Cell Invasion Capability Research Articles

LncRNA ZNF674-AS1 Hinders Proliferation and Invasion of Hepatic Carcinoma Cells through the Glycolysis Pathway

Purpose Long noncoding RNAs (lncRNAs) play important roles in regulating various functions of cells at the levels of transcription and posttranscription. Extensive investigations have illustrated that lncRNAs are critical in the glucose metabolism of tumor cells, but their mechanisms of action need to be further explored. This study evaluates the role of lncRNA ZNF674-AS1 on the apoptosis and proliferation of human hepatic carcinoma cells in vitro through the glucose metabolism and its related mechanisms. Methods Real-time quantitative PCR was employed for detecting the level of expressions for lncRNA ZNF674-AS1 in liver cancer tissues (25 cases), paracancerous tissues, and liver cancer cell lines. The lncRNA ZNF674-AS1 high expression cell strain was constructed by the lentiviral overexpression vector. CCK-8, plate colony formation, transwell assay, lactate production, glucose consumption, and ATP levels were used to detect the change of cell proliferation, colony formation, migration, and invasion, as well as glycolytic capability. Western blot was carried out to detect the expression of HK2, PFKL, PKM2, GLUT1, and PKM1, which are the key proteins of glycolysis in cells. Result The lncRNA ZNF674-AS1 was undesirably expressed in liver cancer cell lines and tissues. Cell function assessments showed that compared with the blank control group (vector), overexpression of lncRNA ZNF674-AS1 could substantially hinder the proliferation, colony formation, migration, and invasion capability of liver cancer cells. Furthermore, overexpression of lncRNA ZNF674-AS1 could inhibit cell glycolysis (inhibit glucose consumption and reduce intracellular lactate and ATP levels) by inhibiting the expression of key proteins (such as PFKL, HK2, PKM2, and GLUT1) in the process of glycolysis. Conclusion As a tumor repressor gene, lncRNA ZNF674-AS1 inhibits the expression of key proteins in glycolysis to inhibit glycolysis level, thereby inhibiting cell migration and proliferation. Therefore, lncRNA ZNF674-AS1 could be a potent therapeutic target or a novel diagnostic molecule for patients suffering from liver cancer.

Endoplasmic reticulum stress triggered autophagy and regulated the phenotype transformation of rheumatoid arthritis synovial fibroblasts via the IRE1/JNK pathway.

BackgroundPrevious studies have indicated that endoplasmic reticulum (ER) stress may actively promote the pathogenesis of rheumatoid arthritis (RA) by evoking autophagy. However, the underlying mechanism remains largely unknown. This study aimed to explore the mechanism of the ER stress-autophagy pathway in regulating the phenotype transformation of rheumatoid arthritis synovial fibroblasts (RASFs).MethodsSynovial tissue was obtained from RA and osteoarthritis (OA) patients during joint replacement surgery. ER stress/autophagy signature markers were examined in synovial tissue by real-time quantitative polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Phenotype transformation of RASFs, including increased cell proliferation and invasion capability, was measured by CCK-8 assay and transwell invasion assay. Signaling pathways were further investigated and inositol requiring enzyme 1 (IRE1) was down-regulated in RASFs by transfecting specific short hairpin RNA-ERN1 (shRNA-ERN1) carried by lentiviral vectors.ResultsThe expression of ER stress/autophagy pathway-associated proteins, including GRP78, IRE1, protein kinase R-like endoplasmic reticulum kinase (PERK), and LC3, was significantly increased in RA synovium compared with OA synovium. After stimulation with tumor necrosis factor alpha (TNF-α) in vitro, the proliferation and invasion ability of RASFs were upregulated, while this phenomenon could be inhibited by 4-PBA (ER stress inhibitor) or 3-MA (autophagy inhibitor). The expression of IRE1 and p-JNK in particular, occurred in an obviously time-dependent manner after stimulation with TNF-α. Moreover, the proliferation and invasion of RASFs were inhibited after transfection with sh-RNA-ERN1 to downregulate IRE1 expression.ConclusionsER stress triggered autophagy via the IRE1/JNK pathway to regulate the phenotype transformation of RASFs, indicating an important role of the ER stress-autophagy pathway in the pathological process of synovitis in RA.

