Exosome-mediated circGMPS facilitates the development of gastric cancer cells through miR-144-3p/PUM1.

In recent years, gastric cancer (GC) is still one of the major public health burdens in the world. It is reported that exosome circular RNA (circRNA) is involved in the GC progression. However, the function and potential mechanism of circGMPS in GC remains unclear and needs further exploration. In this study, we isolated and identified exosomes from serum by TEM, NTA analysis and Western blot. RNA expression was evaluated by qRT-PCR. Western blot was employed to examine protein expression. Cell proliferation was measured using CCK-8. Transwell assay was adopted to analyze cell migration and invasion. The relationship between genes was explored through bioinformatics analysis, dual-luciferase reporter gene assay and spearman correlation coefficient. We found that circGMPS was elevated in GC exosomes, tissues and cells. Poor prognosis of GC patients was related to high circGMPS expression. Both exosome co-culture with cells and insertion of circGMPS clearly promoted cell progression. Mechanically, circGMPS sponged miR-144-3p to regulate PUM1. Inhibition of PUM1 or miR-144-3p overexpression inhibited the malignant GC cell progression. Our data confirmed that exosome-derived circGMPS boosted malignant progression by miR-144-3p/PUM1 axis in GC cells, providing strong evidences for circGMPS as a clinical biomarker of GC treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00597-9.

Spatial Distribution of Inhibitory Innervations of Excitatory Pyramidal Cells by Major Interneuron Subtypes in the Auditory Cortex.

Mental disorders, characterized by the National Institute of Mental Health as disruptions in neural circuitry, currently account for 13% of the global incidence of such disorders. An increasing number of studies suggest that imbalances between excitatory and inhibitory neurons in neural networks may be a crucial mechanism underlying mental disorders. However, the spatial distribution of inhibitory interneurons in the auditory cortex (ACx) and their relationship with excitatory pyramidal cells (PCs) remain elusive. In this study, we employed a combination of optogenetics, transgenic mice, and patch-clamp recording on brain slices to investigate the microcircuit characteristics of different interneurons (PV, SOM, and VIP) and the spatial pattern of inhibitory inhibition across layers 2/3 to 6 in the ACx. Our findings revealed that PV interneurons provide the strongest and most localized inhibition with no cross-layer innervation or layer specificity. Conversely, SOM and VIP interneurons weakly regulate PC activity over a broader range, exhibiting distinct spatial inhibitory preferences. Specifically, SOM inhibitions are preferentially found in deep infragranular layers, while VIP inhibitions predominantly occur in upper supragranular layers. PV inhibitions are evenly distributed across all layers. These results suggest that the input from inhibitory interneurons to PCs manifests in unique ways, ensuring that both strong and weak inhibitory inputs are evenly dispersed throughout the ACx, thereby maintaining a dynamic excitation-inhibition balance. Our findings contribute to understanding the spatial inhibitory characteristics of PCs and inhibitory interneurons in the ACx at the circuit level, which holds significant clinical implications for identifying and targeting abnormal circuits in auditory system diseases.

Effect of thalidomide combined with TP chemotherapy on serum VEGF and NRP-1 levels advanced esophageal cancer patients.

OBJECTIVE: This study aimed to investigate the effect of thalidomide combined with pacilitaxel plus cisplatin (TP) chemotherapy on serum vascular endothelial growth factor (VEGF) and neuropilin-1 (NRP-1) levels in advanced esophageal cancer patients. METHOD: A total of 133 patients with advanced esophageal cancer receiving treatment in Danzhou People's Hospital from February 2017 to July 2019 were recruited and divided into a control group (CG, n = 53) and a study group (SG, n = 80) randomly. Patients in the CG (53 cases) were treated with TP chemotherapy, and patients in the SG (80 cases) were treated with thalidomide on the basis above. The general data of the two groups of patients was observed, as well as the therapeutic effect, chemotherapy-related toxicity, and quality of life. Serum vascular endothelial growth factor (VEGF) and neuropilin-1 (NRP-1) levels were tested before and after treatment. RESULTS: There was no difference in general data between the two groups (P>0.05), and the occurrence of nausea and vomiting in SG was significantly lower than those in CG (P<0.05). The therapeutic effect was better in SG than CG (P<0.05). The Karnofsky Performance Scale (KPS) score improvement rate, appetite increase rate and body weight increase rate in SG were better than those in CG (P<0.05). After treatment, compared with CG, SG had lower serum VEGF and NRP-1 levels (P<0.05) and better quality of life (P<0.05). CONCLUSION: Thalidomide combined with TP chemotherapy is safe and effective in treating advanced esophageal cancer patients, which reduces patients' serumlevels of VEGF and NRP-1.

Disintegrin Tablysin-15 Suppresses Cancer Hallmarks in Melanoma Cells by Blocking FAK/Akt/ERK and NF-κB Signaling.

