Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation

BackgroundEzrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear.MethodsDistribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis.ResultsEzrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo.ConclusionEzrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.     

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.     

The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression

BackgroundOsteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma.MethodsMiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human).ResultsMiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells.ConclusionsDown-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients.     

STIM1调控细胞运动促进骨肉瘤转移的研究

目的:明确STIM1是否参与调控细胞运动促进骨肉瘤的转移。方法:应用靶向STIM1的si RNA沉默MG-63骨肉瘤细胞中STIM1的表达,然后用侵袭实验、迁移实验以及黏附实验检测骨肉瘤细胞侵袭、迁移与黏附能力的变化,采用Western Blot检测细胞FAK和paxillin的表达及Rac1和RhoA信号通路的活性。结果:转染靶向STIM1的si RNA后,MG-63骨肉瘤细胞中STIM1的蛋白表达和m RNA表达均明显降低(P0.05),细胞的侵袭、迁移与黏附能力均显著下降(P0.05),细胞伪足与细胞骨架的重要组分FAK和paxillin的表达及调控细胞运动的Rac1和RhoA信号通路活性均显著降低(P0.05)。结论:STIM1可能通过激活RhoA和Rac1的信号通路,增加FAK和paxillin的表达,从而调控骨肉瘤细胞运动,促进骨肉瘤转移。     

钙通道蛋白Orai1在骨肉瘤细胞转移中的作用研究

目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,并对维持肿瘤细胞粘附、侵袭、迁移等恶性表型有非常重要的作用。然而,钙通道蛋白Orai1是否参与了骨肉瘤的转移过程,目前未见相关报道。本研究的目的是探究钙通道蛋白Orai1是否在骨肉瘤转移过程中的发挥作用。方法:利用合成的靶向Orai1的小干扰RNA(Orai1 si RNA)片段,转染至人骨肉瘤细胞系Saos-2细胞。在Saos-2细胞中抑制Orai1的表达。采用细胞黏附实验、细胞划痕实验和细胞Transwell实验检测骨肉瘤细胞的黏附、迁移及侵袭等肿瘤细胞转移能力;Western-blot实验检测Saos-2细胞的中黏着斑激酶(FAK)和桩蛋白(Paxillin)的表达水平和磷酸化水平。结果:靶向Orai1 si RNA瞬时转染至骨肉瘤细胞系Saos-2细胞后,Saos-2细胞中Orai1蛋白表达水平和m RNA转录水平均显著下降。并且,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的黏附能力、迁移能力、及侵袭能力均显著下降。进一步研究发现,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的FAK和Paxillin磷酸化水平明显下降。结论:Orai1可以促进骨肉瘤细胞的黏附、迁移和侵袭,增加黏着斑的形成,从而促进骨肉瘤的转移。因此,深入研究钙通道蛋白Orai1调控骨肉瘤转移的分子机制,可为骨肉瘤转移的治疗提供新的新方向和新策略。     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.     

NGX6a Is Degraded through a Proteasome-dependent Pathway without Ubiquitination Mediated by Ezrin,a Cytoskeleton-Membrane Linker

Our previous study demonstrated that the NGX6b gene acts as a suppressor in the invasion and migration of nasopharyngeal carcinoma (NPC). Recently, we identified the novel isoform NGX6a, which is longer than NGX6b. In this study, we first found that NGX6a was degraded in NPC cells and that this degradation was mediated by ezrin, a linker between membrane proteins and the cytoskeleton. Specific siRNAs against ezrin increase the protein level of NGX6a in these cells. During degradation, NGX6a is not ubiquitinated but is degraded through a proteasome-dependent pathway. The distribution pattern of ezrin was negatively associated with NGX6a in an immunochemistry analysis of a nasopharyngeal carcinoma tissue microarray and fetus multiple organ tissues and Western blot analysis in nasopharyngeal and NPC cell lines, suggesting that ezrin and NGX6a are associated and are involved in the progression and invasion of NPC. By mapping the interacting binding sites, the seven-transmembrane domain of NGX6a was found to be the critical region for the degradation of NGX6a, and the amino terminus of ezrin is required for the induction of NGX6a degradation. The knockdown of ezrin or transfection of the NGX6a mutant CO, which has an EGF-like domain and a transmembrane 1 domain, resulted in no degradation, significantly reducing the ability of invasion and migration of NPC cells. This study provides a novel molecular mechanism for the low expression of NGX6a in NPC cells and an important molecular event in the process of invasion and metastasis of nasopharyngeal carcinoma cells.     

Matriptase-2 inhibits HECV motility and tubule formation in vitro and tumour angiogenesis in vivo

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.     

