Peroxiredoxin II regulates exosome secretion from dermal mesenchymal stem cells through the ISGylation signaling pathway.

BACKGROUND: Exosomes are small extracellular vesicles that play important roles in intercellular communication and have potential therapeutic applications in regenerative medicine. Dermal mesenchymal stem cells (DMSCs) are a promising source of exosomes due to their regenerative and immunomodulatory properties. However, the molecular mechanisms regulating exosome secretion from DMSCs are not fully understood. RESULTS: In this study, the role of peroxiredoxin II (Prx II) in regulating exosome secretion from DMSCs and the underlying molecular mechanisms were investigated. It was discovered that depletion of Prx II led to a significant reduction in exosome secretion from DMSCs and an increase in the number of intracellular multivesicular bodies (MVBs), which serve as precursors of exosomes. Mechanistically, Prx II regulates the ISGylation switch that controls MVB degradation and impairs exosome secretion. Specifically, Prx II depletion decreased JNK activity, reduced the expression of the transcription inhibitor Foxo1, and promoted miR-221 expression. Increased miR-221 expression inhibited the STAT signaling pathway, thus downregulating the expression of ISGylation-related genes involved in MVB degradation. Together, these results identify Prx II as a critical regulator of exosome secretion from DMSCs through the ISGylation signaling pathway. CONCLUSIONS: Our findings provide important insights into the molecular mechanisms regulating exosome secretion from DMSCs and highlight the critical role of Prx II in controlling the ISGylation switch that regulates DMSC-exosome secretion. This study has significant implications for developing new therapeutic strategies in regenerative medicine. Video Abstract.

Bioinformatics Analysis of Novel Targets for Treating Cervical Cancer by Immunotherapy Based on Immune Escape.

BACKGROUND/AIM: Cervical cancer (CC) is a high-risk disease in women, and advanced CC can be difficult to treat even with surgery, radiotherapy, and chemotherapy. Hence, developing more effective treatment methods is imperative. Cancer cells undergo a renewal process to escape immune surveillance and then attack the immune system. However, the underlying mechanisms remain unclear. Currently, only one immunotherapy drug has been approved by the Food and Drug Administration for CC, thus indicating the need for and importance of identifying key targets related to immunotherapy. MATERIALS AND METHODS: Data on CC and normal cervical tissue samples were downloaded from the National Center for Biotechnology Information database. Transcriptome Analysis Console software was used to analyze differentially expressed genes (DEGs) in two sample groups. These DEGs were uploaded to the DAVID online analysis platform to analyze biological processes for which they were enriched. Finally, Cytoscape was used to map protein interaction and hub gene analyses. RESULTS: A total of 165 up-regulated and 362 down-regulated genes were identified. Among them, 13 hub genes were analyzed in a protein-protein interaction network using the Cytoscape software. The genes were screened out based on the betweenness centrality value and average degree of all nodes. The hub genes were as follows: ANXA1, APOE, AR, C1QC, CALML5, CD47, CTSZ, HSP90AA1, HSP90B1, NOD2, THY1, TLR4, and VIM. We identified the following 12 microRNAs (miRNAs) that target the hub genes: hsa-miR-2110, hsa-miR-92a-2-5p, hsa-miR-520d-5p, hsa-miR-4514, hsa-miR-4692, hsa-miR-499b-5p, hsa-miR-5011-5p, hsa-miR-6847-5p, hsa-miR-8054, hsa-miR-642a-5p, hsa-miR-940, and hsa-miR-6893-5p. CONCLUSION: Using bioinformatics, we identified potential miRNAs that regulated the cancer-related genes and long noncoding RNAs (lncRNAs) that regulated these miRNAs. We further elucidated the mutual regulation of mRNAs, miRNAs, and lncRNAs involved in CC occurrence and development. These findings may have major applications in the treatment of CC by immunotherapy and the development of drugs against CC.

Induction of Targeted Differentiation of Dermal Mesenchymal Stem Cells Into Neural Lineage According to Peroxiredoxin II Expression.