The CBP/β-Catenin Antagonist, ICG-001, Inhibits Tumor Metastasis via Blocking of the miR-134/ITGB1 Axis-Mediated Cell Adhesion in Nasopharyngeal Carcinoma.

Simple SummaryMetastatic nasopharyngeal carcinoma (NPC) is incurable and remains the main cause of NPC death. Our previous studies found that the CBP/β-catenin Wnt antagonist, IGC-001, could inhibit the primary tumor formation of NPC tumor cells. Here, we further explored the anti-metastatic activity of ICG-001. We started by screening a panel of microRNAs that are related to epithelial–mesenchymal transition and cancer stem cell phenotypes; both properties can contribute to tumor metastasis. MicroRNA-134 was found to be consistently upregulated by ICG-001. The role of miR-134 in NPC is largely unknown but some studies found an association between low expression of miR-134 and poor prognosis. We examined the role of miR-134 in NPC with both in vitro and in vivo models and found that miR-134 could inhibit cancer cell adhesion, migration, and invasion. Our study provided a functional explanation for the poor prognosis observed in NPC patients with low or loss of miR-134 expression in their tumors and showed that modulation of the Wnt signaling by ICG-001 could effectively inhibit NPC metastasis via the miR-134/ITGB1 axis.Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/β-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial–mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/β-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin β1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3′-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/β-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

A Novel Matrisomal-Related LncRNA Signature Associated With Survival Outcome and Immune Evasion in Patients With Gastric Cancer.

BackgroundDifferent matrisomal patterns are shared across carcinomas. However, little is known about whether there exists a unique tumor matrisome that modulates GC progression and immune regulation.MethodsWe conducted a genome-wide analysis based on matrisomal-related lncRNAs (MRLs) in 375 patients with GC from the Cancer Genome Atlas (TCGA) database. Patients were split into the training set and validation set at a ratio of 1:1 using the R package cart. Pearson correlation analysis (PCA) was performed to identify lncRNAs that correlated with matrisome based on differential expression genes. Subsequently, we performed univariate Cox regression analyses and lasso Cox analysis on these lncRNAs to construct a risk model. Considering the primary effect of GRASLND on the GC prognosis, we chose it for further validation in an experimental setting.ResultsWe identified a 15-MRL signature to predict overall survival and immune cell infiltration of patients with GC. The AUC values to predict 5-year outcome in three sets were 0.89, 0.65, and 0.78, respectively. Further analyses suggested that the high-risk group showed more obvious immune cell infiltration, and demonstrated an immunologically “cold” profile. In vitro, knockdown of GRASLND could inhibit the invasion capability of GC cells, and downregulate the protein expression of crucial matrisomal-related gene MMP9.ConclusionsThe 15-MRL gene signature might serve as a relatively good predictive tool to manage patients with GC.

Abstract 5474: A new class of histone deacetylase inhibitors alleviate breast cancer cells growth and invasion capacity by targeting TIMP/MMP signaling

Abstract Background: Mounting epidemiological and experimental evidences strongly suggest that epigenetic processes including histone modifications are involved in the development of drug resistance in breast cancer. Histone deacetylases (HDACs) selective inhibition is an important strategy of anticancer drug discovery field. However, lack of HDACs isozymes selective inhibitors associated with adverse side effects that limits their clinical applications. We have recently reported new and selective HDACs inhibitors displaying varying HDAC isozyme selectivity degrees that resulted in increased histones acetylation and apoptosis induction in cancer cells. In the present study, we investigated molecular mechanisms underlying anticancer potential of these molecules in breast cancer cells. Experimental methods: Proliferation ability, invasion capacity, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) major group’s gene expressions and protein levels, and the gelatinolytic activity of MMPs were analyzed after treatment of MCF-7 and MDA-MB-231 breast cancer cell lines with HDACs inhibitors compounds. Results and Discussions: Two compounds, identified as MAL-4 and MAL-5, were selected based on the half-maximal inhibitory concentration (IC50) in both breast cancer cells. Treatment of MCF-7 and MDA-MB-231 breast cancer cells with 5 µM MAL-4 and MAL-5 significantly induces TIMP-2 mRNA and protein levels whereas MMP-2 and MMP-9 content and activities were reduced. Further analyses showed that MAL-4 and MAL-5 attenuated invasion capability of breast cancer cells. Conclusion: We have demonstrated that the new compounds can be used as potential lead drug candidates for an effective anti-metastatic therapy mediated by HDAC inhibition in breast cancer. Citation Format: Khuloud Bajbouj, Lina Sahnoon, Abeer AL-Ali, Jasmin Shafarin, Leonie Binder, Reem Abdul Rehman, Rafat El-Awady, Taleb H. Al-Tel. A new class of histone deacetylase inhibitors alleviate breast cancer cells growth and invasion capacity by targeting TIMP/MMP signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5474.

