathways. The luciferase reporter assay confirmed that miR-138-1-3p targeted CRIPTO and downregulated the expression of CRIPTO. Furthermore, miR-138-1-3p affected the stability of the CRIPTO-GRP78 complex on the cell membrane and also reversed the radioresistant characteristics of NPC stem cells, which affected EMT and the JAK2/STAT3 signaling pathway.ConclusionsThe miR-138-1-3p is a small molecule that can modulate radiosensitivity in the radioresistant C666-IR and HK-1R NPC cell lines by inhibiting EMT and targeting CRIPTO to reduce the activation of the JAK2/STAT3 pathway.

PKCα is a Potentially Useful Marker for Planning Individualized Radiotherapy for Nasopharyngeal Carcinoma.

PurposeTo examine the expression of protein kinase C alpha (PKCα) in nasopharyngeal carcinoma (NPC) and determine its relationship to the radio-sensitivity of NPC in order to evaluate its potential as a molecular marker for the guidance of individualized radiation therapy for NPC.Materials and MethodsPKCα expression levels were detected in tumor samples from patients and in NPC cell lines with varying degrees of radio-sensitivity. A survival analysis was performed to analyze the association of PKCα expression with the 5-year overall survival (OS), progression-free survival (PFS), locoregional recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) in patients. In vitro and in vivo experiments using NPC cell lines were performed to study the effects of down-regulation of PKCα by short hairpin RNA treatment on the radio-sensitivity of NPC.ResultsPKCα expression was up-regulated in the well-differentiated NPC tissues of patients and in the more radio-resistant NPC cell lines. Moreover, high PKCα expression was associated with a worse 5-year PFS and LRFS of patients. shRNA-mediated knockdown of PKCα led to an increase in the sensitivity of NPC cells to radiation therapy, both in vitro as cultured cells and in vivo as tumor xenografts.ConclusionThe elevated expression of PKCα in NPC and its association with patient PFS indicates that PKCα is a potential molecular marker for guiding precision radiotherapy in NPC patients. Also, the increased radiosensitivity of NPC cells after loss of PKCα identifies PKCα as a promising therapeutic target for enhancing the radio-sensitivity of NPC.

PADI4, negatively regulated by miR-335-5p, participates in regulating the proliferation, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells.

Peptidyl arginine deiminase 4 (PADI4), an enzyme that converts arginine residues to citrulline residues in the presence of calcium ions, affects the biochemical activities of proteins. The biological function of PADI4 as well as its mechanism in nasopharyngeal carcinoma (NPC) necessitates further investigation. PADI4 expression in NPC tissues and cells was detected using Western blot. qRT-PCR was used to determine the expression of miR-335-5p and PADI4 mRNA in NPC tissues and cells. BrdU assay and CCK-8 assay were employed to detect cell proliferation. Cell migration and invasion were evaluated using Transwell assay. NPC cells were exposed to different doses of radiation in vitro, and then colony formation assays were used to detect colony survival. The target relationship between miR-335-5p and PADI4 was verified using Western blot, qRT-PCR, and dual-luciferase reporter gene assays. Compared with normal mucosal epithelial tissues and cell lines, the expression level of PADI4 in NPC tissues and cells was significantly up-regulated. PADI4 overexpression promoted the proliferation, migration, and invasion of NPC cells. Under radiation, NPC cell survival was significantly promoted by the up-regulation of PADI4. Conversely, knock-down of PADI4 suppressed the above-mentioned malignant phenotypes. MiR-335-5p could bind with the 3' UTR of PADI4 mRNA, and suppressed the expression of PADI4. PADI4 down-regulated the expression of p21 and activated the mTOR signaling pathway. PADI4, which is negatively regulated by miR-335-5p, promotes the proliferation, migration, invasion and radioresistance of NPC cells by regulating the p21 and mTOR signaling pathways.

LCN2 Is a Potential Biomarker for Radioresistance and Recurrence in Nasopharyngeal Carcinoma.