BACKGROUND: Integrins are crucial anti-cancer therapy targets. We previously showed that tablysin-15 is an integrin antagonist with its Arg-Gly-Asp motif in a novel structural context. OBJECTIVE: Here we investigated the anti-cancer effects and mechanisms of action of tablysin-15 in melanoma cells. METHODS: Cell adhesion, competitive binding, cell viability, and ATP chemiluminescence assays were used to analyze the binding of tablysin-15 to αvß3 integrin and its phenotypic effects. Wound healing, transwells, and zymography were performed to detect motility and matrix metalloproteinase- 2/-9 activities. PARP and caspase-3 cleavage were used as apoptosis assays, while LDH release and flow cytometry were used for necrosis and cell cycle analysis. The expression of mRNAs and proteins of target molecules was measured by qRT-PCR and western blotting, respectively. RESULTS: Tablysin-15 dose-dependently inhibited the proliferation, migration, and invasion of M21 cells through integrin αvß3. The proliferation inhibition caused by tablysin-15 was attributable to G0/G1 phase arrest rather than apoptosis or necrosis. Furthermore, tablysin-15 suppressed MMP-2/- 9 activities and the mRNA expression of MMP-2/-9 and COX-2 but was upregulated TIMP-1 in M21 cells. Meanwhile, tablysin-15 suppressed the expression of cyclin D1/E and CDK 2/6, the phosphorylation of FAK, Akt, and ERK, and nuclear translocation of NF-κB, while increasing the expression of the CDK inhibitor p21waf1/C1. Taken together, tablysin-15 might inhibit melanoma cell metastasis and proliferation by competing with αvß3 integrin, thereby blocking FAK-associated signaling pathways and nuclear translocation of NF-κB. CONCLUSION: Tablysin-15 has reliable anti-cancer effects against M21 melanoma cells, suggesting tablysin-15 is a promising anti-tumor drug.

Patient-shared TCRß-CDR3 clonotypes correlate with favorable prognosis in chronic hepatitis B.

The presence of shared T-cell clonotypes was found in several different diseases, but its relationship with the progression of disease remains unclear. By sequencing the complementary determining region 3 of T-cell receptor (TCR) ß chains from the purified antigen-experienced CD8+ T cells, we characterized the T-cell repertoire in a prospective cohort study among 75 patients with chronic hepatitis B in China, as well as a healthy control and a validation cohort. We found that most T-cell clones from patients harbored the "patient-specific" TCR sequences. However, "patient-shared" TCR clonotypes were also widely found, which correlated with the favorable turnover of disease. Interestingly, the frequency of the "patient-shared" clonotypes can serve as a biomarker for favorable prognosis. Based on the clonotypes in those patients with favorable outcomes, we created a database including several clusters of protective anti-HBV CD8+ T-cell clonotypes that might be a reasonable target for therapeutic vaccine development or adoptive cell transfer therapy. These findings were validated in an additional independent cohort of patients. These results suggest that the "patient-shared" TCR clonotypes may serve as a valuable prognostic tool in the treatment of chronic hepatitis B and possibly other chronic viral diseases.

Analysis of T cell receptor repertoire in monozygotic twins concordant and discordant for chronic hepatitis B infection.

Due to their identical inheritance and shared surroundings, identical twins have been the recommended group for studying the susceptibility and prognosis of diseases. Here, CD8+ T cell receptor beta (TCRß) chains were analyzed by high-throughput sequencing in three pairs of healthy identical twins and chronic hepatitis B patients. The data showed a high level of similarity in the TCR repertoire of each pair in terms of average TCR Vß segment expression and frequency of the complementary determining region 3 (CDR3) pattern and skewed or oligoclonal clonotypes. Notably, the level of similarity in TCR Vß expression between the twins appeared to be independent of the consistency or inconsistency of chronic HBV infection, although the detailed CDR3 pattern and frequency were related to disease prognosis. There were more immunodominant clonotypes in patients with HBV antigen seroconversion, which showed an increased abundance. These immunodominant clonotypes may be used as favorable prognostic biomarkers and potential targets for immunotherapy. Thus, delineating the CD8+ T cell repertoire of identical twins with concordant chronic viral infections provides a promising means to screen protective TCR genes for immunotherapy.

Nestin overexpression promotes the embryonic development of heart and brain through the regulation of cell proliferation.

Nestin, an intermediate filament protein, is a key regulator of various extracellular proteins that play important roles in cell growth and differentiation. In recent years, nestin has been widely accepted as a molecular marker for neural stem/progenitor cells. However, its function during embryogenesis remains largely unknown since its depletion is lethal after stage embryonic day 8.5 (E8.5). In order to understand the role of this protein in vivo, we compared the heart and brain tissues of control mice with those of mice overexpressing a human nestin cDNA transgene under the control of a ROSA26 promoter. In these tissues we examined the general histology and cell size, the presence of apoptotic cells by TUNEL assay, and the presence of progenitor cell markers like SOX2. Compared to controls, mouse embryos overexpressing the human nestin transgene have a larger size and display characteristic morphological changes including a larger heart and forebrain. In these tissues we found corresponding increases in the size of cardiomyocytes and brain cells, as well as indications of augmented cell proliferation. In contrast, apoptosis was not significantly altered. Co-staining brain sections with SOX2 and Ki67 showed that most of the proliferating cells in the forebrain were neural stem cells. Moreover, nestin overexpression was responsible for a marked activation of the PI3K/Akt signaling pathway. Taken together, the results of this study indicate that nestin plays an important role in the embryonic development of at least two mouse organs (heart and brain) through the regulation of cell proliferation.

