Identification and preliminary analysis of hub genes associated with bladder cancer progression by comprehensive bioinformatics analysis.

Bladder cancer (BC) is a crisis to human health. It is necessary to understand the molecular mechanisms of the development and progression of BC to determine treatment options. Publicly available expression data were obtained from TCGA and GEO databases to spot differentially expressed genes (DEGs) between cancer and normal bladder tissues. Weighted co-expression networks were constructed, and Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Associations in hub genes, immune infiltration, and immune therapy were evaluated separately. Protein-protein interaction (PPI) networks for the genes identified in the normal and tumor groups were launched. 3461 DEGs in the TCGA dataset and 1069 DEGs in the GSE dataset were identified, including 87 overlapping genes between cancer and normal bladder groups. Hub genes in the tumor group were mainly enriched for cell proliferation, while hub genes in the normal group were related to the synthesis and secretion of neurotransmitters. Based on survival analysis, CDH19, RELN, PLP1, and TRIB3 were considerably associated with prognosis (P < 0.05). CDH19, RELN, PLP1, and TRIB3 may play important roles in the development of BC and are potential biomarkers in therapy and prognosis.

Hypertension Promotes the Proliferation and Migration of ccRCC Cells by Downregulation of TIMP3 in Tumor Endothelial Cells through the miR-21-5p/TGFBR2/P38/EGR1 Axis.

Recent studies have demonstrated that hypertension correlates with tumorigenesis and prognosis of clear-cell renal cell carcinoma (ccRCC); however, the underlying molecular mechanisms remain unclear. By analyzing bulk and single-cell RNA sequencing data and experimental examining of surgical excised ccRCC samples, we found that tissue inhibitors of metalloproteinases 3 (TIMP3), a pivotal paracrine factor in suppressing tumor progression, was significantly reduced in the tumor endothelial cells of patients with hypertensive ccRCC. Besides, in tumor xenograft of NCG mouse model, compared with saline normotensive group the expression of TIMP3 was significantly decreased in the angiotensin II-induced hypertension group. Treating human umbilical vein endothelial cells (HUVEC) with the plasma of patients with hypertensive ccRCC and miR-21-5p, elevated in the plasma of patients with hypertensive ccRCC, reduced the expression of TIMP3 compared with normotensive and control littermates. We also found that the inhibition of TIMP3 expression by miR-21-5p was not through directly targeting at 3'UTR of TIMP3 but through suppressing the expression of TGFß receptor 2 (TGFBR2). In addition, the knockout of TGFBR2 reduced TIMP3 expression in HUVECs through P38/EGR1 (early growth response protein 1) signaling axis. Moreover, via coculture of ccRCC cell lines with HUVECs and mouse tumor xenograft model, we discovered that the TIMP3 could suppress the proliferation and migration of ccRCC. IMPLICATIONS: Overall, our findings shed new light on the role of hypertension in promoting the progression of ccRCC and provide a potential therapeutic target for patients with ccRCC with hypertension.

Single-cell landscape reveals active cell subtypes and their interaction in the tumor microenvironment of gastric cancer.

Background: Gastric cancer remains the third most common cause of cancer-related death worldwide. The development of novel therapeutic strategies for gastric cancer requires a deep understanding of the tumor cells and microenvironment of gastric cancer. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine untreated non-metastatic gastric cancer patients. The transcriptomic atlas and ligand-receptor-based intercellular communication networks of the single cells were characterized. Results: Here, we profiled the transcriptomes of 47,304 cells from nine patients with gastric cancer. Tregs cells were significantly enriched in the gastric tumor tissues with increased expression of immune suppression related genes, which suggest a more immunosuppressive microenvironment. We also observed the absence of separate exhausted CD8+ T cell cluster, and the low expression level of exhaustion markers PDCD1, CTLA4, HAVCR2, LAG-3, and TIGIT in those specific cells. These may serve as molecular-level evidence for the limited benefit of immunotherapy among gastric cancer patients. In addition, we found ACKR1 specifically expressed in tumor endothelial cells, associated with poor prognosis in the cohort data and potentially provided a novel target of gastric cancer treatment. Furthermore, the tight interaction between endothelial cells and fibroblast implied the important roles of fibroblast in tumor angiogenesis and the maintenance of tumor vasculature. Conclusions: In conclusion, this single-cell atlas provide understanding the cellular heterogeneity from molecular level in gastric cancer and will serve as a valuable resource for developing innovative early and companion diagnostics, as well as discovering novel targeted therapies for gastric cancer.

NEDD4L suppresses PD-L1 expression and enhances anti-tumor immune response in A549 cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) poses a salient threat to public health. E3 ubiquitin ligase commonly functions as an anti-tumor role. OBJECTIVE: This study probed the effect of E3 ligase NEDD4L on A549 cells. METHODS: NEDD4L expression in NSCLC and its correlation with NSCLC patient's prognosis were predicted and verified. PD-L1 protein level was measured, and the correlation between NEDD4L and PD-L1 was analyzed. The effects of NEDD4L overexpression on the binding of NEDD4L to PD-L1 and ubiquitination level of PD-L1 were examined. Xenograft tumor model was established in mice. The volume and weight of xenograft tumors were recorded. The proportion of CD8+ T cells and contents of IL-2 and INF-γ were detected. RESULTS: NEDD4L expression was downregulated in NSCLC tissues and A549 cells, and correlated with poor prognosis of NSCLC patients. PD-L1 was upregulated in NSCLC and negatively correlated with NEDD4L. Overexpression of NEDD4L upregulated ubiquitination level of PD-L1 and reduced protein level of PD-L1. Overexpression of NEDD4L decreased tumor volume and weight and enhanced proportion of CD8+ T cells and contents of IL-2 and INF-γ. CONCLUSIONS: Collectively, overexpression of NEDD4L suppressed PD-L1 protein level through ubiquitination, thereby enhancing anti-tumor immune response and retarding NSCLC progression.

Targeted Methylation of the LncRNA NEAT1 Suppresses Malignancy of Renal Cell Carcinoma.

Long-chain non-coding RNA (LncRNA) has been found to play an important role in the regulation of the occurrence and progression of renal cell carcinoma (RCC). In this study, we demonstrated that LncRNA NEAT1 expression and m6A methylation level was decreased in RCC tissues. Further, the downregulated expression level of LncRNA NEAT1 was associated with poor prognosis for RCC patients. Then we used CRIPSR/dCas13b-METTL3 to methylate LncRNA NEAT1 in RCC cells. The results showed that the expression level of LncRNA NEAT1 was upregulated after methylated by dCas13b-METTL3 in RCC cells. And the proliferation and migration ability of RCC cells was decreased after methylated LncRNA NEAT1. Finally, we examined the effect of LncRNA NEAT1 hypermethylation on the transcriptome. We found differentially expressed genes in RCC cells were associated with "cGMP-PKG signaling pathway", "Cell adhesion molecules" and "Pathways in cancer". In conclusion, CRISPR/Cas13b-METTL3 targeting LncRNA NEAT1 m6A methylation activates LncRNA NEAT1 expression and provides a new target for treatment of RCC.

Prognostic role of ACTL10 in Cytogenetic Normal Acute Myeloid Leukemia.

ACTL10 is a member of the actin family; however, despite previous studies suggesting that certain proteins in this family may be related to the pathogenesis of leukemia, to the best of our knowledge, no studies to date have demonstrated any association between ACTAL10 and leukemia. Thus, the present study aimed to determine the association between ACTL10 expression levels, DNA methylation levels and the clinical prognosis in cytogenic normal acute myeloid leukemia (CN-AML). Data from seventy-five patients with CN-AML and patients with AML treated with chemotherapy or allogeneic hematopoietic stem cell transplantation were obtained from The Cancer Genome Atlas (TCGA) dataset and were used to analyze the clinical prognosis of ACTL10 RNA expression levels and DNA methylation levels. In addition, the study also investigated the combined clinical prognosis of ACTL10 RNA expression levels and ACTL10 DNA methylation levels in 74 patients with CN-AML from the TCGA dataset. ACTL10 RNA expression levels were observed to be highly expressed in patients with CD34+/CD38+ AML (P<0.01). Both ACTL10 RNA expression levels and DNA methylation were found to be independent prognostic factors for patients with CN-AML; patients with CN-AML in the ACTL10 RNA-high expression group had an increased EFS (P=0.0016) and OS (P=0.014) and patients in ACTL10 DNA methylation-low group also demonstrated a long EFS (P<0.0001) and OS (P=0.004). Notably, integrating ACTL10 RNA expression levels and ACTL10 DNA methylation levels could more accurately predict the prognosis of patients with CN-AML (EFS and OS, P<0.0001). In conclusion, the findings of the present study suggested that the high RNA expression levels and low DNA methylation levels of ACTL10 may predict a good prognosis in patients with CN-AML.

CRISPR-Cas13-Mediated Knockdown of lncRNA-GACAT3 Inhibited Cell Proliferation and Motility, and Induced Apoptosis by Increasing p21, Bax, and E-Cadherin Expression in Bladder Cancer.

The current study is to investigate the expression pattern and biological function of long non-coding RNA Focally gastric cancer-associated transcript3 (GACAT3) in bladder cancer. Real-time quantitative qPCR was used to detect the expression level of GACAT-3 in tumor tissues and paired normal tissues. Human bladder cancer T24 and 5637 cell lines were transiently transfected with specific CRISPR-Cas13 or negative control CRISPR-Cas13. Cell migration, proliferation, and apoptosis were measured by using wound healing assay CCK-8 assay and Caspase-3 ELISA assay, respectively. The expression changes of p21, Bax, and E-cadherin after knockdown of GACAT3 were detected by using Western blot. The results demonstrated that GACAT3 was up-regulated in bladder cancer tissues than that in the paired normal tissues. Inhibition of cell proliferation, increased apoptosis, and decreased motility were observed in T24 and 5637 cell lines transfected by CRISPR-Cas13 targeting GACAT3. Downregulation of GACAT3 increased p21, Bax, and E-cadherin expression and silencing these genes could eliminate the phenotypic changes induced by knockdown of GACAT3. A ceRNA mechanism for GACAT3 was also revealed. By using CRISPR-Cas13 biotechnology, we suggested that GACAT3 may be a novel target for diagnosis and treatment of bladder cancer.

[Medication versus health education for patients with type â ¢ prostatitis-like symptoms: A prospective randomized control study].

OBJECTIVE: To investigate the necessity of medication for patients with type Ⅲ prostatitis-like symptoms for less than 3 months. METHODS: We enrolled in this study 171 outpatients with type Ⅲ prostatitis-like symptoms for less than 3 months in our hospital from November 2016 to October 2017, and randomly divided them into groups A (n = 57), B (n = 57) and C (n = 57). The patients of group A received tamsulosin, levofloxacin and health education, those of group B tamsulosin and health education, and those of group C health education only. Three months later, we evaluated the therapeutic effects according to the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) scores of the patients, 4-point reduction in the total score indicating effectiveness. RESULTS: After 3 months of treatment, the total NIH-CPSI scores of the patients in groups A, B and C were decreased by (9.0 ± 2.9), (8.2 ± 3.4) and (8.6 ± 3.2) points respectively, all indicating effectiveness, the pain scores (4.2 ± 1.8), (4.0 ± 1.9) and (4.2 ± 1.6) points, the urinary symptom scores decreased by decreased by (2.4 ± 1.2), (2.4 ± 1.4) and (2.2 ± 1.2) points, and quality of life scores decreased by (2.4 ± 1.4), (1.9 ± 1.4) and (2.2 ± 1.3) points, none with statistically significant difference among the three groups (P > 0.05). CONCLUSIONS: Health education is proved to have a therapeutic effect on type Ⅲ prostatitis-like symptoms similar to that of alpha receptor blockers.

A revolutionary tool: CRISPR technology plays an important role in construction of intelligentized gene circuits.

With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence-specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR-associated RNA-guided endonuclease Cas9 (CRISPR-associated protein 9)-targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties-operability, high efficiency and programmability. The traditional single-targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti-tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour-specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch-inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR-based gene circuit development.

Lentivirus-mediated shRNA targeting MUTYH inhibits malignant phenotypes of bladder cancer SW780 cells.

OBJECTIVES: MUTYH is a protein-coding gene that takes part in base excision repair. Many previous studies have reported that MUTYH is directly related to hereditary adenomatous polyposis and colorectal cancer and is also associated with other cancers. However, the relationship between MUTYH and bladder cancer (BC) is unknown. MATERIALS AND METHODS: The expression of MUTYH and clinical characteristics of BC were collected from databases including The Cancer Genome Atlas and Cancer Cell Line Encyclopedia. RNA sequencing and quantitative real-time PCR were used to detect MUTYH expression in SW780 BC cells. The level of MUTYH was stably downregulated by lentivirus-mediated vector in SW780 cells. Cell proliferation was evaluated using Cell Counting Kit-8 assay and 5-ethynyl-20-deoxyuridine assay, migration was detected using scratch assay and Transwell assay, and apoptosis was determined using ELISA. RESULTS: MUTYH was upregulated in BC tissues and SW780 cells and its expression level was positively associated with the stage and grade of carcinomas. MUTYH was successfully downregulated in SW780 cells by transducing with a lentivirus-mediated shRNA targeting MUTYH. MUTYH knockdown inhibited the proliferation and migration and induced apoptosis in SW780 cells. CONCLUSION: Our data suggest that MUTYH is a new participant in bladder urothelial carcinoma. MUTYH may play a role as a biomarker and therapeutic target in BC.

Oestrogen promotes tumorigenesis of bladder cancer by inducing the enhancer RNA-eGREB1.

In recent years, studies have shown that enhancer RNAs (eRNAs) can be transcribed from enhancers. Increasing evidence has revealed that eRNAs play critical roles in the development of various cancers. Oestrogen-associated eRNAs are closely related to breast cancer. In view of the gender differences in bladder cancer (BCa), we suppose that oestrogen-associated eRNAs are also involved in tumorigenesis of BCa. In our study, we first demonstrated that eGREB1 derived from the enhancer of an oestrogen-responsive gene-GREB1 was up-regulated in BCa tissues, and the expression level of eGREB1 is positively associated with the histological grade and TNM stage of BCa. Knockdown of eGREB1 by CRISPR-Cas13a could inhibit cell proliferation, migration and invasion and induce apoptosis in BCa cells T24 and 5637. Besides, we exhibited the promoting effect of oestrogen on BCa cells. What's more, down-regulation of eGREB1 could improve the malignant biological characteristics of BCa cells induced by oestrogen. In conclusion, our data indicated that eGREB1 plays oncogenic role and oestrogen may promote the occurrence and progression of BCa by inducing eGREB1 production. Our findings provide new insights into the prevention of BCa and develop a novel therapeutic target for the treatment of BCa.

Enhancer RNA - P2RY2e induced by estrogen promotes malignant behaviors of bladder cancer.

Enhancers are transcriptional regulatory elements that increase target gene expression. It has reported that enhancers could universally transcribe into enhancer RNAs (eRNAs) with stimulation. Increasing evidence showed eRNAs participated in various disease processes including malignant tumors. P2RY2 enhancer RNA (P2RY2e) is an estrogen-responsive eRNA and involved in the development of breast cancer. However, the relationship between P2RY2e and bladder cancer (BCa) is unclear. In the study, we discovered that P2RY2e was upregulated in BCa tissues and estrogen-treated cells. Estrogen promoted the malignant abilities of BCa cells. P2RY2e knockdown by CRISPR-Cas13a inhibit the cell multiplication, invasion and migration. Additionally, the cell apoptosis was facilitated. What's more, downregulation of P2RY2e could weaken the cancer-promoting effects of estrogen on BCa. Our study revealed that P2RY2e played a carcinogenic role in BCa and estrogen might promote the initiation of BCa by inducing P2RY2e. We provide a potential therapeutic target for BCa and a new perspective for the tumorigenesis of bladder cancer.

Enhancer RNAs (eRNAs): New Insights into Gene Transcription and Disease Treatment.

Enhancers are cis-acting elements that have the ability to increase the expression of target genes. Recent studies have shown that enhancers can act as transcriptional units for the production of enhancer RNAs (eRNAs), which are hallmarks of activity enhancers and are involved in the regulation of gene transcription. The in-depth study of eRNAs is of great significance for us to better understand enhancer function and transcriptional regulation in various diseases. Therefore, eRNAs may be a potential therapeutic target for diseases. Here, we review the current knowledge of the characteristics of eRNAs, the molecular mechanisms of eRNAs action, as well as diseases related to dysregulation of eRNAs.

High expression of enhancer RNA MARC1 or its activation by DHT is associated with the malignant behavior in bladder cancer.

Enhancer RNAs (eRNAs), a subclass of noncoding RNA from enhancers, have biological functions in gene expression. However, their potential role in bladder cancer (BCa) remains largely unknown. The present study investigated the functional role of androgen-associated androgen receptor (AR) mediated-eRNA MARC1 (eMARC1) in BCa progression. Cell proliferation, migration, and apoptosis of BCa cell lines (5637 and T24) with different eMARC1 expression levels or treated with 5α-dehydrotestosterone (DHT) were investigated. In the current study, we discovered that eMARC1 was highly expressed in BCa tissues and cell lines, and eMARC1 overexpression promoted the progression of BCa cells, while knockdown of eMARC1 suppressed tumorigenesis. DHT treatment significantly elevated eMARC1 expression levels, which also facilitated cell proliferation, motility, and inhibited cell apoptosis. We further found that eMARC1 silencing impaired the androgenic effect of DHT in BCa cells. These results suggested that eMARC1 exerted its effects on BCa cell progression, and DHT promoted bladder cancer progression by activating eMARC1.

rs11614913 polymorphism in miRNA-196a2 and cancer risk: an updated meta-analysis.

Several epidemiological studies have reported that polymorphisms in microRNA-196a2 (miR-196a2) were associated with various cancers. However, the results remained unverified and were inconsistent in different cancers. Therefore, we carried out an updated meta-analysis to elaborate the effects of rs11614913 polymorphism on cancer susceptibility. A total of 84 articles with 35,802 cases and 41,541 controls were included to evaluate the association between the miR-196a2 rs11614913 and cancer risk by pooled odds ratios (ORs) and 95% confidence intervals (CIs). The results showed that miR-196a2 rs11614913 polymorphism is associated with cancer susceptibility, especially in lung cancer (homozygote comparison, OR =0.840, 95% CI =0.734-0.961; recessive model, OR =0.858, 95% CI =0.771-0.955), hepatocellular carcinoma (allelic contrast, OR =0.894, 95% CI =0.800-0.998; homozygote comparison, OR =0.900, 95% CI =0.813-0.997; recessive model, OR =0.800, 95% CI =0.678-0.944), and head and neck cancer (allelic contrast, OR =1.076, 95% CI =1.006-1.152; homozygote comparison, OR =1.214, 95% CI =1.043-1.413). In addition, significant association was found among Asian populations (allele model, OR =0.847, 95% CI =0.899-0.997, P=0.038; homozygote model, OR =0.878, 95% CI =0.788-0.977, P=0.017; recessive model, OR =0.895, 95% CI =0.824-0.972, P=0.008) but not in Caucasians. The updated meta-analysis confirmed the previous results that miR-196a2 rs11614913 polymorphism may serve as a risk factor for patients with cancers.

Long non-coding PANDAR as a novel biomarker in human cancer: A systematic review.

OBJECTIVES: Long non-coding RNAs (lncRNAs) are characterized as a group of RNAs that more than 200 nucleotides in length and have no protein-coding function. More and more evidences provided that lncRNAs serve as key molecules in the development of cancer. Deregulation of lncRNAs functions as either oncogenes or tumour suppressor genes in various diseases. Recently, increasing studies about PANDAR in cancer progression were reported. In our review, we will focus on the current research on the character of PANDAR include the clinical management, tumour progression and molecular mechanisms in human cancers. MATERIALS AND METHODS: We summarize and analyze current studies concerning the biological functions and mechanisms of lncRNA PANDA in tumour development. The related studies were obtained through a systematic search of Pubmed. RESULTS: PANDAR was a well-characterized oncogenic lncRNA and widely overexpressed in many tumours. PANDAR is upregulated in many types of cancer, including colorectal cancer, lung cancer, renal cell carcinoma, cholangiocarcinoma, osteosarcoma, thyroid cancer and other cancers. Upregulation of PANDAR was significantly associated with advanced tumour weights, TNM stage and overall survival. Furthermore, repressed of PANDAR would restrain proliferation, migration and invasion. CONCLUSION: PANDAR may act as a powerful tumour biomarker for cancer diagnosis and treatment.

Knockdown of long noncoding RNA FGFR3- AS1 induces cell proliferation inhibition, apoptosis and motility reduction in bladder cancer.

OBJECTIVES: To study the expression pattern of long non-coding RNA FGFR3 antisense transcript 1(FGFR3-AS1) and the cell proliferation inhibition, apoptosis, and motility changes induced by silencing FGFR3-AS1 in bladder cancer. METHODS: The differential expression levels of FGFR3-AS1 and FGFR3 in tumor tissues and paired normal tissues were determined using Real-Time qPCR in a total of 36 patients diagnosed with bladder cancer (urothelial carcinoma). Pearson's coefficient correlation was used for expression correlation assay. Expression differences of FGFR3-AS1 were analyzed according to grading and staging. FGFR3 protein was detected by western blot assay. Human bladder cancer T24 and 5637 cell lines were transiently transfected with FGFR3-AS1-specific siRNA or negative control siRNA. The cell proliferation changes of transfected bladder cancer cells were determined using CCK-8 assay. Apoptosis caused by knockdown of FGFR3-AS1 was evaluated using ELISA assay. Motility changes induced by knockdown of FGFR3-AS1 were measured using wound healing assay and transwell assay. RESULTS: Both FGFR3-AS1 and FGFR3 were overexpressed in bladder cancer tissues compared to matched normal tissues. They were also positively expressed in bladder cancer. FGFR3-AS1 expression levels were higher in high grade tumors than those in low grade tumors. FGFR3-AS1 expression levels were higher in invasive tumors than those in non-invasive tumors. Cell proliferation inhibition, increased apoptosis, and decreased motility were observed in FGFR3-AS1 siRNA-transfected T24 and 5637 cell lines. CONCLUSIONS: FGFR3-AS1 plays an oncogenic role in human bladder cancer. Knockdown of FGFR3-AS1 may provide a potential new therapeutic approach to this disease.

LncRNA MALAT1 Inhibits Apoptosis and Promotes Invasion by Antagonizing miR-125b in Bladder Cancer Cells.

Accumulating evidences suggest that longnon-coding RNAs (lncRNAs) play functional roles in development of different cancers, including cancer initiation and progression. Metastasis associated lung adenocarcinoma transcript 1(MALAT1) is a well-known lncRNA which was previously shown to be a direct target of miR-125b in bladder cancer (BCa) and to promote cancer progression and invasion. However, little is known whether MALAT1 can also target miR-125b. In the present study, using CRISPR-based technologies and qRT-PCR, we show that MALAT1 is capable of suppressing mature miR-125b and increasing the expression of its target genes (Bcl-2 and MMP-13), but has no effect on pri-miR-125b and pre-miR-125b. We observe that the biotin-labeled MALAT1-RNA probe is able to pull down Ago2 and miR-125b and that the negative regulation of miR-125b by MALAT1 is dependent on Ago2. Importantly, the results of flow cytometry assay and transwell assay reveal that the MALAT1-mediated cancer progression is in part due to specific suppression of miR-125b and activation of its two target genes. All together, these data suggest that the "MALAT1-miR-125b-Bcl-2 / MMP-13" axis plays an important role in the progression of BCa, thereby may provide a potential therapeutic strategy for the treatment of human BCa.