TRIM69 suppressed the anoikis resistance and metastasis of gastric cancer through ubiquitin‒proteasome-mediated degradation of PRKCD.

The tripartite motif (TRIM) protein family has been investigated in multiple human cancers, including gastric cancer (GC). However, the role of TRIM69 in the anoikis resistance and metastasis of GC cells remains to be elucidated. We identified the differentially expressed genes in anoikis-resistant GC cells using RNA-sequencing analysis. The interaction between TRIM69 and PRKCD was analyzed by coimmunoprecipitation and mass spectrometry. Our results have shown that TRIM69 was significantly downregulated in anoikis-resistant GC cells. TRIM69 overexpression markedly suppressed the anoikis resistance and metastasis of GC cells in vitro and in vivo. TRIM69 knockdown had the opposite effects. Mechanistically, TRIM69 interacted with PRKCD through its B-box domain and catalyzed the K48-linked polyubiquitination of PRKCD. Moreover, TRIM69 inhibited BDNF production in a PRKCD-dependent manner. Importantly, overexpression of PRKCD or BDNF blocked the effects of TRIM69 on the anoikis resistance and metastasis of GC cells. Interestingly, a TRIM69-PRKCD+BDNF+ cell subset was positively associated with metastasis in GC patients. TRIM69-mediated suppression of the anoikis resistance and metastasis of GC cells via modulation of the PRKCD/BDNF axis, with potential implications for novel therapeutic approaches for metastatic GC.

MiR-200/183 family-mediated module biomarker for gastric cancer progression: an AI-assisted bioinformatics method with experimental functional survey.

BACKGROUND: Gastric cancer (GC) is a major cancer burden throughout the world with a high mortality rate. The performance of current predictive and prognostic factors is still limited. Integrated analysis is required for accurate cancer progression predictive biomarker and prognostic biomarkers that help to guide therapy. METHODS: An AI-assisted bioinformatics method that combines transcriptomic data and microRNA regulations were used to identify a key miRNA-mediated network module in GC progression. To reveal the module's function, we performed the gene expression analysis in 20 clinical samples by qRT-PCR, prognosis analysis by multi-variable Cox regression model, progression prediction by support vector machine, and in vitro studies to elaborate the roles in GC cells migration and invasion. RESULTS: A robust microRNA regulated network module was identified to characterize GC progression, which consisted of seven miR-200/183 family members, five mRNAs and two long non-coding RNAs H19 and CLLU1. Their expression patterns and expression correlation patterns were consistent in public dataset and our cohort. Our findings suggest a two-fold biological potential of the module: GC patients with high-risk score exhibited a poor prognosis (p-value < 0.05) and the model achieved AUCs of 0.90 to predict GC progression in our cohort. In vitro cellular analyses shown that the module could influence the invasion and migration of GC cells. CONCLUSIONS: Our strategy which combines AI-assisted bioinformatics method with experimental and clinical validation suggested that the miR-200/183 family-mediated network module as a "pluripotent module", which could be potential marker for GC progression.

Combined application of multiple biomarkers for early auxiliary diagnosis of silicosis.

Silicosis is an important industrial health problem for those workers exposed to silica. The present study aimed to investigate the sensitivity and specificity of combined detection of biomarkers in early auxiliary diagnosis of silicosis, the risk factors of silicosis were also studied. The study sample comprised 65 workers who had clinical silicosis and 70 matched control subjects who were exposed to silica but did not have clinical silicosis. The levels of superoxide dismutase, malondialdehyde, interleukin 6 (IL-6), tumor necrosis factor-alpha, and cholinesterases in the serum of 135 subjects were measured. After completing the biochemical assays, a logistic regression model based on the above biochemical determination results was established, and the receiver operating characteristic curve was used for judging the discrimination ability of different statistical indexes. The expression levels of MDA, IL-6, and TNF-alpha in serum samples of patients with stage I silicosis and MDA and IL-6 in serum samples of patients with stage II silicosis were all significantly higher. Results from logistic regression analysis showed that ChEs were protective factors for silicosis, while age, chronic respiratory symptoms, IL-6, and MDA were risk factors. The areas under the ROC curve (AUC) were 0.86 (IL-6), 0.81 (MDA), and 0.65 (TNF-alpha or ChEs). AUC-ROC = 0.90 (95%CI:0.84-0.95). The diagnostic efficiency of IL-6 combined with MDA and TNF-alpha was better than that of any single biomarker.

Circular RNA circLDLR facilitates cancer progression by altering the miR-30a-3p/SOAT1 axis in colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy worldwide. Circular RNAs (circRNAs) have been reported to play critical regulatory roles in tumorigenesis, serving as tumor biomarkers and therapeutic targets. However, the contributions of circRNAs to CRC tumorigenesis are unclear. In our study, high expression of circLDLR was found in CRC tissues and cells and was closely associated with the malignant progression and poor prognosis of CRC patients. We demonstrated that circLDLR boosts growth and metastasis of CRC cells in vitro and in vivo, and modulates cholesterol levels in vitro. Mechanistically, we showed that circLDLR competitively binds to miR-30a-3p and prevents it from reducing the SOAT1 level, facilitating the malignant progression of CRC. In sum, our findings illustrate that circLDLR participates in CRC tumorigenesis and metastasis via the miR-30a-3p/SOAT1 axis, serving as a potential biomarker and therapeutic target in CRC.

Gastric cancer-derived exosomal miR-135b-5p impairs the function of Vγ9Vδ2 T cells by targeting specificity protein 1.

