Identification of KDM4C as a gene conferring drug resistance in multiple myeloma.

Bortezomib (BTZ), a proteasome inhibitor, is a promising therapeutic option for multiple myeloma (MM) patients. However, drug resistance often occurs, leading to disease relapse and poor prognosis. In this study, we aimed to identify novel genes associated with drug resistance and investigate their roles in BTZ resistance. Through the screening of 26 genes frequently associated with chemosensitivity or drug resistance, we discovered that KDM4C, a histone demethylase, exhibited increased expression in BTZ-resistant MM cells compared to their sensitive counterparts. Overexpression of KDM4C enhanced the tolerance of a MM cell line to the drug, whereas the knockdown of KDM4C, using shRNA, increased the sensitivity of resistant cells to BTZ treatment. This suggests that KDM4C plays a pivotal role in conferring BTZ resistance. Our study offers fresh insights into BTZ resistance in MM and highlights KDM4C as a potential target for overcoming drug resistance.

Glycyrrhetinic Acid Mitigates Radiation-Induced Pulmonary Fibrosis via Inhibiting the Secretion of TGF-ß1 by Treg Cells.

PURPOSE: Radiation-induced pulmonary fibrosis (RIPF) is a common side effect of radiation therapy for thoracic tumors without effective prevention and treatment methods at present. The aim of this study was to explore whether glycyrrhetinic acid (GA) has a protective effect on RIPF and the underlying mechanism. METHODS AND MATERIALS: A RIPF mouse model administered GA was used to determine the effect of GA on RIPF. The cocultivation of regulatory T (Treg) cells with mouse lung epithelial-12 cells or mouse embryonic fibroblasts and intervention with GA or transforming growth factor-ß1 (TGF-ß1) inhibitor to block TGF-ß1 was conducted to study the mechanism by which GA alleviates RIPF. Furthermore, injection of Treg cells into GA-treated RIPF mice to upregulate TGF-ß1 levels was performed to verify the roles of TGF-ß1 and Treg cells. RESULTS: GA intervention improved the damage to lung tissue structure and collagen deposition and inhibited Treg cell infiltration, TGF-ß1 levels, epithelial mesenchymal transition (EMT), and myofibroblast (MFB) transformation in mice after irradiation. Treg cell-induced EMT and MFB transformation in vitro were prevented by GA, as well as a TGF-ß1 inhibitor, by decreasing TGF-ß1. Furthermore, reinfusion of Treg cells upregulated TGF-ß1 levels and exacerbated RIPF in GA-treated RIPF mice. CONCLUSIONS: GA can improve RIPF in mice, and the corresponding mechanisms may be related to the inhibition of TGF-ß1 secreted by Treg cells to induce EMT and MFB transformation. Therefore, GA may be a promising therapeutic candidate for the clinical treatment of RIPF.

Single-cell sequencing reveals homogeneity and heterogeneity of the cytopathological mechanisms in different etiology-induced AKI.

Homogeneity and heterogeneity of the cytopathological mechanisms in different etiology-induced acute kidney injury (AKI) are poorly understood. Here, we performed single-cell sequencing (scRNA) on mouse kidneys with five common AKI etiologies (CP-Cisplatin, IRI-Ischemia-reperfusion injury, UUO-Unilateral ureteral obstruction, FA-Folic acid, and SO-Sodium oxalate). We constructed a potent multi-model AKI scRNA atlas containing 20 celltypes with 80,689 high-quality cells. The data suggest that compared to IRI and CP-AKI, FA- and SO-AKI exhibit injury characteristics more similar to UUO-AKI, which may due to tiny crystal-induced intrarenal obstruction. Through scRNA atlas, 7 different functional proximal tubular cell (PTC) subtypes were identified, we found that Maladaptive PTCs and classical Havcr1 PTCs but not novel Krt20 PTCs affect the pro-inflammatory and pro-fibrotic levels in different AKI models. And cell death and cytoskeletal remodeling events are widespread patterns of injury in PTCs. Moreover, we found that programmed cell death predominated in PTCs, whereas apoptosis and autophagy prevailed in the remaining renal tubules. We also identified S100a6 as a novel AKI-endothelial injury biomarker. Furthermore, we revealed that the dynamic and active immune (especially Arg1 Macro_2 cells) -parenchymal cell interactions are important features of AKI. Taken together, our study provides a potent resource for understanding the pathogenesis of AKI and early intervention in AKI progression at single-cell resolution.

