Overexpressing Cells Research Articles

A Multifunctional Aptamer Decorated Lipid Nanoparticles for the Delivery of EpCAM-targeted CRISPR/Cas9 Plasmid for Efficacious In Vivo Tumor Regression.

Epithelial cell adhesion molecule (EpCAM) gene encodes a type-I trans-membrane glycoprotein that is overexpressed in many cancerous epithelial cells and promotes tumor progression by regulating the expression of several oncogenes like c-myc and other cyclins. Because of this tumorigenic association, the EpCAM gene has been a potential target for anti-cancer therapy in recent days. Herein, it is attempted to knockout the proto-oncogenic EpCAM expression by efficiently delivering an all-in-one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) plasmid via a lipid nanoparticle system made out of synthetic stimuli-sensitive lipids. The plasmid possesses the necessary information in the form of a guide RNA targeted to the EpCAM gene. The aptamer decorated system selectively targets EpCAM overexpressed cells and efficiently inhibits the genetic expression. It has explored the pH-responsive property of the developed lipid nanoparticles and monitored their efficacy in various cancer cell lines of different origins with elevated EpCAM levels. The phenomenon has further been validated in vivo in non-immunocompromised mouse tumor models. Overall, the newly developed aptamer decorated lipid nanoparticle system has been proven to be efficacious for the delivery of EpCAM-targeted CRISPR/Cas9 plasmid.

PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells.

Overcoming temozolomide (TMZ)-resistance is a major challenge in glioblastoma therapy. Therefore, identifying the key molecular player in chemo-resistance becomes urgent. We previously reported the downregulation of PDCD10 in primary glioblastoma patients and its tumor suppressor-like function in glioblastoma cells. Here, we demonstrate that the loss of PDCD10 causes a significant TMZ-resistance during treatment and promotes a rapid regrowth of tumor cells after treatment. PDCD10 knockdown upregulated MGMT, a key enzyme mediating chemo-resistance in glioblastoma, accompanied by increased expression of DNA mismatch repair genes, and enabled tumor cells to evade TMZ-induced cell-cycle arrest. These findings were confirmed in independent models of PDCD10 overexpressing cells. Furthermore, PDCD10 downregulation led to the dedifferentiation of glioblastoma cells, as evidenced by increased clonogenic growth, the upregulation of glioblastoma stem cell (GSC) markers, and enhanced neurosphere formation capacity. GSCs derived from PDCD10 knockdown cells displayed stronger TMZ-resistance and regrowth potency, compared to their parental counterparts, indicating that PDCD10-induced stemness may independently contribute to tumor malignancy. These data provide evidence for a dual role of PDCD10 in tumor suppression by controlling both chemo-resistance and dedifferentiation, and highlight PDCD10 as a potential prognostic marker and target for combination therapy with TMZ in glioblastoma.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Effects of TET2-mediated methylation reconstruction on A2058 melanoma cell sensitivity to matrix stiffness in a 3D culture system

Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.

CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis: CREB1 promotes cholangiocarcinoma metastasis through regulating LAYN/TLN1/β1 integrin axis

BackgroundLayilin (LAYN) plays an important role in tumor progression, invasion, and metastasis; however, its role in cholangiocarcinoma (CHOL) has not been elucidated. MethodsWe utilized the GEPIA, STRING, and hTFtarget databases for bioinformatics analysis. Overexpression or knockdown cell lines were constructed by transfecting the cells with different plasmids. Western blot (WB) was performed to detect LAYN, TLN1, and CREB1 expression. Cell proliferation, migration, and invasiveness were assessed using CCK-8 and Transwell assays. Immunofluorescence and WB were used to detect epithelial-mesenchymal transition (EMT) markers. The CHOL metastasis model was established by injecting RBE cells into the tail veins of nude mice. Metastatic lesions were identified using hematoxylin and eosin staining. Co-immunoprecipitation and Chromatin immunoprecipitation were used to validate the interactions. ResultsLAYN was highly expressed in the CHOL cells. Knockdown of LAYN significantly inhibited proliferation, migration, invasion, and EMT in both QBC-939 and RBE human CHOL cells, while overexpression of LAYN had the opposite effect. Furthermore, in a CHOL metastasis model using nude mice, knocking down LAYN expression markedly suppressed CHOL liver and lung metastases. LAYN interacts with TLN1, and CREB1 binds to the LAYN promoter, with all three showing a positive correlation. Additionally, bioinformatics analysis revealed high expression of both TLN1 and CREB1 in CHOL. Knockdown of TLN1 or CREB1 in QBC-939 and RBE cells inhibited cell proliferation, migration, invasion, and EMT, reversing the effects of LAYN overexpression. Moreover, knockdown of TLN1 or CREB1 also suppressed the expression of ITGB1 and the phosphorylation levels of c-Jun, p38 MAPK, and ERK, further reversing the effects of LAYN overexpression. ConclusionOur results suggest that CREB1 promotes CHOL metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis.

