Cell Invasion Capacity Research Articles

Diallyl disulfide downregulating RhoGDI2 induces differentiation and inhibit invasion via the Rac1/Pak1/LIMK1 pathway in human leukemia HL-60 cells.

Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.

LncRNA CASC2 Regulate Cell Proliferation and Invasion by Targeting miR-155/SOCS1 Axis in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNAs) have been reported to be involved in the development and progression of various human malignancies. However, the role of lncRNA CASC2 in hepatocellular carcinoma (HCC) remains mostly unknown. The aim of this study was to investigate the potential roles and underlying mechanisms of CASC2 in HCC progression. We found that CASC2 expressions were downregulated in HCC tissue samples and cell lines. The clinical assays revealed that lower levels of CASC2 were associated with the TNM stage, lymph node metastasis, and a poorer prognosis specific to HCC patients. Overexpression of CASC2 inhibited the proliferating, migratory, and invasion capacity of HCC cells. Bioinformatics analysis and the luciferase reporter assay revealed that CASC2 worked as a molecular sponge for miR-155. And CASC2 could upregulate SOCS1 expression by inhibiting miR-155 expression in HCC cells. Furthermore, SOCS1 inhibition partially inverses the suppression effect of cell proliferation, migration, and invasion regulated by CASC2 in Huh7 and HepG2 cells. Taken together, our findings identified CASC2 as a tumor suppressor to inhibit HCC development by regulating the miR-155/SOCS1 axis, and CASC2 might be a potential therapeutic target of HCC for future clinical treatment.

Hsa_circ_0017956 Acts as miR-758-3p Sponge to Facilitate the Progression of Non-small-Cell Lung Cancer by Regulating FOXP4 Expression.

Accumulating studies have demonstrated the important role of circular RNAs (circRNAs) in the progression of different human tumors, including non-small-cell lung cancer (NSCLC). The purpose of this study was to deeply study the function and mechanism of circ_0017956 in NSCLC. Real-time quantitative polymerase chain reaction (RT-qPCR) was applied to detect the expression of circ_0017956, microRNA-758-3p (miR-758-3p), and Forkhead Box P4 (FOXP4). Western blot was performed to determine the protein levels. Cell proliferation was examined by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was used to evaluate the apoptosis of NSCLC cells. Transwell assay was applied to detect cell migratory and invasive capacities. The angiogenesis ability was evaluated by tube formation experiment. The target relationship between miR-758-3p and circ_0017956 or FOXP4 was confirmed by dual-luciferase reporter assay. Animal experiment was conducted to assess the effect of circ_0017956 in vivo. Circ_0017956 and FOXP4 were upregulated, while miR-758-3p was downregulated in NSCLC tissues and cells. Silencing of circ_0017956 significantly suppressed cell proliferation, migration, invasion, and angiogenesis, but promoted cell apoptosis in NSCLC cells. Mechanically, circ_0017956 functioned as a sponge for miR-758-3p and miR-758-3p could directly interact with FOXP4. Moreover, silencing of miR-758-3p or overexpression of FOXP4 could overturn the anticancer influence of circ_0017956 interference on NSCLC cells. Besides that, circ_0017956 knockdown hindered tumor growth in vivo. Altogether, circ_0017956 promoted the progression of NSCLC by regulating FOXP4 through sponging miR-758-3p.

Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling

BackgroundIn the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the “amoeboid angiogenesis” in the absence of proteases activity.MethodsWe first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies.ResultsOur data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn’t boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF.ConclusionsWe underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

Complex mixtures of pesticides and metabolites modulate the malignant phenotype of murine melanoma B16-F1 cells.

Pesticides use increased worldwide with a record in Brazil. Although several works addressed the effects of pesticides on living organisms, only a few considered their mixture, and even fewer tried to unravel their role in tumoral progression. Due to the relevance of cancer, in the present study, the effects of the mixture of pesticides widely used in Brazil (Glyphosate, 2,4-dichlorophenoxyacetic acid, Mancozeb, Atrazine, Acephate, and Paraquat) and their main metabolites (Aminomethylphosphonic Acid, 2,4-diclorophenol, Ethylenethiourea, Desethylatrazine, Methamidophos, and Paraquat) were investigated on the malignancy phenotype of murine melanoma B16-F1 cells after acute (24h) and chronic (15days) exposures. The tested concentrations were based on the Acceptable Daily Intake (ADI) value established by Brazilian legislation. The set of results showed that these chemicals modulate important parameters of tumor progression, affecting the expression of genes related to tumor aggressiveness (Mmp14 and Cd44) and multidrug resistance (Abcb1, Abcc1, and Abcc4), as well as tissue inhibitors of metalloproteinases (Timp1, Timp2, and Timp3). These findings revealed an absence of cytotoxicity but showed modulation of migration, invasion, and colonization capacity of B16-F1 cells. Together, the results point to some negative ways that exposure to pesticides can affect the progression of melanoma and raise a concern related to the increasing trend in pesticide use in Brazil, as the country is one of the major world food suppliers.