A Subset of Breg Cells, B10, Contributes to the Development of Radiation-Induced Pulmonary Fibrosis.

PURPOSE: Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiation therapy, but the underlying mechanisms are unknown. B10 cells, as negative B regulatory cells, play important roles in regulating inflammation and autoimmunity. However, the role of B10 cells in RIPF progression is unclear. The aim of this study was to determine the role of B10 cells in aggravating RIPF and the underlying mechanism. METHODS AND MATERIALS: The role of B10 cells in RIPF was studied by constructing mouse models of RIPF and depleting B10 cells with an anti-CD22 antibody. The mechanism of B10 cells in RIPF was further explored through cocultivation of B10 cells and MLE-12 or NIH3T3 cells and administration of an interleukin (IL)-10 antibody to block IL-10. RESULTS: B10 cell numbers increased significantly during the early stage in the RIPF mouse models compared with the controls. In addition, depleting B10 cells with the anti-CD22 antibody attenuated the development of lung fibrosis in mice. Subsequently, we confirmed that B10 cells induced epithelial-mesenchymal transition and the transformation of myofibroblasts via activation of STAT3 signaling in vitro. After blockade of IL-10, it was verified that IL-10 secreted by B10 cells mediates the epithelial-mesenchymal transition of myofibroblasts, thereby promoting RIPF. CONCLUSIONS: Our study uncovers a novel role for IL-10-secreting B10 cells that could be a new target of research for relieving RIPF.

Bioinformatics analysis of potential pathogenesis and risk genes of immunoinflammation-promoted renal injury in severe COVID-19.

Renal injury secondary to COVID-19 is an important factor for the poor prognosis of COVID-19 patients. The pathogenesis of renal injury caused by aberrant immune inflammatory of COVID-19 remains unclear. In this study, a total of 166 samples from 4 peripheral blood transcriptomic datasets of COVID-19 patients were integrated. By using the weighted gene co-expression network (WGCNA) algorithm, we identified key genes for mild, moderate, and severe COVID-19. Subsequently, taking these genes as input genes, we performed Short Time-series Expression Miner (STEM) analysis in a time consecutive ischemia-reperfusion injury (IRI) -kidney dataset to identify genes associated with renal injury in COVID-19. The results showed that only in severe COVID-19 there exist a small group of genes associated with the progression of renal injury. Gene enrichment analysis revealed that these genes are involved in extensive immune inflammation and cell death-related pathways. A further protein-protein interaction (PPI) network analysis screened 15 PPI-hub genes: ALOX5, CD38, GSF3R, LGR, RPR1, HCK, ITGAX, LYN, MAPK3, NCF4, SELP, SPI1, WAS, TLR2 and TLR4. Single-cell sequencing analysis indicated that PPI-hub genes were mainly distributed in neutrophils, macrophages, and dendritic cells. Intercellular ligand-receptor analysis characterized the activated ligand-receptors between these immune cells and parenchyma cells in depth. And KEGG enrichment analysis revealed that viral protein interaction with cytokine and cytokine receptor, necroptosis, and Toll-like receptor signaling pathway may be potentially essential for immune cell infiltration leading to COVID-19 renal injury. Finally, we validated the expression pattern of PPI-hub genes in an independent data set by random forest. In addition, we found that the high expression of these genes was correlated with a low glomerular filtration rate. Including them as risk genes in lasso regression, we constructed a Nomogram model for predicting severe COVID-19. In conclusion, our study explores the pathogenesis of renal injury promoted by immunoinflammatory in severe COVID-19 and extends the clinical utility of its key genes.

Intermittent administration of tacrolimus enhances anti-tumor immunity in melanoma-bearing mice.

