Actin Filament-associated Protein Research Articles

Roles of AFAP1-AS1 in Gynecology and Urogenital System.

Human disease onset and progression are strongly associated with aberrant long noncoding RNA (lncRNA) expression, highlighting the functional regulatory role of lncRNA. Actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1), a member of lncRNAs, is located on the antisense strand of Actin filament-associated protein 1 (AFAP1). We conducted a comprehensive review of AFAP1-AS1's functions in gynecology and urogenital systems using the "PubMed" database. Our analysis reveals that AFAP1-AS1 is overexpressed and engages in the initiation and process of gynecological and urogenital diseases. The regulatory mechanisms employed by AFAP1-AS1 involve four major strategies: gene-level effects, competition for microRNA (miRNA) repression, protein binding, participation in signaling networks that influence cellular processes such as proliferative phenotype, migration, invasiveness, epithelial-mesenchymal transition (EMT), cycle regulation, drug resistance, and more. Furthermore, AFAP1-AS1 is implicated in guiding clinicopathological characteristics. AFAP1-AS1 holds promise as a potent diagnostics and treatment option for gynecological and genitourinary systems in the future.

Hypoxia-induced AFAP1L1 regulates pathological neovascularization via the YAP-DLL4-NOTCH axis

BackgroundPathological neovascularization plays a pivotal role in the onset and progression of tumors and neovascular eye diseases. Despite notable advancements in the development of anti-angiogenic medications that target vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), the occurrence of adverse reactions and drug resistance has somewhat impeded the widespread application of these drugs. Therefore, additional investigations are warranted to explore alternative therapeutic targets. In recent years, owing to the swift advancement of high-throughput sequencing technology, pan-cancer analysis and single-cell sequencing analysis have emerged as pivotal methodologies and focal areas within the domain of omics research, which is of great significance for us to find potential targets related to the regulation of pathological neovascularization.MethodsPan-cancer analysis and scRNA-seq data analysis were employed to forecast the association between Actin filament-associated protein 1 like 1 (AFAP1L1) and the development of tumors and endothelial cells. Tumor xenograft model and ocular pathological neovascularization model were constructed as well as Isolectin B4 (IsoB4) staining and immunofluorescence staining were used to assess the effects of AFAP1L1 on the progression of neoplasms and neovascular eye diseases in vivo. Transwell assay, wound scratch assay, tube forming assay, three-dimensional germination assay, and rhodamine-phalloidin staining were used to evaluate the impact of AFAP1L1 on human umbilical vein endothelial cells (HUVECs) function in vitro; Dual luciferase reporting, qRT-PCR and western blot were used to investigate the upstream and downstream mechanisms of pathological neovascularization mediated by AFAP1L1.ResultsOur investigation revealed that AFAP1L1 plays a crucial role in promoting the development of various tumors and demonstrates a strong correlation with endothelial cells. Targeted suppression of AFAP1L1 specifically in endothelial cells in vivo proves effective in inhibiting tumor formation and ocular pathological neovascularization. Mechanistically, AFAP1L1 functions as a hypoxia-related regulatory protein that can be activated by HIF-1α. In vitro experiments demonstrated that reducing AFAP1L1 levels can reverse hypoxia-induced excessive angiogenic capacity in HUVECs. The principal mechanism of angiogenesis inhibition entails the regulation of tip cell behavior through the YAP-DLL4-NOTCH axis.ConclusionIn conclusion, AFAP1L1, a newly identified hypoxia-related regulatory protein, can be activated by HIF-1α. Inhibiting AFAP1L1 results in the inhibition of angiogenesis by suppressing the germination of endothelial tip cells through the YAP-DLL4-NOTCH axis. This presents a promising therapeutic target to halt the progression of tumors and neovascular eye disease.

Prognostic value of the long noncoding RNA AFAP1-AS1 in cancers*

Abstract Objective This meta-analysis explored whether the expression of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) is related to the prognosis and clinicopathological features of patients with cancer. Methods PubMed, EMBASE, and Cochrane Library were systematically searched. Hazard ratios (HRs) with 95% confidence intervals (CIs) were used to assess the prognostic value based on overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). Odds ratios (ORs) with 95% CIs were used to determine the relationships between AFAP1-AS1 and clinicopathological features, such as large tumor size (LTS), high tumor stage (HTS), poor histological grade (PHG), lymph node metastasis (LNM), and distant metastasis (DM). Results Thirty-five eligible articles and 3433 cases were analyzed. High AFAP1-AS1 expression, compared to low AFAP1-AS1 expression, correlated with significantly shorter OS (HR = 2.15, 95% CI = 1.97-2.34, P < 0.001), DFS (HR = 1.37, 95% CI = 1.19-1.57, P < 0.001), and PFS (HR = 1.97, 95% CI = 1.56-2.50, P < 0.001) in patients with cancer. In various cancers, elevated AFAP1-AS1 expression was significantly associated with LTS (OR = 2.76, 95% CI = 2.16-3.53, P < 0.001), HTS (OR = 2.23, 95% CI = 1.83-2.71, P < 0.001), and PHG (OR = 1.39, 95% CI = 1.08-1.79, P = 0.01) but not LNM (OR = 1.59, 95% CI = 0.88-2.85, P = 0.12) or DM (OR = 1.81, 95% CI = 0.90-3.66, P = 0.10). Conclusion High AFAP1-AS1 expression was associated with prognostic and clinicopathological features, suggesting that AFAP1-AS1 is a prognostic biomarker for human cancers.

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocytophilum protein) and found that it dynamically changed its pattern and subcellular location in cells and enhanced cell adhesion. Tandem affinity purification combined with mass spectrometry identified host nucleolin as an AFAP-interacting protein. Further study showed the disruption of nucleolin by RNA interference, and the treatment of a nucleolin-binding DNA aptamer AS1411 attenuated AFAP-mediated cell adhesion, indicating that AFAP enhanced cell adhesion in a nucleolin-dependent manner. The characterization of cell adhesion-enhancing AFAP and the identification of host nucleolin as its interaction partner may help understand the mechanism underlying A. phagocytophilum-promoting cell adhesion, facilitating the elucidation of HGA pathogenesis.

AFAP1L1 promotes gastric cancer progression by interacting with VAV2 to facilitate CDC42-mediated activation of ITGA5 signaling pathway

BackgroundThe actin filament-associated protein (AFAP) family genes include AFAP1/AFAP-110, AFAP1L1 and AFAP1L2/XB130. Increasing evidence indicates these three AFAP family members participate in tumor progression, but their clinical significance and molecular mechanisms in gastric cancer (GC) remain unclear.MethodsWe first analyzed expression of AFAP family genes using public datasets and verified the results. The clinical significance of AFAP family genes in GC patients was also analyzed. In vitro and in vivo experiments were applied to explore the function of AFAP1L1. Enrichment analysis was used to explore potential molecular mechanisms. We then performed additional experiments, such as cell adhesion assay, co-immunoprecipitation and so on to confirm the downstream molecular mechanisms of AFAP1L1.ResultsPublic data analyses and our verification both showed AFAP1L1 was the only AFAP family members that was significantly upregulated in GC compared with normal gastric tissues. Besides, only AFAP1L1 could predict poor prognosis and act as an independent risk factor for GC patients. In addition, AFAP1L1 promotes GC cells proliferation, migration, invasion in vitro and tumor growth, metastasis in vivo by inducing epithelial-to-mesenchymal transition (EMT). In terms of mechanism, AFAP1L1 interacts with VAV guanine nucleotide exchange factor 2 (VAV2) to activate Rho family GTPases CDC42, which finally promotes expression of integrin subunit alpha 5 (ITGA5) and activation of integrin signaling pathway.ConclusionAFAP1L1 promotes GC progression by inducing EMT through VAV2-mediated activation of CDC42 and ITGA5 signaling pathway, indicating AFAP1L1 may be a promising prognostic biomarker and therapeutic target for GC patients.

Role of Semaphorin 3D in Inhibition of Colorectal Cancer Cell Migration by Regulating Actin Filament Associated Protein 1 Like 1

Role of Semaphorin 3D in Inhibition of Colorectal Cancer Cell Migration by Regulating Actin Filament Associated Protein 1 Like 1

Regulations of Exosomal-Transmitted AFAP1-AS1 LncRNA on Ovarian Cancer Cell Migration and Invasion.

Adolescent ovarian cancer (OC) has high malignancy. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of various malignancies, but their role in adolescent OC remains poorly understood. This study aims to assess the modulatory role of Exosome-transmitted lncRNA Actin filament-associated protein 1 Antisense RNA 1 (AFAP1-AS1) on the activity of OC cells. We recruited a cohort of 40 adolescent patients diagnosed with OC and a control group of 40 healthy individuals. Serum samples were collected from both groups prior surgical intervention. Exosomes from peripheral blood and ascites were collected via differential centrifugation. The expression levels of AFAP1-AS1 in OC tissues and cell lines (IOSE-80, CAOV3, and SKOV3) were quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The exosomal particle size and surface markers were characterized through nanoparticle tracking analysis and transmission electron microscopy. Furthermore, siRNA-mediated knockdown of AFAP1-AS1 was performed in IOSE-80, CAOV3, and SKOV3 cell lines. Functional assays, including wound-healing experiments and Transwell migration assays, were conducted to evaluate cellular migration and metastasis. Our findings demonstrate that the expression of AFAP1-AS1 is significantly upregulated in OC patients' serum exosomes and ascitic fluid, correlating with unfavorable pathological features such as advanced federation international of gynecology and obstetrics (FIGO) stage and larger tumor diameter. In-vitro experiments revealed that OC cell lines and primary human OC cells showed enhanced proliferation and metastasis when exposed to ascites-derived exosomes enriched in AFAP1-AS1. Importantly, we observed that AFAP1-AS1 can be transmitted to neighboring cells via exosomal pathways. Additionally, exosomes isolated from ascites treated with siRNA targeting AFAP1-AS1 can inhibit cellular migration and invasion. Our data provide evidence for the oncogenic role of AFAP1-AS1, which is transmitted via exosomes. This finding has significant implications for understanding the molecular mechanisms of AFAP1-AS1 in the pathogenesis of adolescent ovarian cancer.

High expression of AFAP1-AS1 is associated with poor prognosis of digestive system cancers: A meta-analysis.

Background:Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) is associated with prognosis in many cancers. The aim of this study was to systematically evaluate the potential correlation between AFAP1-AS1 and the prognosis of digestive system cancers (DSC).Methods:EMBASE, Web of Science, Cochrane Library, PubMed, Wanfang Data (Chinese), and CNKI (Chinese) were comprehensively searched for literature published from the establishment of the database to September 2021.All case-control studies that met the inclusion criteria were retrieved; additionally manual retrieval and literature tracing was performed. After extracting the relevant data, Revman 5.3.5 software was used for meta-analysis.Results:Eighteen studies were included in analyses, high expression of AFAP1-AS1 was significantly correlated with poor prognosis in DSC, including overall survival (HR = 1.93, 95% CI: 1.72–2.17, P < .001) and disease-free survival/progression-free survival (HR = 1.87, 95% CI: 1.56–2.26, P < .001). In addition, the expression of AFAP1-AS1 was significantly correlated with tumor size, tumor stage, and lymph node metastasis.Conclusion:High expression of AFAP1-AS1 was associated with poor prognosis in DSC. Therefore, it could be used as a potential marker for evaluating prognosis in DSC

AFAP1 antisense RNA 1 promotes retinoblastoma progression by sponging microRNA miR-545-3p that targets G protein subunit beta 1