Recent studies have shown that tumor-derived exosomes participate in the communication between tumor cells and their microenvironment and mediate malignant biological behaviors including immune escape. In this study, we found that gastric cancer (GC) cell-derived exosomes could be effectively uptaken by Vγ9Vδ2 T cells, decrease the cell viability of Vγ9Vδ2 T cells, induce apoptosis, and reduce the production of cytotoxic cytokines IFN-γ and TNF-α. Furthermore, we demonstrated that exosomal miR-135b-5p was delivered into Vγ9Vδ2 T cells. Exosomal miR-135b-5p impaired the function of Vγ9Vδ2 T cells by targeting specificity protein 1 (SP1). More importantly, blocking the SP1 function by Plicamycin, an SP1 inhibitor, abolished the effect of stable miR-135b-5p knockdown GC cell-derived exosomes on Vγ9Vδ2 T cell function. Collectively, our results suggest that GC cell-derived exosomes impair the function of Vγ9Vδ2 T cells via miR-135b-5p/SP1 pathway, and targeting exosomal miR-135b-5p/SP1 axis may improve the efficiency of GC immunotherapy based on Vγ9Vδ2 T cells.

Myeloid cell subsets that express latency-associated peptide promote cancer growth by modulating T cells.

Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-ß on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-ß inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer.

B7-H5 blockade enhances CD8+ T-cell-mediated antitumor immunity in colorectal cancer.

Negative immune checkpoint blockade immunotherapy has shown potential for multiple malignancies including colorectal cancer (CRC). B7-H5, a novel negative immune checkpoint regulator, is highly expressed in tumor tissues and promotes tumor immune escape. However, the clinical significance of B7-H5 expression in CRC and the role of B7-H5 in the tumor microenvironment (TME) has not been fully clarified. In this study, we observed that high B7-H5 expression in CRC tissues was significantly correlated with the lymph node involvement, AJCC stage, and survival of CRC patients. A significant inverse correlation was also observed between B7-H5 expression and CD8+ T-cell infiltration in CRC tissues. Kaplan-Meier analysis showed that patients with high B7-H5 expression and low CD8+ T-cell infiltration had the worst prognosis in our cohort of CRC patients. Remarkably, both high B7-H5 expression and low CD8+ T infiltration were risk factors for overall survival. Additionally, B7-H5 blockade using a B7-H5 monoclonal antibody (B7-H5 mAb) effectively suppressed the growth of MC38 colon cancer tumors by enhancing the infiltration and Granzyme B production of CD8+ T cells. Importantly, the depletion of CD8+ T cells obviously abolished the antitumor effect of B7-H5 blockade in the MC38 tumors. In sum, our findings suggest that B7-H5 may be a valuably prognostic marker for CRC and a potential target for CRC immunotherapy.

B7-H3 suppresses doxorubicin-induced senescence-like growth arrest in colorectal cancer through the AKT/TM4SF1/SIRT1 pathway.

Emerging evidence suggests that cellular senescence induced by chemotherapy has been recognized as a new weapon for cancer therapy. This study aimed to research novel functions of B7-H3 in cellular senescence induced by a low dose of doxorubicin (DOX) in colorectal cancer (CRC). Here, our results demonstrated that B7-H3 knockdown promoted, while B7-H3 overexpression inhibited, DOX-induced cellular senescence. B7-H3 knockdown dramatically enhanced the growth arrest of CRC cells after low-dose DOX treatment, but B7-H3 overexpression had the opposite effect. By RNA-seq analysis and western blot, we showed that B7-H3 prevented cellular senescence and growth arrest through the AKT/TM4SF1/SIRT1 pathway. Blocking the AKT/TM4SF1/SIRT1 pathway dramatically reversed B7-H3-induced resistance to cellular senescence. More importantly, B7-H3 inhibited DOX-induced cellular senescence of CRC cells in vivo. Therefore, targeting B7-H3 or the B7-H3/AKT/TM4SF1/SIRT1 pathway might be a new strategy for promoting cellular senescence-like growth arrest during drug treatment in CRC.

H19 promotes aerobic glycolysis, proliferation, and immune escape of gastric cancer cells through the microRNA-519d-3p/lactate dehydrogenase A axis.

Long noncoding RNAs (lncRNAs) have been investigated in multiple human cancers including gastric cancer (GC). Our research aims to explore the role of H19 in aerobic glycolysis, proliferation, and immune escape of GC cells. The expression of H19 in GC samples was analyzed using Gene Expression Profiling Interactive Analysis, Gene Expression Omnibus data, and real-time quantitative PCR analysis. Relative quantification of glucose consumption and lactate production from cell supernatant were applied to assess the aerobic glycolysis of GC cells. Subcellular fractionation, luciferase reporter, and western blot assays certified the binding between genes. Cell Counting Kit-8 and colony formation assays were used to determine GC cell proliferation. Flow cytometry, ELISA, and real-time quantitative PCR assays were applied to analyze the immunosuppressive effect of H19. H19 was highly expressed in samples of patients with GC, and associated with tumor growth in vivo. H19 knockdown suppressed glucose consumption, lactate production, and proliferation of GC cells by regulating the microRNA (miR)-519d-3p/lactate dehydrogenase A (LDHA) axis. Both miR-519d-3p depletion and LDHA overexpression could reverse the H19 knockdown-induced decrease in aerobic glycolysis and proliferation. Moreover, conditioned medium from stable knockdown H19 GC cells modulated the activity of immune cells including γδT cells, Jurkat cells, and tumor-associated macrophages in a miR-519d-3p/LDHA/lactate axis-dependent manner. The H19/miR-519d-3p/LDHA axis mainly contributed to aerobic glycolysis, proliferation, and immune escape of GC cells.

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations.