A robust immune-related lncRNA signature for the prognosis of human colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent malignant cancers worldwide. Immune-related long non-coding RNAs (IRlncRNAs) are proved to be essential in the development and progression of carcinoma. The purpose of the present study was to develop and validate a prognostic IRlncRNA signature for CRC patients. METHODS: Gene expression profiles of CRC samples were downloaded from The Cancer Genome Atlas (TCGA) database. Immune-related genes were obtained from the ImmPort database and were used to identify IRlncRNA by correlation analysis. Through LASSO Cox regression analyses, a prognostic signature was constructed. Functional enrichment analysis was performed by gene set enrichment analysis (GSEA). TIMER2.0 web server and tumor immune dysfunction and exclusion (TIDE) algorithm were employed to analyze the association between our model and tumor-infiltrating immune cells and immunotherapy response. The expression levels of IRlncRNAs in cell lines were detected by quantitative real-time PCR (qPCR). RESULTS: A 9-IRlncRNA signature was developed by a LASSO Cox proportional regression model. Based on the signature, CRC patients were divided into high- and low-risk groups with different prognoses. GSEA results indicated that patients in high-risk group were associated with cancer-related pathways. In addition, patients in low-risk group were found to have more infiltration of anti-tumor immune cells and might show a favorable response to immunotherapy. Finally, the result of qPCR revealed that most IRlncRNAs were differently expressed between normal and tumor cell lines. CONCLUSION: The constructed 9-IRlncRNA signature has potential to predict the prognosis of CRC patients and may be helpful to guide personalized immunotherapy.

Increased interleukin-11 associated with disease activity and development of interstitial lung disease in patients with rheumatoid arthritis.

OBJECTIVES: To investigate the association of serum interleukin-11 (IL-11) with disease activity and occurrence of interstitial lung disease (ILD) in patients with rheumatoid arthritis (RA). METHODS: One hundred and six RA patients were included, including 31 with ILD. All patients were divided into two groups according to the 28-joint Disease Activity Score (DAS28), active-disease group (DAS28>3.2) and target-achieved group (DAS28≤3.2). Serum IL-11 was detected by ELISA. Serum autoantibodies [anticitrullinated protein antibody (ACPA) and rheumatoid factor (RF)], inflammatory markers [C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)], and complete blood count were measured with routine methods. RESULTS: Serum IL-11 was upregulated in RA patients compared with healthy controls (HC), and increased more significantly in patients with ILD (RA-ILD) than patients without ILD (RA-nonILD). In both RA-ILD and RA-nonILD patients, serum level of IL-11 was higher in the active-disease group than that in the target-achieved group. Pearson correlation analysis confirmed that IL-11 was positively correlated with DAS28. No significant correlation was found between serum level of IL-11 and ACPA or RF. IL-11 was positively correlated with ESR and CRP levels and PLT count in RA patients. CONCLUSIONS: IL-11 was found to be involved in the development of arthritis and ILD in RA patients, and might constitute a potential target for the treatment of RA-ILD.

Role of oncogenic KRAS in the prognosis, diagnosis and treatment of colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease at the cellular and molecular levels. Kirsten rat sarcoma (KRAS) is a commonly mutated oncogene in CRC, with mutations in approximately 40% of all CRC cases; its mutations result in constitutive activation of the KRAS protein, which acts as a molecular switch to persistently stimulate downstream signaling pathways, including cell proliferation and survival, thereby leading to tumorigenesis. Patients whose CRC harbors KRAS mutations have a dismal prognosis. Currently, KRAS mutation testing is a routine clinical practice before treating metastatic cases, and the approaches developed to detect KRAS mutations have exhibited favorable sensitivity and accuracy. Due to the presence of KRAS mutations, this group of CRC patients requires more precise therapies. However, KRAS was historically thought to be an undruggable target until the development of KRASG12C allele-specific inhibitors. These promising inhibitors may provide novel strategies to treat KRAS-mutant CRC. Here, we provide an overview of the role of KRAS in the prognosis, diagnosis and treatment of CRC.