Correlation of overexpression of nestin with expression of epithelial-mesenchymal transition-related proteins in gastric adenocarcinoma.

BACKGROUND: Nestin is associated with neoplastic transformation. However, the mechanisms by which nestin contributes regarding invasion and malignancy of gastric adenocarcinoma (GAC) remain unknown. Recent studies have shown that the epithelial-mesenchymal transition (EMT) is important in invasion and migration of cancer cells. In the present study, we aimed to investigate the expression of nestin and its correlation with EMT-related proteins in GAC. MATERIALS AND METHODS: The expression of nestin and EMT-related proteins was examined in GAC specimens and cell lines by immunohistochemistry and Western blotting. Clinicopathological features and survival outcomes were retrospectively analyzed. RESULTS: Positive nestin immunostaining was most obviously detected in the cytoplasm, nucleus or both cytoplasm and nucleus of tumor cells in 19.2% (24/125) of GAC tissues, which was significantly higher than that in normal gastric mucosa tissues (1.7%, 1/60) (p=0.001). Nestin expression was closely related to several clinicopathological factors and EMT-related proteins (E-cadherin, vimentin and Snail) and displayed a poor prognosis. Interestingly, simultaneous cytoplasmic and nuclear nestin expression correlated with EMT-related proteins (E-cadherin, vimentin and Snail) (p<0.05) and lymph node metastasis (p=0.041) and a shorter survival time (p<0.05), but this was not the case with cytoplasmic or nuclear nestin expression. CONCLUSIONS: Nestin, particularly expression in both cytoplasm and nucleus, might be involved in regulating EMT and malignant progression in GAC, with potential as an unfavorable indicator in tumor diagnosis and a target for clinical therapy.

[Establishment of nestin transgenic mice and nestin expression in the organs].

OBJECTIVE: To develop a nestin transgenic mice and study the distribution of nestin expression in the organs. METHODS: The three segments of nestin in full-length cDNA was amplified using human glioma cell line U251 cDNA as the template and cloned into the vector pBROAD3 containing a strong promoter ROSA26. The constructed vector, after identification with restriction enzyme and sequencing and removal of the prokaryotic sequences, were purified and injected into the fertilized eggs of mice. Transgenic mice were identified by PCR and the founder was maintained. Western blot analysis was used to detect the expression of nestin of the F3 transgenic mice and the wild-type ones in the vital organs (heart, lung, brain and kidney). RESULTS: The Nestin transgenic vector controlled by ROSA26 promoter was successfully constructed and validated by sequencing. Among the 34 newborn mice, 2 founders were tested to be nestin-positive by PCR. Westem blot analysis showed that the F3 transgenic mice expressed high levels of nestin in the brain and lungs. CONCLUSION: Nestin transgenic mice have been successfully established with stable nestin expression in the brain and lungs of the offspring mice, which can be useful for studying the functions of nestin in tumor metastasis, stemness maintenance and differentiation of cells.

[Roles of miR-590-5p and miR-590-3p in the development of hepatocellular carcinoma].

OBJECTIVE: To study the effect of the two arms of miR-590, miR-590-5p and miR-590-3p, on hepatoma cell proliferation and their roles in tumor development. METHODS: We analyzed and verified the expression pattern of miR-590 in liver cancer specimens and cell lines by miRNA microarrays and QPCR. MiR-590 mimic or inhibitor was transfected into normal liver cells or liver cancer cells via liposome, and the changes in cell proliferation and survival were determined by MTT assay and soft agar colony formation assay. The target genes of miR-590-5p and miR-590-3p were predicated with Targetscan and validated by luciferase reporter system and Western blotting. RESULTS: The expressions of miR-590-5p and miR-590-3ps were up-regulated in 3 hepatocellular carcinoma (HCC) tissues and their synchronization was significantly up-regulated in 8 out of 10 HCC tissues as compared with the adjacent tissues. QPCR further showed that miR-590-5p/3p was up-regulated in 3 HCC cell lines (HepG2, Hep3B, and Huh7) in comparison with the normal liver cell line L-O2. L-O2 cells over-expressing miR-590-5p and miR-590-3p exhibited significantly increased proliferation (P<0.05), while down-regulation of miR-590-5p and miR-590-3p caused significantly suppressed proliferation in HepG2, Hep3B, and Huh7 cells. Targetscan predicted PDCD4 and PTEN as the potential target genes of miR-590-5p and miR-590-3p, which was verified by luciferase reporter system and Western blotting. miR-590-3p was found to activate PI3K-AKT signaling pathway by down-regulating PTEN to promote AKT1-S473 phosphorylation. CONCLUSION: MiR-590 is an important tumorigenic factor for HCC, and its two arms can both promote tumorigenesis by regulating the expression of their target tumor suppressor gene, PDCD4 and PTEN, to promote HCC cell proliferation and survival and activate the core tumor signal pathway PI3K-AKT.