One key reason for T cell exhaustion is continuous antigen exposure. Early exhausted T cells can reverse exhaustion and differentiate into fully functional memory T cells if removed from persisting antigen stimulation. Therefore, this study viewed T cell exhaustion as an over-activation status induced by chronic antigen stimuli. This study hypothesized that blocking TCR signal intermittently to terminate over-activation signal can defer the developmental process of T cell exhaustion. In this study, melanoma-bearing mice were treated with tacrolimus (FK506) every 5 days. The tumor size and tumor-infiltrating lymphocytes (TILs) were analyzed. We found that intermittent administration of tacrolimus significantly inhibited tumor growth, and this effect was mediated by CD8+T cells. Intermittent tacrolimus treatment facilitated the infiltration of CD8+TILs. RNA-seq and quantitative RT-PCR of sorted CD8+TILs showed the expression of Nr4a1 (an exhaustion-related transcription factor) and Ctla4 (a T cell inhibitory receptor) was remarkably downregulated. These results indicated that intermittently blocking TCR signal by tacrolimus can promote anti-tumor immunity and inhibit the tumor growth in melanoma-bearing mice, inhibiting the transcription of several exhaustion-related genes, such as Nr4a1 and Ctla4.

Prioritization of Diagnostic and Prognostic Biomarkers for Lupus Nephritis Based on Integrated Bioinformatics Analyses.

Lupus nephritis (LN) is an important driver of end-stage renal disease (ESRD). However, few biomarkers are available for evaluating the diagnosis and prognosis of LN. For this study, we downloaded microarray data of multiple LN expression profiles from the GEO database. We used the WGCNA and R limma packages to identify LN hub genes and differentially-expressed genes (DEGs). We identified nine co-DEGs in the intersection with LN-related genes from the Genecards database. We found DEGs that are primarily associated with immune-related functions and pathways (including with the complement pathway, primary immunodeficiency markers, and MHC-like protein complexes) through our comprehensive GSEA, GO, and KEGG enrichment analyses. We used other LN and SLE validation datasets and discovered six explicitly expressed co-DEGs: HLA-DMA, HLA-DPA1, HLA-DPB1, HLA-DRA, IL10RA, and IRF8 in the LN set; ROC and Precision-Recall curve analyses revealed that these six genes have a good diagnostic efficacy. The correlation analysis with prognostic data from the Nephroseq database indicates that the differential expression of these co-DEGs is associated with a low glomerular filtration rate in that cohort. Additionally, we used a single-cell LN database of immune cells (for the first time) and discovered these co-DEGs to be predominantly distributed in different types of macrophages and B cells. In conclusion, by integrating multiple approaches for DEGs discovery, we identified six valuable biomarkers that are strongly correlated with the diagnosis and prognosis of LN. These markers can help clarify the pathogenesis and improve the clinical management of LN.

PNMA5 Promotes Bone Metastasis of Non-small-Cell Lung Cancer as a Target of BMP2 Signaling.

Bone metastases frequently occur in NSCLC patients at the late stage, indicating poor survival. However, mechanisms about the initiation of NSCLC bone metastases remain largely unclear. In our previous reports, BMP2 signaling activation has been found to enhance NSCLC bone metastases through enhancing carcinoma cells migration, invasion, osteoclasts differentiation and osteoblasts immature differentiation. Nevertheless, downstream target genes of BMP2 contributing to those processes still remain unknown. In this project, we find that the expression of Pnma5 is higher in metastatic bone tumors of Lewis lung carcinoma than in metastatic lung tumors and parental Lewis lung cells. Pnma5 overexpression not only can promote cell migration and invasion of NSCLC cells but also tumor-induced osteoclasts differentiation. Interestingly, knockdown of Pnma5 in Lewis lung cells blocks BMP2 signaling from inducing Lewis lung cells migration and invasion. Although BMP2 signaling can promote Lewis lung cells-induced osteoclasts differentiation from macrophages, this effect can also be blocked when Pnma5 is knocked down in Lewis lung cells. Moreover, Pnma5 can promote NSCLC bone metastases in vivo as the downstream target of BMP2. Those results above indicate that BMP2 signaling enhances NSCLC bone metastases via its direct downstream target gene Pnma5. This research reveals the detailed molecular mechanism about how BMP2 signaling contributes to NSCLC bone metastases via PNMA5 and provides a new potential therapeutic target for the treatment of NSCLC bone metastases.