ABSTRACT The oncogenic role of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) has been reported in retinoblastoma (RB). However, the underlying regulatory mechanisms remain poorly understood. In this study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were performed to analyze the expression of AFAP1-AS1, microRNA miR-545-3p, or G protein subunit beta 1 (GNB1). Cell Counting Kit-8 (CCK-8) and Transwell migration assays were used to detect cell proliferation and migration. In addition, caspase-3 activity was monitored by caspase-3 activity assay. Luciferase reporter assays combined with RNA immunoprecipitation (RIP) and pull-down assays were performed to elucidate the target relationship between miR-545-3p and AFAP1-AS1 or GNB1. Xenograft tumor experiments were performed to evaluate RB cell growth in vivo. Increased AFAP1-AS1 and GNB1 expression in RB tissues and cells was confirmed by RT-qPCR; conversely, miR-545-3p was found to be downregulated in RB tissues and cells. AFAP1-AS1 overexpression resulted in increased proliferation and migration of RB cells, whereas AFAP1-AS1 silencing resulted in decreased proliferation and migration of RB cells. Moreover, AFAP1-AS1 was found to target miR-545-3p. The anti-miR-545-3p treatment phenocopied the effect of AFAP1-AS1 overexpression and promoted RB cell growth in vivo. miR-545-3p was found to directly target GNB1. GNB1 silencing resulted in reduced proliferation and migration of RB cells and attenuated the oncogenic effect of the miR-545-3p inhibitor. Thus, in this study, a novel ceRNA regulation network of AFAP1-AS1 in RB was identified, where AFAP1-AS1 regulated GNB1 expression by targeting miR-545-3p, ultimately driving RB progression.

Long non-coding ribonucleic acid AFAP1-AS1 promotes chondrocyte proliferation via the miR-512-3p/matrix metallopeptidase 13 (MMP-13) axis

ABSTRACT Long-chain non-coding RNAs are reported to be involved in cartilage damage. However, less research on the role of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) in osteoarthritis. To investigate AFAP1-AS1 function in osteoarthritis development, AFAP1-AS1 and miR-512-3p expression levels in osteoarthritis cartilage and cells were evaluated using RT-qPCR. The downstream target genes of AFAP1-AS1 and miR-512-3p were predicted and validated using luciferase reporter assays. Moreover, a knee osteoarthritis model was established by injecting monoiodoacetate into the knee joints of mice. The effects of AFAP1-AS1 and miR-512-3p on osteoarthritis chondrocyte proliferation and MMP-13, collagen II, and collagen IV expressions were detected in vivo using CCK-8 assay and Western blotting and RT-qPCR, respectively. AFAP1-AS1 expression was upregulated in osteoarthritis cartilage and cells. MiR-512-3p expression was downregulated in osteoarthritis cartilage. AFAP1-AS1 overexpression inhibited miR-512-3p expression in chondrocytes. Furthermore, AFAP1-AS1 over-expression promoted chondrocyte proliferation, and miR-512-3p mimic inhibited chondrocyte proliferation in vivo. AFAP1-AS1 overexpression reduced type II and type IV collagen expression, while miR-512-3p overexpression promoted type II and type IV collagen in vivo. AFAP1-AS1 overexpression enhanced MMP-13 expression in vivo. AFAP1-AS1 overexpression regulated chondrocyte proliferation by inhibiting miR-512-3p expression in vivo. AFAP1-AS1 could be a potential target to treat osteoarthritis by inhibiting miR-512-3p and subsequently inducing chondrocyte proliferation and regulating matrix synthesis.

Role of calponin 2 in cell migration and mechanoregulation

Calponin is a family of actin filament-associated regulatory proteins and functions via inhibiting actin-activated myosin ATPase activity. Among three calponin isoforms in vertebrates, calponin 2 encoded by Cnn2 gene expresses in smooth muscles and non-muscle cells such as monocytes/macrophages, endothelial cells, epithelial cells, fibroblasts, myoblasts, and lung alveolar cells. Our group previously reported that calponin 2 regulates the rate of cell proliferation and migration by stabilizing the actin cytoskeleton.

XB130 Plays an Essential Role in Folliculogenesis Through Mediating Interactions Between Microfilament and Microtubule Systems in Thyrocytes.

Background: XB130 (actin filament-associated protein 1-like 2, AFAP1L2) is a thyroid-abundant adaptor/scaffold protein. Xb130-/- mice exhibit transient growth retardation postnatally due to congenital hypothyroidism with diminished thyroglobulin iodination and release at both embryonic and early postnatal stages due to disorganized thyroid apical membrane structure and function. We hypothesized that XB130 is crucial for polarity and folliculogenesis by mediating proper cytoskeletal structure and function in thyrocytes. Methods: Primary thyrocytes isolated from thyroid glands of Xb130-/- mice and their wild-type littermates at postnatal week 2 were cultured in 10% Matrigel for different time periods. Folliculogenesis was studied with immunofluorescence staining, followed by confocal microscopy. Cells were also transfected to express human XB130 fused Green Fluorescent Protein (XB130-GFP) or Green Fluorescent Protein (GFP) only before morphological analysis. Cytoskeletal structures from embryo and postnatal thyroid glands were also studied. Results: In three-dimensional cultures of thyrocytes, XB130, aligned with actin filaments, participated in defining the site of apical membrane formation and coalescence to form a thyroid follicle lumen. Xb130-/- thyrocytes displayed delayed folliculogenesis, reduced recruitment of a microtubule (MT)-associated proteins, and disorganized acetylated tubulin under the apical membrane, resulting in delayed folliculogenesis with reduced efficiency in formation of the thyroid follicle lumen. Conclusions: XB130 critically regulates thyrocyte polarization by functioning as a link between the actin filament cortex and MT network at the apical membrane of thyrocytes. Defects of adaptor scaffold proteins may affect cellular polarity and cytoskeletal structure and function and result in disorders of epithelial function, such as congenital hypothyroidism.

Long non-coding RNA AFAP1-AS1 promotes thyroid cancer progression by sponging miR-204-3p and upregulating DUSP4.

Long non-coding RNA actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1) shows crucial regulatory function in tumor progression. Nonetheless, the biological function and underlying mechanism of AFAP1-AS1 in the progression of thyroid cancer is still unclear. Expressions of AFAP1-AS1, miR-204-3p and DUSP4 were quantified utilizing quantitative real-time polymerase chain reaction and/or western blot. In loss-of-function and gain-of-function assays, cell proliferation, migration and invasion were appraised by CCK-8 assay, wound healing assay, Transwell migration and invasion assays, respectively. Luciferase reporter assay was employed for validating the interaction between miR-204-3p and AFAP1-AS1 or the 3'UTR of dual specificity phosphatase 4 (DUSP4). AFAP1-AS1 was highly expressed in thyroid cancer tissues and cell lines. Highly expressed AFAP1-AS1 was in association with advanced TNM stage and positive lymph node metastasis. Knockdown of AFAP1-AS1 suppressed the proliferation, migration and invasion of thyroid cancer cells, and overexpression of AFAP1-AS1 induced a reversed effect. MiR-204-3p was targetedly repressed by AFAP1-AS1, and miR-204-3p could negatively regulate DUSP4 expression. AFAP1-AS1 augmented the expression of DUSP4 via repressing miR-204-3p, and the effects of AFAP1-AS1 overexpression on thyroid cancer cells were also partly abolished by miR-204-3p restoration. In summary, AFAP1-AS1 facilitates thyroid cancer cell proliferation, migration and invasion by regulating miR-204-3p/DUSP4 axis.

LncRNA AFAP1-AS1/miR-27b-3p/VEGF-C axis modulates stemness characteristics in cervical cancer cells.

Background:Long non-coding RNA (lncRNA) actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) functions as a competing endogenous RNA to regulate target genes expression by sponging microRNAs (miRs) to play cancer-promoting roles in cancer stem cells. However, the regulatory mechanism of AFAP1-AS1 in cervical cancer (CC) stem cells is unknown. The present study aimed to provide a new therapeutic target for the clinical treatment of CC.Methods:Hyaluronic acid receptor cluster of differentiation 44 variant exon 6 (CD44v6)(+) CC cells were isolated by flow cytometry (FCM). Small interfering RNAs of AFAP1-AS1 (siAFAP1-AS1) were transfected into the (CD44v6)(+) cells. The levels of AFAP1-AS1 were measured by quantitative real-time PCR (qRT-PCR). Sphere formation assay, cell cycle analysis, and Western blotting were used to detect the effect of siAFAP1-AS1. RNA pull-down and luciferase reporter assay were used to verify the relationship between miR-27b-3p and AFAP1-AS1 or vascular endothelial growth factor (VEGF)-C.Results:CD44v6(+) CC cells had remarkable stemness and a high level of AFAP1-AS1. However, AFAP1-AS1 knockdown with siAFAP1-AS1 suppressed the cell cycle transition of G(1)/S phase and inhibited self-renewal of CD44v6(+) CC cells, the levels of the stemness markers octamer-binding transcription factor 4 (OCT4), osteopontin (OPN), and cluster of differentiation 133 (CD133), and the epithelial-mesenchymal transition (EMT)-related proteins Twist1, matrix metalloprotease (MMP)-9, and VEGF-C. In the mechanism study, miR-27b-3p/VEGF-C signaling was demonstrated to be a key downstream of AFAP1-AS1 in the CD44v6(+) CC cells.Conclusions:LncRNA AFAP1-AS1 knockdown inhibits the CC cell stemness by upregulating miR-27b-3p to suppress VEGF-C.

Identification of Actin Filament-Associated Proteins in Giardia lamblia.

ABSTRACTThe deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti-GlActin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial.IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin (GlActin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining GlActin’s importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia’s host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

LncRNA AFAP1-AS1 Promotes the Progression of Colorectal Cancer through miR-195-5p and WISP1.

Objective Colorectal cancer (CRC) is the most common cancer. But, the molecular mechanisms of CRC progression are not fully understood. This study was conducted to explore how the long noncoding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) participates in CRC progression through the regulation of microRNA-195-5p (miR-195-5p) and wingless-type inducible signaling pathway protein-1 (WISP1). Methods The expressions of AFAP1-AS1, miR-195-5p, and WISP1 were detected by RT-qPCR or western blot. A dual-luciferase assay confirmed the target relationship of AFAP1-AS1, miR-195-5p, and WISP1. Colony formation, wound-healing, and Transwell assays were used to detect the growth, migration, and invasion abilities of cells, respectively. Results AFAP1-AS1 and WISP1 expressions were notably increased, and miR-195-5p expression was markedly reduced in CRC. The dual-luciferase assay verified that AFAP1-AS1 could bind to miR-195-5p. AFAP1-AS1 knockdown could inhibit the malignant behavior of CRC cells. miR-195-5p could target and regulate WISP1 expression. Overexpression of WISP1 or miR-195-5p inhibition reversed the inhibition effect of AFAP1-AS1 knockdown on the biological activity of CRC cells. Conclusions AFAP1-AS1 knockdown may inhibit the proliferation, migration, and invasion of CRC cells through the miR-195-5p/WISP1 axis.

Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer

Long noncoding ribonucleic acids (LncRNAs) have been found to be involved in the proliferation, apoptosis, invasion, migration, and other pathological processes of triple-negative breast cancer (TNBC). Expression of the lncRNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) has been found to be significantly higher in TNBC than in other subtypes or in normal tissue samples, but the specific mechanism by which AFAP1-AS1 affects the occurrence and development of TNBC is yet to be revealed. In this study, we used Cell Counting Kit-8 (CCK-8), colony formation, wound healing migration, Transwell invasion, and nude mouse xenograft assays to confirm the role of AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. In addition, we performed bioinformatics analyses, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and dual-luciferase reporter assays (dual-LRA) to confirm interaction among AFAP1-AS1, micro-RNA 2110 (miR-2110), and Sp1 transcription factor (Sp1). We found that silencing AFAP1-AS1 and Sp1 or upregulating miR-2110 suppressed the proliferation, migration, and invasion of MDA–MB-231 and MDA–MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-LRA highlighted that miR-2110 was an inhibitory target of AFAP1-AS1, and that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reversed by joint transfection with miR-2110 inhibitor. Our findings demonstrated that AFAP1-AS1 could modulate the progression of breast cancer cells and affect tumorigenesis in mice by acting as a miR-2110 sponge, resulting in regulation of Sp1 expression. Therefore, AFAP1-AS1 could play a pivotal role in the treatment of TNBC.