BACKGROUND/AIM: To optimize the therapeutic potential of stem cells in stem cell therapy for neurological diseases, it is crucial to enhance the differentiation, migration, and neural network formation of stem cells, and to eliminate uncertain cell differentiation and proliferation factors. Several studies have shown that reactive oxygen species (ROS) are important factors in the regulation of neurogenesis, and Prx II (Peroxiredoxin II) is a gene that regulates ROS. MATERIALS AND METHODS: As the entry point in this study to conduct a bioinformatics analysis of the sequencing results of Prx II+/+ dermal mesenchymal stem cells (DMSCs) and Prx II-/- DMSCs. lncRNA/miRNA/mRNA networks were then constructed and preliminarily verified in RT-qPCR experiments. RESULTS: In this study, a total of 11 hub genes (Gria1, Nrcam, Sox10, Snap25, Cntn2, Dlg2, Ngf, Ntrk3, Amph, Syt1, and Cd24a), eight miRNAs (miRNA-4661, miRNA-34a, miRNA-185, miRNA-34b-5p, miRNA-34c, miRNA-449a, miRNA-449b, miRNA-449c) and 12 lncRNAs (Dubr, Gas5, Gm20427, Gm26917, Gm42547, Gm8066, Kcnq1ot1, Malat1, Mir17hg, Neat1, Rian, and Tug1) were predicted in lncRNA/miRNA/mRNA network. CONCLUSION: The regulatory mechanism of Prx II in the differentiation of DMSCs into neurons through ROS was explored, and a theoretical basis was determined that can be applied in future research on nervous system diseases and the clinical applications of stem cells.

Identification of Hub Genes and Upstream Regulatory Factors Based on Cell Adhesion in Triple-negative Breast Cancer by Integrated Bioinformatical Analysis.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is characterized by metastasis and invasion, as well as poor prognosis, with chemotherapy being the main treatment option. Cell adhesion regulates tumorigenesis and new blood vessel formation. Thus, accurately identifying effective targets for TNBC and cell adhesion is challenging. Herein, we screened for differentially expressed genes between TNBC and normal cancer-free tissues to identify genes contributing to TNBC. MATERIALS AND METHODS: Microarray data were obtained using a comprehensive gene-expression database. We used Database for Annotation, Visualization and Integrated Discovery, Kyoto Encyclopedia of Genes and Genomes and Functional Enrichment (FunRich) to perform Gene Ontology functional enrichment and predict signal pathways. The protein interaction network was predicted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape v. 3.8.2. for visualization of results. TargetScan, miRanda, miRDB, miRWalk and RNA22 were used to predict miRNAs regulating key genes, and long non-coding RNAs (lncRNAs) regulating miRNAs were predicted using StarBase V2.0 from a comprehensive gene-expression database. RESULTS: Differentially expressed genes were mainly concentrated in the biological process of cell-cell adhesion. The protein-protein interaction network identified eight hub genes: Fibronectin 1 (FN1), Rac family small GTPase 1 (RAC1), heat-shock protein 90 alpha family class B member 1 (HSP90AB1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), heat-shock protein family A member 8 (HSPA8), IQ motif containing GTPase-activating protein 1 (IQGAP1), CD44 molecule (CD44), and catenin beta 1 (CTNNB1). miRNAs related to TNBC occurrence and development were hsa-miR-142-5p, hsa-miR-144, hsa-miR-28-5p, hsa-miR-548d-3p, hsa-miR-587, hsa-miR-641, and hsa-miR-708. StarBase v2.0 predicted 12 lncRNAs, namely NEAT1, XIST, OIP5-AS1, MALAT1, AL035425.3, NORAD, AL391069.4, AC118758.3, AC026362.1, AC009065.4, AC016876.2, and AC093010.3, as upstream molecules that regulate miRNAs and which may regulate TNBC. CONCLUSION: Overall, mRNA-miRNA-lncRNA interactions appear to play a role in TNBC development.

Network analysis for the identification of hub genes and related molecules as potential biomarkers associated with the differentiation of bone marrow-derived stem cells into hepatocytes.

The incidence of liver diseases has been increasing steadily. However, it has some shortcomings, such as high cost and organ donor scarcity. The application of stem cell research has brought new ideas for the treatment of liver diseases. Therefore, it is particularly important to clarify the molecular and regulatory mechanisms of differentiation of bone marrow-derived stem cells (BMSCs) into liver cells. Herein, we screened differentially expressed genes between hepatocytes and untreated BMSCs to identify the genes responsible for the differentiation of BMSCs into hepatocytes. GSE30419 gene microarray data of BMSCs and GSE72088 gene microarray data of primary hepatocytes were obtained from the Gene Expression Omnibus database. Transcriptome Analysis Console software showed that 1896 genes were upregulated and 2506 were downregulated in hepatocytes as compared with BMSCs. Hub genes were analyzed using the STRING and Cytoscape v 3.8.2, revealing that twenty-four hub genes, play a pivotal role in the differentiation of BMSCs into hepatocytes. The expression of the hub genes in the BMSCs and hepatocytes was verified by reverse transcription-quantitative PCR (RT-qPCR). Next, the target miRNAs of hub genes were predicted, and then the lncRNAs regulating miRNAs was discovered, thus forming the lncRNA-miRNA-mRNA interaction chain. The results indicate that the lncRNA-miRNA-mRNA interaction chain may play an important role in the differentiation of BMSCs into hepatocytes, which provides a new therapeutic target for liver disease treatment.

