OncoSplicing: an updated database for clinically relevant alternative splicing in 33 human cancers.

Alternative splicing (AS) represents a crucial method in mRNA level to regulate gene expression and contributes to the protein complexity. Abnormal splicing has been reported to play roles in several diseases, including cancers. We developed the OncoSplicing database for visualization of survival-associated and differential alternative splicing in 2019. Here, we provide an updated version of OncoSplicing for an integrative view of clinically relevant alternative splicing based on 122 423 AS events across 33 cancers in the TCGA SpliceSeq project and 238 558 AS events across 32 cancers in the TCGA SplAdder project. The new version of the database contains several useful features, such as annotation of alternative splicing-associated transcripts, survival analysis based on median and optimal cut-offs, differential analysis between TCGA tumour samples and adjacent normal samples or GTEx normal samples, pan-cancer views of alternative splicing, splicing differences and results of Cox'PH regression, identification of clinical indicator-relevant and cancer-specific splicing events, and downloadable splicing data in the SplAdder project. Overall, the substantially updated version of OncoSplicing (www.oncosplicing.com) is a user-friendly and registration-free database for browsing and searching clinically relevant alternative splicing in human cancers.

Loss of SETD2-mediated downregulation of intracellular and exosomal miRNA-10b determines MAPK pathway activation and multidrug resistance in renal cancer.

SET domain-containing 2 (SETD2) is the most frequently mutated gene among all the histone methyltransferases in clear cell renal cell carcinoma (ccRCC). Microarrays, RNA sequencing analysis and exosomes analysis of cellular supernatant were performed after transfection A498 cells with si-SETD2 or siRNA of negative control. Chromatin immunoprecipitation and Luciferase reporter assay were conducted to evaluate the interaction between SETD2 and miR-10b. Functional and drug experiments in vitro and in vivo were performed to verify the role of SETD2, miR-10b and MAP4K4. The results showed that loss of SETD2 mediated downregulation of intracellular and exosomal microRNA-10b. MAP4K4 were relevant to oncogenesis of ccRCC caused by loss of SETD2 and miR-10b. SETD2 could directly target miR-10b and regulate the expression of multidrug resistance (MDR)-1 (P-gp170) through JNK pathway, which was one of the downstream pathways of MAP4K4. The coordinated expression of SETD2/H3K36me3/miR-10b/MAPKs/JNK/MDR pathway was revealed to the progression of ccRCC.

Comprehensive characterization of alternative splicing in renal cell carcinoma.

Irregular splicing was associated with tumor formation and progression in renal cell carcinoma (RCC) and many other cancers. By using splicing data in the TCGA SpliceSeq database, RCC subtype classification was performed and splicing features and their correlations with clinical course, genetic variants, splicing factors, pathways activation and immune heterogeneity were systemically analyzed. In this research, alternative splicing was found useful for classifying RCC subtypes. Splicing inefficiency with upregulated intron retention and cassette exon was associated with advanced conditions and unfavorable overall survival of patients with RCC. Splicing characteristics like splice site strength, guanine and cytosine content and exon length may be important factors disrupting splicing balance in RCC. Other than cis-acting and trans-acting regulation, alternative splicing also differed in races and tissue types and is also affected by mutation conditions, pathway settings and the response to environmental changes. Severe irregular splicing in tumor not only indicated terrible intra-cellular homeostasis, but also changed the activity of cancer-associated pathways by different splicing effects including isoforms switching and expression regulation. Moreover, irregular splicing and splicing-associated antigens were involved in immune reprograming and formation of immunosuppressive tumor microenvironment. Overall, we have described several clinical and molecular features in RCC splicing subtypes, which may be important for patient management and targeting treatment.

Anthocyanins accumulation analysis of correlated genes by metabolome and transcriptome in green and purple peppers (Capsicum annuum).

