Causal relationships between gut microbiota and programmed cell death protein 1/programmed cell death-ligand 1: A bidirectional Mendelian randomization study.

Background: Multiple clinical studies have indicated that the gut microbiota influences the effects of immune checkpoint blockade (ICB) therapy comprising PD-1/PD-L1 inhibitors, but the causal relationship is unclear. Because of numerous confounders, many microbes related to PD-1/PD-L1 have not been identified. This study aimed to determine the causal relationship between the microbiota and PD-1/PD-L1 and identify possible biomarkers for ICB therapy. Method: We used bidirectional two-sample Mendelian randomization with two different thresholds to explore the potential causal relationship between the microbiota and PD-1/PD-L1 and species-level microbiota GWAS to verify the result. Result: In the primary forward analysis, genus_Holdemanella showed a negative correlation with PD-1 [ßIVW = -0.25; 95% CI (-0.43 to -0.07); PFDR = 0.028] and genus_Prevotella9 showed a positive correlation with PD-1 [ßIVW = 0.2; 95% CI (0.1 to 0.4); PFDR = 0.027]; order_Rhodospirillales [ßIVW = 0.2; 95% CI (0.1 to 0.4); PFDR = 0.044], family_Rhodospirillaceae [ßIVW = 0.2; 95% CI (0 to 0.4); PFDR = 0.032], genus_Ruminococcaceae_UCG005 [ßIVW = 0.29; 95% CI (0.08 to 0.5); PFDR = 0.028], genus_Ruminococcus_gnavus_group [ßIVW = 0.22; 95% CI (0.05 to 0.4); PFDR = 0.029], and genus_Coprococcus_2 [ßIVW = 0.4; 95% CI (0.1 to 0.6); PFDR = 0.018] were positively correlated with PD-L1; and phylum_Firmicutes [ßIVW = -0.3; 95% CI (-0.4 to -0.1); PFDR = 0.031], family_ClostridialesvadinBB60group [ßIVW = -0.31; 95% CI (-0.5 to -0.11), PFDR = 0.008], family_Ruminococcaceae [ßIVW = -0.33; 95% CI (-0.58 to -0.07); PFDR = 0.049], and genus_Ruminococcaceae_UCG014 [ßIVW = -0.35; 95% CI (-0.57 to -0.13); PFDR = 0.006] were negatively correlated with PD-L1. The one significant species in further analysis was species_Parabacteroides_unclassified [ßIVW = 0.2; 95% CI (0-0.4); PFDR = 0.029]. Heterogeneity (P > 0.05) and pleiotropy (P > 0.05) analyses confirmed the robustness of the MR results.

Silencing c-Jun inhibits autophagy and abrogates radioresistance in nasopharyngeal carcinoma by activating the PI3K/AKT/mTOR pathway.

BACKGROUND: Radioresistance plays an important role in the failure of radiotherapy (RT) for nasopharyngeal carcinoma (NPC), leading to poor prognosis. The purpose of this study was to explore the relationship between the expression of the c-Jun oncogene and the prognosis of NPC. In addition, we investigated the potential mechanisms of c-Jun in the regulation of tumor growth and radioresistance in NPC. METHODS: c-Jun expression in NPC tissues and nasopharyngeal mucosa tissues was evaluated using immunochemistry. c-Jun and its downstream targets were verified by dual-luciferase reporter assays. Inhibitors or activators were used to interfere with the PI3K/AKT/mTOR pathway. Protein expression was analyzed by western blotting. NPC nude mouse xenograft models were used to investigate the potential effects of c-Jun and ionizing radiation in vivo. RESULTS: The expression of c-Jun in NPC tissues was significantly higher than that in normal nasopharyngeal mucosa (NNM) tissues, and Cox regression analysis revealed that c-Jun overexpression was an independent risk factor for poor prognosis in NPC patients. Both in vitro and in vivo experiments verified that c-Jun targeted PI3K/AKT signaling. We also performed an in vivo study showing that c-Jun knockdown effectively suppressed NPC growth in a xenograft tumor model by autophagy inhibition, and these effects were accompanied by the upregulation of p-PI3K p-AKT, p-mTOR, and P62 and downregulation of LC3-II expression. CONCLUSIONS: High expression of c-Jun was correlated with poor prognosis in NPC patients. c-Jun knockdown increased cell sensitivity to radiation by inhibiting autophagy activation via the PI3K/AKT/mTOR signaling pathway. The present study provides a theoretical basis for a promising treatment for radioresistant NPC by inhibiting c-Jun expression.