Inhibitory Effect of Bovine Adipose-Derived Mesenchymal Stem Cells on Lipopolysaccharide Induced Inflammation of Endometrial Epithelial Cells in Dairy Cows.

Endometritis is a disease that affects reproductive health in dairy cows and causes serious economic damage to the dairy industry world-wide. Although in recent years, the application of mesenchymal stem cell (MSC) therapy for the treatment of inflammatory diseases has attracted much attention, there are few reports of the use of MSCs in dairy cows. In the present study, our objective was to explore the inhibitory effects of bovine adipose-derived mesenchymal stem cells (bAD-MSCs) on lipopolysaccharide (LPS) induced inflammation in bovine endometrial epithelial cells (bEECs) along with the potential underlying molecular mechanisms. We characterized isolated bAD-MSCs using cell surface marker staining and adipogenic/osteogenic differentiation, and analyzed them using immunofluorescence, flow cytometry (surface marker staining), and adipogenic and osteogenic differentiation. Furthermore, to understand the anti-inflammatory effects of bAD-MSCs on LPS induced bEEC inflammation, we used a bAD-MSC/bEEC co-culture system. The results showed that bAD-MSC treatments could significantly decrease LPS induced bEEC apoptosis and pro-inflammatory cytokine expression levels, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Furthermore, our results showed that bAD-MSC treatments could also significantly downregulate LPS induced p38, IkB-a, and JAK1 phosphorylation and Bax protein expression levels, which are closely related to inflammatory progress and cellular apoptosis in bEECs. Our findings demonstrate that bAD-MSCs play an inhibitory role in LPS induced bEEC inflammation and provide new insights for the clinical therapy of endometritis in dairy cows.

Peroxiredoxin II with dermal mesenchymal stem cells accelerates wound healing.

Peroxiredoxin II (Prx II) is involved in proliferation, differentiation, and aging in various cell types. However, Prx II-mediated stem cell regulation is poorly understood. Here, dermal mesenchymal stem cells (DMSCs), cell-growth factor-rich conditioned medium from DMSCs (DMSC-CM), and DMSC-derived exosomes (DMSC-Exos) were used to explore the regulatory role of Prx II in DMSC wound healing. Following treatment, wound healing was significantly decelerated in Prx II-/- DMSCs than in Prx II+/+ DMSCs. In vitro stimulation with 10 µM H2O2 significantly increased apoptosis in Prx II-/- DMSCs compared with Prx II+/+ DMSCs. The mRNA expression levels of EGF, b-FGF, PDGF-B, and VEGF did not significantly differ between Prx II-/- and Prx II+/+ DMSCs. Fibroblasts proliferated comparably when treated with Prx II+/+ DMSC-CM or Prx II-/- DMSC-CM. Wound healing was significantly higher in the Prx II-/- DMSC-Exos-treated group than in the Prx II+/+ DMSCs-Exos-treated group. Moreover, microRNA (miR)-21-5p expression levels were lower and miR-221 levels were higher in Prx II-/- DMSCs than in Prx II+/+ DMSCs. Therefore, our results indicate that Prx II accelerated wound healing by protecting DMSCs from reactive oxygen species-induced apoptosis; however, Prx II did not regulate cell/growth factor secretion. Prx II potentially regulates exosome functions via miR-21-5p and miR-221.

Cystic duct cancer: Should it be deemed as a type of gallbladder cancer?