Irradiated whole-cell vaccine suppresses hepatocellular carcinoma growth in mice via Th9 cells.

Liver cancer is one of the most common malignant tumors with no available satisfactory treatment. The aim of the present study was to investigate the anti-tumor effect of an irradiated hepatocellular carcinoma (HCC) whole-cell vaccine and its underlying mechanisms. Hepa1-6 and H22 HCC cell lines were irradiated in preparation for whole-cell vaccine production. Subsequently, two HCC tumor-bearing mouse models were created by injecting these Hepa1-6 and H22 cells into the abdominal skin of C57BL/6 and ICR mice, respectively. The mice were immunized with the corresponding whole-cell vaccine the next day, and then once a week until the end of the experimental period. Tumor growth, blood T helper (Th)9 cells and plasma interleukin (IL)-9 levels were monitored during the immunization period. Th9 cells were also induced by in vitro co-culture of the whole-cell vaccine with lymphocytes from the spleen and lymph nodes of the corresponding mice. Alterations of gene expression in transcription factor (TF) were determined by reverse transcription-quantitative PCR, and Th9 cells were detected using flow cytometry. The whole-cell vaccine effectively suppressed HCC tumor growth, as indicated by slower tumor growth and a smaller tumor size in the immunized group compared with the control. The percentage of blood Th9 cells and the concentration of plasma IL-9 were significantly increased in the immunized group. The whole-cell vaccine also induced Th9 cell differentiation and upregulated the expression of TFs PU.1, interferon regulatory factor 4 and basic leucine zipper transcriptional factor ATF-like. These results suggest that the irradiated HCC whole-cell vaccine inhibited tumor growth by increasing Th9 cell numbers in HCC mice.

GSDME-mediated pyroptosis promotes inflammation and fibrosis in obstructive nephropathy.

Renal tubular cell (RTC) death and inflammation contribute to the progression of obstructive nephropathy, but its underlying mechanisms have not been fully elucidated. Here, we showed that Gasdermin E (GSDME) expression level and GSDME-N domain generation determined the RTC fate response to TNFα under the condition of oxygen-glucose-serum deprivation. Deletion of Caspase-3 (Casp3) or Gsdme alleviated renal tubule damage and inflammation and finally prevented the development of hydronephrosis and kidney fibrosis after ureteral obstruction. Using bone marrow transplantation and cell type-specific Casp3 knockout mice, we demonstrated that Casp3/GSDME-mediated pyroptosis in renal parenchymal cells, but not in hematopoietic cells, played predominant roles in this process. We further showed that HMGB1 released from pyroptotic RTCs amplified inflammatory responses, which critically contributed to renal fibrogenesis. Specific deletion of Hmgb1 in RTCs alleviated caspase11 and IL-1ß activation in macrophages. Collectively, our results uncovered that TNFα/Casp3/GSDME-mediated pyroptosis is responsible for the initiation of ureteral obstruction-induced renal tubule injury, which subsequentially contributes to the late-stage progression of hydronephrosis, inflammation, and fibrosis. This novel mechanism will provide valuable therapeutic insights for the treatment of obstructive nephropathy.

BMP2 signalling activation enhances bone metastases of non-small cell lung cancer.

Distant metastases occur when non-small cell lung cancer (NSCLC) is at late stages. Bone metastasis is one of the most frequent metastases of NSCLC and leads to poor prognosis. It has been reported that high expression of BMP2 in NSCLC correlates with poor survival, but whether BMP2 contributes to NSCLC bone metastasis remains largely unknown. The activation of BMP signalling is found in metastatic bone tumours of mice Lewis lung carcinoma and predicts poor survival in human NSCLC. BMP2 signalling activation can enhance bone metastasis of Lewis lung carcinoma. Moreover, BMP2 secreted by stroma fibroblasts can promote the migration and invasion of NSCLC cells. Besides, in combination with pre-osteoblast and LLCs, BMP2 could enhance the differentiation of macrophages into osteoclasts to play roles in the osteolytic mechanism of NSCLC bone metastasis. Interestingly, NSCLC cells can also enrich BMP2 to pre-osteoblasts to function in the osteoblastic mechanism. Our results firstly demonstrate the detailed mechanisms about what roles BMP2 signalling play in enhancing NSCLC bone metastases. These findings provide a new potential therapy choice for preventing bone metastases of NSCLC via the inhibition of BMP2 signalling.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear. Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry. Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an "in situ tumor vaccine" and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells. Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

TREX1 suppression imparts cancer-stem-cell-like characteristics to CD133- osteosarcoma cells through the activation of E2F4 signaling.

There is ongoing debate whether cancer stem cells (CSCs) could arise from the transformation of non-CSCs under specific conditions. In the present study, the role of the three prime repair exonuclease 1 (TREX1) in regulating CSC generation form human osteosarcoma cells was investigated. High, intermediate and low levels of TREX1 expression were respectively observed in low-grade, high-grade and metastatic human osteosarcoma samples, while the opposite tendency was observed for E2F4, a transcription factor associated with G2 arrest. Luciferase assay proved that TREX1 had a negative impact on the activity of E2F4 promoter. TREX1 was highly expressed in CD133- HOS cells (non-CSC osteosarcoma cells) compared to CD133+ ones; whereas TREX1 knockdown endowed the CD133- non-CSCs with CSC-like characteristics in vitro relying on E2F4 activation, as demonstrated by enlarged proportion of the subset expressing CSC markers in flow cytometry analysis, enhanced self-renewal ability in osteosphere formation assay, increased metastasis capacity in migration and invasion assays, together with improved chemoresistance to cisplatin. Furthermore, TREX1 knockdown and subsequent E2F4 activation could promote the tumorigenicity of CD133- non-CSCs in vivo. With respect to underlying mechanisms, it was found that in CD133- HOS cells, TREX1 suppression would allow the activation of ß-catenin signaling in the dependence of E2F4, thus possibly leading to the up-regulation of the transcription factor OCT4. These findings suggested that TREX1 was probably a negative regulator of CSC formation and hence worth to be further studied for developing new treatments in cancer therapies targeting CSCs.

Hypoxia induced δ-Catenin to enhance mice hepatocellular carcinoma progression via Wnt signaling.

Hypoxia frequently occurs in solid tumors, hepatocellular carcinoma included. Hypoxia-inducible factors (HIFs) upregulated in hypoxia can induce various downstream target genes to resist hypoxia stress, resulting in tumor growth, angiogenesis and metastasis in vivo. Therefore, hypoxia associated genes are usually cancer progression associated genes and can be potential therapy targets for cancer therapy. In our present work, we find that the hypoxia-inducible transcriptional factor, HIF1α, can directly upregulate the expression of the gene Ctnnd2, which codes the protein δ-Catenin. Then, δ-Catenin can stabilize ß-Catenin by disrupting the destruction complex, which leads to the activation of Wnt signaling. As a result, δ-Catenin can promote the proliferation and migration of HCC cells in vitro, further enhance mice HCC tumorigenesis in vivo. In summary, our work reveals that δ-Catenin is a direct downstream target gene of HIF1α. It can activate Wnt signaling via ß-Catenin stabilization. δ-Catenin can enhance HCC progression.

Effects of CKMT1 on radiosensitivity of nasopharyngeal carcinoma cells.

PURPOSE: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear. The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity. MATERIAL AND METHODS: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2. Biological changes were detected after cells exposing to different doses of X-ray to determine the role of CKMT1 on NPC cell radiosensitivity. RESULTS: CKMT1 promotes proliferation and migration in NPC cell lines CNE-1 and CNE-2. Overexpression of CKMT1 in CNE-1 cells enhanced colony formation rates, reduced G2/M phase cell cycle arrest, lowered apoptosis rate and c-PARP level, and elevated STAT3 phosphorylation level after radiation treatment. While knocking out CKMT1 using the CRISPR/Cas9 system in CNE-2 cells lowered colony formation rates, increased G2/M phase cell cycle arrest, apoptosis rates, and c-PARP levels, and decreased STAT3 phosphorylation in response to radiation treatment. CONCLUSIONS: NPC cells with higher CKMT1 exhibited lower radiosensitivity through promoting phosphorylation of STAT3. Our findings suggest that CKMT1 may be an alternative radiotherapeutic target in NPC therapy.