BACKGROUND: In order to clarify the the molecular mechanism of anthocyanin accumulation in green and purple fruits of pepper using metabolomics and transcriptomics,to identify different anthocyanin metabolites,and to analyze the differentially expressed genes involved in anthocyanin biosynthesis.. RESULTS: We analyzed the anthocyanin metabolome and transcriptome data of the fruits of 2 purple pepper and 1 green pepper. A total of 5 anthocyanin metabolites and 2224 differentially expressed genes were identified between the green and purple fruits of pepper. Among the 5 anthocyanin metabolites,delphin chloride was unique to purple pepper fruits,which is the mainly responsible for the purple fruit color of pepper. A total of 59 unigenes encoding 7 enzymes were identified as candidate genes involved in anthocyanin biosynthesis in pepper fruit. The six enzymes (PAL,C4H,CHI,DFR,ANS,UFGT) had higher expression levels except the F3H gene in purple compared with green fruits. In addition,seven transcription factors were also found in this study. These transcription factors may contribute to anthocyanin metabolite biosynthesis in the fruits of pepper. One of differentially expressed gene novel.2098 was founded. It was not annotated in NCBI. Though blast analysis we preliminarily considered that this gene related to MYB transcription factor and was involved in anthocyanin biosynthesis in pepper fruit. CONCLUSIONS: Overall, the results of this study provide useful information for understanding anthocyanin accumulation and the molecular mechanism of anthocyanin biosynthesis in peppers.

Two-tier manipulation of holographic information.

Here we demonstrate the two-tier manipulation of holographic information using frequency-selective metasurfaces. Our results show that these devices can diffract light efficiently at designed frequency and environmental conditions. By changing the frequency and refractive index of the surrounding environment, the metasurfaces produce two different holographic images. We anticipate that these environmental dependent, frequency-selective metasurfaces will have practical applications in holographic encryption and sensing.

Pioglitazone decreased renal calcium oxalate crystal formation by suppressing M1 macrophage polarization via the PPAR-γ-miR-23 axis.

Interaction of pioglitazone (PGZ) and macrophages (Mps) in renal crystal formation remains unclear. In the present study, we investigated the possible mechanisms involved with Mps of PGZ in suppressing renal crystal formation. Crystal formation in the mouse kidney was detected using polarized light optical microscopy and Pizzolato staining. Gene expression was detected by Western blot analysis, quantitative RT-PCR, immunohistochemistry, immunofluorescence, and ELISA. Mp phenotypes were identified by flow cytometric analysis. Cell apoptosis was detected with TUNEL assay, and tubular injury was detected with periodic acid-Schiff staining. Interaction of peroxisome proliferator-activated receptor (PPAR)-γ and promoter was determined by chromatin immunoprecipitation assay. Luciferase reporter assay was performed to authenticate target genes of miRNA-23 (miR-23). Recombinant adenovirus was used to elucidate the role of miR-23 in vivo. Renal crystal formation, inflammation, tubular injury, and cell apoptosis were significantly marked in glyoxylic acid-treated groups and significantly decreased in PGZ-treated groups. PGZ significantly reduced Mp infiltration and M1 Mp polarization in the kidney. In vitro, PGZ shifted crystal-stimulated M1-predominant Mps to M2-predominant Mps, which were anti-inflammatory. PPAR-γ could directly bind to one PPAR-γ regulatory element in the promoter of pre-miR-23 to promote expression of miR-23 in Mps. We identified two downstream target genes of miR-23, interferon regulatory factor 1 and Pknox1. Moreover, miR-23 decreased crystal deposition, M1 Mp polarization, and injury in the kidney. This study has proven that PGZ decreased renal calcium oxalate crystal formation and renal inflammatory injury by suppressing M1 Mp polarization through a PPAR-γ-miR-23-interferon regulatory factor 1/Pknox1 axis. PGZ is liable to be a potential therapeutic medicine for treating urolithiasis.

Integrated Analysis of Genetic Abnormalities of the Histone Lysine Methyltransferases in Prostate Cancer.

BACKGROUND The histone methyltransferase (HMT) family includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). The role of HMT gene variants in prostate cancer remains unknown. Therefore, this study aimed to evaluate HMT gene variants in the pathogenesis and prognosis of human prostate cancer, using in vitro cell studies and bioinformatics analysis. MATERIAL AND METHODS Integrative bioinformatics analysis of the expression of 51 HMT genes in human prostate cancer was based on datasets from the Cancer Genome Atlas (TCGA). Correlation and regression analysis were used to identify critical HMTs in prostate cancer. Kaplan-Meier and the area under the receiver operating characteristics curve (AUROC) were performed to evaluate the function of the HMTs on prognosis. Gene expression and function of 22Rv1 human prostate carcinoma cells were studied. RESULTS The HMT genes identified to have a role in the pathogenesis of prostate cancer included the EZH2, SETD5, PRDM12, NSD1, SETD6, SMYD1, and the WHSC1L1 gene. The EZH2, SETD5, and SMYD1 genes were selected as a prognostic panel, with the SUV420H2 HMT gene. SETD2, NSD1, and ASH1L were identified as critical genes in the development of castration-resistant prostate cancer (CRPC), similar to mixed-lineage leukemia (MLL) complex family members. Knockdown of the SETD5 gene in 22Rv1 prostate carcinoma cells in vitro inhibited cancer cell growth and migration. CONCLUSIONS HMT gene variants may have a role in the pathogenesis of prostate cancer. Future studies may determine the role of HMT genes as prognostic biomarkers in patients with prostate cancer.

Androgen-receptor splice variant-7-positive prostate cancer: a novel molecular subtype with markedly worse androgen-deprivation therapy outcomes in newly diagnosed patients.

Androgen-deprivation therapy has been the standard treatment for metastatic and locally advanced prostate cancer, but the majority of patients will progress to castration-resistant prostate cancer within 2-3 years. Unlike the case in breast cancer, no clinically validated biomarker has been used to predict the outcomes of androgen-deprivation therapy. To evaluate androgen-receptor splice variant-7 (AR-V7) detection in newly diagnosed advanced prostate cancer and describe the distinctive prognosis of this novel molecular subtype, this study retrospectively enrolled 168 newly diagnosed prostate cancer patients from 2003 to 2015 who received androgen-deprivation therapy. AR-V7 immunohistochemical staining was performed with a monoclonal antibody, and AR-V7 expression was determined using Immune-Reactive Score data. The association between nuclear AR-V7 expression and prognosis was determined. Multiple cause-specific Cox regression and stratified cumulative incidences were used to analyze the prognosis risk. Among the 168 patients, 32 (19%) were AR-V7-positive. Compared with the AR-V7-negative patients, the AR-V7-positive patients had significantly lower prostate-specific antigen response rates (P<0.001) to androgen-deprivation therapy and a much shorter time to castration-resistant prostate cancer (P<0.0001). In Kaplan-Meier analysis, the AR-V7-positive group showed markedly lower castration-resistant prostate cancer progression-free survival (P<0.0001) and much lower cancer-specific (P<0.0001) and overall survival (P<0.0001) both in all enrolled patients and in patients with metastases. AR-V7 positivity was a significant predictor of castration-resistant prostate cancer progression in multiple Cox regression (hazard ratio: 4.826; 95% CI: 2.960-7.869; P<0.001). AR-V7 immunohistochemical detection in newly diagnosed prostate cancer patients who are planning to receive androgen-deprivation therapy, especially those with metastases, is necessary and valuable for prognostic assessment. AR-V7-positive prostate cancer should be considered a novel prostate cancer subtype that should be distinguished upon initial biopsy. The main limitation of this study is its observational nature.

Efficacy and safety of PDE5-Is and α-1 blockers for treating lower ureteric stones or LUTS: a meta-analysis of RCTs.

BACKGROUND: Lower ureteric stones and lower urinary tract symptoms are common in urology.Drug treatment is one of standard therapy,but the efficacy was controversial.Thus we aimed to investigate the efficacy and safety of monotherapy or combination therapy of adrenoceptor1 blockers and phosphodiesterase5 inhibitors for treatment. METHODS: Randomized controlled trials up to November 2016 were retrieved from PubMed, the Cochrane Library, Web of Science and Embase. A total of 17 studies were included. We analyzed data through random or fixed effect models. The heterogeneity between studies was assessed by the I2 test statistic. RESULTS: As for lower ureter stones, our analysis demonstrated tadalafil had a significantly lower incidence of abnormal ejaculation than adrenoceptor1 blockers (2.31 95%CI 0.22to0.84, P = 0.01),while combination therapy had a higher expulsion rate (2.49 95%CI 1.44to4.29, P = 0.001) and shorter expulsion time (- 1.98 95%CI -3.08to0.88, P = 0.0004) than tamsulosin. As for lower urinary tract symptoms, our analysis indicated adrenoceptor1 blockers was more effective than phosphodiesterase5 inhibitors on decreasing International Prostate Symptom Score (1.96 95%CI 0.03to3.89, P = 0.05) and Post-Void Residual (9.41 95%CI 1.40to14.41, P = 0.02) and phosphodiesterase5 inhibitors showed a greater effect than adrenoceptor1 blockers on improving Erectile Dysfunction (2.23 95%CI 1.24to3.22, P<0.0001).Combination therapy had a significantly better effect on International Prostate Symptom Score (1.47 95%CI 1.25to1.69, P<0.0001), Maximum flow rate (0.87 95%CI 0.71to1.04, P<0.0001), Post-Void Residual (10.74 95%CI 3.53to17.96,P = 0.004) and Quality of life (0.59 95%CI 0.22to0.97, P = 0.002) but was associated with higher incidences of adverse events (3.40 95%CI 1.82to6.36, P = 0.0001) than adrenoceptor1 blockers. Combination therapy had a significantly better effect on International Prostate Symptom Score (4.19 95%CI 3.34to5.04, P<0.0001), Maximum flow rate (1.86 95%CI 1.32to2.39, P<0.0001), Post-Void Residual (22.58 95%CI 9.13to36.04, P = 0.001) and Quality of life (0.68 95%CI 0.37to1.00, P<0.0001) without higher incidences of adverse events than PDE5-Is. CONCLUSIONS: In conclusion, this meta-analysis suggested combination therapy had a best efficacy of therapy for lower ureteric stones or lower urinary tract symptoms correlated with benign prostatic hyperplasia than monotherapy. Adrenoceptor1 blockers was more effective than phosphodiesterase5 inhibitors on International Prostate Symptom Score and Post-Void Residual. Both monotherapy and combination therapy were safe.

Pure Small Cell Carcinoma of Prostate: A Report of 8 Cases.

PURPOSE: To describe characteristics of pure small cell carcinoma of prostate (SCCP) and assess the prognostic factors. PATIENTS AND METHODS: We summarized data of pure SCCP from published studies and ours and made Kaplan-Meier analysis and Cox regression to evaluate prognosis factors. RESULTS: A total of 2,213 patients with prostate cancer was identified, of which eight (0.36%) patients were pure SCCP. The mean age at diagnosis was 61 years old. And there were 2 patients diagnosed at 34 and 50 years old respectively. Symptoms of these patients were similar to patients with prostate adenocarcinoma. Serum prostate specific antigen of 7 patients was at normal level. Five patients received chemotherapy, average overall survival (OS) was 9.75 months; 3 only received conservative treatment, average OS was 4 months. By univariate and multivariate Cox analysis, chemotherapy is an independent predictor of survival. Kaplan-Meier analysis demonstrated that chemotherapy was associated with longer OS. CONCLUSION: Clinical characteristics, examination and treatment strategy of pure SCCP are very different from prostate adenocarcinoma. According to the data from published studies and from our studies, the average survival of patients receiving chemotherapy is longer than those who received other treatment modalities.

Erratum: Inhibition of SMYD2 suppresses tumor progression by down-regulating microRNA-125b and attenuates multi-drug resistance in renal cell carcinoma: Erratum.

[This corrects the article DOI: 10.7150/thno.37628.].

Erratum: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis: Erratum.

[This corrects the article DOI: 10.7150/thno.44054.].

Corrigendum: Transcription factors BARX1 and DLX4 contribute to progression of clear cell renal cell carcinoma via promoting proliferation and epithelial-mesenchymal transition.

[This corrects the article DOI: 10.3389/fmolb.2021.626328.].

Clinicopathological characteristics of androgen receptor splicing variant 7 (AR-V7) expression in patients with castration resistant prostate cancer: A systematic review and meta-analysis.