MiR-513b-5p represses autophagy during the malignant progression of hepatocellular carcinoma by targeting PIK3R3.

Hepatocellular carcinoma (HCC) serves as a prevailing global malignancy with severe mortality and extremely unsatisfactory prognosis, in which autophagy is a fundamental process in liver cancer pathogenesis, but the mechanisms are poorly understood. MicroRNAs (miRNAs) serve as a type of well-recognized non-coding regulators and contribute to the modulation of liver cancer development, from the aspects of diagnosis, progression, and therapy. Here, we aimed to investigate the function of hsa_microRNA-513b-5p (miR-513b-5p) in regulating autophagy during HCC progression. Specifically, our data showed that miR-513b-5p mimic reduced the LC3-II and beclin1 expression but enhanced p62 expression in HCC cells. MiR-513b-5p repressed liver cancer cell proliferation, migration/invasion, and induced apoptosis in vitro. Crucially, miR-513b-5p attenuated tumor growth of liver cancer cells in vivo. In the mechanical investigation, we identified that PIK3R3 mRNA 3'UTR was targeted by miR-513b-5p and miR-513b-5p suppressed PIK3R3 expression. PIK3R3 overexpression partly reversed miR-513b-5p-mediated autophagy, proliferation, and apoptosis of liver cancer cells. Consequently, we concluded that miR-513b-5p repressed autophagy during the malignant progression of HCC by targeting PIK3R3. MiR-513b-5p may be applied as a therapeutic target for HCC.

VEGF promotes migration and invasion by regulating EMT and MMPs in nasopharyngeal carcinoma.

Background: Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. Accumulating data have indicated that VEGF is involved in tumour metastasis. However, the mechanism through which VEGF regulates nasopharyngeal carcinoma (NPC) metastasis is largely unknown. This study aimed to examine the biological function of VEGF in NPC metastasis and its underlying mechanism. Methods: We used western blotting and qPCR to examine the difference in VEGF expression between NPC cells and the immortalized nasopharyngeal epithelial cell line NP69. Wound healing assays, transwell assays and animal experiments were used to further verify the role of VEGF in the invasion and migration of NPC cells. The protein levels of the epithelial-mesenchymal transition (EMT) and matrix metalloproteinase (MMP) family were analysed by immunofluorescence (IF) and western blotting. Enzyme-linked immunosorbent assay (ELISA) and transwell assays were used to determine whether VEGF enhanced the invasion and migration of NPC cells in an autocrine manner. Western blotting was used to examine how autocrine VEGF-VEGFR2 signalling regulated EMT and MMPs. Results: We observed higher levels of VEGF in NPC cells than that in NP69 cells and identified an association between high VEGF levels and tumour invasion and migration. Mechanistically, the VEGF-mediated increase in EMT markers, MMP2 and MMP9 promoted NPC cell invasion and migration. Additionally, NPC cells secreted VEGF to promote cell invasion, migration and angiogenesis. Autocrine VEGF-VEGFR2 signalling increased ERK1/2 phosphorylation, promoted EMT process and MMPs at the indicated times. Conclusion: This study revealed that VEGF plays a role in controlling NPC cell metastasis by regulating EMT markers and MMPs in an autocrine manner.

VEGF knockdown enhances radiosensitivity of nasopharyngeal carcinoma by inhibiting autophagy through the activation of mTOR pathway.

Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced the radioresistance of tumors. The purpose of this study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells and the underlying mechanisms. The radiosensitivity of NPC cells after VEGF silencing was detected by cell counting kit 8 (CCK-8) and clonogenic assay, while cell cycle and apoptosis were detected by flow cytometry. The processes of DNA damage, repair and autophagy were examined by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation studies. The effect of VEGF on radiosensitivity of NPC cells was investigated in vivo using a xenograft model. Furthermore, immunohistochemistry and TUNEL assays were used to verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion. Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of NPC cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in NPC cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through activation of the mTOR pathway. VEGF depletion increased radiosensitivity of NPC cells by suppressing autophagy via activation of the mTOR pathway.

Downregulation of CD166 inhibits invasion, migration, and EMT in the radio-resistant human nasopharyngeal carcinoma cell line CNE-2R.

Objective: CD166 is known as a tumor stem cell specific marker, associating with tumor metastasis. The purpose of this study was to further discuss CD166 gene on cell proliferation, invasion, metastasis, and the epithelial-mesenchymal transition (EMT) in CNE-2R cell line of nasopharyngeal carcinoma (NPC). Materials and methods: CNE-2R cells were transfected with lentivirus CD166-shRNA, and quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting were used to confirm the silencing effects. The wound healing test and transwell test were carried out to assess cell invasive and migratory abilities in vitro. With the establishment of xenograft nude mouse model, Western blotting and immunohistochemistry were undertaken to detect the expression level of E-cadherin, N-cadherin, and vimentin. In vivo metastasis detection was carried out by injecting tumor cells into nude mice via the tail vein. Results: The invasive and migratory abilities of CNE-2R cells were significantly reduced after CD166 was downregulated. In addition, silencing of CD166 of CNE-2R cells increased the expression of E-cadherin, while down-regulated the expression of N-cadherin and vimentin. Immunohistochemistry of tumors showed consistent results with in-situ tumor formation experiment. Additionally, the growth of transplanted tumor was inhibited. In addition, in vivo metastasis test proved that knockdown of CD166 suppressed pulmonary metastasis and liver metastasis according to hematoxylin and eosin (H&E) staining. Expression of E-cadherin increased, while expression of N-cadherin and vimentin decreased, as revealed by Western blotting of metastatic lung tumors. Conclusion: Silencing of CD166 in CNE-2R cells evidently inhibited proliferation, invasion, metastasis, and EMT process in vivo and in vitro.

The role of autophagy in the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R.

PURPOSE: The present study aimed to study the role of autophagy in the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R. METHODS: Before being irradiated, CNE-2R cells were treated with the autophagy inhibitor chloroquine diphosphate (CDP) or the autophagy inducer rapamycin (RAPA). Microtubule-associated protein light chain 3 (LC3-II) and p62 were assessed using Western blotting analysis 48 hours after CNE-2R cells were irradiated. The percentage of apoptotic cells was assessed via flow cytometry. CNE-2R cell viability was evaluated using the Cell Counting Kit-8 (CCK8). The radiosensitivity of cells was assessed via clone formation analysis. RESULTS: The level of autophagy in CNE-2R cells improved as the radiation dose increased, reaching the maximum at a dose of 10 Gy. Autophagy was most significantly inhibited by 60 µmol/L CDP in CNE-2R cells, but was obviously enhanced by 100 nmol/L RAPA. Compared with the irradiation (IR) alone group, in the IR + CDP group, autophagy was significantly inhibited, viability was low, the rate of radiation-induced apoptosis was increased, and radiosensitivity was upregulated. In contrast, cells of the IR + RAPA group exhibited greater autophagy, higher viability, a lower rate of radiation-induced apoptosis, and downregulated radiosensitivity. CONCLUSION: The autophagy level is negatively correlated with radiosensitivity for the radio-resistant human nasopharyngeal carcinoma cell line CNE-2R.