BackgroundRadioresistance-induced local failure, which can result in residual or recurrent tumors, remains one of the major causes of treatment failure in nasopharyngeal carcinoma (NPC). Lipocalin 2 (LCN2) is known to play important roles in cancer initiation, progression, and treatment responses. However, its role in the radioresistance of NPC remains unclear.MethodsMicroarray data from the Gene Expression Omnibus (GEO) was screened for candidate biomarkers relating to the radioresistance of NPC. The expression of LCN2 in NPC cell lines was verified by quantitative real-time PCR (RT-qPCR) and western blotting. The effects of knockdown or overexpression of LCN2 on NPC radiosensitivity were examined using a soft agar colony formation assay and a γH2AX assay. LCN2 expression in NPC specimens was evaluated by immunohistochemistry. Survival outcomes were analyzed. A possible correlation between LCN2 and hypoxia-inducible factor 1-alpha (HIF-1A) was examined by western blotting and a tissue microarray.ResultsLCN2 was highly expressed in the radioresistant NPC cell line CNE2R. Knocking down LCN2 enhanced the radiosensitivity of NPC cells by impairing their ability to repair DNA damage or proliferate, while ectopic expression of LCN2 conferred additional radioresistance to NPC cells. Immunohistochemical analysis of 100 NPC specimens revealed that LCN2 expression was significantly upregulated in radioresistant NPC tissues and was associated with NPC recurrence. Furthermore, a significant correlation between the expression of LCN2 and HIF-1A was detected.ConclusionLCN2 is associated with radioresistance and recurrence in NPC and may facilitate the development of a radioresistant phenotype through interacting with HIF-1A. Our data indicate that LCN2 is a promising target for predicting and overcoming radioresistance in NPC.

Radiation induces epithelial to mesenchymal transition via upregulation of PD-L1 in nasopharyngeal carcinoma cell.

BackgroundThe acquisition of radioresistance by nasopharyngeal carcinoma (NPC) cells during radiotherapy may lead to tumor metastasis and poor survival. This study aimed to explore the mechanism of long-term radiation-induced NPC metastasis.MethodsThe radioresistant NPC cell, Hone-1R, was established for further study. A colony-forming assay was selected for the evaluation of radioresistant capacity, while a scratch wound healing assay was used to detect tumor cell migration. The expression of relative protein levels were detected by Western blot (WB) analysis and immunofluorescence. Cell morphology was acquired by microscopy. The programmed cell death ligand-1 (PD-L1) expression level in NPC tumor tissues was evaluated based on the publicly available datasets of NPC patients.ResultsA radioresistant NPC cell, Hone-1R, was established with a total dose of 180 Gy, and verified by radioresistant capacity testing. The morphology of Hone-1R cells showed obvious mesenchymal-like cells. WB and wound healing assays indicated that Hone-1R cells exhibited an epithelial-mesenchymal transition (EMT) phenotype with high migration ability and upregulation of PD-L1. Knockdown of PD-L1 reversed EMT status and reduced the migration ability of Hone-1R cells. Further analysis indicated that PD-L1 was overexpressed in more advanced stages and was positively correlated with the EMT score in NPC patients based on in silico analysis.ConclusionsOur study revealed that long-term radiation induces EMT and increases migration ability of NPC radioresistant cells through upregulation of PD-L1. These results advance our investigation of the underlying mechanism of ionizing radiation (IR)-induced migration, and suggest potential interventions to reverse EMT-induced acquisition of radioresistance in NPC.

Inhibition of cell proliferation and radioresistance by miR-383-5p through targeting RNA binding protein motif (RBM3) in nasopharyngeal carcinoma.

BackgroundRNA binding protein motif (RBM3) is associated with radioresistance in nasopharyngeal carcinoma (NPC), and miR-383-5p was predicted to target the 3'-untranslated region (3'UTR) of RBM3 messenger RNA (mRNA). Our study aimed to investigate the role and the mechanisms of miR-383-5p targeting RBM3 in NPC cell proliferation and radioresistance (RR).MethodsThe expression of miR-383-5p was detected by Real-time quantitative PCR (qRT-PCR) between RS (Radiosensitivity) and RR (Radioresistance) NPC patient- tissue specimens and cell lines. Cell Counting Kit-8 (CCK-8) and Clonogenic survival assay were applied to analyze the effect of miR-383-5p on NPC cell proliferation and radioresistance. Possible downstream target of miR-383-5p in NPC cells, RBM3was evaluated by luciferase assay and qRT-PCR. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3 by rescue experiments. The effect of miR-383-5p on radiation-induced apoptosis was explored through Flow cytometric analysis and Western blotting. Western blotting was analyzed the molecular of RBM3-mediated Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling pathwaysResultsThe expression of miR-383-5p was decreased in radioresistant NPC tissues and cells. miR-383-5p inhibited cell proliferation and radioresistance in CNE1/IR cells. We also observed that therapeutic administration of a miR-383-5p agomir dramatically sensitized NPC xenografts to radiation in a mouse model. Conversely, in the same xenograft model, administration of a miR-383-5p antagomir dramatically increased NPC resistance to radiation. miR-383-5p targeted the 3'UTR of RBM3. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3. Finally, we found that miR-383-5p increased radiation-induced apoptosis, activated JNK signaling, and inhibited ERK signaling.ConclusionsOur study revealed that miR-383-5p targeted the 3'UTR of RBM3 and contributed to the efficacy of NPC radiation therapy by altering the RBM3-mediated JNK and ERK signaling pathways.