Casp8 acts through A20 to inhibit PD-L1 expression: The mechanism and its implication in immunotherapy.

Immunotherapy targeting the PD-L1/PD-1 pathway is a novel type of clinical cancer treatment, but only small subsets of patients can benefit from it because of multiple factors. PD-L1/PD-1 expression is a biomarker for predicting the efficacy of anti-PD-L1/PD-1 therapy, which highlights the importance of understanding the regulatory mechanisms of PD-L1 expression in cancer cells. Casp8 is an apical caspase protease involved in mediating cell apoptosis, but it also has multiple nonapoptotic functions. Casp8 mutations are associated with increased risks of cancer, and low expression of Casp8 is closely connected with poor prognosis in patients with cancer. In addition, mutations of Casp8 in lymphocytes also lead to human immunodeficiency, thereby causing dysfunction of the innate immune system, but the roles of Casp8 in antitumor immunity remain unclear. Here, we found that knocking down Casp8 in mouse melanoma cells promoted tumor progression in an immune system-dependent manner. Mechanistically, Casp8 induced PD-L1 degradation by upregulating TNFAIP3 (A20) expression, a ubiquitin-editing enzyme that results in PD-L1 ubiquitination. In addition, compared with Casp8fl/fl mice, mice with conditional deletion of Casp8 in natural killer (NK) cells (Ncr1iCre/+ Casp8fl/fl mice) showed a decreased frequency of IFN-γ+ and CD107a+ NK cells but an increased frequency of PD-1+ and CTLA-4+ NK cells. Melanoma cells with Casp8 knocked down exhibited sensitivity to anti-PD-1 or anti-CTLA-4 antibody treatments, particularly in Ncr1iCre/+Casp8fl/fl mice. Together, the results indicate that Casp8 induces PD-L1 degradation by upregulating A20 expression and that decreased Casp8 expression is a potential biomarker for predicting the sensitivity to anti-PD-L1/PD-1 immunotherapy.

An epithelial-mesenchymal transition-related 5-gene signature predicting the prognosis of hepatocellular carcinoma patients.

BACKGROUND: Tumor metastasis is one of the leading reasons of the dismal prognosis of hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is closely associated with tumor metastasis including HCC. The purpose of this study is to construct and validate an EMT-related gene signature for predicting the prognosis of HCC patients. METHODS: Gene expression data of HCC patients was downloaded from The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) was performed to found the EMT-related gene sets which were obviously distinct between normal samples and paired HCC samples. Cox regression analysis was used to develop an EMT-related prognostic signature, and the performance of the signature was evaluated by Kaplan-Meier curves and time-dependent receiver operating characteristic (ROC) curves. A nomogram incorporating the independent predictors was established. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of the hub genes in HCC cell lines, and the role of PDCD6 in the metastasis of HCC was determined by functional experiments. RESULTS: An EMT-related 5-gene signature (PDCD6, TCOF1, TRIM28, EZH2 and FAM83D) was constructed using univariate and multivariate Cox regression analysis. Based on the signature, the HCC patients were classified into high- and low-risk groups, and patients in high-risk group had a poor prognosis. Time-dependent ROC and Cox regression analyses suggested that the signature could predict HCC prognosis exactly and independently. The predictive capacity of the signature was also validated in two external cohorts. GSEA results showed that many cancer-related signaling pathways such as PI3K/Akt/mTOR pathway and TGF-ß/SMAD pathway were enriched in high-risk group. The result of qRT-PCR revealed that PDCD6, TCOF1 and FAM83D were highly expressed in HCC cancer cells. Among them, PDCD6 were found to promote cell migration and invasion. CONCLUSION: The EMT-related 5-gene signature can serve as a promising prognostic biomarker for HCC patients and may provide a novel mechanism of HCC metastasis.

C1QTNF6 Overexpression Acts as a Predictor of Poor Prognosis in Bladder Cancer Patients.