BACKGROUND:  Studies have shown that AR-V7 may be correlated with the poor prognosis of castration resistant prostate cancer (CRPC), however, clinicopathological characteristics of AR-V7 have not been fully elucidated. OBJECTIVE: This study aimed at evaluating the clinicopathological features of AR-V7 in CRPC patients. MATERIALS AND METHODS: To evaluate the clinicopathological features of AR-V7 in CRPC patients. A search of PubMed, Embase, and Web of Science was performed using the keywords prostate cancer, prostate tumor, prostate neoplasm, prostate carcinoma, AR-V7, AR3, androgen receptor splicing variant-7, or androgen receptor-3. Twenty-four trials published by February 2020 were included in this study. RESULTS: The proportion of Gleason score ≥ 8 was found to be significantly higher in AR-V7-positive CRPC (69.5%) than negative (54.9%) (OR 1.68, 95% CI 1.25-2.25, p < 0.001), while the rates of T3/T4 stage (OR 1.16, 95% CI 0.60-2.24, p = 0.65) and N1 stage (OR 0.99, 95% CI 0.65-1.51, p = 0.96) were not statistically correlated with AR-V7 status. The AR-V7-positive patients exhibited a significantly higher proportion of any site metastasis (61.3% versus 35.0%; OR 2.19, 95% CI 1.57-3.05, p < 0.001) and bone metastasis (81.7% versus 69.0%; OR 1.97, 95% CI 1.44-2.69, p < 0.001), and a trend close to significance was expected in visceral metastasis (28.8% versus 22.1%; OR 1.29, 95% CI 0.96-1.74, p = 0.09). Incidences of pain in AR-V7-positive CRPC (54.6%) were significantly higher than in negative CRPC (28.1%; OR 4.23, 95% CI 2.52-7.10, p < 0.001), line with worse ECOG performance status (56.7% versus 35.0%, OR 2.18, 95% CI 1.51-3.16, P < 0.001). Limitations of the study include differences in sample sizes and designs, AR-V7 detection assays, as well as disease characteristics of the included studies. CONCLUSIONS: AR-V7 positivity is associated with a higher Gleason score, bone or any site metastasis, pain and worse ECOG performance scores in CRPC. However, it is not correlated with tumor stage or lymph node metastasis. More studies are needed to confirm these findings.

Transcription Factors BARX1 and DLX4 Contribute to Progression of Clear Cell Renal Cell Carcinoma via Promoting Proliferation and Epithelial-Mesenchymal Transition.

Dysregulation of transcription factors contributes to the carcinogenesis and progression of cancers. However, their roles in clear cell renal cell carcinoma remain largely unknown. This study aimed to evaluate the clinical significance of TFs and investigate their potential molecular mechanisms in ccRCC. Data were accessed from the cancer genome atlas kidney clear cell carcinoma cohort. Bioinformatics algorithm was used in copy number alterations mutations, and differentially expressed TFs' analysis. Univariate and multivariate Cox regression analyses were performed to identify clinically significant TFs and construct a six-TF prognostic panel. TFs' expression was validated in human tissues. Gene set enrichment analysis (GSEA) was utilized to find enriched cancer hallmark pathways. Functional experiments were conducted to verify the cancer-promoting effect of BARX homeobox 1 (BARX1) and distal-less homeobox 4 (DLX4) in ccRCC, and Western blot was performed to explore their downstream pathways. As for results, many CNAs and mutations were identified in transcription factor genes. TFs were differentially expressed in ccRCC. An applicable predictive panel of six-TF genes was constructed to predict the overall survival for ccRCC patients, and its diagnostic efficiency was evaluated by the area under the curve (AUC). BARX1 and DLX4 were associated with poor prognosis, and they could promote the proliferation and migration of ccRCC. In conclusion, the six-TF panel can be used as a prognostic biomarker for ccRCC patients. BARX1 and DLX4 play oncogenic roles in ccRCC via promoting proliferation and epithelial-mesenchymal transition. They have the potential to be novel therapeutic targets for ccRCC.

Androgen Receptor Splice Variant 7 Predicts Shorter Response in Patients with Metastatic Hormone-sensitive Prostate Cancer Receiving Androgen Deprivation Therapy.

BACKGROUND: Whether AR-V7 expression can predict the response in patients with metastatic hormone-sensitive prostate cancer (mHSPC) who receive androgen deprivation therapy (ADT) remains to be explored. OBJECTIVE: To evaluate the predictive value of AR-V7 expression in the prognosis of mHSPC patients receiving ADT. DESIGN, SETTING, AND PARTICIPANTS: In this multicenter prospective cohort study, 310 mHSPC patients commencing ADT were enrolled. Standard immunohistochemical staining was used to assess AR-V7 protein expression in biopsy tissues collected before initiation of ADT. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Kaplan-Meier survival estimates and Cox regression analyses were used to evaluate associations of AR-V7 status (positive vs negative) with progression-free survival (PFS) and overall survival (OS). RESULTS AND LIMITATIONS: Sixty-four (21%) patients were AR-V7-positive and 246 (79%) patients were AR-V7-negative. The median follow-up for patients not confirmed dead was 25 mo (interquartile range 10-30). Compared to AR-V7-negative patients, AR-V7-positive patients had significantly shorter PFS (hazard ratio [HR] 47.39, 95% confidence interval [CI] 25.83-86.94) and OS (HR 3.57, 95% CI 1.46-8.72). In multivariable analysis, AR-V7 was an independent predictive factor (HR 7.61, 95% CI 5.24-11.06) for shorter PFS. Limitations include the sample size and follow-up period. CONCLUSIONS: AR-V7 expression in primary cancer tissue is correlated with poor prognosis for mHSPC patients receiving ADT. PATIENT SUMMARY: In this study of men with metastatic hormone-sensitive prostate cancer, AR-V7 protein expression in primary cancer tissue was associated with poor outcomes on androgen deprivation therapy.

The clinicopathological significance and prognostic value of programmed death-ligand 1 in prostate cancer: a meta-analysis of 3133 patients.

BACKGROUND: Programmed death-ligand 1 (PD-L1) is considered an adverse factor predicting poor prognosis in various cancers, but the significance of PD-L1 expression for the prognosis of prostate cancer (PCa) is still unclear. We aimed to investigate the clinicopathological significance and prognostic value of PD-L1 expression in PCa. METHODS: Studies were retrieved from PubMed, Web of Science, Cochrane Library and Embase before March 23, 2020. Odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CIs) were obtained to assess the results. Begg's test was applied to evaluate publication bias. RESULTS: Fourteen studies involving 3133 cases were analyzed. The pooled data showed that both PD-L1 protein expression and PD-L1 DNA methylation (mPD-L1) were negatively associated with biochemical recurrence-free survival, with HRs of 1.67 (95% CI = 1.38-2.06, p < 0.001) and 2.23 (95% CI = 1.51-3.29, p < 0.001), respectively. In addition, PD-L1 overexpression was significantly related to advanced tumor stage (OR = 1.40, 95% CI= 1.13-1.75, p = 0.003), positive surgical margin (OR = 1.36, 95% CI = 1.03-1.78, p = 0.028), higher Gleason score (OR = 1.81, 95% CI = 1.35-2.42, p < 0.001) and androgen receptor positivity (OR = 2.20, 95% CI = 1.61-3.01, p < 0.001), while no significant correlation with age (p = 0.122), preoperative PSA (p = 0.796) or nodal status (p = 0.113) was observed. CONCLUSIONS: The study revealed that high expression of PD-L1 was related to unfavorable prognosis and advanced clinicopathological factors in PCa patients.

Delayed-onset adrenal hypoplasia congenita and hypogonadotropic hypogonadism caused by a novel mutation in DAX1.

In this study, we described a male who presented with delayed-onset adrenal hypoplasia congenita (AHC) and mild hypogonadotropic hypogonadism (HHG) without a relevant family history. A novel mutation in the DAX1 (dosage-sensitive sex reversal, congenital adrenal hypoplasia critical region on the X chromosome, gene 1) gene was shown to cause X-linked AHC and HHG. Genetic analysis revealed a novel nonsense mutation, c.154G > T (p.Glu52Term), in the DAX1 gene. Molecular testing demonstrated that the milder phenotype caused by this mutation was due to expression of a partially functional, amino-truncated DAX1 protein generated from an alternate in-frame translation start site (methionine at codon 83). This unusual case revealed a potential mechanism for a novel mutation that resulted in an unusual delayed-onset mild clinical phenotype. It expands the spectrum of adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis.

Intrarenal calcium oxalate (CaOx) crystals induce renal tubular epithelial cells (TECs) injury and inflammation, which involve Toll-like receptor 4 (TLR4)/interferon regulatory factor 1 (IRF1) signaling. Additionally, infiltrating macrophages (Mϕs) might influence intrarenal CaOx crystals and CaOx-induced renal injury. Although the roles of nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating inflammation and macrophage polarization are well characterized, its potential mechanisms in regulating CaOx nephrocalcinosis remain undefined. Methods: We used a Gene Expression Omnibus dataset to analyze gene-expression profiles. Luciferase reporter, western blot, quantitative polymerase chain reaction, immunofluorescence staining, fluorescence in situ hybridization, positron emission tomography computed tomography imaging, flow cytometry, and chromatin immunoprecipitation assays were employed to study the mechanism of miR-93-TLR4/IRF1 regulation by Nrf2. Anti-inflammatory activity and regulation of macrophage polarization by Nrf2 were investigated in vitro and in vivo. Results: We found that stone-mediated kidney inflammation significantly affected stone growth, and that sulforaphane attenuated CaOx nephrocalcinosis-induced kidney injury and renal CaOx crystals deposition. Additionally, Nrf2 levels significantly increased and negatively correlated with TLR4 and IRF1 levels in a mouse model of CaOx nephrocalcinosis following sulforaphane treatment. Moreover, Nrf2 suppressed TLR4 and IRF1 levels and decreased M1-macrophage polarization which induced by supernatants from COM-stimulated TECs in vitro. In terms of mechanism, transcription factor analyses, microRNA microarray, and chromatin immunoprecipitation assays showed that Nrf2 exhibited positive transcriptional activation of miR-93-5p. In addition, Luciferase reporter, qRT-PCR, and western blot validated that miR-93-5p targets TLR4 and IRF1 mRNA. Furthermore, suppressed miR-93-5p expression partially reversed Nrf2-dependent TLR4/IRF1 downregulation. Conclusions: The results suggested that sulforaphane might promote M2Mϕ polarization and inhibit CaOx nephrocalcinosis-induced inflammatory injury to renal tubular epithelial cells via the Nrf2-miR-93-TLR4/IRF1 pathway in vitro and in vivo.

HnRNP A1 - mediated alternative splicing of CCDC50 contributes to cancer progression of clear cell renal cell carcinoma via ZNF395.

BACKGROUND: Aberrant alternative splicing events play critical roles in carcinogenesis and progression of many cancers, while sparse studies regarding to alternative splicing are available for clear cell renal cell carcinoma (ccRCC). We identified that alternative splicing of coiled-coil domain containing 50 (CCDC50) was dysregulated in ccRCC, whereas the clinical significance of this splicing event and its splicing regulation mechanisms were still elusive. METHODS: Bioinformatic algorithm was utilized to identify significant exon skipping events in ccRCC via exon sequencing data from The Cancer Genome Atlas. Semi-quantitative real-time polymerase chain reaction and western blot were used to validate the aberrant expression of different transcripts in renal cancer tissues, cell lines and corresponding noncancerous controls. Short hairpin RNA targeting CCDC50 and overexpressing plasmids for each transcript were introduced into ccRCC cell lines, followed by a series of in vitro and in vivo functional experiments. Moreover, a panel of splicing factors were identified and their roles on splicing regulation of CCDC50 precursor mRNA (pre-mRNA) were studied. Furthermore, RNAseq data were analyzed to elucidate downstream molecules of CCDC50. Two-way analysis of variance and unpaired Student t test were used in statistical analysis. RESULTS: Pre-mRNA of CCDC50 generated two transcripts, full-length transcript (CCDC50-FL) and truncated transcript (CCDC50-S) with exon 6 skipped. CCDC50-S was overexpressed in ccRCC tissues and cell lines compared to noncancerous counterparts, but CCDC50-FL was only detected in noncancerous tissues and normal renal epithelial cells. Higher percent spliced-in index was associated with better survival in ccRCC patients. In vitro and in vivo functional experiments indicated that CCDC50-S transcript promoted the proliferation, migration, invasion and tumorigenesis of ccRCC, while CCDC50-FL exerted opposite tumor suppressive functions. Besides, we identified that heterogeneous nuclear ribonucleoprotein A1 (HnRNP A1) could promote the skipping of exon 6, which resulted in higher portion of CCDC50-S and oncogenic transformation. Moreover, zinc finger protein 395 (ZNF395) was identified as a downstream protein of CCDC50-S, and the interaction initiated oncogenic pathways which were involved in ccRCC progression. CONCLUSIONS: Aberrant alternative splicing of CCDC50 is regulated by HnRNP A1 in ccRCC. This splicing event contributes to cancer progression through the downstream pathway involving ZNF395.