Modulation of PD-L1 by Astragalus polysaccharide attenuates the induction of melanoma stem cell properties and overcomes immune evasion

BackgroundMelanoma is a highly aggressive form of skin cancer. The existence of cancer stem cells (CSCs) and tumor immune evasion are two major causes of melanoma progression, but no effective treatment has been found at present. Astragalus polysaccharide (APS) is a principal active component derived from Astragalus membranaceus, showing anti-tumor effects in various tumors including melanoma. However, the underlying mechanism is still unclear.MethodsThe regulation of APS on self-renewal ability and CSC markers expression in melanoma stem cells (MSCs) was measured by tumor sphere formation and tumorigenicity assays, RT-qPCR, and western blot. Flow cytometry was conducted to evaluate the activation of immune system by APS in melanoma mice. Further, the mechanism was explored based on PD-L1 overexpression and knock-down B16 cells.ResultsAPS attenuated the tumor sphere formation of MSCs in vitro as well as the tumorigenicity in vivo. It also decreased the expression of CD133, BMI1 and CD47. Based on the PD-L1 overexpression and knock-down B16 cells, it was confirmed that APS inhibited the induction of MSCs by down-regulating PD-L1 expression. Meanwhile, APS increased the infiltration of CD4+ and CD8+T cells in tumor tissues because of its inhibitory effect on PD-L1.ConclusionsAPS inhibited MSC induction and overcame tumor immune evasion through reducing PD-L1 expression. This study provided compelling evidence that APS could be a prospective therapeutic agent for treating melanoma.

Curcumin suppresses copper accumulation in non-small cell lung cancer by binding ATOX1

BackgroundNon-small cell lung cancer (NSCLC) is associated with intracellular copper accumulation. Antioxidant 1 (ATOX1) is a copper chaperone. This study aimed to analyze the anti-cancer effects of curcumin on the ATOX1-mediated copper pathway in NSCLC.MethodsA binding activity between curcumin and ATOX1 was measured using molecular docking. NSCLC cells, A549 and H1299, were treated with different doses of curcumin (10, 20, 40 µM) or DC-AC50 (5, 10, 20 µM) for 24 h. The cell viability and levels of ATOX1, ATP7A and COX17 proteins were observed in cells. Overexpressing ATOX1 in cells was established by pcDNA3.1-ATOX1 transfection for 24 h. The ATOX1 overexpressing cells were treated with 40 µM curcumin or 20 µM DC-AC50 for 24 h to analyze the mechanism of curcumin in NSCLC treatment. Cell viability was measured by CCK-8, and levels of proteins were measured by western blotting. The copper level in cells was labeled by copper sensor-1. Moreover, nude mice models were induced by injection of A549 cells and treated with 20 mg/kg/d DC-AC50 or 40 mg/kg/d curcumin. Tumor growth was observed by measuring tumor volume and tumor weight. The levels of ATOX1, ATP7A and COX17 in tumors were measured by immunohistochemistry and western blotting.ResultsCurcumin bound to ATOX1 (score = −6.1 kcal/mol) and decreased the levels of ATOX1, ATP7A and COX17 proteins in NSCLC cells. The curcumin or DC-AC50 treatment suppressed cell viability by inhibiting the ATOX1-mediated copper signaling in NSCLC cells. The ATOX1 overexpression in cells significantly weakened the effects of curcumin on suppressing copper accumulation and the ATOX1-mediated copper pathway (p < 0.05). In mice models, curcumin or DC-AC50 treatment also suppressed tumor growth by suppressing the ATOX1-mediated copper pathway in tumors.ConclusionThis study demonstrated that curcumin bound ATOX1 to suppress copper accumulation in NSCLC cells, providing a new mechanism of curcumin for NSCLC treatment.