Development of leucine rich repeat containing G protein-coupled receptor 5 aptamer based on magnetic assisted rapid aptamer selection platform

Objective: This study aims to screen the aptamers of LGR5 through MARAS platform to regulate the migration, proliferation, and invasion in breast cancer cells. Methods: MARAS platform was used to screen adaptation of LGR5. The expressions of LGR5, β-catenin, c-Myc, Cyclin D1 and DKK1 were detected by RT-qPCR and Western blot. Cell viability was analyzed by MTT assay. The capacity of tumor cell migration and invasion were evaluated using wound healing and transwell assays. Results: Anti-LGR5-APtamer was obtained by screening on MARAS platform. Anti-LGR5-aptamer can remarkably inhibit MDA-MB-231 cells growth, such as proliferation, migration and invasion Mechanically, anti-LGR5-aptamer inhibits β-catenin, C-MyC, Cyclin D1 expression and promotes the expression of DKK1. In addition, the Dox-anti-LGR5-aptamer system can enhance the ability of Dox to enter MDA-MB-231 cells, enabling them to exert tumor suppressive function. Conclusions: Screening of LGR5 aptamers through MARAS platform can effectively inhibit the function of LGR5 in breast cancer cells. In addition, using LGR5 aptamers as biological probes loaded with chemotherapy drugs may provide a future strategy for thereapy of breast cancer.

PVT1/miR-16/CCND1 axis regulates gastric cancer progression.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be a vital modulator in tumorigenesis of gastric cancer (GC). However, the detailed regulatory mechanism of PVT1 in GC remains largely unclear. In this work, the expressions of PVT1 and microRNA-16 (miR-16) were detected by quantitative real-time PCR (qRT-PCR) in GC tissues and cell lines. GC cell lines NCI-N87 and MKN45 cell lines were chosen for the following assays. After PVT1 was overexpressed or depleted, CCK-8 and Transwell assays were performed to examine the cell viability and invasive capacity. Cell cycle was analyzed by flow cytometry. The expression of cyclin D1 (CCND1) at mRNA and protein levels was measured by qRT-PCR and western blot. The competitive endogenous RNA molecular mechanism among PVT1, miR-16 and CCND1 was verified by bioinformatics analysis, luciferase-reporter gene assay and RNA immunoprecipitation assay. In the present study, it was revealed that PVT1 expression was remarkably evaluated in GC tissues and cell lines than that in the corresponding control group. PVT1 positively regulated the proliferation, migration and cell cycle progression of GC cells. Besides, miR-16 was identified as a target of PVT1, and CCND1 was identified as a target of miR-16. The depletion of PVT1 promoted the expression of miR-16 and suppressed CCND1 expression. Moreover, either miR-16 inhibitor or CCND1 overexpression plasmid could reverse the promoting effects of PVT1 on the malignant biological behaviors of GC cells. In conclusion, PVT1 promoted CCND1 expression by negatively regulating miR-16 expression to enhance the viability, invasion and cell cycle progression of GC cells.

Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas' migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis-trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas' migration and invasion abilities.

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation

BackgroundMolecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood.MethodsWe analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems.ResultsWe observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p.DiscussionOur data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.

M2 macrophages enhance endometrial cell invasiveness by promoting collective cell migration in uterine adenomyosis

Research questionAre M2 macrophages implicated in endometrial invasiveness in adenomyosis? DesignSeventeen formalin-fixed paraffin-embedded uterine samples and 16 fresh endometrial biopsies were collected from women with or without adenomyosis. Double immunofluorescence was performed to determine the predominant macrophage population in adenomyosis between M1 and M2 phenotypes. The invasion capacity of endometrial cells was assessed by invasion assays and quantitative polymerase chain reaction for genes involved in cell motility and epithelial–mesenchymal transition (EMT). Specific mechanisms of invasion were investigated by immunohistochemistry for E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). ResultsOnly M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both eutopic endometrium (P = 0.0109) and lesions (P = 0.0267) than healthy tissue. Co-culture with M2 macrophages significantly boosted invasion capacity in endometrial epithelial (P = 0.0002; P = 0.002) and stromal cells (P = 0.0469; P = 0.0047) from both adenomyosis patients and healthy controls. No gene expression differences indicating EMT were noted, either between co-cultured and control cells, or between healthy and adenomyotic cells. E- and N-cadherin protein expression did not differ significantly between endometrium from adenomyosis subjects and healthy tissue but MMP9 expression was increased in eutopic stroma from adenomyosis patients (P = 0.0492). In adenomyosis, both E-cadherin (P = 0.0379) and N-cadherin (P = 0.0196) were more extensively expressed in basal glands than functional glands. ConclusionsM2 macrophages accumulate in adenomyosis and enhance invasion capacity of adenomyotic and even healthy endometrial cells, implying that macrophage infiltration alone may be sufficient to promote the disease. This study failed to detect any changes pointing to EMT, suggesting an alternative mode of invasion. Strong E- and N-cadherin-positive intercellular junctions in basal (invasive) glands suggest the involvement of collective cell migration in the invasion process of endometrium.

Circ_0001686 knockdown suppresses tumorigenesis and enhances radiosensitivity in esophagus cancer through regulating miR-876-5p/SPIN1 axis.

Abnormal expression of circular RNAs (circRNAs) plays an important role in tumorigenesis and radiosensitivity of many cancers. Nevertheless, it is not clear whether circ_0001686 is associated with the development and radiosensitivity of esophagus cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circ_0001686, microRNA-876-5p (miR-876-5p) and spindlin 1 (SPIN1). Counting Kit-8 (CCK-8) assay, EdU assay, flow cytometry and transwell assay were applied to evaluate cell viability, cell proliferation, cell apoptosis and cell invasion capacities. Radiosensitivity was monitored by colony formation assay. The target relationship between miR-876-5p and circ_0001686 or SPIN1 was identified by dual-luciferase reporter assay. The protein level of SPIN1 was measured by western blot assay. Xenograft tumor models were used to analyze the influence of circ_0001686 on radiosensitivity and tumor growth in vivo. The expression levels of circ_0001686 and SPIN1 were increased, while miR-876-5p was decreased in esophagus cancer tissues and cells. Interference of circ_0001686 constrained cell proliferation and invasion, but promoted cell apoptosis and radiosensitivity. Additionally, miR-876-5p was the target of circ_0001686 and miR-876-5p inhibition effectively ameliorated the impacts of circ_0001686 deficiency on tumorigenesis and radiosensitivity. Moreover, SPIN1 was a direct target of miR-876-5p and SPIN1 overexpression partially overturned the effects of miR-876-5p transfection on tumor progression and radiosensitivity. Importantly, circ_0001686 could sponge miR-876-5p to regulate SPIN1 expression. In addition, circ_0001686 silencing also constrained tumor growth and increased radiosensitivity in vivo. Circ_0001686 contributed to the progression and radioresistance of esophagus cancer cells via regulating SPIN1 expression by targeting miR-876-5p, providing a new therapeutic target for improving the prognosis of esophagus cancer patients.

Abstract C043: Aberrant mRNA splice variants as novel prognostic biomarkers for tumor aggressiveness and treatment resistance in African American prostate cancer

Abstract African Americans (AAs) have among the highest incidence of prostate cancer (PCa) and mortality attributable to this disease, and AA PCa disparities remains a clinical challenge. Specifically, AAs are 1.6 times more likely to develop PCa, and 2.4 times more likely to die from this disease compared to European Americans (EAs). A number of studies have shown that higher mortality and recurrence rates of PCa in AA men were observed even after adjustment for socioeconomic factors, suggesting that intrinsic biological difference may account for part of the PCa disparities. Our recent genomic study has identified global splicing events as critical drivers for promoting AA and EA PCa disparities. In this study, we utilized population-specific cell lines as in-vitro AA and EA PCa models to evaluate the molecular functions of the aberrant splice variants involved in cancer aggressiveness and drug resistance. Comprehensive functional assays (such as MTT, apoptosis, invasion, kinase activity assays, and western blotting analysis in PCa cells treated with vehicle or small molecule drugs, etc.) were employed to elucidate the functional roles of an oncogenic splice variant, PIK3CD-S, in AA and EA PCa cells in response to drug treatments. Immunohistochemistry and RNAScope assays were performed to examine the expression profile of PIK3CD-L (full length) and PIK3CD-S variants in an independent PCa cohort derived from AA and EA patients. Higher PIK3CD-S/PIK3CD-L expression ratios were detected in AA PCa cell lines and patient specimens. In addition, the functional validations have indicated that the higher PIK3CD-S/PIK3CD-L expression ratio in AA PCa cells potentially contributes to the higher cell proliferative and invasive capacities. By targeting splicing mechanism and/or PI3K isoforms using specific small molecule inhibitors in AA and EA cell models, we further explored the functional mechanisms of these oncogenic splice variants underlying the drug resistance. In conclusion, our molecular mapping/validation and functional analysis suggest that high PIK3CD-S/PIK3CD-L expression profile may contribute to the PCa aggressiveness and treatment resistance in AA PCa, potentially serving as a precision prognostic biomarker and novel drug target in advanced PCa Citation Format: Siyoung Ha, Himali Gujrati, Bi-Dar Wang. Aberrant mRNA splice variants as novel prognostic biomarkers for tumor aggressiveness and treatment resistance in African American prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C043.

Hyperin promotes proliferation, migration, and invasion of HTR-8/SVneo trophoblast cells via activation of JAK1/STAT3 pathway in recurrent spontaneous abortions

The proliferation of extravillous trophoblasts (EVT) and their further migration, invasion, and differentiation into the decidual and myometrial vasculature are vital for spiral artery remodeling. These physiological functions of EVT are also essential steps in the implantation of the human embryo and the formation of the placenta and are closely related to pregnancy maintenance and the occurrence of abortion. Hyperin is a flavonoid with anti-inflammatory, pro-proliferative, and anti-apoptotic properties. Consequently, we investigated the previously unexplored effects of hyperin on the proliferation, migration, and invasion of HTR-8/SVneo cells. Human extravillous trophoblast-derived HTR-8/SVneo cells were incubated with different concentrations of hyperin (0, 5, 10, 25, 50, and 100 μM) to observe the changes in cell proliferation, migration, invasive capacity, and pathway activation. Proliferation, migration, and invasion were promoted by activating the JAK1/STAT3 pathway in HTR-8/SVneo cells treated with hyperin. In addition, brepocitinib (PF-06700841) significantly inhibited the proliferation, migration, and invasion effects of hyperin on HTR-8/SVneo cells. In vivo experiments confirmed that hyperin reduces the embryo loss rate in recurrent spontaneous abortion (RSA) model mice. Furthermore, our study revealed that hyperin promoted the proliferation, migration, and invasion of HTR-8/SVneo cells via activation of the JAK1/STAT3 pathway, further improving pregnancy outcomes in RSA.

Transwell In Vitro Cell Migration and Invasion Assays.

Cell migration and invasion have essential roles in both normal physiology and disease. As such, methodologies to assess cell migratory and invasive capacities are necessary to elucidate normal cell processes and underlying mechanisms of disease. Here, we describe commonly used transwell in vitro methods for the study of cell migration and invasion. The transwell migration assay involves the chemotaxis of cells through a porous membrane after the establishment of a chemoattractant gradient using two medium-filled compartments. The transwell invasion assay involves the addition of an extracellular matrix on top of the porous membrane which only permits chemotaxis of cells which possess invasive properties such as tumor cells.

Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma

Gliomas are highly aggressive intracranial tumors that are difficult to resect and have high lethality and recurrence rates. According to WHO grading criteria, glioblastoma with wild-type IDH1 has a poorer prognosis than WHO grade 4 IDH-mutant astrocytomas. To date, no effective therapeutic strategies have been developed to treat glioblastoma. Clinical trials have shown that herpes simplex virus (HSV)-1 is the safest and most efficacious oncolytic virus against glioblastoma, but the molecular antitumor mechanism of action of HSV-1 has not yet been determined. Deletion of the γ34.5 and ICP47 genes from a strain of HSV-1 yielded the oncolytic virus, oHSV-1, which reduced glioma cell viability, migration, and invasive capacity, as well as the growth of microvilli. Infected cell polypeptide 4 (ICP4) expressed by oHSV-1 was found to suppress the expression of the transcription factor Sp1, reducing the expression of host invasion-related genes. In vivo, oHSV-1 showed significant antitumor effects by suppressing the expression of Sp1 and invasion-associated genes, highly expressed in high-grade glioblastoma tissue specimens. These findings indicate that Sp1 may be a molecular marker predicting the antitumor effects of oHSV-1 in the treatment of glioma and that oHSV-1 suppresses host cell invasion through the ICP4-mediated downregulation of Sp1.

Single-cell profiling reveals that SAA1+ epithelial cells promote distant metastasis of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers globally, with significant cell heterogeneity and poor prognosis. Distant metastasis in ESCC is one of the key factors that affects the prognosis of patients. Starting with the analysis of ESCC single-cell sequencing data, we constructed a single-cell atlas of ESCC in detail and clarified the cell heterogeneity within tumor tissues. Through analysis of epithelial-mesenchymal transition (EMT) levels, gene expression, and pathway activation, we revealed the existence of a novel subpopulation of SAA1+ malignant cells in ESCC that are highly aggressive and closely associated with distant metastasis of ESCC. In vitro wound healing and transwell assays confirmed a strong invasion capacity of ESCC tumor cells with high expression of SAA1. Then, we constructed an effective and reliable prediction model based on the gene expression pattern of SAA1+ malignant cell subpopulations and confirmed that patients in the high-risk group had significantly worse prognosis than those in the low-risk group in the training cohort, internal verification cohort and external verification cohort. This manuscript contributes to exploration of the heterogeneity of ESCC tumor tissues and the search for new ESCC subpopulations with special biological functions. These results contribute to our understanding of the underlying mechanisms of distant metastasis of ESCC and thus provide a theoretical basis for improved therapies.

MiRNA-576-5p promotes endometrial cancer cell growth and metastasis by targeting ZBTB4

PurposeMicroRNAs (miRNAs) have already been shown to have a strong correlation with the invasion and metastasis capacity of tumor cells. The present research examined the function of miRNA-576-5p (miR-576-5p) in the development of endometrial cancer (EC).MethodsmiR-576-5p and ZBTB4 expression in EC and benign endometrial tissues was measured using quantitative real-time PCR (qRT-PCR) and western blot. To evaluate the proliferation ability of tumor cells in vitro, 2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays were carried out. The effect of miR-576-5p on the proliferation ability of EC cells in vivo was measured by the tumor formation in nude mice. The migration and invasion ability of tumor cells was determined using the transwell assay. To confirm the association between expressions of miR-576-5p and zinc finger and BTB domain containing four (ZBTB4), western blot, qRT-PCR, and luciferase assay were carried out.ResultsmiR-576-5p expression increased significantly in EC samples than in benign endometrial tissues. The level of miR-576-5p was significantly higher in the polymerase ε (POLE) ultramutated subgroup compared to the other three subgroups. High levels of miR-576-5p expression were linked to a shorter progression-free interval time in the copy number high subgroup. Furthermore, upregulated miR-576-5p facilitated EC cell invasion and migration in vitro and promoted the proliferation of EC tumor cell lines both in vitro and in vivo. Moreover, this study showed that the expression of ZBTB4 decreased in patients with EC, and the dual-luciferase reporter assay confirmed that miR-576-5p binds directly to the 3′-UTR of ZBTB4 and inhibits the expression of ZBTB4. An increase in miR-576-5p expression leads to a decrease in the mRNA and protein expression level of ZBTB4. The effects of miR-576-5p can be reversed by overexpression of ZBTB4.ConclusionmiR-576-5p promoted proliferation and metastasis capacity of EC cells by inhibiting ZBTB4 expression. We hypothesized that miR-576-5p could be a prospective therapeutic target for EC.

CircSTK39 suppresses the proliferation and invasion of bladder cancer by regulating the miR-135a-5p/NR3C2-mediated epithelial-mesenchymal transition signaling pathway.

Circular RNAs (circRNAs) serve as novel noncoding RNAs that have crucial functions in the development of tumors, including those from bladder cancer (BCa). However, the role and underlying molecular mechanism of circRNAs in mediating the epithelial-mesenchymal transition (EMT) processes in BCa have yet to be studied. In this research, we first found a novel circRNA, circSTK39 (termed as has_circ_0001079), which was a downregulated gene based on the results of high-throughput RNA sequencing. Subsequently, we determined that the expression of circSTK39 in BCa tissues and their cell lines was significantly reduced. In addition, lower circSTK39 expression was strongly related to a worse prognosis for BCa patients. Next, we detected the biological functions of circSTK39 by using loss and gain experiments in vitro and in vivo. Ectopic expression of circSTK39 decreased cell proliferation, colony formation, and invasion capacities, while circSTK39 knockdown prevented the above phenotypes. Mechanically, circSTK39 could sponge with miR-135a-5p, thus inhibiting NR3C2-mediated EMT processes in the BCa progression. In conclusion, our results revealed that circSTK39 inhibited EMT of BCa cells through the miR-135a-5p/NR3C2 axis and may provide promising biomarkers for the diagnosis or prospective therapeutic targets for BCa.

DARS-AS1 modulates cell proliferation and migration of gastric cancer cells by regulating miR-330-3p/NAT10 axis.

The long noncoding RNA DARS-AS1 was aberrantly expressed and participated in several human cancer progressions, whereas whether DARS-AS1 is involved in human gastric cancer remains unclear. This study aimed to investigate the influence of DARS-AS1 on gastric cancer progression and explore the potential regulatory network of DARS-AS1/miR-330-3p/NAT10. The expression levels of DARS-AS1, miR-330-3p, and NAT10 were measured by quantitative real-time polymerase chain reaction. The CCK-8 assay and Transwell assay were used to determine the cell viability, migration, and invasion capacities, respectively. The target association between miR-330-3p and DARS-AS1 or NAT10 was confirmed using a luciferase reporter assay. In result, DARS-AS1 levels were elevated in tumor tissues and associated with shorter overall survival in patients with gastric cancer. Knockdown of DARS-AS1 could hamper cell viability, migration, and invasion in gastric cancer cells. DARS-AS1 acts as a competitive endogenous RNA to regulate the NAT10 expression by sponging miR-330-3p in gastric cancer cells. In conclusion, DARS-AS1 was elevated in gastric cancer, and DARS-AS1/miR-330-3p/NAT10 signaling offered some new horizons for predicting prognosis and a novel therapeutic method for the treatment of gastric cancer.

Identification of VASH1 as a Potential Prognostic Biomarker of Lower-Grade Glioma by Quantitative Proteomics and Experimental Verification.

VASH1 is a novel angiogenic regulatory factor, that participates in the process of carcinogenesis and the development of diverse tumors. Our study aimed to investigate the expression and prognostic value of the VASH1 in Lower-Grade Glioma (LGG), to explore its functional network in LGG and its effects on biological behaviors. LGG transcriptome data, somatic mutation profiles and clinical features analyzed in the present study were obtained from the TCGA, GTEx, CCLE, CGGA, UALCAN, and GEPIA2 databases, as well as clinical data and tissue sections of 83 LGG patients in our hospital. The expression characteristics of VASH1 in LGG were investigated by univariate, multivariate, immunohistochemistry, qRT-PCR, and western-blot. Subsequently, we analyzed the prognostic significance of VASH1 in LGG patients by survival analysis, subject operation characteristic curve, correlation analysis, external validation, independent prognostic significance analysis, and clinical stratification, and confirmed its biological effect on glioma cell lines in vitro. Finally, we performed GO, KEGG, and GSEA to clarify biological functions and related pathways. CIBERSORT and ESTIMATE algorithms were used to calculate the proportion of immune cells and immune microenvironment fraction in LGG. We found that VASH1 is highly expressed in LGG tissues and is associated with poor prognosis, WHO grade, IDH1 wild-type, and progressive disease (P < 0.05). Multivariate and the Nomogram model showed that high VASH1 expression was an independent risk factor for glioma prognosis and had better prognostic prediction efficacy in different LGG Patient cohorts (HR = 4.753 and P=0.002). In vitro experiments showed that knockdown of VASH1 expression in glioma cell lines caused increased glioma cell proliferation, invasion, and migration capacity. The mechanism may be related to VASH1 promoting microtubule formation and remodeling of immune microenvironment. Our study firstly found that high VASH1 expression was associated with poor prognosis. In addition, We identified the possible mechanism by which VASH1 functioned in LGG. VASH1 inhibits the invasion and migration of tumor cells by affecting microtubule formation and immune infiltration in the tumor microenvironment. May be an important endogenous anti-tumor factor for LGG and provide a potential biomarker for individualized treatment of LGG.