Calponin 2 Regulates the Rate of Cytokinesis

Actin microfilament is a major component of the cytoskeleton and the basis of various cell motility functions. Calponin 2 is an actin filament-associated regulatory protein that inhibits the rates of cell migration, phagocytosis and proliferation. To investigate the mechanisms of calponin 2 inhibition of cell proliferation, here we demonstrate that the levels of calponin 2 in NIH/3T3 and HEK-293 cells rapidly decrease prior to cell division with a removal of calponin 2 at the actin contractile ring during cytokinesis.

Identification and characterization of an actin filament-associated Anaplasma phagocytophilum protein

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA) is an obligate intracellular Gram-negative bacterium. During intracellular replication, A. phagocytophilum interacts with many host cell components including actin cytoskeleton. However the bacterial factors contributing to the interaction between A. phagocytophilum and actin filaments remain unknown. In this study we identified a novel type IV secretion system substrate of A. phagocytophilum by employing TEM-1 β-lactamase based protein translocation assay, and found it is an actin filament-associated protein. Here, we name this protein as an actin filament-associated Anaplasma phagocytophilumprotein (AFAP). Further analysis showed that the middle region of AFAP harboring four tandem repeats is involved in its interaction with actin filaments. The identification and characterization of an actin filament-associated A. phagocytophilum protein in this study may help understand the interaction between A. phagocytophilum and actin cytoskeleton of its host cells, facilitating the elucidation of HGA pathogenesis.

Silencing lncRNA AFAP1-AS1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma Cells via Regulating the miR-498/VEGFA Axis.

PurposeIn view of the continuous increase of the mortality rate, esophageal squamous cell carcinoma (ESCC) develops into a major health concern. In this study, we aimed to investigate the underlying mechanism of long noncoding RNA (lncRNA) actin filament-associated protein 1 antisense RNA (AFAP1-AS1)/microRNA-498 (miR-498)/vascular endothelial growth factor A (VEGFA) in ESCC cells.MethodsThe expression levels of AFAP1-AS1, miR-498 and VEGFA in ESCC tissues and cells were detected using quantitative real-time polymerase chain reaction (qRT-PCR). The effects of AFAP1-AS1 on ESCC cells proliferation and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. Transwell assay was carried out to determine cell migration. In addition, VEGFA and cell behaviors-related proteins were determined by Western blot analysis. The targeted relationships of AFAP1-AS1 were verified by dual-luciferase reporter and RNA pull-down assays.ResultsThe expression levels of lncRNA AFAP1-AS1 and VEGFA mRNA were upregulated, but miR-498 was downregulated in ESCC tissues and cells. Moreover, miR-498 was directly targeted by AFAP1-AS1 and there was a negative correlation between miR-498 and AFAP1-AS1. Functionally, AFAP1-AS1 silencing inhibited the proliferation and migration and induced apoptosis of ESCC cells. Interestingly, miR-498 inhibition rescued the effects of AFAP1-AS1 knockdown on cell proliferation, apoptosis and migration and restored the expression levels of tumor-developing marker proteins of AFAP1-AS1 silencing in Eca109 and KYSE-30 cells. Furthermore, VEGFA was verified as a direct target of miR-498 and reversed the effects of miR-498 overexpression on cell behaviors of ESCC in vitro.ConclusionDownregulation of AFAP1-AS1 impeded the proliferation and migration and induced apoptosis of ESCC cells by regulating miR-498/VEGFA axis, which might serve as a novel biomarker for the diagnosis and treatment of ESCC.