BACKGROUND: According to the latest American Joint Committee on Cancer and Union for International Cancer Control manuals, cystic duct cancer (CC) is categorized as a type of gallbladder cancer (GC), which has the worst prognosis among all types of biliary cancers. We hypothesized that this categorization could be verified by using taxonomic methods. AIM: To investigate the categorization of CC based on population-level data. METHODS: Cases of biliary cancers were identified from the Surveillance, Epidemiology, and End Results 18 registries database. Together with routinely used statistical methods, three taxonomic methods, including Fisher's discriminant, binary logistics and artificial neuron network (ANN) models, were used to clarify the categorizing problem of CC. RESULTS: The T staging system of perihilar cholangiocarcinoma [a type of extrahepatic cholangiocarcinoma (EC)] better discriminated CC prognosis than that of GC. After adjusting other covariates, the hazard ratio of CC tended to be closer to that of EC, although not reaching statistical significance. To differentiate EC from GC, three taxonomic models were built and all showed good accuracies. The ANN model had an area under the receiver operating characteristic curve of 0.902. Using the three models, the majority (75.0%-77.8%) of CC cases were categorized as EC. CONCLUSION: Our study suggested that CC should be categorized as a type of EC, not GC. Aggressive surgical attitude might be considered in CC cases, to see whether long-term prognosis could be immensely improved like the situation in EC.

Irbesartan, an FDA approved drug for hypertension and diabetic nephropathy, is a potent inhibitor for hepatitis B virus entry by disturbing Na(+)-dependent taurocholate cotransporting polypeptide activity.

The liver-specific Na(+)-dependent taurocholate cotransporting polypeptide (NTCP) was recently identified as an entry receptor for hepatitis B virus (HBV) hepatotropic infection. In this study, an NTCP-overexpressing HepG2 cell line named HepG2.N9 susceptible to HBV infection was established using transcription activator-like effector nucleases (TALEN) technology. Using this cell line, irbesartan, the new NTCP-interfering molecule reported recently, was demonstrated here to effectively inhibit HBV infection with an IC50 of 3.3µM for hepatitis B e antigen (HBeAg) expression and exhibited no obvious cytotoxicity up to 1000µM. Irbesartan suppressed HBV uptake weakly but inhibited HBV covalently closed circular DNA (cccDNA) formation efficiently at physiological temperature. These results suggested that irbesartan targeted HBV infection at a post-uptake prior to cccDNA formation step such as the cell membrane fusion. Based on these findings, irbesartan, an FDA approved drug for hypertension and diabetic nephropathy, could be a potential candidate for treatment of HBV infection although further in vivo experiments are required.

CpG oligodeoxynucleotide inhibits HBV replication in a hydrodynamic injection murine model.

BACKGROUND: Chronic HBV infection is a significant public health problem and one major cause of liver cirrhosis and hepatocellular carcinoma (HCC). HBV impairs the host immune system and results in immunotolerance, which is a major obstacle to HBV therapy. CpG oligodeoxynucleotide (ODN) is a strong immunostimulant which activates the innate immune response rapidly and has been shown to be an efficient therapy agent in viral infection treatment. Here, we report the anti-HBV activity of CpG-1826 in a hydrodynamic injection murine model. METHODS: CpG-1826 was administrated intraperitoneally every other day in HBV carrier mice established by tail vein hydrodynamic injection of HBV plasmids. The serum concentrations of HBV surface antigen (HBsAg), HBV e antigen (HBeAg), HBV surface antibody (HBsAb), HBV core antibody (HBcAb), interferon-α (IFN-α) and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA). The activities of alanine aminotransferase (ALT) were determined by ALT kit using a Spectramax Plus spectrophotometer. Hepatic HBV DNA was quantified by quantitative real-time PCR. The expression of HBV core antigen (HBcAg) in liver was detected by immunohistochemistry. Drug toxicity of CpG-1826 was evaluated by body weighting and liver histopathology confirmation. RESULTS: CpG-1826 administration inhibited HBV replication efficiently with significant reduction of serum HBsAg and HBeAg, hepatic HBcAg and HBV DNA levels. The serum levels of IFN-α, IFN-γ and HBsAb increased but the HBcAb level declined in the CpG-1826 group compared to CpG-1982 and PBS control groups. Results of ALT activity indicated no significant difference among CpG-1826 group, CpG-1982 and PBS control groups. Body weighting and histopathology examination showed no obvious toxicity. CONCLUSIONS: Given the stimulation activity of a host immune system, CpG ODN is a promising strategy for HBV therapy with more relevant research needed.

Associations between hsa-miR-603 polymorphism, lifestyle-related factors and colorectal cancer risk.

