RhoB Expression Research Articles

Downregulation of RhoB Inhibits Cervical Cancer Progression and Enhances Cisplatin Sensitivity.

RhoB, a member of the Rho GTPase family, has been implicated in the malignant progression of various cancer types. However, its role in cervical cancer (CC) remains unclear. Therefore, this study aims to elucidate the biological function of RhoB in CC and its relationship with cisplatin sensitivity. We analyzed data from the TCGA, GTEx, and GEO databases, revealing that RhoB mRNA expression is downregulated in CC tissues compared to normal cervical tissues. The further analysis of the TCGA database and Tongji samples showed that CC patients with a high RhoB expression had a shorter overall survival (OS). Subsequently, we found that the knockdown of RhoB inhibited the proliferation, migration, and invasion of cancer cells, while increasing apoptosis. Through Western blot (WB) analysis, we found that knocking down RhoB resulted in an increased expression of the epithelial marker E-cadherin, while the levels of N-cadherin, MMP2, MMP9, Vimentin, and Snail1 were reduced. Additionally, RhoB mRNA expression was upregulated in CC tissues after chemotherapy compared to CC tissues before chemotherapy. In CC cells, RhoB expression increased with cisplatin concentration, and the IC50 value decreased following RhoB knockdown. Moreover, the knockdown of RhoB could enhance the cellular apoptosis triggered by cisplatin. This study demonstrated that RhoB plays an oncogenic role in CC and that its knockdown could enhance the sensitivity of CC cells to cisplatin.

Enhancing Cell Aggregation and Migration via Double-Click Cross-Linking with Azide-Modified Hyaluronic Acid.

We present a novel approach to the formation of cell aggregates by employing click chemistry with water-soluble zwitterionic dibenzo cyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N3) as a linker to facilitate rapid and stable cell aggregation. By optimizing the concentrations of HA-N3 and WS-CODY, we achieved efficient cross-linking between azide-modified cell surfaces and HA-N3, generating cell aggregates within 10 min, and the resulting aggregates remained stable for up to 5 days, with cell viability maintained at approximately 80%. Systematic experiments revealed that a stoichiometric balance between HA-N3 and WS-CODY is important for effective cross-linking, highlighting the roles of both cell-surface azide modification and HA in the aggregate formation. We also investigated the genetic basis of altered cell behavior within these aggregates. Transcriptome analysis (RNA-seq) of aggregates postcultivation revealed a marked fluctuation of genes associated with 'cell migration' and 'cell adhesion', including notable changes in the expression of HYAL1, ICAM-1, CEACAM5 and RHOB. These findings suggest that HA-N3-mediated cell aggregation can induce intrinsic cellular responses that not only facilitate cell aggregate formation but also modulate cell-matrix interactions. We term this phenomenon 'chemo-resilience', The simplicity and efficacy of this click chemistry-based approach suggest it may have broad applicability for forming cell aggregates and modulating cell-matrix interactions in tissue engineering and regenerative medicine.

Abstract 2038: Statin Targeted Treatment Against Intimal Hyperplasia Using Unique Chitosan-PLGA Nanoparticles