MicroRNA-21在卵巢癌病灶转移过程中的作用及机制研究

目的:探讨micro RNA-21在卵巢癌病灶转移过程中的作用及其机制。方法:选取本院2016年6月-2018年5月收治的138例卵巢癌患者,其中63例出现结直肠转移,75例未发现有转移。q RT-PCR分别检测两组患者肿瘤组织、癌旁组织和正常组织中micro RNA-21的表达;Western blot检测两组患者肿瘤组织中PGDH、PGE2、Twist表达。通过转染过表达载has-micro RNA-21上调A2780细胞中micro RNA-21的表达,采用平板克隆实验检测细胞克隆形成能力,Trans-well细胞迁移实验和侵袭实验分别检测细胞迁移和侵袭能力。Western blot检测PGDH、PGE2、Twist蛋白表达。结果:卵巢癌转移组肿瘤组织中micro RNA-21表达高于未转移组、癌旁组织和正常卵巢组织(P0.05),卵巢癌转移组肿瘤组织中PGDH表达低于未转移组,而PGE2、Twist表达高于未转移组(P0.05)。micro RNA-21过表达的A2780细胞平板克隆形成能力、迁移和侵袭能力及上皮间质转化相关蛋白PGE2和Twist表达均明显高于阴性对照组(P0.05),而PGDH表达的表达明显降低(P0.05)。结论:micro RNA-21可能通过抑制PGDH的表达增加PGE2的表达,进而激活上皮间质转化,促进卵巢癌转移。     

miR-155靶向SOCS1对骨肉瘤Saos2细胞的增殖、侵袭和迁移能力的影响

目的:探讨microRNA-155(miR-155)对骨肉瘤Saos2细胞增殖、侵袭和迁移的影响以及其作用机制。方法:利用实时荧光定量(qRT-PCR)实验检测miR-155在正常成骨细胞与骨肉瘤Saos2细胞中的表达水平,以及miR-155-mimic、miR-155-inhibitor的转染效率。采用CCK-8实验检测细胞的增殖能力,Transwell实验和划痕实验分别检测Saos2细胞的侵袭和迁移能力,Western blot检测细胞内的STAT3磷酸化水平以及SOCS1表达水平,双荧光素酶报告基因实验进行靶基因验证。结果:miR-155在骨肉瘤Saos2细胞中表达明显高于正常成骨细胞(P0.001)。在分别转染miR-155-mimic和miR-155-inhibitor后,Saos2细胞内miR-155表达水平明显上调和下降(P0.001)。过表达miR-155可促进Saos2细胞增殖、侵袭和迁移,降低SOCS1的蛋白水平,上调STAT3的磷酸化水平,差异均具有统计学意义。相反,降低miR-155水平可抑制Saos2细胞的增殖、侵袭和迁移能力,差异均具有统计学意义。结论:骨肉瘤Saos2细胞中高表达的miR-155可以通过抑制SOCS1表达来激活STAT3信号通路进而促进细胞的增殖、侵袭和迁移,因此,靶向抑制miR-155表达可以作为潜在治疗骨肉瘤的途径。     

Design and pharmacophore modeling of biaryl methyl eugenol analogs as breast cancer invasion inhibitors

Cell invasion and migration are required for the parent solid tumor cells to metastasize to distant organs. Microtubules form a polarized network, enabling organelle and protein movement throughout the cell. Cytoskeletal elements coordinately regulate cell’s motility, adhesion, migration, exocytosis, endocytosis, and division. Thus, microtubule disruption can be a useful target to control cancer cell invasion and metastasis. The phenolic ether methyl eugenol (1), the major component of the essential oil of the leaves of Melaleuca ericifolia Sm. (Myrtaceae), was used as a starting scaffold to design eleven new and three known anti-tubulin agents 2–15 using carbon–carbon coupling reactions. A computer-assisted approach was used to design these new biaryl derivatives using colchicine-binding site of tubulin as the molecular target and colchicine as an active ligand. Several derivatives showed potent inhibitory activity against MDA-MB-231 cell migration at the 1–4 μM dose range. The Z isomers, 4 and 15 were more active as invasion inhibitors compared to their structurally related E isomers, 2 and 14. The cytotoxic activities of compounds 2–15 against two breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated. Anti-invasive activity of the semisynthetic derivatives is not due to a direct cytotoxic effect on MDA-MB-231. Analogs 2–15 may promote their anti-invasive activity through the induction of changes in cell morphology. A pharmacophore model was generated involving seven essential features for activity, which was consistent with a previously generated colchicine site inhibitors model.     