BACKGROUND: Accumulated studies have suggested that single nucleotide polymorphisms (SNPs) in microRNAs are associated with risk of colorectal cancer (CRC). OBJECTIVE: We tested our hypothesis that rs11014002 in hsa-miR-603 may be associated with CRC risk with a crosstalk of life-related factors. METHODS: We conducted a case-control study which included 102 CRC patients and 204 matched cancer-free controls in Xiaoshan County. RESULTS: We observed that subjects with rs11014002 CT/TT genotype had an increased susceptibility for CRC (CT vs. CC: odds ratio (OR)=2.352, 95% confidence interval (CI): 1.142-4.840, P=0.020; CT+TT vs. CC: OR=2.031, 95% CI: 1.063-3.883, P=0.032). After stratification by lifestyle-related factors, similar results were found among nonsmokers (CT vs. CC: OR=2.753, 95% CI: 1.085-6.983, P=0.033; CT+TT vs. CC: OR=2.971, 95% CI: 1.188-7.435, P=0.020) and non-alcohol drinkers (CT+TT vs. CC: OR=3.279, 95% CI: 1.071-10.033, P=0.037). CONCLUSIONS: Our data suggest that hsa-miR-603 may be involved in colorectal tumorigenesis, and the genetic polymorphism in hsa-miR-603 is associated with CRC susceptibility.

A genetic variant in MiR-146a modifies digestive system cancer risk: a meta-analysis.

MicroRNAs (miRNAs) negatively regulate gene expression and act as tumor suppressors or oncogenes in oncogenesis. The association between a single nucleotide polymorphism (SNP) in miR-146a rs2910164 and susceptibility to digestive system cancers was inconsistent in previous studies. In this study, we conducted a literature search of PubMed to identify all relevant studies published before August 31, 2013. A total of 21 independent case-control studies were included in this updated meta-analysis with 9,558 cases and 10,614 controls. We found that the miR-146a rs2910164 polymorphism was significantly associated with decreased risk of digestive system cancers in an allele model (OR=0.90, 95%CI 0.87-0.94), homozygote model (OR=0.84, 95%CI 0.77-0.91), dominant model (OR=0.90, 95%CI 0.84-0.96), and recessive model (OR=0.85, 95%CI 0.79-0.91), while in a heterozygous model (OR = 0.99, 95% CI 0.89-1.11) the association showed marginal significance. Subgroup analysis by cancer site revealed decreased risk in colorectal cancer above allele model (OR=0.90, 95%CI 0.83- 0.97) and homozygote model (OR=0.85, 95%CI 0.72-1.00). Similarly, decreased cancer risk was observed when compared with allele model (OR=0.87, 95%CI 0.81-0.93) and recessive model (OR=0.81, 95%CI 0.72-0.90) in gastric cancer. When stratified by ethnicity, genotyping methods and quality score, decreased cancer risks were also observed. This current meta-analysis indicated that miR-146a rs2910164 polymorphism may decrease the susceptibility to digestive system cancers, especially in Asian populations.

rs12904 polymorphism in the 3'UTR of EFNA1 is associated with colorectal cancer susceptibility in a Chinese population.

Accumulated evidence has indicated that Ephrin A1 (EFNA1) is associated with angiogenesis and tumorigenesis in various types of malignancies, including colorectal cancer (CRC). In the current study, we performed an online search using the public microarray database to investigate whether EFNA1 expression might be altered in CRC tissues. We then conducted a case-control study including 306 subjects (102 cases and 204 well-matched controls) in Xiaoshan County to assess any association between genetic polymorphisms in EFNA1 and CRC susceptibility. Searches in the Oncomine expression profiling database revealed EFNA1 to be overexpressed in CRC tissue compared with adjacent normal tissue. The rs12904 G-A variant located in the 3' untranslated region (UTR) of EFNA1 was observed to be associated with CRC susceptibility. Compared with the AA homozygous genotype, those carrying GA genotype had a decreased risk of developing CRC (odds ratio (OR) =0.469, 95% confidence interval (CI): 0.225-0.977, and P =0.043). The association was stronger among smokers and tea drinkers, however, no statistical evidence of interaction between rs12904 polymorphism and smoking or tea drinking on CRC risk was found. Our results suggest that EFNA1 is involved in colorectal tumorigenesis, and rs12904 A>G polymorphism in the 3' UTR of EFNA1 is associated with CRC susceptibility. Larger studies and further mechanistic investigations are warranted to confirm our findings.