δ-Catenin peptide vaccines repress hepatocellular carcinoma growth via CD8+ T cell activation.

As classical therapy method of advanced hepatocellular carcinoma (HCC) is not effective enough, HCC immunotherapy is a hot spot for research in recent years. Although in recent years, immune checkpoint inhibitors are focused in cancer therapy, vaccines and adoptive cell therapy (ACT), as traditional immunotherapy methods for HCC are still promising. We found that δ-Catenin might be a new tumor-associated antigen for HCC, for it could be upregulated as a stress associated protein under hypoxia and irradiation treatment. δ-Catenin peptide vaccines could inhibit the growth of subcutaneous hepatocellular tumors in vivo. According to our work, δ-Catenin peptide vaccines could stimulate the activation of cytotoxic T lymphocytes (CTLs) and enhance the infiltration of CD8+ T cells into tumors. Moreover, δ-Catenin peptide vaccines could enhance the secretion of IFN-γ and the killing of tumor cells by T cells. Mechanistically, δ-Catenin peptide vaccines, presented by antigen-presenting cells to T cells, could enhance the activation of T cells via MAPK/ERK signaling and the transcriptional factors Eomes and T-bet. Our research results indicate new potential peptide vaccines, which can be applied in clinical HCC therapy.

Influence of POLG on Radiosensitivity of Nasopharyngeal Carcinoma Cells.

BACKGROUND AND OBJECTIVE: There is a high incidence of nasopharyngeal carcinoma (NPC), malignant head and neck tumors, in southern China. Radioresistance is the main cause affecting the efficacy of NPC treatments. The POLG gene particularly plays an important role in radiation-induced damage repair. In this study, the authors established RNAi CNE-1 and CNE-2 knockdown in two NPC cell lines to observe whether this gene affects the radiosensitivity of NPC cells. MATERIALS AND METHODS: Four short hairpin RNA (shRNA) expression plasmids targeting POLG gene were constructed and transfected into the NPC cell lines CNE-1 and CNE-2. Screening was performed to evaluate the stable expression of cloned cells, which were named CNE-1/POLG-shRNA1, CNE-1/POLG-shRNA2, CNE-2/POLG-shRNA1, and CNE-2/POLG-shRNA2. The negative controls CNE-1/Neg-shRNA and CNE-2/Neg-shRNA were additionally used. The MTT method, flow cytometry, clone formation analysis, cell migration, and other experimental methods were employed to verify changes in the radiosensitivity of the NPC cells. RESULTS: Fluorescent quantitative PCR and Western blot confirmed the downregulation of the PLOG gene through diminished PLOG messenger RNA and protein levels. Consequently, the authors report the stable knockdown of the POLG gene in an NPC model. Dose-dependent radiation exposure of POLG inhibited NPC cell growth and increased apoptosis compared with control cells (p < 0.01), as demonstrated through colony formation assay and flow cytometry. Functional assays indicated that knockdown of the POLG in CNE-1 and CNE-2 cells remarkably reduced cell viability and proliferation. Specifically, POLG knockdown led to G1 phase arrest and apoptosis. CONCLUSIONS: Overall, the authors conclude that POLG downregulation alters the radiosensitivity of NPC cells, indicating that the gene is likely involved in conferring the radiation response of the cells. In addition, findings in this study suggest a novel role for POLG as a potential predictive marker for NPC radiotherapy efficiency. POLG gene can be used as a potential clinical target to effectively improve the radiosensitivity of NPC.

18ß-Glycyrrhetinic acid mitigates radiation-induced skin damage via NADPH oxidase/ROS/p38MAPK and NF-κB pathways.