CD166 promotes cancer stem cell-like phenotype via the EGFR/ERK1/2 pathway in the nasopharyngeal carcinoma cell line CNE-2R

AimsThe present study aimed to investigate the role and underlying mechanisms of CD166 in cancer stem cell-like (CSCs) phenotype of the radioresistant nasopharyngeal carcinoma cell CNE-2R. Main methodsEstablished CD166-shRNA- CNE-2R cell line by lentivirus-mediated silencing CD166. Then, CSC-related genes mRNAs and proteins, and EGFR/ERK1/2 signaling pathway were detected using RT-PCR and western blot. Sphere formation assay was performed to evaluate the sphere formation capacity in CD166-shRNA- CNE-2R cells. The tumorigenesis ability in vivo was examined in nude mice mode. Key findingsDownregulation of CD166 inhibited the expression of the CSC-related genes, pEGFR and pERK in vitro and vivo. The capacity to form spheres and tumorigenesis was significantly decreased in CD166-shRNA cells. Furthermore, EGF-stimulated CD166-shRNA cells exhibited an increase in CSC-like traits by activating EGFR/ERK1/2 signaling. SignificanceCD166 induced CSCs formation by activating the EGFR/ERK1/2 signaling pathway in nasopharyngeal carcinoma, which may serve as a critical molecular target for NPC therapeutic strategies.

IncRNA ZFAS1 contributes to the radioresistance of nasopharyngeal carcinoma cells by sponging hsa-miR-7-5p to upregulate ENO2

ABSTRACT Previous research revealed that lncRNA ZFAS1 could promote nasopharyngeal carcinoma (NPC) by inhibiting its downstream target axis. However, the association between ZFAS1 and radioresistant NPC cells is unclear. This study aimed to explore the roles of ZFAS1 in the radioresistance of NPC. Bioinformatics analysis was conducted to identify the significant factors (ENO2 and miR-7-5p) that contributed to the radioresistance of NPC cells. After performing qRT-PCR analysis, we found that the expression of ZFAS1 and ENO2 was upregulated in NPC cells but that the miR-7-5p expression was downregulated in the same samples. Apart from that, we noticed that ZFAS1 inhibition enhanced the sensitivity of NPC cells to radiation therapy by repressing cell proliferation and promoting cell apoptosis. Subsequently, we found that ZFAS1 could sponge miR-7-5p to upregulate ENO2, which was the target of miR-7-5p. Experimental results also indicated that the suppression of miR-7-5p inhibited the sensitivity of NPC cells to radiation therapy, thereby suppressing ENO2 expression. Overall, our findings suggested that ZFAS1 contributed to the radioresistance of NPC cells by regulating the miR-7-5p/ENO2 axis and that ZFAS1 might be a potential therapeutic target for addressing the radioresistance of NPC cells.

MiR‑182‑5p contributes to radioresistance in nasopharyngeal carcinoma by regulating BNIP3 expression.

Radioresistance is the primary roadblock limiting the success of treatment of nasopharyngeal carcinoma (NPC). microRNA (miRNA/miR)-182-5p has been reported to affect the sensitivity of cancer cells to irradiation; however, the role of miR-182-5p in NPC has not been assessed. The aim of the present study was to investigate the contribution of miR-182-5p to the radioresistance of NPC cells. The key mRNA and miRNA involved in NPC radioresistance were identified using bioinformatics analysis. The two cell lines used in the present study were 5–8F cells (radio-sensitive) and 5–8F-R cells (radioresistant). A dual-luciferase reporter assay system was used to validate the binding between BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) mRNA and miR-182-5p. Reverse transcription-quantitative PCR and western blotting were used to determine the RNA and protein expression levels. To obtain a deeper insight into the effects of the BNIP3/miR-182-5p axis on NPC radioresistance, Cell Counting Kit-8, wound healing, Transwell invasion and colony formation assays, as well as flow cytometry analysis were performed. The results showed that miR-182-5p and BNIP3 were up and downregulated, respectively, in 5–8F-R cells. BNIP3 was also confirmed to be the target of miR-182-5p, and miR-182-5p reversed the inhibitory effect of BNIP3 in 5–8F-R cells. The cellular experiments showed that upregulation of BNIP3 not only inhibited cell proliferation, viability, invasion and migration, but also promoted the apoptosis of 5–8F-R cells. However, the effects of BNIP3 were attenuated by the simultaneous upregulation of miR-182-5p. Thus, through downregulation of BNIP3, miR-182-5p contributed to radiation resistance of NPC cells.

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.

Induction of interleukin-6 by irradiation and its role in epithelial mesenchymal transition and radioresistance of nasopharyngeal carcinoma cells.

Epithelial mesenchymal transition (EMT) participates in the radioresistance of cancer cells. In this study, we evaluated whether interleukin-6 (IL-6) regulates EMT and subsequent radioresistance in nasopharyngeal carcinoma (NPC) cells. Western blot was used to analyze the expression of proteins. Immunofluorescent assay was performed to detect the position and content of specific proteins. Colony formation assay was applied to measure the proliferation ability of NPC cells. Flow cytometry was adopted to measure cell apoptosis. NPC cells seen with typical morphological transition and unique proteins change of EMT when exposed to irradiation. IL-6 and its downstream signal molecules elevated in irradiated cells. Blocking IL-6 with anti-IL-6 receptor antibody hampered EMT and radioresistance. Cell radiosensitivity and apoptosis rates in irradiated NPC cells declined significantly upon IL-6 stimulation. Irradiation could induce EMT and activate IL-6 signaling in NPC cell lines. Blocking IL-6 was able to inhibit EMT and cellular radioresistance.

Long non-coding RNA FAM133B-2 represses the radio-resistance of nasopharyngeal cancer cells by targeting miR-34a-5p/CDK6 axis.

Long non-coding RNAs (lncRNAs) were found to play roles in various cancers, including nasopharyngeal carcinoma. In this study, we focused on the biological function of the lncRNA FAM133B-2 in the radio-resistance of nasopharyngeal carcinoma. The RNA-seq and qRT-PCR analysis showed that FAM133B-2 is highly expressed in the radio-resistant nasopharyngeal carcinoma cells. The following biochemical assays showed that FAM133B-2 represses the nasopharyngeal carcinoma radio-resistance and also affects the apoptosis and proliferation of nasopharyngeal carcinoma cells. Further investigations suggested that miR-34a-5p targets FAM133B-2 and also regulates the cyclin-dependent kinase 6 (CDK6). All these results suggested that the lncRNA FAM133B-2 might function as a competitive endogenous RNA (ceRNA) for miR-34a-5p in nasopharyngeal carcinoma radio-resistance, thus it may be regarded as a novel prognostic biomarker and therapeutic target in nasopharyngeal carcinoma diagnosis and treatment.

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.

FOXD1, negatively regulated by miR-186, promotes the proliferation, metastasis and radioresistance of nasopharyngeal carcinoma cells.

Foxhead box D1 (FOXD1) is validated to be over-expressed in a variety of human malignancies and promotes cancer progression. Nevertheless, the role of FOXD1 and the associated mechanism in nasopharyngeal carcinoma (NPC) remain largely unknown. A total of seventy-five cases of NPC tissue samples were collected. FOXD1 expression in NPC tissues and cells (SUNE1, CNE1, CNE2, and HONE1) was detected using immunohistochemistry and Western blot, respectively. The relationship between FOXD1 expression and clinicopathological parameters of NPC patients was analyzed. FOXD1 mRNA and miR-186 expression in NPC tissues and cells was detected using quantitative polymerase chain reaction (qPCR). The cell viability of NPC cells was detected using CCK-8 assay. Colony survival of NPC cells exposed to different doses of radiation was detected using colony formation assay. Transwell assay was used to evaluate the migration and invasion of NPC cells. The dual-luciferase reporter gene assay was employed to verify the targeting relationship between miR-186 and FOXD1. FOXD1 was over-expressed in NPC tissues (average fold change on mRNA level = 4.72), and its high expression was correlated to NPC positive lymph node metastasis and tissue differentiation. The over-expression of FOXD1 promoted the proliferation, migration, invasion and radio-resistance of NPC cells. On the contrary, the knock-down of FOXD1 inhibited the malignant phenotypes of the above cells. It was verified that FOXD1 was one of the downstream targets of miR-186 and was negatively regulated by it. FOXD1, which is negatively regulated by miR-186, acts as a novel oncogene in NPC and serves as potential biomarker and therapeutic target for NPC. The research will provide great theoretical basis for further clinical diagnosis and therapy.