Introduction: Statins have beneficial pleiotropic effects, including reducing intimal hyperplasia (IH) after intervention for peripheral arterial disease. Using a unique nanoparticle (NP) to locally maximize statin’s pleiotropic benefits, we hypothesized chitosan-functionalized polylactic-co-glycolic nanoparticles loaded with simvastatin (SL-cNPs) would: 1) be taken up by endothelial cells (ECs) and vascular smooth muscle cells (VSMCs); 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin. Methods: cNPs were loaded with simvastatin (2% w/w ) and encapsulated with lipophilic tracer, DiD. In vitro , human aortic ECs and VSMCs (passage 3-5) were cultured with 0.2mg SL-cNPs or basal media (30 min). After 24 hours, uptake of SL-cNPs was assessed by immunostaining and flow cytometry. Functional effect of SL-cNPs was determined using qPCR for mRNA expression of RhoA, RhoB and Ras. In vivo, male Sprague-Dawley rats (n=6-9/group) underwent carotid artery balloon injury in the following groups: 1) intraluminal therapy using normal saline control, empty cNPs or SL-cNPs; and 2) oral simvastatin (10mg/kg/day, 7 days pre-operative until sacrifice) plus intraluminal saline or SL-cNPs. Rats were sacrificed (day 14), perfusion fixed, arteries harvested, and IH quantified with morphometric analysis. Student’s t-test and one-way ANOVA was performed (p <0.05 for significance). Results: In vitro , SL-cNPs were readily taken up by ECs (79.6%, 38,606/48,501) and VSMCs (46.4%, 25,774/55,549). SL-cNPs induced a compensatory increase in expression of EC RhoA (1.3-fold) and VSMC RhoA (1.7-fold) and RhoB (5.4-fold) (p<0.05). In vivo, oral simvastatin plus intraluminal SL-cNPs reduced IH compared to controls (0.44 ± 0.04 vs 0.34 ± 0.03, p<0.05). Although oral simvastatin and SL-cNPs alone trended toward IH reduction, the effect was not significant (0.35 ± 0.03 and 0.377 ± 0.03, respectively). No difference existed between oral simvastatin and SL-cNP therapy. Conclusion: cNPs can be used as a novel vehicle to locally deliver statins to vascular cells. Although only the combination of oral simvastatin and SL-cNPs effectively reduced IH, different routes and/or concentration of SL-cNPs may allow for a more robust effect on IH prevention.

Nuclear respiratory factor 1 regulates super enhancer-controlled SPIDR to protect hepatocellular carcinoma cells from oxidative stress

BackgroundCellular response to oxidative stress plays significant roles in hepatocellular carcinoma (HCC) development, yet the exact mechanism by which HCC cells respond to oxidative stress remains poorly understood. This study aimed to investigate the role and mechanism of super enhancer (SE)-controlled genes in oxidative stress response of HCC cells.MethodsThe GSE112221 dataset was used to identify SEs by HOMER. Functional enrichment of SE-controlled genes was performed by Metascape. Transcription factors were predicted using HOMER. Prognosis analysis was conducted using the Kaplan-Meier Plotter website. Expression correlation analysis was performed using the Tumor Immune Estimation Resource web server. NRF1 and SPIDR expression in HCC and normal liver tissues was analyzed based on the TCGA-LIHC dataset. ChIP-qPCR was used to detect acetylation of lysine 27 on histone 3 (H3K27ac) levels of SE regions of genes, and the binding of NRF1 to the SE of SPIDR. To mimic oxidative stress, HepG2 and Hep3B cells were stimulated with H2O2. The effects of NRF1 and SPIDR on the oxidative stress response of HCC cells were determined by the functional assays.ResultsA total of 318 HCC-specific SE-controlled genes were identified. The functions of these genes was significant association with oxidative stress response. SPIDR and RHOB were enriched in the “response to oxidative stress” term and were chosen for validation. SE regions of SPIDR and RHOB exhibited strong H3K27ac modification, which was significantly inhibited by JQ1. JQ1 treatment suppressed the expression of SPIDR and RHOB, and increased reactive oxygen species (ROS) levels in HCC cells. TEAD2, TEAD3, NRF1, HINFP and TCFL5 were identified as potential transcription factors for HCC-specific SE-controlled genes related to oxidative stress response. The five transcription factors were positively correlated with SPIDR expression, with the highest correlation coefficient for NRF1. NRF1 and SPIDR expression was up-regulated in HCC tissues and cells. NRF1 activated SPIDR transcription by binding to its SE. Silencing SPIDR or NRF1 significantly promoted ROS accumulation in HCC cells. Under oxidative stress, silencing SPIDR or NRF1 increased ROS, malondialdehyde (MDA) and γH2AX levels, and decreased superoxide dismutase (SOD) levels and cell proliferation of HCC cells. Furthermore, overexpression of SPIDR partially offset the effects of NRF1 silencing on ROS, MDA, SOD, γH2AX levels and cell proliferation of HCC cells.ConclusionNRF1 driven SPIDR transcription by occupying its SE, protecting HCC cells from oxidative stress-induced damage. NRF1 and SPIDR are promising biomarkers for targeting oxidative stress in the treatment of HCC.

RhoB expression associated with chemotherapy response and prognosis in colorectal cancer

PurposeTo examine the role of RhoB expression in relation to chemotherapy response, clinical outcomes and associated signaling pathways in colorectal cancer patients.Materials and methodsThe study included 5 colon cancer cell lines, zebrafish embryos and 260 colorectal cancer patients treated with 5-fluorouracil (5-FU) and oxaliplatin (OXL). The methods consisted of CRISPR/Cas9, reactive oxygen species (ROS), caspase-3 activity, autophagy flux, in-silico RNA sequencing and immunohistochemistry. Gene expression analysis and pathway analysis were conducted using RNA-seq data.ResultsAll cancer lines tested, including SW480, SW480-KO13 (RhoB knockout), SW480-KO55 (RhoB knockout), HCT116 and HCT116-OE (RhoB overexpressed), exhibited cytotoxicity to 5-FU and OXL. RhoB knockout cell lines demonstrated significantly reduced migration compared to the control cell lines. Furthermore, RhoB played a role in caspase-3-dependent apoptosis, regulation of ROS production and autophagic flux. The mRNA sequencing data indicated lower expression levels of oncogenes in RhoB knockout cell lines. The zebrafish model bearing SW480-KO showed a light trend toward tumor regression. RhoB expression by immunohistochemistry in patients was increased from normal mucosa to tumor samples. In patients who received chemotherapy, high RhoB expression was related to worse survival compared to low RhoB expression. Furthermore, the molecular docking analysis revealed that OXL had a higher binding affinity for RhoB than 5-FU, with a binding affinity of -7.8 kcal/mol and HADDOCK predicted molecular interactions between RhoB and caspase 3 protein. Gene-set enrichment analysis supported these findings, showing that enrichment of DNA damage response pathway and p53 signaling in RhoB overexpression treatment group, while the RhoB knockout treatment group exhibited enrichment in the negative regulation pathway of cell migration.ConclusionRhoB was negatively associated with chemotherapy response and survival in colorectal cancers. Therefore, RhoB inhibition may enhance chemotherapeutic responses and patient survival.

Fluvastatin prevents lung metastasis in triple-negative breast cancer by triggering autophagy via the RhoB/PI3K/mTOR pathway

Triple-negative breast cancer is more common among younger than older women and is associated with the poorest survival outcomes of all breast cancer types. Fluvastatin inhibits tumour progression and induces the autophagy of breast cancer cells; however, the role of autophagy in fluvastatin-induced inhibition of breast cancer metastasis is unknown. Therefore, this study aimed to determine this mechanism. The effect of fluvastatin on human hormone receptor-negative breast cancer cells was evaluated in vitro via migration and wound healing assays, western blotting, and morphological measurements, as well as in vivo using a mouse xenograft model. Chloroquine, a prophylactic medication used to prevent malaria in humans was used as an autophagy inhibitor. We found that fluvastatin administration effectively prevented the migration/invasion of triple-negative breast cancer cells, an effect that was largely dependent on the induction of autophagy. Administration of the autophagy inhibitor chloroquine prevented the fluvastatin-induced suppression of lung metastasis in the nude mouse model. Furthermore, fluvastatin increased Ras homolog family member B (RhoB) expression, and the autophagy and anti-metastatic activity induced by fluvastatin were predominantly dependent on the regulation of RhoB through the protein kinase B–mammalian target of rapamycin (Akt–mTOR) signaling pathway. These results suggest that fluvastatin inhibits the metastasis of triple-negative breast cancer cells by modulating autophagy via the up regulation of RhoB through the AKT-mTOR signaling pathway. Fluvastatin may be a promising therapeutic option for patients with triple-negative breast cancer.

The effect of circulating iron on barrier integrity of primary human endothelial cells

Iron is hypothesized to be one of the contributors to cardiovascular disease and its levels in the circulation may correlate with cardiovascular risk. The aim of this study is to investigate the mechanisms that underlie the effects of iron on the barrier function of primary human endothelium. We used Human Umbilical Vein Endothelial Cells (HUVEC) to investigate the effects of Fe3+ using electric cell-substrate impedance sensing, microscopy, western blot and immunofluorescence microscopy. Exposure to Fe3+ caused EC elongation and upregulation of stress-induced proteins. Analysis of barrier function showed a dose-dependent drop in endothelial integrity, which was accompanied by Reactive Oxygen Species (ROS) production and could partly be prevented by ROS scavengers. Inhibition of contractility by the ROCK inhibitor Y27632, showed even more effective rescue of barrier integrity. Using western blot, we detected an increase in expression of the small GTPase RhoB, an inducer of EC contraction, and a small decrease in VE-cadherin, suggestive for an iron-induced stress response. Co-stimulation by TNFα and iron, used to investigate the role of low-grade inflammation, revealed an additive, negative effect on barrier integrity, concomitant with an upregulation of pro-inflammatory markers ICAM-1 and RhoB. Iron induces a response in HUVEC that leads to endothelial activation and a pro-inflammatory state measured by loss of barrier integrity which can be reversed by ROS scavengers, combined with inhibition of contractility. These data suggest that ROS-mediated damage of the vascular endothelium could contribute to the increased cardiovascular risk which is associated with elevated levels of circulating iron.

Prediction of Five-Year Survival Rate for Rectal Cancer Using Markov Models of Convolutional Features of RhoB Expression on Tissue Microarray.

The ability to predict survival in cancer is clinically important because the finding can help patients and physicians make optimal treatment decisions. Artificial intelligence in the context of deep learning has been increasingly realized by the informatics-oriented medical community as a powerful machine-learning technology for cancer research, diagnosis, prediction, and treatment. This paper presents the combination of deep learning, data coding, and probabilistic modeling for predicting five-year survival in a cohort of patients with rectal cancer using images of RhoB expression on biopsies. Using about one-third of the patients' data for testing, the proposed approach achieved 90% prediction accuracy, which is much higher than the direct use of the best pretrained convolutional neural network (70%) and the best coupling of a pretrained model and support vector machines (70%).

CERS4 predicts positive anti-PD-1 response and promotes immunomodulation through Rhob-mediated suppression of CD8+Tim3+ exhausted T cells in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is one of the main malignant tumors with high mortality and short survival time. Immunotherapy has become the standard treatment for advanced NSCLC, but it has the problems of drug resistance and low response rate. Therefore, obtaining effective biomarkers to predict and enhance immune checkpoint inhibitors (ICIs) efficacy in NSCLC is important. Sphingolipid metabolism is recently found to be closely involved in tumor immunotherapy. CERS4, an important sphingolipid metabolizing enzyme, is positively correlated with the efficacy of anti-PD-1 therapy for NSCLC. Upregulation of CERS4 expression could improve the efficacy of anti-PD-1 therapy for NSCLC. High expression of CERS4 could downregulate the expression of Rhob in tumor. Significantly, the ratio of CD4+/CD8+ T cell increased and the ratio of Tim-3+/CD8+ T cell decreased in spleen and peripheral blood cells. When Rhob was knocked out, the efficacy of PD-1 mAb treatment increased, and the frequency of Tim-3+ CD8+ T cell decreased. This finding further confirmed the role of sphingolipid metabolites in regulating the immunotherapeutic function of NSCLC. These metabolites may improve the efficacy of PD-1 mAb in NSCLC by regulating the CERS4/Rhob/Tim-3 axis. Overall, this study provided a potential and effective target for predicting and improving the efficacy of ICIs for NSCLC. It also provided a new perspective for the study on the mechanisms of ICIs resistance for NSCLC.

Artificial intelligence fusion for predicting survival of rectal cancer patients using immunohistochemical expression of Ras homolog family member B in biopsy.

The process of biomarker discovery is being accelerated with the application of artificial intelligence (AI), including machine learning. Biomarkers of diseases are useful because they are indicators of pathogenesis or measures of responses to therapeutic treatments, and therefore, play a key role in new drug development. Proteins are among the candidates for biomarkers of rectal cancer, which need to be explored using state-of-the-art AI to be utilized for prediction, prognosis, and therapeutic treatment. This paper aims to investigate the predictive power of Ras homolog family member B (RhoB) protein in rectal cancer. This study introduces the integration of pretrained convolutional neural networks and support vector machines (SVMs) for classifying biopsy samples of immunohistochemical expression of protein RhoB in rectal-cancer patients to validate its biologic measure in biopsy. Features of the immunohistochemical expression images were extracted by the pretrained networks and used for binary classification by the SVMs into two groups of less and more than 5-year survival rates. The fusion of neural search architecture network (NASNet)-Large for deep-layer feature extraction and classifier using SVMs provided the best average classification performance with a total accuracy = 85%, prediction of survival rate of more than 5 years = 90%, and prediction of survival rate of less than 5 years = 75%. The finding obtained from the use of AI reported in this study suggest that RhoB expression on rectal-cancer biopsy can be potentially used as a biomarker for predicting survival outcomes in rectal-cancer patients, which can be informative for clinical decision making if the patient would be recommended for preoperative therapy.

Classification of IHC Images of NATs With ResNet-FRP-LSTM for Predicting Survival Rates of Rectal Cancer Patients

Over a decade, tissues dissected adjacent to primary tumors have been considered "normal" or healthy samples (NATs). However, NATs have recently been discovered to be distinct from both tumorous and normal tissues. The ability to predict the survival rate of cancer patients using NATs can open a new door to selecting optimal treatments for cancer and discovering biomarkers. This paper introduces an artificial intelligence (AI) approach that uses NATs for predicting the 5-year survival of pre-operative radiotherapy patients with rectal cancer. The new approach combines pre-trained deep learning, nonlinear dynamics, and long short-term memory to classify immunohistochemical images of RhoB protein expression on NATs. Ten-fold cross-validation results show 88% accuracy of prediction obtained from the new approach, which is also higher than those provided from baseline methods. Preliminary results not only add objective evidence to recent findings of NATs' molecular characteristics using state-of-the-art AI methods, but also contribute to the discovery of RhoB expression on NATs in rectal-cancer patients. The ability to predict the survival rate of cancer patients is extremely important for clinical decision-making. The proposed AI tool is promising for assisting oncologists in their treatments of rectal cancer patients.

LncRNA DUXAP8 induces breast cancer radioresistance by modulating the PI3K/AKT/mTOR pathway and the EZH2-E-cadherin/RHOB pathway

ABSTRACT Radiation resistance poses a major clinical challenge in breast cancer (BC) treatment, but little is known about how long noncoding RNA (lncRNA) may regulate this phenomenon. Here, we reported that DUXAP8 was highly expressed in radioresistant BC tissues, and high expression of DUXAP8 was associated with poor prognosis. We found that the overexpression of DUXAP8 promoted radioresistance, while the knockdown of DUXAP8 expression increased radiosensitivity. Further studies revealed that DUXAP8 enhanced the radioresistance of BC cells by activating the PI3K/AKT/mTOR pathway and by repressing the expression of E-cadherin and RHOB through interaction with EZH2. Together, our work demonstrates that the overexpression of DUXAP8 promotes the resistance of BC cells toward radiation through modulating PI3K/AKT/mTOR pathway and EZH2-E-cadherin/RHOB axis. Targeting DUXAP8 may serve as a potential strategy to overcome radioresistance in BC treatment.

RhoB Promotes Endometrial Stromal Cells Decidualization Via Semaphorin3A/PlexinA4 Signaling in Early Pregnancy.

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

MicroRNA-223 Attenuates Stretch-Injury-Induced Apoptosis in Brain Microvascular Endothelial Cells by Regulating RhoB Expression.

MiR-223 is a miRNA with important functions in apoptosis, carcinogenesis, and inflammation, and it was demonstrated to be over-expressed in brain tissue after traumatic brain injury (TBI). However, few studies have focused on its role in protecting brain microvascular endothelial cells (BMECs). This study evaluated the protective effect of miR-223 on BMECs after stretch injury (SI). bEnd.3 cells (BMECs of mouse) were transfected with overexpressing and blocking lentivirus of miR-223, then were subjected to SI. After immunofluorescence assay, it was demonstrated that miR-223 overexpression significantly rescued the SI-induced loss of ZO-1 (Zonula Occludens 1, tight junction protein) (p < 0.01), while miR-223 blocking exacerbated the loss of ZO-1 (p < 0.05). Flow cytometry confirmed a significant increase in the proportion of apoptotic bEnd.3 cells after SI, and miR-223 overexpression reduced this proportion (p < 0.001). The result of Western blot revealed that miR-223 overexpression significantly reduced the expression of cleaved caspase-3 (cl-caspase 3) (p < 0.05) and RhoB (p < 0.01), while miR-223 blocking increased the expression of these proteins (p < 0.05, p < 0.001). Additionally, knockdown of RhoB significantly reduced the expression of cl-caspase 3 (p < 0.001). These findings suggested that miR-223 can alleviate SI-induced apoptosis of BMECs, and this anti-apoptotic effect is at least partially achieved by inhibiting the expression of RhoB. Moreover, miR-223 may play a role in maintaining the integrity of BBB during TBI.

Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer

BackgroundAlthough gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro.MethodsImmunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178.ResultsWe found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan–Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters.ConclusionsOur results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

Brain and Breast Cancer Cells with PTEN Loss of Function Reveal Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Utilizing 3D Scaffolds and Aligned Microfiber Tracts.

Simple SummaryGlioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations are associated with brain tumor spreading and poor patient outcomes. GBM, and possibly TNBC, migrate on axons and blood vessels to disseminate in the brain; however, the mechanism is unresolved. There is a need for new therapeutic targets to blunt brain tumor spreading. Using 3D aligned printed microfibers mimicking brain structures proved that RHOB, in addition to ROCK and PTEN signaling, were essential for GBM and TNBC 3D cell migration. GBM and TNBC cell lines with PTEN loss of function and high RHOB expression exhibited amoeboid morphology with increased durotaxis, binding and migration speed on 3D microfibers, in contrast to the PTEN wildtype. Depending on the PTEN genotype, RHO-ROCK-PTEN inhibitors or PTEN rescue significantly regulated these properties. Regarding GBM and brain metastasizing TNBC, we conclude that RHOB inhibitors could play a novel role for improved therapy response and patient outcome.Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

Intestinal Metabolites Influence Macrophage Phagocytosis and Clearance of Bacterial Infection

The metabolite-rich environment that is the intestinal lumen contains metabolic by-products deriving from microbial fermentation and host cell metabolism, with resident macrophages being constantly exposed to this metabolic flux. Succinate, lactate and itaconate are three metabolites secreted by primed macrophages due to a fragmented tri-carboxylic acid (TCA) cycle. Additionally, succinate and lactate are known by-products of microbial fermentation. How these metabolites impact biological functioning of resident macrophages particularly in response to bacterial infection remains poorly understood. We have investigated the potential influence of these metabolites on macrophage phagocytosis and clearance of Escherichia coli (E. coli) infection. Treatment of murine bone-marrow-derived macrophages (BMDMs) with succinate reduced numbers of intracellular E. coli early during infection, while lactate-treated BMDMs displayed no difference throughout the course of infection. Treatment of BMDMs with itaconate lead to higher levels of intracellular E. coli early in the infection with bacterial burden subsequently reduced at later time-points compared to untreated macrophages, indicative of enhanced engulfment and killing capabilities of macrophages in response to itaconate. Expression of engulfment mediators MARCKS, RhoB, and CDC42 were reduced or unchanged following succinate or lactate treatment and increased in itaconate-treated macrophages following E. coli infection. Nitric oxide (NO) levels varied while pro- and anti-inflammatory cytokines differed in secretory levels in all metabolite-treated macrophages post-infection with E. coli or in response to lipopolysaccharide (LPS) stimulation. Finally, the basal phenotypic profile of metabolite-treated macrophages was altered according to marker gene expression, describing how fluid macrophage phenotype can be in response to the microenvironment. Collectively, our data suggests that microbe- and host-derived metabolites can drive distinct macrophage functional phenotypes in response to infection, whereby succinate and itaconate regulate phagocytosis and bactericidal mechanisms, limiting the intracellular bacterial niche and impeding the pathogenesis of infection.