Radiation-induced inflammation plays an important role in radiation-induced tissue injury. 18ß-glycyrrhetinic acid (18ß-GA) has shown an anti-inflammatory activity. This study aimed to assess the activity of 18ß-GA against radiation-induced skin damage, and explore the underlying mechanisms. In vitro assay revealed 18ß-GA treatment decreased the production of IL-1ß, IL-6, PGE2 and decreased p38MAPK phosphorylation, DNA-binding activity of AP-1, and NF-κB activation in irradiated RAW264.7 macrophages. Additionally, 18ß-GA suppressed NF-κB activation by inhibiting NF-κB/p65 and IκB-α phosphorylation and alleviated ROS overproduction in irradiated RAW264.7 macrophages. In vivo assay showed 18ß-GA alleviated severity of radiation-induced skin damage, reduced inflammatory cell infiltration and TNF-α, IL-1ß and IL-6 levels in cutaneous tissues. Our findings demonstrate that 18ß-GA exhibits anti-inflammatory actions against radiation-induced skin damage probably by inhibiting NADPH oxidase activity, ROS production, activation of p38MAPK and NF-κB signaling, and the DNA binding activities of NF-κB and AP-1, consequently suppressing pro-inflammatory cytokine production.

DcR3, a new biomarker for sepsis, correlates with infection severity and procalcitonin.

Early diagnosis of sepsis is critical for successful treatment. The clinical value of DcR3 in early diagnosis of sepsis was determined in a dynamic follow-up study. Alterations in plasma levels of DcR3, PCT, CRP, and IL-6 were measured by ELISA and compared among patients with sepsis (n = 134), SIRS (n = 60) and normal adults (n = 50). Correlations and dynamic patterns among the biomarkers, APACHE II scores, clinical outcomes, and pathogens were also examined. Plasma DcR3 was significantly increased in sepsis compared to SIRS and normal adults (median 3.87 vs. 1.28 and 0.17 ng/ml). The elevated DcR3 could be detected in 97.60% sepsis patients 1-2 days prior to the result of blood culture reported. For diagnosis of sepsis, the sensitivity was 97.69% and specificity 98.04%; and for differential diagnosis of sepsis from SIRS, the sensitivity was 90.77% and specificity 98.40%. DcR3 level was positively correlated with severity of sepsis (rs = 0.82). In 41 patients who died of sepsis, DcR3 elevated as early as 1-2 days before blood culture and peaked on day 3 after blood culture performed. In 90% of sepsis patients, the dynamic alteration pattern of DcR3 was identical to that of PCT, while pattern of 10% patients differed in which clinical data was consistent with DcR3. In 13% sepsis patients, while PCT remained normal, DcR3 levels were at a high level. DcR3 levels had no difference among various pathogens infected. DcR3, a new biomarker, will aid in early diagnosis of sepsis and monitoring its outcome, especially when sepsis patients were PCT negative.

δ-Catenin promotes tumorigenesis and metastasis of lung adenocarcinoma.

δ-Catenin coded by gene CTNND2 has been found to be overexpressed in various types of cancers, including prostate, breast, lung and ovarian cancers. However, the function of δ-catenin in lung carcinoma remains largely unknown. In the present study, we revealed that δ-catenin acts as an oncogene promoting the malignancy of lung adenocarcinoma. When δ-catenin proteins of Lewis lung cells were depleted by knocking out Ctnnd2 via CRISPR/Cas9 technology, the cells lost the tumorigenic and metastatic abilities in vivo. Consistently, overexpression of Ctnnd2 enhances the subcutaneous tumorigenesis and distant metastasis of Lewis lung cells in vivo. However, δ-catenin promotes cell proliferation and cell cycle progression of Lewis lung cells. Mechanistically, δ-catenin enhances G1-S phase transition in cooperation with canonical Wnt signaling in Lewis lung cells. Moreover, δ-catenin promotes oncosphere formation of lung adenocarcinoma cells and is associated with the expression of cancer stem cell markers, which indicates δ-catenin enhances colonization and invasion via cancer stem cell maintenance. Taken together, our data suggest that δ-catenin may serve an important role in the malignancy of lung adenocarcinoma through activating canonical Wnt signaling and cancer stem cell maintenance. Our research indicates that δ-catenin can be a new potential target for the treatment of lung adenocarcinoma.