BACKGROUND: Bladder cancer is one of the most common urinary malignancies. This study is aimed at providing some promising molecular biomarkers for bladder cancer (BC) by investigating the correlation between C1QTNF6 expression and clinical characteristics as well as prognosis in patients with bladder cancer. METHODS: Sequencing profiles of C1QTNF6 mRNA in BC patients were collected to evaluate the distinctive gene expression, between normal bladder mucosa and BC, according to the TCGA and GEO databases. The association between C1QTNF6 expression and the clinical features as well as the disease prognosis was evaluated using two independent cohorts. The expression of C1QTNF6 in normal bladder and BC cells was examined by western blotting and PCR, so the underlying molecular mechanism could be further investigated. RESULTS: C1QTNF6 mRNA levels were found to be differentially expressed in two independent public cohorts, including the TCGA database and GSE13507 dataset from GEO. The protein and RNA levels of C1QTNF6 in BC cells were both elevated when compared to normal bladder cell lines. High C1QTNF6 expression was detected in advanced T/M stages, pathological grade, and AJCC stage when compared to the low C1QTNF6 expression group. The underlying mechanism related to this differential expression could be explained by cell migration and invasion assays, where bladder cancer cells 5637 and T24 had a significant reduction on migration and invasion ability upon knockdown of C1QTNF6 expression. The low C1QTNF6 expression group presented a more prominent OS advantage over the high-expression group in both TCGA and GSE13507 cohorts. Moreover, the protein content in tissues was further validated using the HPA database and TMA. Survival analyses also indicated that the high C1QTNF6 expression group had an unfavorable OS when compared to the low-expression group. CONCLUSIONS: High C1QTNF6 expression may serve as a predictor of poor prognosis in bladder cancer patients, and the underlying mechanism is possibly associated with changes on cancer cell migration and invasion ability.

EP300 mutation is associated with tumor mutation burden and promotes antitumor immunity in bladder cancer patients.

Bladder cancer is a leading cause of morbidity and mortality worldwide. Currently, immunotherapy has become a worthwhile therapy for bladder cancer. Tumor mutation burden (TMB) has been regarded as the most prevalent biomarker to predict immunotherapy. Bladder cancer is reported to have the third highest mutation rate. However, whether these gene mutations are related to TMB and immune response remain unknown. In this study, we downloaded somatic mutation data of bladder cancer from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets, and found 11 frequently mutated genes were covered by both two cohorts including FGFR3, TTN, XIRP2, CREBBP, PIK3CA, TP53, MUC16, EP300 (E1A binding protein P300), ARID1A, ERBB2, and KDM6A. Among them, EP300 mutation was associated with higher TMB and indicated a favorable clinical prognosis. Furthermore, based on Gene set enrichment analysis (GSEA) and CIBERSORT algorithm, we observed that EP300 mutation upregulated signaling pathways involved in immune system and enhanced antitumor immune response. In conclusion, EP300 is frequently mutated in bladder cancer, and its mutation is associated with increased TMB and promotes antitumor immunity, which may serve as a biomarker to predict immune response.

Bladder cancer cell­secreted exosomal miR­21 activates the PI3K/AKT pathway in macrophages to promote cancer progression.

Tumour­associated macrophages (TAMs) compose a major component of the tumour microenvironment and form in this microenvironment prior to cancer metastasis. However, the detailed mechanisms of TAM remodelling in the context of bladder cancer have not been clearly defined. The present study collected exosomes from the conditioned medium of human bladder T24 cancer cells. The effects of macrophages treated with exosomes derived from T24 cells on bladder cancer cell migration and invasion were analysed by Transwell assays. The expression levels of endogenous and exosomal microRNA­21 (miR­21) were examined by reverse transcription­quantitative PCR, while the expression level of the target protein was analysed by western blot analysis. Luciferase reporter plasmids and mutants were used to confirm direct targeting. The effects of miR­21 on bladder cancer cell migration and invasion were analysed by Transwell and Matrigel assays following miR­21 transfection. It was identified that exosomes derived from bladder cancer cells polarized THP­1 cell­derived macrophages into the M2 phenotype, and TAM­mediated pro­migratory and pro­invasive activity was determined. Moreover, it was found that miR­21 was highly expressed in exosomes derived from bladder cancer cells as well as in macrophages treated with exosomes. In addition, macrophages transfected with miR­21 exhibited M2 polarization and promoted T24 cell migratory and invasive ability. Mechanistically, exosomal miR­21 derived from bladder cancer cells inhibited phosphatase and tensin homolog activation of the PI3K/AKT signalling pathway in macrophages and enhanced STAT3 expression to promote M2 phenotypic polarization. The present results suggest that exosomal miR­21 can promote cancer progression by polarizing TAMs.

Profiles of tumor-infiltrating immune cells in renal cell carcinoma and their clinical implications.

Tumor-infiltrating immune cells (TIICs) are crucial for the clinical outcome of renal cell carcinoma (RCC), as they regulate cancer progression. TIICs have therefore the potential to become novel targets of immunotherapies. The present study used CIBERSORT analytical tool, which is a deconvolution algorithm, to comprehensively analyze the composition of immune cells in RCC and normal tissues from The Cancer Genome Atlas (TCGA) cohort, and to determine the prognostic value of TIICs in RCC. A landscape of infiltrating immune cells was determined as containing 13 subpopulations of immune cells, with significant differences between normal and tumor tissues. Subsequently, Kaplan-Meier analysis and log-rank test were used to estimate the prognostic value of TIICs in RCC. The results demonstrated that a higher proportion of regulatory T cells (Tregs) [hazard ratio (HR)=1.596; 95% confidence interval (CI), 1.147-2.222; P=0.006] and follicular helper T cells (HR=1.516; 95% CI, 1.089-2.111; P=0.014) were associated with poor outcome in patients with RCC. Conversely, resting mast cells (HR=0.678; 95% CI, 0.487-0.943; P=0.021) and monocytes (HR=0.701; 95% CI, 0.503-0.977; P=0.036) were associated with a favorable prognosis in patients with RCC. Furthermore, the results from multivariate Cox regression analysis indicated that Tregs and monocytes represented independent risk factors for prognosis in patients with RCC. These findings demonstrated that gene profiling deconvolution by CIBERSORT served to determine the composition of immune cells infiltrated in RCC and may provide some crucial information for the development of immunotherapies.

Increased expression of caspase 1 during active phase of connective tissue disease.

Key factors of pyroptosis play an important role in the inflammatory response to connective tissue disease (CTD). However, information on active and stable stages of CTD is scarce. To distinguish the differences of concentrations of C-reactive protein (CRP), caspase 1, caspase 4, caspase 5 and sCD14 in plasma between the patients with active and stable stages of CTD. A cohort study was conducted to recruit patients diagnosed with CTD of active phase and stable phase as well as health control. These data included the analysis of the concentration of sCD14, caspase 1, caspase 4 and caspase 5 in peripheral plasma by ELISA. The Wilcoxon rank-sum test was used to compare the two groups. The sex ratio and ages of the three groups were not different statistically. The concentrations of sCD14, caspase4 and caspase5 of plasma in the CTD of active phase and the stable phase as well as the health control. The concentration of caspase 1 in active phase of CTD (470.19 [422.33-513.14] pmol/L) was significantly higher than that in stable group (203.95 [160.94-236.12] pmol/L) and healthy control (201.65 [191.11-240.35] pmol/L] pmol/L) (p < 0.001, both), but there was no significant difference between stable group and healthy control (p = 0.2312). Similarly, the concentration of CRP in the active phase of CTD (8.96 [3.06-20.28] mg/L) was significantly higher than that in the stable group (3.00 [1.30-11.40] mg/L) and the healthy control (3.70 [2.30-4.73] mg/L) (p = 0.0013, p = 0.0006, respectively), but there was no significant difference between the stable group and the healthy control (p = 0.3205). However, there were no significant differences in the concentration of sCD14, caspase 4 and caspase 5 in the active phase of CTD and the stable group as well as the health group. Consequently, the patients of the active phase of CTD showed increased expression of caspase 1.

Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR-199a-3p.

Renal ischemia/reperfusion (I/R) injury is the main reason for acute kidney injury (AKI) and is closely related to high morbidity and mortality. In this study, we found that exosomes from human-bone-marrow-derived mesenchymal stem cells (hBMSC-Exos) play a protective role in hypoxia/reoxygenation (H/R) injury. hBMSC-Exos were enriched in miR-199a-3p, and hBMSC-Exo treatment increased the expression level of miR-199a-3p in renal cells. We further explored the function of miR-199a-3p on H/R injury. miR-199a-3p was knocked down in hBMSCs with a miR-199a-3p inhibitor. HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs were more susceptible to H/R injury and showed more apoptosis than those cocultured with hBMSCs or miR-199a-3p-overexpressing hBMSCs. Meanwhile, we found that HK-2 cells exposed to H/R treatment incubated with hBMSC-Exos decreased semaphorin 3A (Sema3A) and activated the protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways. However, HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs restored Sema3A expression and blocked the activation of the AKT and ERK pathways. Moreover, knocking down Sema3A could reactivate the AKT and ERK pathways suppressed by a miR-199a-3p inhibitor. In vivo, we injected hBMSC-Exos into mice suffering from I/R injury; this treatment induced functional recovery and histologic protection and reduced cleaved caspase-3 and Sema3A expression levels, as shown by immunohistochemistry. On the whole, this study demonstrated an antiapoptotic effect of hBMSC-Exos, which protected against I/R injury, via delivering miR-199a-3p to renal cells, downregulating Sema3A expression and thereby activating the AKT and ERK pathways. These findings reveal a novel mechanism of AKI treated with hBMSC-Exos and provide a therapeutic method for kidney diseases.

BMSCs protect against renal ischemia-reperfusion injury by secreting exosomes loaded with miR-199a-5p that target BIP to inhibit endoplasmic reticulum stress at the very early reperfusion stages.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been recently reported to play a variety of vital roles in organ and tissue damage repair, mainly via potent paracrine activity, including secreting extracellular vesicles, such as exosomes, that serve as mediators facilitating intercellular communication and reprogramming recipient cells by delivering their contents to target cells. However, the underlying mechanisms are diverse and complex, and the influencing characteristics have rarely been studied. Accordingly, we designed this study to explore the time dependence of the effects of exosomes derived from BMSCs (BMexos) on renal ischemia-reperfusion (I/R) injury and the underlying mechanisms associated with the reperfusion time. Impressively, our study is the first to find that BMexos protected against renal I/R injury in vitro and in vivo at the very early reperfusion stages, especially 4-8 h after reperfusion in vitro and 8-16 h after reperfusion in vivo. Interestingly, we simultaneously found that endoplasmic reticulum (ER) stress was significantly suppressed following the administration of BMexos in vitro and in vivo with a similar time dependence. Additionally, we discovered that miR-199a-5p, which was abundant in the BMSCs, was transferred into renal tubular epithelial cells (NRK-52E) in a time-dependent manner and significantly inhibited I/R-induced ER stress by targeting binding immunoglobulin protein (BIP). Cocultivation with miR-199a-5p-overexpressing BMSCs amplified the suppression of ER stress and further protected against I/R injury. However, coculture with miR-199a-5p-knockdown BMSCs obviously increased ER stress and reversed the BMexos-induced protection, and silencing BIP by small interfering RNA-1098 in NRK-52E inhibited these effects. This study provides evidence that administering BMexos at the very early reperfusion stages significantly protects against renal I/R injury, and ER stress is closely linked to this protection. These results suggest a novel therapeutic strategy during the very early reperfusion stages of renal I/R injury.-Wang, C., Zhu, G., He, W., Yin, H., Lin, F., Gou, X., Li, X. BMSCs protect against renal ischemia-reperfusion injury by secreting exosomes loaded with miR-199a-5p that target BIP to inhibit endoplasmic reticulum stress at the very early reperfusion stages.

Negative feedback loop of autophagy and endoplasmic reticulum stress in rapamycin protection against renal ischemia-reperfusion injury during initial reperfusion phase.

Rapamycin, an immunosuppressant, is widely used in patients with kidney transplant. However, the therapeutic effects of rapamycin remain controversial. Additionally, previous studies have revealed deleterious effects of rapamycin predominantly when administered for ≥24 h. Few studies, however, have focused on the short-term effects of rapamycin administered only during the initial reperfusion phase. As such, we designed this study to explore the potential effects and mechanisms of rapamycin under a specific therapeutic regimen in which rapamycin is mixed in the perfusate during the initial reperfusion phase (within 24 h). Interestingly, we found that rapamycin maintained renal function and attenuated ischemia-reperfusion (I/R)-induced apoptosis in vivo and in vitro during the initial reperfusion phase, especially at 8 h after reperfusion. Simultaneously, rapamycin activated autophagy and inhibited endoplasmic reticulum (ER) stress and 3 pathways of unfolding protein response: ATF6, PERK, and IRE1α. Interestingly, we further found that the protective effects of rapamycin were suppressed when autophagy was inhibited by chloroquine and 3-methyladenine or when ER stress was induced by thapsigargin. Moreover, in terms of the regulatory effects of rapamycin, a negative-feedback loop between autophagy and ER stress occurred, with autophagy inhibiting ER stress and increased ER stress promoting autophagy during the initial reperfusion phase of renal I/R injury. Our study provides evidence that immediate reperfusion with rapamycin during the initial reperfusion phase repairs renal function and reduces apoptosis via activating autophagy, which could further inhibit ER stress. These results suggest a novel treatment modality for application during the initial reperfusion phase of renal I/R injury caused by kidney transplantation.-Li, X., Zhu, G., Gou, X., He, W., Yin, H., Yang, X., Li, J. Negative feedback loop of autophagy and endoplasmic reticulum stress in rapamycin protection against renal ischemia-reperfusion injury during initial reperfusion phase.

Risk factors, follow-up, and treatment of urethral recurrence following radical cystectomy and urinary diversion for bladder cancer: a meta-analysis of 9498 patients.

PURPOSE: Patients frequently undergo radical cystectomy and urinary diversion for treatment of bladder cancer. However, they remain at risk of urethral recurrence (UR). Studies have determined various risk factors leading to urethral recurrence. However, no publications have weighed the predictive values of these factors. MATERIALS AND METHODS: Studies published between 1971 and 2016 were retrieved from PubMed, EMBASE and MEDLINE. We used STATA software (Version 12.0) to estimate the pooled risk ratio. RESULTS: Twenty-five publications with 9498 patients were included. Overall, male patients, especially those with concomitant carcinoma in situ, superficial or intravesical bladder cancer, non-orthotopic diversion, prostatic involvement, bladder neck involvement, positive urethral margins or multifocal bladder cancer were at higher risk of urethral recurrence. The overall risks of recurrence, reported as risk ratios, varied widely. Among all 25 studies, 118 (60.2%) cases in 9 studies were diagnosed through routine follow-up. Another 82 (40.8%) patients in 11 studies first reported symptomatic abnormalities. Prognoses were worse for patients with symptomatic recurrence. Urethral cytology was the most common diagnostic method. Treatment after UR was reported for 272 cases in 14 publications, and 190 patients underwent urethrectomy and 52 underwent urethra-sparing treatments. Outcomes after UR were described in 12 studies reporting 180 cases, and 41 patients were alive through the end of follow-up and 65 patients died of bladder cancer. CONCLUSIONS: UR following radical cystectomy for bladder cancer was closely related to risk factors. Precautions, strict follow-up protocols and rational therapies were critical to patients with high risks of urethral recurrences.