Cancer stem cell-like characteristics and telomerase activity of the nasopharyngeal carcinoma radioresistant cell line CNE-2R.

The radioresistance of nasopharyngeal carcinoma (NPC) may be related to cancer stem cells (CSCs), and the characteristics of CSCs may be maintained by telomerase activity. In this study, we explored the CSC-like characteristics and telomerase activity of the NPC radioresistant cell line CNE-2R. This work provides a foundation for future studies on stem cell-targeted therapies by targeting the radioresistance of NPC. The expression of stem cell-related genes/proteins and the hTERT gene/protein in CNE-2R and its parent radiosensitive cell line CNE-2 were detected using qPCR/Western Blot. Label-retaining cells (LRCs) were detected through immunocytochemistry, and telomerase activity was detected using a PCR-ELISA kit. CD133 expression was detected with flow cytometry. CNE-2R-CD133+ and CNE-2R-CD133- cells were separated with magnetic-activated cell sorting. The proliferation and tumorigenesis capacities of CNE-2R-CD133+, CNE-2R-CD133-, and CNE-2R cells were compared with a CCK-8 assay, sphere formation assay, and an in vivo experiment. Our results showed that the expression of stem cell-related genes and the hTERT gene in CNE-2R cells was higher than those in CNE-2 cells. Similarly, the expression of stem cell-related proteins and the hTERT protein in CNE-2R cells was markedly higher than those in CNE-2 cells. The proportion of LRCs in CNE-2R and CNE-2 cells was (3.10 ± 0.63%) vs (0.40 ± 0.35%; P < 0.001), respectively. Telomerase activity in CNE-2R cells was remarkably higher than that in CNE-2 cells. Flow cytometry suggested that the CD133 positive rates in CNE-2R and CNE-2 cells were (2.49 ± 0.56%) vs (0.76 ± 0.25%; P = 0.008), respectively. Meanwhile, the proliferation capacity, tumorigenesis capacity, and telomerase activity of CNE-2R-CD133+ cells were notably higher than those of CNE-2R-CD133- and CNE-2R cells. Collectively, CNE-2R displayed CSC-like characteristics; our results also showed that CNE-2R cells, especially the sorted CSCs, had high telomerase activity levels.

Silencing of c-jun decreases cell migration, invasion, and EMT in radioresistant human nasopharyngeal carcinoma cell line CNE-2R.

BACKGROUND: Previously, we found that c-jun was highly expressed in radiation-resistant human nasopharyngeal carcinoma cells (CNE-2R) compared with human nasopharyngeal carcinoma cells (CNE-2). MATERIALS AND METHODS: In this study, we first used the scratch assays and transwell assays to detect the migration and invasion of CNE-2R and CNE-2 cells and tested the epithelial mesenchymal transformation (EMT)-related proteins E-cadherin and N-cadherin by Western blot analysis. Subsequently, c-jun was knocked down to establish the effect of c-jun on EMT, migration, and invasion of CNE-2R cells both in vitro and in vivo. RESULTS: A high EMT level, CNE-2R cells were more capable of migration and invasion than CNE-2 cells. Moreover, silencing of c-jun has upregulated the expression of E-cadherin and downregulated N-cadherin in CNE-2R cells, and subsequently the migration and invasion capacity of the cells was decreased. Consistent with in vitro results, in vivo studies indicated that the c-jun silencing reduced pulmonary migration of CNE-2R cells. Immunohistochemistry of lung metastatic tumor tissue showed that E-cadherin was upregulated, and N-cadherin was downregulated. CONCLUSION: These findings suggest that silencing of c-jun in CNE-2R cells reduces cells migration, invasion, and EMT both in vitro and in vivo.

Cofilin-2 Acts as a Marker for Predicting Radiotherapy Response and Is a Potential Therapeutic Target in Nasopharyngeal Carcinoma.