The association of transcription factor Prox1 with the proliferation, migration, and invasion of lung cancer

BackgroundThe current study investigates the effect of transcription factor Prox1 on the proliferation, migration, and invasion ability of lung cancer.MethodsLung cancer cell lines (A549 and H446 cells) were transfected with Prox1NAD and siRNA, respectively. Thus, the A549 and H446 cells overexpressed Prox1 after transfection of Prox1NAD plasmids, and A549 and H446 cells have low expression of Prox1 after transfection with siRNA. Reverse transcriptase quantitative PCR and western blot analyses were used to detect Prox1 mRNA and protein expression in cells. Plate clone formation experiments and MTT experiments were used to detect cell proliferation. Western blot was used to detect the expression of Rho family-related proteins in cells.ResultsCompared to untransfected wild-type A549 and H446 that served as blank controls, the expression level of Prox1mRNA and protein in A549 and H446 cells overexpressing Prox1 after plasmid transfection was high, while the expression level of Prox1mRNA and protein in A549 and H446 cells with low expression of Prox1 after siRNA transfection was low. With the increase of Prox1 expression, the expression of RhoA and RhoC increased, while the expression of RhoB decreased.ConclusionThe finding of this study may provide a new approach for the treatment of lung cancer using targeted gene therapy.

CircRNA Hsa_circ_0001017 Inhibited Gastric Cancer Progression via Acting as a Sponge of miR-197.

Gastric cancer (GC) is one of the most common digestive system diseases and yet lacks effective therapeutic regimen. The aim of our present research was to probe the value of hsa_circ_0001017 in GC treatment. qRT-PCR and Western blot were performed to detect gene and protein expressions, respectively. CCK-8 assay and clone formation assay were used to ensure the proliferation of GC cell lines. Transwell assay was performed to measure the migration and invasion of GC cell lines. The relationship between hsa_circ_0001017 and miR-197 and that between miR-197 and RHOB 3'-UTR were ensured using the luciferase reporter assay. Decreased hsa_circ_0001017 was discovered in GC, and upregulation of hsa_circ_0001017 notably repressed proliferation, migration, and invasion of GC cell lines. We further certificated that hsa_circ_0001017 served as miR-197 sponge and suppressed the expression of miR-197. Moreover, hsa_circ_0001017 upregulation meaningfully accelerated RHOB expression in both gene and protein levels, and RHOB was a downstream target of miR-197. Overexpression of miR-197 could markedly restrain hsa_circ_0001017-induced RHOB increasing and stifle inhibition of hsa_circ_0001017 to the malignant phenotype of GC cell lines. Next, our results further confirmed that hsa_circ_0001017 increasing notably inhibited tumor growth, impeded miR-197 production, while it enhanced the expression of RHOB in vivo. Our data demonstrated that upregulation of hsa_circ_0001017 could notably muffle the proliferation as well as the metastasis of GC cell lines and impede the formation of GC tumor via targeting to miR-197/RHOB signaling pathway. Our results evidenced that hsa_circ_0001017 may act as a rising biomarker for GC treatment.

Abnormal neuronal morphology and altered synaptic proteins are restored by oxytocin in autism-related SHANK3 deficient model

Oxytocin has been suggested as a potential therapeutic agent in autism and other neuropsychiatric conditions. Although, the link between the deficit in “SH3 domain and ankyrin repeat containing protein 3” (SHANK3) and autism spectrum disorders is highly studied topic, developmental mechanisms are still poorly understood. In this study, we clearly confirm that SHANK3 deficiency is accompanied with abnormalities in neurite number and length, which are reversed by oxytocin treatment (1 μM, 48h) in primary hippocampal neurons. Transient silencing for the SHANK3 gene (siSHANK3) in neuron-like cell line (SH-SY5Y) revealed a significant decrease in the expression levels of Neurexins 1α, 1β, 2α and 2β. Oxytocin treatment compensated reduced levels of Synapsin I, PSD95 and Neuroligin 3 in siSHANK3 cells suggesting a marked potential of oxytocin to ameliorate defects present in conditions of SHANK3 deficiency. Further analysis of hippocampal tissue revealed that oxytocin application (0.1 μg/μl, s.c. at P2 and P3 day) affects levels of synaptic proteins and GTPases in both WT and SHANK3 deficient mice on day P5. Oxytocin stimulated the mRNA expression of RhoB and Rac1 in both WT and SHANK3 deficient mice. Our data suggest that autism relevant synaptic pathologies could be reversed by oxytocin treatment.