TRG16, targeted by miR-765, inhibits breast cancer stem cell-like properties via regulating the NF-κB pathway.

Previous studies reported that cancer stem cells (CSCs) might be responsible for drug resistance and cancer progression. Transformation-Related Gene 16 Protein (TRG16), a pseudokinase, was reported to be a suppressor in some types of cancer and its overexpression impaired hepatocellular carcinoma cell stemness. However, the function of TRG16 in BC remains unclear. We found that TRG16 expression was significantly downregulated in BC tissues compared with adjacent tissues (n = 40; P < 0.001) and BC patients with lower expression of TRG16 had a worse prognosis. Forced expression of TRG16 inhibited BC stem cell-like properties as evidenced by decreased CD44-positive cells (CSC marker), reduced mammosphere quantity, and downregulated Nanog, aldehyde dehydrogenase, octamer-binding transcription factor 4, and SRY-box transcription factor 2 expression (CSC markers). Moreover, TRG16 overexpression inhibited self-renewal and invasion capabilities of BC cells in vitro as well as tumor growth in vivo but increased cisplatin sensitivity. However, TRG16 silencing had the opposite effects. Further mechanistic studies revealed that TRG16 was targeted and negatively regulated by miR-765, a facilitator of BC progression. TRG16 could suppress the activation of the NF-κB pathway in BC cells, which is a positive pathway in BC progression and contributes to the maintenance of cancer cell stemness. In conclusion, the results above demonstrate that TRG16, negatively regulated by miR-765, may inhibit the BC progression by regulating BC stem cell-like properties and this inhibition may be mediated by the NF-κB pathway. Our findings indicate that TRG16 may be a potential therapeutic targetable node for BC. TRG16, negatively regulated by miR-765, may inhibit the BC progression through regulating BC stem cell-like properties and this inhibition may be mediated by the NF-κB pathway.

Extracellular Vesicles-Encapsulated miR-153-3p Potentiate the Survival and Invasion of Lung Adenocarcinoma.

Extracellular vesicles (EVs) play an essential role in the communication between cells and the tumor microenvironment. However, the effect of tumor-derived EVs on the growth and metastasis of lung adenocarcinoma (LUAD) remains to be explored. This study aimed to elucidate the role of miR-153-3p-EVs in the invasion and migration capabilities of LUAD cells and explore its mechanism through in vivo and in vitro experiments. We found that miR-153-3p was specifically and highly expressed in LUAD and its secreted EVs. Furthermore, the expression of BANCR was negatively regulated by miR-153-3p and identified as a target gene of miR-153-3p using luciferase reporter assays. Through further investigation, we found that the downregulation of BANCR activates the PI3K/AKT pathway and accelerates the process of epithelial-mesenchymal transition (EMT), which ultimately leads to the aggravation of LUAD. The orthotopic xenograft mouse model was established to illustrate the effect of miR-153-3p-EVs on LUAD. Animal studies showed that miR-153-3p-EVs accelerated tumor growth in mice. Besides, we found that miR-153-3p-EVs could damage the respiratory ability of mice and produce a mass of inflammatory cells around the lung tissue of mice. Nevertheless, antagomir-153-3p treatment could inhibit the deterioration of respiratory function and inhibit the growth of lung tumors in mice. In conclusion, our study reveals the potential molecular mechanism of miR-153-3p-EVs in the development of LUAD and provides a potential strategy for the treatment of LUAD.

Pseudogene CLEC4GP1 modulates trophoblast cell apoptosis and invasion via IL-15 inhibition

Preeclampsia (PE) is a pregnancy-associated complication accompanied by gestational hypertension and proteinuria, affecting 2–8% of pregnancies globally. The placental trophoblast cell invasion of decidua and myometrium during early gestation is crucial for healthy placentation. Thus, trophoblast dysfunction might contribute to PE onset. Therefore, further investigations are needed to elucidate the underlying mechanism of trophoblast cell functions. In the present study, we identified a novel pseudogene named C-Type Lectin Domain Family 4 Member G Pseudogene 1 (CLEC4GP1), which was aberrantly expressed in PE placental tissues. In vitro analyses showed that CLEC4GP1 overexpression significantly increased the cell viability and invasiveness and decreased the apoptosis rate of HTR-8/SVneo and JEG-3 cells, while CLEC4GP1 knockdown exerted opposite effects, suggesting the beneficial role of CLEC4GP1 in trophoblast cells. Next, co-expression analysis found that CLEC4GP1 was negatively correlated with Interleukin 15 (IL-15). The expression of IL-15 dramatically increased in PE placental tissues. In HTR-8/SVneo and JEG-3 cells, IL-15 exhibited detrimental effects, opposite to CLEC4GP1, and they were negatively correlated. In addition, CLEC4GP1 attenuates the mRNA stability of IL-16 by inhibiting the binding between human antigen R (HuR) protein and IL-15 RNA. Finally, the obverse effects of CLEC4GP1 and IL-15 were investigated, and results showed that IL-15 reverted CLEC4GP1 induced cellular functions. In brief, these data suggest that CLEC4GP1/IL-15 axis might modulate the occurrence and progression of PE via influencing the trophoblast cell viability, apoptosis, and invasive capability. This study provided cognizance of targeting the CLEC4GP1/IL-15 axis as a novel therapeutic approach to mitigate PE progression.

Engineering tumor-derived small extra cellular vesicles to encapsulate miR-34a, effectively inhibits 4T1 cell proliferation, migration, and gene expression.

Tumor cells produce small extra cellular vesicles-(tsEV) massively, which act as cancer messengers that may also have anti-cancer effects. Based on this knowledge, we hypothesized that we can benefit from 4T1-derived sEVs to amplify the anti-cancer effects of miR-34a-replacement therapy in 4T1 cells. Supernatant of 4T1 cultured cells gathered after 24h of exposure to serum-free media. tsEVs purified by commercial kit and characterized by transmission and scanning electron microscopy, dynamic light scattering, and bicinchoninic acid assay. Modified CaCl2 method applied for miR-34a loading in tsEV (tsEV-miR) and loading confirmation evaluated by the relative expression of miR-34a. MTT, annexin V/PI, cell cycle, scratch test, and real-time PCR were performed for proliferation, apoptosis, invasion, and relative expression of miR-34a target genes after treatment with tsEV/tsEV-miR, respectively. The results indicated that tsEV-miR provides a time-dose-dependent anti-proliferative effect versus tsEV/control group. tsEV-miR could induce apoptosis and arrest the cell cycle at G0/G1 phase, and moreover, it effectively halted the invasion capability of 4T1 cells. Treatment with tsEV-miR down-regulated miR-34a target genes, including B-cell lymphoma-2, vascular endothelial growth factor and its receptor, matrix metalloproteinase-2 and -9, and interleukin-6. Engineered tsEVs can affect different aspects of 4T1 cancer cells including proliferation, apoptosis, cell cycle, migration, and cancer-related gene expression profile. In this regard, tsEV could be considered a proper vehicle for miR-34a replacement therapy and could exacerbate its anti-cancer effects in triple-negative breast cancer. Indeed, TNBC can be targeted by multiple angles by its weapon.

Discoidin domain receptor 1 promotes lung adenocarcinoma migration via the AKT/snail signaling axis.

Discoidin domain receptor 1 (DDR1), a member of receptor tyrosine kinase, has been implicated in tumor progression. However, the function and underlying mechanism of DDR1 in lung adenocarcinoma (LUAD) progression is unclear. Thus, we explored the molecular regulatory mechanism of DDR1 in the migration of LUAD. Transwell assays, wound healing assays and xenograft tumor assays were performed to study the function of DDR1 in the progression of LUAD. Immunoblotting and quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression levels of genes. Co-immunoprecipitation (co-IP) assays were performed to detect the interaction between DDR1 and AKT. Immunofluorescence and immunohistochemistry assays were used to determine the expression level of proteins in cells and tissues, respectively. DDR1 expression was significantly higher in LUAD tissues than in normal lung tissues, and the level of DDR1 was inversely correlated with prognosis in patients. We found that DDR1 promoted the migration and invasion of LUAD cells in vitro. Furthermore, ectopic expression of DDR1 in LUAD cells altered EMT-related markers expression. Importantly, the DDR1 protein interacted with AKT and phosphorylated AKT. The AKT inhibitor MK2206 interrupted Snail upregulation in DDR1-overexpressing LUAD cells. Finally, our study revealed that depletion of DDR1 attenuated LUAD cell migration in a tumor xenograft mouse model. Our findings uncovered that a high abundance of DDR1 increased the migration and invasion capability of LUAD cells via the AKT/Snail signaling axis and indicated that DDR1 could be a potential target for treating LUAD.

Celastrol Inhibits the Proliferation and Induces Apoptosis of Colorectal Cancer Cells via Downregulating NF-κB/COX-2 Signaling Pathways

Colorectal cancer (CRC) is the third-ranked malignant tumor in the world that contributes to the death of a major population of the world. Celastrol, a bioactive natural product isolated from the medicinal plant Tripterygium wilfordii Hook F, has been proved to be an effective anti-tumor inhibitor for multiple tumors. To reveal the therapeutic effect and underlying mechanisms of celastrol on CRC cells. CCK-8 and clonogenic assay were used to analyze the cell proliferation in CRC cells. Flow cytometry analysis was conducted to assess the cell cycle and cell apoptosis. Wound-healing and cell invasion assay were used to evaluate the migrating and invasion capability of CRC cells. The potential antitumor mechanism of celastrol was investigated by qPCR, western blot, and confocal immunofluorescence analyses. Celastrol effectively inhibited CRC cell proliferation by activating caspase-dependent cell apoptosis and facilitating G1 cell cycle arrest in a dose-dependent manner, as well as cell migration and invasion by downregulating the MMP2 and MMP9. Mechanistic protein expression revealed that celastrol suppressed the expression of COX-2 by inhibiting the phosphorylation of NF-κB p65 and subsequently leading to cytoplasmic retention of p65 protein, thereby inhibiting its nuclear translocation and transcription activities. These findings indicate that celastrol is an effective inhibitor for CRC, regulating the NF-κB/COX-2 pathway, leading to the inhibition of cell proliferation characterized by cell cycle arrest and caspase-dependent apoptosis, providing a potential alternative therapeutic agent for CRC patients.

Histone H3K9 methyltransferase SETDB1 augments invadopodia formation to promote tumor metastasis.

Non-small cell lung cancer (NSCLC) is one of leading causes of cancer-related mortality worldwide, which harbors various accumulated genetic and epigenetic abnormalities. Histone methyltransferase SETDB1 is a pivotal epigenetic regulator whose focal amplification and upregulation are commonly detected in NSCLC. However, molecular mechanisms underlying the pro-oncogenic function of SETDB1 remain poorly characterized. Here, we demonstrate that SETDB1 augments the migration and invasion capabilities of NSCLC cells by reinforcing invadopodia formation and mediated ECM degradation. At the molecular level, SETDB1 suppresses the expression of FOXA2, a crucial tumor and metastasis suppressor via coordinated epigenetic mechanisms - SETDB1 not only catalyzes histone H3K9 methylation on FOXA2 genomic locus, but also recruits DNMT3A to regulate DNA methylation on CpG island. Consequently, depletion of Setdb1 in murine lung adenocarcinoma cells completely abolished their full and spontaneous metastatic capabilities in mouse xenograft models. These findings together establish the pro-metastasis activity of SETDB1 in NSCLC and elucidate the underlying cellular and molecular mechanisms.

Circ-CHFR modulates the proliferation, migration, and invasion of ox-LDL-induced human aorta vascular smooth muscle cells through the miR-214-3p/PAPPA axis.

Circular RNAs (circRNAs) are associated with the pathogenesis of human diseases, including atherosclerosis. Here, we undertook to investigate the biological role and mechanism of circRNA E3 ubiquitin-protein ligase (circ-CHFR) in atherosclerosis. The expression levels of circ-CHFR, miR-214-3p, and pregnancy-associated plasma protein A (PAPPA) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in human aorta vascular smooth muscle cells (HA-VSMCs) exposed to oxidized low-density lipoprotein (ox-LDL). Cell proliferation, migration, and invasion capabilities were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), and transwell assays, respectively. The relationship between miR-214-3p and circ-CHFR or PAPPA was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data showed that circ-CHFR was upregulated in HA-VSMCs after stimulation with ox-LDL. Downregulation of circ-CHFR inhibited the proliferation, migration, and invasion of HA-VSMCs exposed to ox-LDL. Mechanistically, circ-CHFR acted as a miR-214-3p sponge, and miR-214-3p was a molecular mediator of circ-CHFR regulation in ox-LDL-stimulated HA-VSMCs. PAPPA was a miR-214-3p target, and circ-CHFR regulated the expression of PAPPA by sponging miR-214-3p. Moreover, overexpression of miR-214-3p repressed the proliferation, migration, and invasion of ox-LDL-induced HA-VSMCs by decreasing PAPPA expression. Our findings suggest that the circ-CHFR/miR-214-3p/PAPPA axis regulates ox-LDL-induced proliferation, migration, and invasion in HA-VSMCs.

Resveratrol Nanoparticles Suppresses Migration and Invasion of Renal Cell Carcinoma Cells by Inhibiting Matrix Metalloproteinase 2 Expression and Extracellular Signal-Regulated Kinase Pathway.

The aim of this study was to examine the impact of Resveratrol nanoparticles on migration/invasion capacity of renal cell carcinoma (RCC) cells and its mechanism. Human RCC cells were exposed to dimethyl sulfoxide or gradient concentrations of Resveratrol nanoparticles respectively, and U0126 were also added in some experiments. We examined renal cell viability by MTT assay, and wound healing test and Transwell assays were used detect invasion and migration capability of RCC cells. We used Western blotting assay to analyze the protein levels in extracellular signal-regulated kinase (ERK) signaling. We also detected the enzymatic capacity of matrix metalloproteinase 2 (MMP-2) in cells by gelatin enzymatic profiling. Resveratrol nanoparticles treatment significantly suppressed cell viability to migrate and invade RCC cells in a dose-dependent manner. Also, notably were reduced MMP-2 activity and expression, and elevated TIMP-2 level were observed in RCC cells exposed with Resveratrol nanoparticles. Further, Resveratrol nanoparticles treatment significantly decreased only the expression of p-ERK1/2, but not p-p38 and p-JNK. Moreover, U0126, which is the ERK inhibitor, exerted similar role as Resveratrol nanoparticles did. Of note was that, combined use of U0126 and Resveratrol nanoparticles displayed a more intense suppression of MMP-2 activity and expression, and also the viability to migrate and invade the RCC cells, compared with Resveratrol nanoparticles treatment alone. The Resveratrol nanoparticles inhibited RCC cells migration and invasion by regulating MMP2 expression and ERK pathways.

MiR-144-3p/miR-451a promotes lymphovascular invasion through repression of PTEN/p19 in rectal neuroendocrine tumors.

Although rectal neuroendocrine tumor (NET-G1) have potential metastatic capability, even among small tumors, no predictive biomarker for invasion and metastasis has been reported. We analyzed microRNA (miRNA) expression profiles in rectal NET-G1 tissues with and without lymphovascular invasion (LVI). Moreover, we then investigated their target genes to clarify the mechanism of invasion/metastasis in NET-G1. miRNA array analysis was performed using seven rectal NET-G1 tissues with LVI and seven without LVI. miRNA expression was confirmed by quantitative real-time PCR. A NET cell line H727 was transfected with miRNA mimic or target gene small interfering RNA, and migration and invasion assays were performed. The expression levels of miR-144-3p and miR-451a were significantly higher in NET-G1 with LVI versus without LVI, as determined by miRNA array analysis and RT-qPCR. A significant correlation was observed between miR-144-3p and miR-451a expression levels, strongly suggesting miR144/451 cluster overexpression in NET-G1 with LVI. Bioinformatic analysis of target genes revealed that miR-144-3p and miR-451a directly interact with PTEN and p19 mRNA, respectively. Immunohistochemistry revealed significantly lower expression of PTEN and p19 in NET-G1 tissues with LVI than in those without LVI. The miR-144-3p and miR-451a mimic significantly increased cell migration/invasion capability, respectively. Knockdown of PTEN and p19 induced significant augmentation of cell invasion and migration capability, respectively. Our data suggest that overexpression of miR-144/miR-451 cluster promotes LVI via repression of PTEN and p19 in rectal NET-G1 cells. miR-144/451 cluster may be a novel biomarker for predicting invasion/metastasis in rectal NET-G1.

ASPM Promotes the Progression of Anaplastic Thyroid Carcinomas by Regulating the Wnt/β-Catenin Signaling Pathway.

Background Abnormal spindle-like microcephaly-associated protein (ASPM) is closely correlated with several malignant tumors, whereas little is known about the role of ASPM in anaplastic thyroid cancer (ATC). Herein, we sought to investigate whether ASPM is involved in the pathogenesis of ATC and the underlying mechanisms. Methods The data from two data sets (GSE76039 and GSE33630) were extracted and analyzed for the expression of ASPM, followed by a further validation in collected ATC patients using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The effect of ASPM on cell proliferation, migration, invasion, and cell cycle was explored in ATC cell lines by in vitro inhibition of ASPM, while ASPM-mediated tumorigenicity was investigated in a xenograft tumor model. The involvement of Wnt/β-catenin signaling pathway was also investigated. Results ASPM was overexpressed in ATC patients and cell lines. In vitro knockdown of ASPM inhibited the proliferation, migration, and invasion capabilities of ATC cells and induced cell cycle arrest. Wnt/β-catenin signaling was suppressed in response to ASPM inhibition, while rescue of β-catenin expression restored the impaired biological functions of ATC cells. In vivo transplantation of ASPM-knockdown cells inhibited the growth of tumors. Conclusions Upregulation of ASPM promotes the malignant properties of ATC cells and contributes to tumorigenesis through the Wnt/β-catenin signaling pathway.

MiR-326 inhibits trophoblast growth, migration, and invasion by targeting PAX8 via Hippo pathway

Preeclampsia (PE), a pregnancy disorder that affects 5–7% of pregnant women, is among the primary causes for maternal and perinatal mortality. PE is believed to be associated with insufficient invasion of villous and extravillous trophoblasts (EVTs), which hampers uterine spiral artery remodeling and finally induces PE. But the mechanism responsible for reduction of trophoblast invasion remains unclear. In this study, placental tissues taken from healthy donors and PE patients were used to evaluate the miR-326 expression; CCK8 and colony formation assays were used to confirm the effect of miR-326 on cell proliferation; transwell assay was used to demonstrate the effect of miR-326 on cell invasion capability; western blot was used to investigate the underlying mechanism; and luciferase assay was used to detect the effect of miR-326 on YAP/TAZ-mediated transcription activity. It was revealed the miR-326 expression was higher in placentas from PE patients than from healthy donors. After transfection of miR-326 mimics, trophoblast proliferation and invasion were impaired. Using TargetScan, we speculated that PAX8 was a target of miR-326, which was later confirmed by western blot. The YAP/TAZ expression was also downregulated after transfection with miR-326. Luciferase assay demonstrated that overexpression of miR-326 suppressed YAP/TAZ-mediated transcription activity by targeting PAX8. Overexpression of PAX8 could partly rescue miR-326-induced suppression of trophoblast proliferation and invasion. Taken together, our result indicated that miR-326 suppresses trophoblast growth, invasion, and migration by means of targeting PAX8 via the Hippo pathway.

Hsa_circ_0005230 is up-regulated and promotes gastric cancer cell invasion and migration via regulating the miR-1299/RHOT1 axis

ABSTRACT Gastric cancer (GC) is one of the most common cancers in the world. Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely expressed in eukaryotic cells. However, their role has been poorly understood in GC. This report aimed to explore the biological functions of hsa_circ_0005230 and its action mechanism in GC. This study validated that hsa_circ_0005230 was significantly up-regulated in 130 cases of GC tissues using qRT-PCR, and clinicopathological feature analysis revealed that its high expression was positively associated with histological grade, lymph node metastasis, TNM stages, and poor prognosis. In vitro, functional experiments showed that silencing hsa_circ_0005230 significantly decreased GC cell proliferation, invasion and migration capabilities. In addition, the major proteins of EMT (epithelial-mesenchymal transition) relevance have changed. In mechanism studies, bioinformatics analyses were used to predict the hsa_circ_0005230/miR-1299/RHOT1 axis and hsa_circ_0005230 may serve as a sponge for miR-1299 and indirectly regulate the expression of RHOT1. The regulated relationships between the molecules on the axis were verified using qRT-PCR and correlation analysis. Dual-luciferase reporter gene assay has been used to verify the binding site between miR-1299 and RHOT1. WB (Western blotting) and IHC (Immunohistochemical) were used to verify that RHOT1 may play the role of oncoprotein and affect the biological behavior of GC. Overall, hsa_circ_0005230 could enhance the EMT phenotype by promoting RHOT1 expression through sponging miR-1299, thus affecting the biological behavior of GC. Hsa_circ_0005230 can be easily identified as a potential diagnostic biomarker and assessment prognosis target for GC.

LncRNA ABHD11-AS1 promotes tumor progression in papillary thyroid carcinoma by regulating EPS15L1/EGFR signaling pathway.

lncRNA ABHD11 antisense RNA 1 (ABHD11-AS1) acts as an oncogene involved in papillary thyroid carcinoma (PTC) occurrence and progression. ABHD11-AS1 exerts biologic functions by some miRNAs and proteins to regulate multiple targets. Identification of novel mechanism of ABHD11-AS1 could be helpful in therapeutic targeting for PTC treatment. Differentially expressed lncRNAs were selected from TCGA database. qRT-PCR analysis was applied to examine the expression of ABHD11-AS1 in PTC cell lines and tissues. The relationship of ABHD11-AS1 expression and clinicopathological features was analyzed by Kaplan-Meier analysis. Two PTC cell lines (TPC-1 and KTC-1) were transfected with pcDNA 3.1, pcDNA3.1-ABHD11-AS1, si-NC and si-ABHD11-AS1, respectively, to verify the ABHD11-AS1 oncogene-regulating capacity to promote tumor progression. The cell metastasis and proliferation had been evaluated both in vitro and in vivo. High expression of ABHD11-AS1 was found in PTC tissues (P < 0.01), which was significantly correlated with lymph node metastasis (P < 0.05). ABHD11-AS1 overexpression noticeably promoted cell proliferation, migration, and invasion capabilities, which were obviously decreased upon ABHD11-AS1 knockdown. ABHD11-AS1 positively regulated EGFR/EPS15L1 pathway, as EGFR, EPS15L1, STAT3, and p-STAT3 were activated. ABHD11-AS1 promotes tumor progression in PTC by regulating EPS15L1/EGFR pathway.

ACADL Functions as a Tumor Suppressor in Hepatocellular Carcinoma Metastasis by Inhibiting Matrix Metalloproteinase 14.

High aggressiveness is the main reason for the poor prognosis of hepatocellular carcinoma (HCC) patients. However, its molecular mechanisms still remain largely unexplored. ACADL, a mitochondrial enzyme that facilitates the primary regulated step in mitochondrial fatty acid oxidation, plays a role in HCC growth inhibition. However, the function of ACADL in tumor metastasis is not well elucidated. We found that the reduced expression of ACADL is closely associated with the loss of tumor encapsulation, extrahepatic metastasis, and poor prognosis in HCC patients. Upregulation of ACADL significantly inhibited HCC migration and invasion ability. Whereas knockdown of ACADL markedly enhanced cell invasive capability. Expression of matrix metalloproteinase-14 (MMP14) was negatively associated with the content of ACADL in HCC specimens. MMP14-positive patients with a low expression of ACADL showed worse outcome. Treatment with MMP14 agonist reversed the inhibitory effect of ACADL on HCC metastasis. In addition, ACADL negatively regulated MMP14 expression by inhibiting the STAT3 signaling pathway, as the sustained activation of STAT3 effectively restored the level of MMP14 in ACADL-overexpressed cells. Collectively, these findings disclose that ACADL represses HCC metastasis via STAT3-MMP14 pathway. This study may propose a promising strategy for the precise treatment of metastatic HCC patients.