BACKGROUND The purpose of this study was to determine whether cofilin-2 could serve as a protein marker for predicting radiotherapy response and as a potential therapeutic target in nasopharyngeal carcinoma (NPC). MATERIAL AND METHODS Cofilin-2 protein levels in serum and tissue samples from patients with NPC were assessed by sandwich ELISA and IHC. In vitro, cofilin-2 levels in CNE-2R cells were significantly higher than those of CNE-2 cells. Meanwhile, CNE-2R cells were silenced for cofilin-2 to obtain a stable cofilin-2-RNAi-LV3 cell line. Then, cell proliferation, radiosensitivity, invasion and migration abilities, cell cycle, and apoptosis were evaluated by Cell Counting Kit 8 assay (CCK-8), flow cytometry (FCM), clone formation assay, and in vitro. RESULTS The secreted levels of the cofilin-2 protein in radioresistant NPC patients were significantly higher than those of radiosensitive cases. After cofilin-2 knockdown in nasopharyngeal carcinoma CNE-2R cells, proliferation was decreased, while apoptosis and radiosensitivity were enhanced; cell cycle distribution was altered, and the transplanted tumors in nude mice grew significantly less. CONCLUSIONS Overall, our findings suggest that cofilin-2 acts as a marker for predicting radiotherapy response and is a potential therapeutic target in nasopharyngeal carcinoma.

Reduced QSOX1 enhances radioresistance in nasopharyngeal carcinoma.

Radioresistance is a major cause leads to treatment failure in nasopharyngeal carcinoma (NPC). In our previous study, we identified that QSOX1 is a differentially expressed protein in NPC cell lines with variable radiosensitivities. The present study aimed to investigate the biological behavior of QSOX1 in nasopharyngeal carcinoma (NPC) and its effect on radiosensitivity. The levels of QSOX1 detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in radioresistant NPC patient sera and tissue samples were markedly lower than those in radiosensitive samples. Small hairpin RNAs (shRNAs) were employed to knock down endogenous QSOX1 expression in CNE-2 cells, and then, radiosensitivity, apoptosis, migration and invasion were assessed using colony formation, Cell Counting Kit-8 (CCK-8), flow cytometry, and transwell assays, respectively. Tumor growth and radioresistance were also evaluated using a xenograft model in nude mice. The shRNA-mediated knockdown of QSOX1 significantly increased cell survival under irradiation (IR) and weakened radiosensitivity, which was likely due to a reduction in the cell apoptosis rate after IR. Moreover, QSOX1 silencing led to the suppression of cellular migration and invasion. Similar results were obtained with the xenograft mouse model. Thus, targeting QSOX1 will provide a new avenue for increasing the sensitivity of NPC to radiotherapy.

Significant survival benefit of adjuvant chemotherapy after concurrent chemoradiotherapy in locally advanced high-risk nasopharyngeal carcinoma.

The present study aimed to define high-risk patients who may benefit from additional adjuvant chemotherapy (AC) after concurrent chemotherapy in combination with intensity-modulated radiotherapy among patients with loco-regionally advanced nasopharyngeal carcinoma (NPC). A cohort of 511 NPC patients who received concomitant chemoradiotherapy (CCRT) with or without AC between January 2007 and December 2012 were retrospectively analysed. One hundred seventy-seven patients received CCRT alone, whereas 334 received CCRT + AC. The survival analysis showed that ages >45 years old, T3-T4 stages, N2-N3 disease and serum albumin levels ≤42 g/L were significant independent prognostic factors for overall survival (OS). Using these four risk factors, a prognostic model for OS was created as follows: (1) low-risk group: 0-1 risk factors; and (2) high-risk group: 2-4 risk factors. In the CCRT alone and CCRT + AC groups, significant differences in survival were found between the high- and low-risk groups. Patients in the high-risk group exhibited improved OS due to the addition of AC to CCRT, but no survival benefits were found in the low-risk group. In conclusion, high-risk patients may benefit from the addition of AC to CCRT regarding OS.