TRPV1 Activation Antagonizes High-Fat Diet-Induced Obesity at Thermoneutrality and Enhances UCP-1 Transcription via PRDM-16.

Body weight is a balance between energy intake and energy expenditure. Energy expenditure is mainly governed by physical activity and adaptive thermogenesis. Adaptive dietary thermogenesis in brown and beige adipose tissue occurs through mitochondrial uncoupling protein (UCP-1). Laboratory mice, when housed at an ambient temperature of 22-24 °C, maintain their body temperature by dietary thermogenesis, eating more food compared to thermoneutrality. Humans remain in the thermoneutral zone (TNZ) without expending extra energy to maintain normal body temperature. TRPV1 activation by capsaicin (CAP) inhibited weight gain in mice housed at ambient temperature by activating UCP-1-dependent adaptive thermogenesis. Hence, we evaluated the effect of CAP feeding on WT and UCP-1-/- mice maintained under thermoneutral conditions. Our research presents novel findings that TRPV1 activation by CAP at thermoneutrality counters obesity in WT mice and promotes PRDM-16-dependent UCP-1 transcription. CAP fails to inhibit weight gain in UCP-1-/- mice housed at thermoneutrality and in adipose tissue-specific PRDM-16-/- mice. In vitro, capsaicin treatment increases UCP-1 transcription in PRDM-16 overexpressing cells. Our data indicate for the first time that TRPV1 activation counters obesity at thermoneutrality permissive for UCP-1 and the enhancement of PRDM-16 is not beneficial in the absence of UCP-1.

Contactin -Associated protein1 Regulates Autophagy by Modulating the PI3K/AKT/mTOR Signaling Pathway and ATG4B Levels in Vitro and in Vivo.

Contactin-associated protein1 (Caspr1) plays an important role in the formation and stability of myelinated axons. In Caspr1 mutant mice, autophagy-related structures accumulate in neurons, causing axonal degeneration; however, the mechanism by which Caspr1 regulates autophagy remains unknown. To illustrate the mechanism of Caspr1 in autophagy process, we demonstrated that Caspr1 knockout in primary neurons from mice along with human cell lines, HEK-293 and HeLa, induced autophagy by downregulating the PI3K/AKT/mTOR signaling pathway to promote the conversion of microtubule-associated protein light chain 3 I (LC3-I) to LC3-II. In contrast, Caspr1 overexpression in cells contributed to the upregulation of this signaling pathway. We also demonstrated that Caspr1 knockout led to increased LC3-I protein expression in mice. In addition, Caspr1 could inhibit the expression of autophagy-related 4B cysteine peptidase (ATG4B) protein by directly binding to ATG4B in overexpressed Caspr1 cells. Intriguingly, we found an accumulation of ATG4B in the Golgi apparatuses of cells overexpressing Caspr1; therefore, we speculate that Caspr1 may restrict ATG4 secretion from the Golgi apparatus to the cytoplasm. Collectively, our results indicate that Caspr1 may regulate autophagy by modulating the PI3K/AKT/mTOR signaling pathway and the levels of ATG4 protein, both in vitro and in vivo. Thus, Caspr1 can be a potential therapeutic target in axonal damage and demyelinating diseases.

Transcriptome and proteome changes triggered by overexpression of the transcriptional regulator Maf1 in the human pathogen Leishmania major.

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Oncogenic fusion of CD63-BCAR4 contributes cancer stem cell-like properties via ALDH1 activity.

Gene fusions are common somatic alterations in cancers, and fusions with tumorigenic features have been identified as novel drivers of cancer and therapeutic targets. Few studies have determined whether the oncogenic ability of fusion genes is related to the induction of stemness in cells. Cancer stem cells (CSCs) are a subset of cells that contribute to cancer progression, metastasis, and recurrence, and are critical components of the aggressive features of cancer. Here, we investigated the CSC-like properties induced by CD63-BCAR4 fusion gene, previously reported as an oncogenic fusion, and its potential contribution for the enhanced metastasis as a notable characteristic of CD63-BCAR4. CD63-BCAR4 overexpression facilitates sphere formation in immortalized bronchial epithelial cells. The significantly enhanced sphere-forming activity observed in tumor-derived cells from xenografted mice of CD63-BCAR4 overexpressing cells was suppressed by silencing of BCAR4. RNA microarray analysis revealed that ALDH1A1 was upregulated in the BCAR4 fusion-overexpressing cells. Increased activity and expression of ALDH1A1 were observed in the spheres of CD63-BCAR4 overexpressing cells compared with those of the empty vector. CD133 and CD44 levels were also elevated in BCAR4 fusion-overexpressing cells. Increased NANOG, SOX2, and OCT-3/4 protein levels were observed in metastatic tumor cells derived from mice injected with CD63-BCAR4 overexpressing cells. Moreover, DEAB, an ALDH1A1 inhibitor, reduced the migration activity induced by CD63-BCAR4 as well as the sphere-forming activity. Our findings suggest that CD63-BCAR4 fusion induces CSC-like properties by upregulating ALDH1A1, which contributes to its metastatic features.

PSAT1 Promotes Metastasis via p-AKT/SP1/ITGA2 Axis in Estrogen Receptor-Negative Breast Cancer Cell.

Accumulating evidence indicates that PSAT1 not only reprogrammed metabolic function but also exhibits "moonlighting" functions in promoting tumor malignancy. However, the underlying molecular mechanisms of PSAT1 promoting ER-negative breast cancer cell migration need further investigation. Briefly, the PSAT1 and ITGA2 expression in cells and tissues was detected using qRT-PCR, immunofluorescence staining and western blot assay. The effect of PSAT1 and ITGA2 was verified both in vitro and in vivo. RNA-seq analysis explored a series of differently expressed genes. The regulation between SP1 and ITGA2 was investigated by ChIP analysis. We reported PSAT1 was highly expressed in ER-breast cancer tissues and tumor cells and positively correlated with metastasis. Moreover, RNA-seq analysis explored a series of differently expressed genes, including ITGA2, in PSAT1 overexpressed cells. Mechanistically, PSAT1 facilitated breast cancer metastasis via the p-AKT/SP1/ITGA2 axis. We further elucidated that PSAT1 promoted the entry of SP1 into the nucleus through the upregulation of p-AKT and confirmed ITGA2 is a target of SP1. In addition, enhanced cell migration was remarkably reversed by ITGA2 depletion or p-AKT inhibitor treatment. This study clarified the mechanism of PSAT1 in promoting ER-negative breast cancer metastasis, which may provide mechanistic clues for attenuating breast cancer metastasis.

CD38 deficiency prevents diabetic nephropathy by inhibiting lipid accumulation and oxidative stress through activation of the SIRT3 pathway.

Diabetic nephropathy (DN) is one of the most common complications of diabetes. Our previous study showed that CD38 knockout (CD38KO) mice had protective effects on many diseases. However, the roles and mechanisms of CD38 in DN remain unknown. Here, DN mice were generated by HFD feeding plus streptozotocin (STZ) injection in male CD38KO and CD38flox mice. Mesangial cells (SV40 MES 13 cells) were used to mimic the injury of DN with palmitic acid (PA) treatment in vitro. Our results showed that CD38 expression was significantly increased in kidney of diabetic CD38flox mice and SV40 MES 13 cells treated with PA. CD38KO mice were significantly resistant to diabetes-induced renal injury. Moreover, CD38 deficiency markedly decreased HFD/STZ-induced lipid accumulation, fibrosis and oxidative stress in kidney tissue. In contrast, overexpression of CD38 aggravated PA-induced lipid accumulation and oxidative stress. CD38 deficiency increased expression of SIRT3, while overexpression of CD38 decreased its expression. More importantly, 3-TYP, an inhibitor of SIRT3, significantly enhanced PA-induced lipid accumulation and oxidative stress in CD38 overexpressing cell lines. In conclusion, our results demonstrated that CD38 deficiency prevented DN by inhibiting lipid accumulation and oxidative stress through activation of the SIRT3 pathway.

The cytoglobin-dependent transcriptome in melanoma indicates a protective function associated with oxidative stress, inflammation and cancer-associated pathways

Cytoglobin (CYGB) is a member of the oxygen-binding globin superfamily. In this study we generated stable CYGB overexpressing A375 melanoma cells and performed RNA-sequencing to comprehensively explore the CYGB-dependent transcriptome. Our findings reveal that ectopic expression of CYGB dysregulated multiple cancer-associated genes, including the mTORC1 and AKT/mTOR signaling pathways, which are frequently overactivated in tumors. Moreover, several cancer-associated pathways, such as epithelial-mesenchymal transition (EMT) mediated by CSPG4, were downregulated upon CYGB overexpression. Intriguingly, ectopic expression suggested anti-inflammatory potential of CYGB, as exemplified by downregulation of key inflammasome-associated genes, including NLRP1, CASP1 and CD74, which play pivotal roles in cytokine regulation and inflammasome activation. Consistent with established globin functions, CYGB appears to be involved in redox homeostasis. Furthermore, our study indicates CYGB's association to DNA repair mechanisms and its regulation of NOX4, reinforcing its functional versatility. Additionally, multiple significantly enriched pathways in CYGB overexpressing cells were consistently dysregulated in opposite direction in CYGB depleted cells. Collectively, our RNA-sequencing based investigations illustrate the diverse functions of CYGB in melanoma cells, pointing to its putative roles in cellular protection against oxidative stress, inflammation, and cancer-associated pathways. These findings pave the way for further research into the physiological role of CYGB and its potential as a candidate therapeutic target in melanoma.

IFN-β Overexpressing Adipose-Derived Mesenchymal Stem Cells Mitigate Alcohol-Induced Liver Damage and Gut Permeability.

Alcoholic liver disease (ALD) is a form of hepatic inflammation. ALD is mediated by gut leakiness. This study evaluates the anti-inflammatory effects of ASCs overexpressing interferon-beta (ASC-IFN-β) on binge alcohol-induced liver injury and intestinal permeability. In vitro, ASCs were transfected with a non-viral vector carrying the human IFN-β gene, which promoted hepatocyte growth factor (HGF) secretion in the cells. To assess the potential effects of ASC-IFN-β, C57BL/6 mice were treated with three oral doses of binge alcohol and were administered intraperitoneal injections of ASC-IFN-β. Mice treated with binge alcohol and administered ASC-IFN-β showed reduced liver injury and inflammation compared to those administered a control ASC. Analysis of intestinal tissue from ethanol-treated mice administered ASC-IFN-β also indicated decreased inflammation. Additionally, fecal albumin, blood endotoxin, and bacterial colony levels were reduced, indicating less gut leakiness in the binge alcohol-exposed mice. Treatment with HGF, but not IFN-β or TRAIL, mitigated the ethanol-induced down-regulation of cell death and permeability in Caco-2 cells. These results demonstrate that ASCs transfected with a non-viral vector to induce IFN-β overexpression have protective effects against binge alcohol-mediated liver injury and gut leakiness via HGF.

CircGNAO1 suppresses hepatocellular carcinoma progression and metastasis via sponging miR-182-5p and regulating FOXO1 expression

BackgroundHepatocellular carcinoma (HCC) is an aggressive malignant tumor with poor prognosis. Using high-throughput sequencing, we identified a novel circRNA, circGNAO1, which is downregulated in HCC tissues compared to adjacent tissues. However, the potential functions and mechanisms of circGNAO1 in HCC metastasis remain unclear. MethodsqRT-PCR was used to detect the expression of circGNAO1, miR-182-5p, and FOXO1 in HCC cells and tissues. Bioinformatics analysis, RNA pull-down assyas, and dual-luciferase reporter assays were employed to verify the interaction between circGNAO1 and miR-182-5p. Functional experiments were conducted using circGNAO1 overexpression and knockdown cell lines, including Transwell, wound healing, and EdU assays. Liver metastasis models and subcutaneous xenograft mouse models were established to analyze the effect of circGNAO1 on HCC metastasis and growth in vivo. ResultsHigh-throughput sequencing and qRT–PCR results showed that the expression of circGNAO1 dramatically decreased in HCC tissues. Functionally, in vivo and in vitro experiments verified that overexpression of circGNAO1 inhibited the proliferation, migration, invasion and EMT of HCC cells, while knockdown of circGNAO1 promoted these behaviors. Mechanistically, we have demonstrated that circGNAO1 functions as a sponge for miR-182-5p to regulate FOXO1 expression, thereby activating the TGF-β/Smad3 signaling pathway and EMT process. ConclusionscircGNAO1 suppresses the progression and metastasis of HCC through the miR-182-5p/FOXO1 axis, and circGNAO1 may be an efficient therapeutic target in HCC.

Nrf2/ASPM axis regulated vasculogenic mimicry formation in hepatocellular carcinoma under hypoxia.

Hypoxic microenvironment is a common feature of most solid tumors including hepatocellular carcinoma (HCC). Vasculogenic mimicry (VM) formation by tumor cells could provide blood supply to tumor cells under hypoxia. NFE2 like basic leucine zipper (bZIP) transcription factor 2 (Nrf2), a regulator of cellular homeostasis, may promote tumor progression in the hypoxic conditions. However, the role and regulatory mechanisms of Nrf2 in HCC are not fully elucidated. Nrf2 and assembly factor for spindle microtubules (ASPM) expression modulations were conducted by lentiviral transfections. Western blot, immunofluorescence, ChIP-qPCR, dual-luciferase reporter gene assay, flow cytometry, RNA sequencing, multiple bioinformatics databases analysis, cell function assays in vitro, mouse model in vivo and human HCC tissues were employed to assess the effect of Nrf2/ASPM axis on HCC progression under hypoxia. Nrf2 and ASPM expression facilitated epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) feature, and VM formation of HCC cells under hypoxia. Furthermore, Nrf2-regulated ASPM expression, via binding directly to the promoter region of ASPM and transcriptionally promoting ASPM expression. ASPM re-expression in Nrf2 knockdown cells or ASPM knockdown in Nrf2 overexpression cells reversed the cellular function caused by Nrf2. Meantime, retinol metabolism pathway was disrupted following abnormal ASPM expression. Nrf2/ASPM axis in murine models accelerated tumor growth and VM, corroborating in vitro findings. All-trans retinoic acid treatment reversed stemness and VM of HCC cells in vitro and in vivo. Clinically, Nrf2 and ASPM expressions were related to poor prognosis of HCC patients. Nrf2 drives EMT, CSCs characteristics and VM in HCC under hypoxia through the modulation of ASPM. Retinol metabolism pathway was dysregulated in HCC cells with ASPM overexpression. Nrf2/ASPM axis and related pathway provided potential therapeutic target for HCC.

Increased imaging ligand hydrophilicity and improved pharmacokinetic properties provides enhanced in vivo targeting of fibroblast activation protein

In this work, the impact of physicochemical modifications on pharmacokinetics and in vivo targeting of a small molecule fibroblast activation protein inhibitor (FAPI) imaging ligand in a murine model of rheumatoid arthritis was evaluated. While similar ligands have been well-reported in oncology for molecular imaging and radiotherapy, there are limited reports of FAPI derivatives in targeted applications in immunology. As inflammation may increase both specific and non-specific delivery of targeted agents in general, we sought to identify the optimal targeted molecular imaging probe characteristics for efficient cell surface engagement. A series of FAPI derivatives were synthesized and their physicochemical properties modified via conjugation of fluorescent dyes and/or an albumin-binding small molecule. The impact of these modifications on cell surface binding affinity was assessed using an overexpressing cell line. Additionally, a thorough mechanistic characterization of fibroblast activation protein (FAP) cell surface internalization was evaluated in both overexpressing and endogenously expressing cells. Lastly, the pharmacokinetics and in vivo uptake in inflamed arthritic paws were characterized via near-infrared (NIR) imaging. All targeted molecular imaging agents tested maintained strong nanomolar binding affinity to cell surface FAP independent of chemical modification. The murine fibroblast-like synoviocytes expressed lower absolute cell-surface FAP compared to a transfected line, and the net internalization half-life measured for the transfected cells via flow cytometry was 7.2 h. The unmodified FAPI ligand exhibited the poorest in vivo targeting, likely resulting from its large apparent volume of distribution (62.7 ml) and rapid systemic clearance (t1/2 = 0.5 h). Conjugation of a charged, hydrophilic AF647 fluorophore decreased systemic clearance (t1/2 = 2.1 h) and demonstrated a 2-fold improvement in blocking FAPI-800CW engagement of FAP in vivo when compared to blocking of FAPI-800CW with FAPI with up to 2.8-fold improvements noted for the equivalent albumin binding construct comparison.

LncRNA PKD1P6 modulates ovarian granulosa cell survival of hyperandrogenic polycystic ovary syndrome by targeting miR-135b-5p and inhibiting ERK1/2 signaling

Polycystic ovary syndrome (PCOS) is the most common and multifactorial endocrine disease among women of reproductive age. Aberrant folliculogenesis is a common pathological characteristic of PCOS, but the underlying molecular mechanism remains unclear. Emerging evidence indicated that aberrant expression of long noncoding RNAs (lncRNAs) may contribute to the pathogenesis of PCOS. In this study, we found that lncRNA PKD1P6 expression was remarkably down-regulated in ovarian granulosa cells (GCs) of hyperandrogenic PCOS (HA-PCOS) patients and negatively correlated with serum testosterone (T) levels. We further showed that overexpression of PKD1P6 markedly reduced cell viability, attenuated DNA synthesis capacity, arrested the cell cycle at G0/G1 phase and promoted apoptosis of KGN cells. Exosomes derived from PKD1P6 overexpression cells exerted similar effects to PKD1P6 overexpression on the function of KGN cells. Mechanistically, PKD1P6 could act as a competing endogenous RNA (ceRNA) by directly binding with miR-135b-5p. Overexpression of PKD1P6 significantly suppressed ERK1/2 activation, whereas up-regulation of miR-135b-5p exerted an opposing effect. Additionally, excessive androgen was showed to diminish PKD1P6 expression while promote miR-135b-5p expression of PCOS models in vitro and vivo. Collectively, our findings delineate the clinical significance of PKD1P6 in HA-PCOS and the new regulatory mechanisms involved in abnormal folliculogenesis, providing a promising therapeutic target for HA-PCOS.

Dual Template Molecularly Imprinted Polymers Targeting Blockade of CD47 for Enhanced Macrophage Phagocytosis and Synergistic Antimetabolic Therapy.

Glycinamide ribonucleotide formyltransferase (GARFT) is an important enzyme in the folate metabolism pathway, and chemical drugs targeting GARFT have been used in tumor treatments over the past few decades. The development of novel antimetabolism drugs that target GARFT with improved performance and superior activity remains an attractive strategy. Herein, we proposed a targeted double-template molecularly imprinted polymer (MIP) for enhancing macrophage phagocytosis and synergistic antimetabolic therapy. The double-template MIP was prepared by imprinting the exposed peptide segment of the extracellular domain of CD47 and the active center of GARFT. Owing to the imprinted cavities on the surface of MIP, it can actively target cancer cells and mask the "do not eat me" signal upon binding to CD47 thereby blocking the CD47-SIRPα pathway and ultimately enhancing phagocytosis by macrophages. In addition, MIP can specifically bind to the active center of GARFT upon entry into the cells, thereby inhibiting its catalytic activity and ultimately interfering with the normal expression of DNA. A series of cell experiments demonstrated that MIP can effectively target CD47 overexpressed 4T1 cancer cells and inhibit the growth of 4T1 cells. The enhanced phagocytosis ability of macrophages-RAW264.7 cells was also clearly observed by confocal imaging experiments. In vivo experiments also showed that the MIP exhibited a satisfactory tumor inhibition effect. Therefore, this study provides a new idea for the application of molecular imprinting technology to antimetabolic therapy in conjunction with macrophage-mediated immunotherapy.