Novel insights into the roles and therapeutic implications of MUC1 oncoprotein via regulating proteins and non-coding RNAs in cancer.

Mucin 1 (MUC1) is a heterodimeric transmembrane glycoprotein that protects epithelial cells in mammals. The transmembrane C-terminal subunit (MUC1-C) plays a crucial role in oncogenesis. As an oncoprotein, MUC1-C regulates a number of proteins that are associated with tumorigenesis by interacting with oncoproteins, transcription factors, coactivators, etc., inducing proliferation, epithelial-mesenchymal transition (EMT), invasion, stemness, immune evasion, and drug resistance. Moreover, MUC1-C modulates the expression of non-coding RNAs (ncRNAs), which further regulate carcinogenesis by directly binding to specific proteins. ncRNAs can also affect MUC1 protein expression by targeting the MUC1 mRNA 3' untranslated region (UTR). A series of ncRNAs can modulate cancer development by regulating MUC1-C. This review focuses on the interaction of MUC1-C with proteins and ncRNAs in cancer progression. We also summarize the recent advances in immunotherapy with a focus on therapeutic approaches based on MUC1-C and nanocarrier complexes for cancer treatment.

Fer exacerbates renal fibrosis and can be targeted by miR-29c-3p.

AIM: Renal fibrosis (RF) is a common clinical condition leading to irreversible renal function loss. Tyrosine kinase proteins and microRNAs (miRs) are associated with pathogenesis and we aim to investigate the role of Fer and its partner miR(s) in RF. METHOD: In silico reproduction of Mouse Kidney FibrOmics browser was performed to identify potential miR(s) and target gene(s). In vivo validation was performed in C57BL/6 mice with unilateral ureteral obstruction (UUO). In vitro validation was performed in rat kidney fibroblast NRK-49F cells. Mimics and inhibitors of miR-29c-3p were constructed. The target gene Fer was monitored by RT-PCR and western blotting. The levels of interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α in serum and media were measured by ELISA. RESULTS: The Fer expression and protein level were gradually increased during 14 days of UUO modeling. miR-29c-3p expression was strongly correlated with that of Fer. In vivo validation showed increased expressions of fibrosis-associated genes and increased phospoho-Smad3 level in the UUO model. Fer-knockdown (KD) significantly decreased expressions of fibrosis-associated genes. Pharmaceutical inhibition of Fer showed similar effects to miR-29c-3p, and miR inhibition showed a significant decrease of excretion of inflammatory factors. CONCLUSION: Dysregulation of miR-29c-3p and Fer plays a role in RF. Pharmaceutical or genetic inhibition of Fer may serve as the potential treatment for RF.

microRNA let-7i-5p aggravates kidney fibrosis via targeting GALNT1.

Renal fibrosis poses critical health problem. We aimed to investigate role of let-7i-5p in renal fibrosis. In silico reproduction of Mouse Kidney FibrOmics browser was used to identify potential target of let-7i-5p. In vivo validation was conducted in C57BL/6 mice with unilateral ureteral obstruction (UUO) and folic acid (FA) induction. In vitro validation was performed in transforming growth factor (TGF)-ß1-treated HK-2 cells. Mimics and inhibitors of let-7i-5p, and target gene polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1) were monitored by RT-PCR and Western blotting. Fibrosis markers, injury markers, and house-keeping genes were evaluated. Levels of interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α in serum and media were measured by ELISA. In silico analysis showed gradual increase of let-7i-5p and decrease of GALNT1 over time and the combination was validated both in mouse and human miR-gene target prediction databases. Expression of GALNT1 decreased while expression of let-7i-5p increased in renal tissues of both UUO and FA mice. Serum IL-6, IL-1ß, and TNF-α levels were elevated in vivo. In vitro models revealed negative correlation between expression levels of let-7i-5p and GALNT1. Overexpression of let-7i-5p inhibited GALNT1 expression and reduced release of inflammatory factors. In conclusion, overexpression of GALNT1 may combat the inflammation induced by let-7i-5p.

Overexpression of enolase 2 is associated with worsened prognosis and increased glycikolysis in papillary renal cell carcinoma.

Papillary renal cell carcinoma (pRCC) is characterized with underlying genetic disorders and the role enolase 2 (ENO2) in ccRCC is unknown. An in silico exploratory analysis using multiple public genetic datasets was used to establish association between ENO2 expression and clinicopathological parameters. Associations of interest were validated using 49 pRCC samples using immunohistochemistry. In vitro and in vivo assays were carried out to validate findings in tissue. ENO2 was overexpressed and prognostic in pRCC. ENO2 expression was significantly higher in younger patients and in CpG island methylator phenotype subtype. ENO2-overexpressed cases showed significant enrichment in glycolysis. Overexpression of ENO2 significantly increased proliferation and silencing of ENO2 significantly inhibited growth of ACHN cells. Glycolytic genes HK1, HK 2, and lactate dehydrogenase A were decreased when ENO2 was silenced in ACHN. Glycolytic inhibitor TT-232 showed minimal inhibitory effect on ACHN cells yet showed synergistic effect in the presence of ENO2 silencing. ENO2 significantly increased and decreased extracellular glucose, respectively in ACHN cells. Xenograft mouse model showed ENO2 silencing and TT-232 combination treatment showed synergistic effect in ACHN tumors. ENO2 is associated with worsened prognosis in pRCC and is related to glycolysis. ENO2-targeted therapy can be of therapeutic potential.

circPRRC2A promotes angiogenesis and metastasis through epithelial-mesenchymal transition and upregulates TRPM3 in renal cell carcinoma.

Background: Circular RNAs (circRNAs) have been identified as essential regulators in a plethora of cancers. Nonetheless, the mechanistic functions of circRNAs in Renal Cell Carcinoma (RCC) remain largely unknown. Methods: In this study, we aimed to identify novel circRNAs that regulate RCC epithelial-mesenchymal transition (EMT), and to subsequently determine their regulatory mechanisms and clinical significance. Results: circPRRC2A was identified by circRNA microarray and validated by qRT-PCR. The role of circPRRC2A in RCC metastasis was evaluated both in vitro and in vivo. We found that increased expression of circPRRC2A is positively associated with advanced clinical stage and worse survivorship in RCC patients. Mechanistically, our results indicate that circPRRC2A prevents the degradation of TRPM3, a tissue-specific oncogene, mRNA by sponging miR-514a-5p and miR-6776-5p. Moreover, circPRRC2A promotes tumor EMT and aggressiveness in patients with RCC. Conclusions: These findings infer the exciting possibility that circPRRC2A may be exploited as a therapeutic and prognostic target for RCC patients.

An impedimetric immunosensor for determination of porcine epidemic diarrhea virus based on the nanocomposite consisting of molybdenum disulfide/reduced graphene oxide decorated with gold nanoparticles.

An electrochemical immunosensor for the determination of porcine epidemic diarrhea virus (PEDV) is described. It was manufactured by using gold nanoparticles/molybdenum disulfide/reduced graphene oxide nanocomposites modified on the surface of a glassy carbon electrode (GCE). The independently developed monoclonal antibody of PEDV-2C11 was immobilized on the modified electrode at site of gold nanoparticles provided in the nanocomposites. The concentration of PEDV was quantified by measuring the changes in the charge transfer resistance of the electrode before and after the immunoreaction between antigen-antibody by using hexacyanoferrate(II)/(III) as the redox probe. The frequency range was 10-1 to 105 Hz at the amplitude of 10 mV and an applied potential of + 0.180 V. Based on the immunoreaction between PEDV antigen and PEDV-2C11 antibody in 0.1 M phosphate buffer containing 0.1 M KCl at 37.5 °C for 140 min, the relative change in impedance was proportional to the logarithmic value of PEDV concentrations in the range of 82.5 to 1.65 × 104 TCID50 mL-1. Good reproducibility, stability, and specificity of the proposed immunosensor were obtained. It was successfully applied to the determination of PEDV in the spiked sample. Graphical abstractSchematic representation. a The preparation of AuNP/MoS2/rGO composites. b Representation of modification and functioning of the label-free electrochemical immunosensor and the electrochemical impedimetric response obtained before (a) and after (b) incubation of PEDV.

URG11 promotes proliferation and induced apoptosis of LNCaP cells.

von Willebrand factor C and EGF domain­containing protein (URG11), a cell growth regulator, is involved in the progression of a variety of types of cancer, including prostate cancer (Pca). However, the functions of the URG11 gene in Pca cells require in­depth investigation. The mRNA and protein levels of URG11 were measured by reverse transcription quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Cell Counting kit­8 (CCK­8), wound­healing and Transwell assays were used to detect cell viability, migration and invasion, respectively. Apoptosis and cell cycle analyses were performed using flow cytometry. The mRNA and protein expression levels of epithelial (E)­cadherin, vimentin, α­smooth muscle actin (α­SMA), cyclin D1 and MYC proto­oncogene protein (c­Myc) were analyzed by RT­qPCR and western blot analysis. In the present study, the mRNA and protein levels of URG11 were markedly upregulated in Pca cell lines compared with those in the normal prostate epithelial cell line. With functional experiments, the cell viability, migration and invasion of Pca cells were markedly promoted by URG11 overexpression. The cell cycle was effectively induced by URG11 and apoptosis was inhibited by the overexpression of URG11. Concomitantly, the epithelial marker E­cadherin was downregulated, and the mesenchymal markers vimentin and α­SMA were upregulated following URG11 overexpression. By contrast, genetic knockout of URG11 elicited the opposite effects. The present study also identified that the downstream effector genes of the Wnt/ß­catenin signal pathway, cyclin D1 and c­Myc, were increased following the overexpression of endogenous URG11, which are known to regulate cell proliferation. In addition, the Wnt/ß­catenin inhibitor FH535 ameliorated the promotive effects of URG11 on LNCaP cells viability, migration and invasion, and the Wnt/ß­catenin agonist LiCl reversed the inhibitory effects of siURG11 in LNCaP cells on cell viability, migration and invasion. The present study demonstrated that URG11 served an oncogenic role in the development of Pca cells and provided evidence that URG11 has potential as a novel therapeutic target in Pca.

EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis.

Aim: BAP1 is frequently mutated in clear cell renal cell carcinoma (ccRCC) with a definitive role still unclear. Methods: In silico analysis of BAP1-mutant and wild-type gene enrichment and functional annotation in TCGA-KIRC dataset was performed. Target gene was studied based on functional clustering and was knowledge-based. Validation using in-house pathological sections were performed immunohistochemically. In vitro and in vivo studies on target gene were performed. Results: The TCGA ccRCC dataset included 534 ccRCC samples. BAP1 was frequently mutated and more frequently downregulated in ccRCC compared to normal kidney tissue or benign renal tumors. In the analysis between samples with BAP1 mutation (N = 33) and pan-negative (N = 33), we found that cancers with BAP1 mutation was significantly enriched for 14 pathways, of which 3 were DNA repair pathways, in which EZH2 played a role. CcRCC patients with lower BAP1 expression had poor prognosis and showed higher EZH2 expression, which also conferred worsened survival. Genetic and pharmaceutical inhibition of EZH2 not only inhibited BAP1-mutatn ccRCC cell viability and invasion but also abrogated genetic replenishing of BAP1 expression. Validation cohort encompassing 62 ccRCC samples confirmed the worsened phenotype for cases with higher EZH2 expression and significant positive correlation between expressions of EZH2 and BAP1. EZH2 inhibitor also inhibited tumor growth in xenograft mouse model with BAP1-mutated ccRCC cells with unremarkable toxicity. Conclusion: CcRCC with decreased BAP1 level has poor prognosis and is associated with higher EZH2 expression. Inhibition of EZH2 in BAP1-mutated entity holds promise for further investigation.

Overexpression of CKAP4 is Associated with Poor Prognosis in Clear Cell Renal Cell Carcinoma and Functions via Cyclin B Signaling.

Aim: We aimed to study the role of CKAP4 in clear cell renal cell carcinoma (ccRCC), which is not reported previously. Method: In silico exploration and validation using immunohistochemistry in ccRCC samples were used to identify the impact of CKAP4 expression on clinicopathological parameters. In vitro and in vivo studies were carried out to recapitulate the role of CKAP4 in ccRCC cell lines and animal models. Results: Overexpression of CKAP4 occurred in 5% of ccRCC patients, who had significantly worsened prognosis. Increased CKAP4 expression was significantly associated with TNM staging and Fuhrman grade. Pathway analysis for genes coexpressed with CKAP4 in ccRCC unanimously revealed significant cell cycle progression at G2/M phase. Expressions of CCNB1 and CCNB2 were correlated with CKAP4 expression. Genetic upregulation of CKAP4 significantly increased proliferation, cell invasion and migration in ccRCC cell lines, and vice versa for CKAP4 silencing. CKAP4 silencing also significantly increased cell population at G2/M phase, while not influencing cell apoptosis. Silencing or upregulation of CKAP4 resulted in decreased or increased CCNB1/2 expressions, respectively. CCNB1/CDK1 inhibitor significantly inhibited colony formation ability and in vivo tumor growth of RCC cells with CKAP4 overexpression. Conclusion: Upregulation of CKAP4 was associated with worsened characteristics of ccRCC. CKAP4 was related with CCNB signaling in ccRCC, which supported a role for CCNB/CDK inhibitor for ccRCC with such genotype.

Genetic alteration in phosphofructokinase family promotes growth of muscle-invasive bladder cancer.

AIMS: Metabolic alterations in cancer, including bladder cancer, have been addressed in recent years. We aimed to study the role of phosphofructokinase (PFK) in muscle-invasive bladder cancer (MIBC). METHOD: By in silico analysis of the bladder cancer data from the Cancer Genome Atlas (TCGA) database using the cBioPortal platform, we studied genetic alteration of genes within the PFK family (PFKL, PFKM, PFKP, PFKFB1, PFKFB2, PFKFB3, and PFKFB4). In vitro studies were carried out using the PFK inhibitor 2,5-anhydro-D-glucitol-6-phosphate. RESULTS: Genetic alterations of PFK family genes were observed in ~44% of MIBC cases in TCGA. The main alterations were amplification and upregulation. Patients with altered PFK gene status were more likely to have a history of noninvasive bladder cancer. Altered PFK status was not associated with survival or disease relapse. Use of the PFK inhibitor significantly decreased the level of glycolysis and inhibited the growth and invasion of bladder cancer cells. CONCLUSIONS: PFKs were critical genes in charge of glycolysis and were upregulated in bladder cancer. Targeting this pathway could inhibit cell growth in bladder cancer.