17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr(567) in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis

Metastatic cancers, once established, are the primary cause of mortality associated with cancer. Previously, we used a genomic approach to identify metastasis-associated genes in cancer. From this genomic data, we selected ezrin for further study based on its role in physically and functionally connecting the actin cytoskeleton to the cell membrane. In a mouse model of osteosarcoma, a highly metastatic pediatric cancer, we found ezrin to be necessary for metastasis. By imaging metastatic cells in the lungs of mice, we showed that ezrin expression provided an early survival advantage for cancer cells that reached the lung. AKT and MAPK phosphorylation and activity were reduced when ezrin protein was suppressed. Ezrin-mediated early metastatic survival was partially dependent on activation of MAPK, but not AKT. To define the relevance of ezrin in the biology of metastasis, beyond the founding mouse model, we examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, we found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients.     

Involvement of intracellular transport in TREK-1c current run-up in 293T cells

The TREK-1 channel, the TWIK-1-related potassium (K⁺) channel, is a member of a family of 2-pore-domain K⁺ (K2P) channels, through which background or leak K⁺ currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335–360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.     

MiR-138 Acts as a Tumor Suppressor by Targeting EZH2 and Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells

Chemotherapeutic insensitivity remains a major obstacle to treating osteosarcoma effectively. Recently, increasing evidence has suggested that microRNAs (miRNAs) are involved in drug resistance. However, the effect of miR-138 on cisplatin chemoresistance in osteosarcoma has not been reported. We used real-time PCR to detect the expression of mature miR-138 in osteosarcoma tissues and cell lines. Cell proliferation, invasion, and migration assays were used to observe changes to the osteosarcoma malignant phenotype. MiR-138 was downregulated in osteosarcoma tissues and cell lines, and miR-138 overexpression negatively regulated osteosarcoma cell proliferation, migration, and invasion. We also verified that EZH2 is a direct target of miR-138. Furthermore, enhancing EZH2 expression reduced the inhibitory effects of miR-138 on osteosarcoma. Proliferation, apoptosis assays and caspase-3 activity assay confirmed that elevated miR-138 expression enhanced osteosarcoma cell chemosensitivity to cisplatin by targeting EZH2. In conclusion, the present study demonstrates that miR-138 acts as a tumor suppressor by enhancing osteosarcoma cell chemosensitivity and supports its potential application for treating osteosarcoma in the future.     

Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis.Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3β^Ser9, and p-GSK3β^Tyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2, and U2-OS) were detected by Western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAK^Y397, PTEN, MMP-2, and MMP-9 were measured by Western blotting.Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells that were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAK^Y397 expressions were observed following GSK3β knockdown by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK.     

LncRNA MEG3 negatively modified osteosarcoma development through regulation of miR-361-5p and FoxM1

This study was aimed to figure out whether long noncoding RNA MEG3/miR-361-5p/FoxM1 signaling would contribute to improved proliferation and metastasis of osteosarcoma cells. We altogether collected 204 pairs of osteosarcoma tissues and adjacent normal tissues, and obtained four human osteosarcoma cell lines. Then pcDNA3.1-MEG3, si-MEG3, miR-361-5p mimic, miR-361-5p inhibitor, pcDNA3.1-FoxM1, si-FoxM1, and negative control (NC) were, respectively, transfected into the osteosarcoma cells. Furthermore, real time polymerase chain reaction was utilized to determine the mRNA expressions of maternally expressed gene 3 (MEG3) and miR-361-5p, and western blot analysis was applied for determining the FoxM1 expression. Besides, dual luciferase reporter gene assay was adopted to verify if MEG3 can be directly targeted by miR-361-5p. Finally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, colony formation assay, flow cytometry, wound healing assay, and transwell assay were conducted to investigate the influence of MEG3, miR-361-5p, and FoxM1 expressions on the viability, proliferation, apoptosis, migration, and invasion of osteosarcoma cells. MEG3 and miR-361-5p were observed to be significantly downregulated within both osteosarcoma tissues and cell lines, whereas FoxM1 was upregulated in osteosarcoma tissues and cell lines (p < 0.05). MEG3 directly bound to miR-361-5p, and significantly upgraded its expression (p < 0.05). The upregulated MEG3 and miR-361-5p or the downregulated FoxM1 appeared to substantially inhibit proliferation, migration, and invasion of osteosarcoma cells (p < 0.05). Finally, the proliferation, migration, invasion, and motility of osteosarcoma cells within the miR-NC + pcDNA3.1-FoxM1 group and pcDNA + pcDNA-FoxM1 group were markedly promoted when compared with the miR-361-5p mimic group and pcDNA3.1-MEG3 group (p < 0.05). The MEG3/miR-361-5p/FoxM1 axis could potentially serve as therapeutic targets or diagnostic biomarkers for osteosarcoma.     

Cytoskeletal Actin Structure in Osteosarcoma Cells Determines Metastatic Phenotype via Regulating Cell Stiffness,Migration, and Transmigration

Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young’s modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis.