Actin-related protein 2/3 complex subunit 5 (ARPC5) contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma

Our expression signatures of human cancers including head and neck squamous cell carcinoma (HNSCC) demonstrated that downregulation of microRNA-133a (miR-133a) were frequently observed in cancer cells. The restoration of miR-133a in cancer cells revealed that it functions as a tumor suppressor. In this study, we investigated the novel molecular targets of miR-133a in HNSCC cancer cells and its oncogenic function, especially as it contributes to cancer cell migration and invasion. The genome-wide gene expression analysis and bioinformatics study showed that actin-related protein 2/3 complex subunit 5 (ARPC5) is a candidate target of miR-133a. Furthermore, luciferase reporter assay demonstrated that ARPC5 is directly regulated by miR-133a. Silencing of ARPC5 revealed significant inhibition of cell migration and invasion in HNSCC cell lines, SAS, HSC3 and IMC-3. In HSC3 cells, restoration of miR-133a or silencing ARPC5 led to a reorganization of the actin cytoskeleton and a subsequent change in cell morphology to a round, bleb-like shape. The expression levels of ARPC5 were significantly higher in HNSCC tissues than in non-cancer tissues. Immunohistochemistry showed that the levels of ARPC5 expression were significantly higher in invasive cancer cells. ARPC5 contributed to cancer cell migration and invasion in HNSCC and this gene was directly regulated by miR-133a. Our analysis of novel tumor-suppressive miR?133a-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis.     

miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.     

miR-218在子宫颈癌组织中的表达及预测的靶基因的生物信息学分析

目的 探讨miR-218在子宫颈癌组织中的表达,并对其预测的靶基因进行生物信息学分析,为miR-218在子宫颈癌发生中的作用机制研究提供理论基础.方法 利用miRNA芯片技术检测3份子宫颈癌组织及3份正常子宫颈组织中miRNA表达谱,用实时定量聚合酶链反应（PCR）法在55例子宫颈组织中进行验证.通过生物信息学预测miR-218的靶基因,并对其靶基因进行基因本体（G0）功能富集分析及KEGG信号转导通路富集分析.结果 芯片结果显示子宫颈癌组织中有15个miRNA表达上调（＞2倍）,10个miRNA表达下调（＜0.5倍）,其中miR-218下调最明显.miR-218预测靶基因集合功能富集于生物学调控、蛋白代谢、细胞迁移和黏附、细胞分化等生物学过程,以及蛋白结合、连接酶活性等分子功能上;KEGG通路分析涉及癌通道、黏附连接、胰岛素信号通路、凋亡等信号转导通路及前列腺癌、急性和慢性髓系白血病等疾病通路.结论 miR-218在子宫颈癌组织中呈低表达,miR-218预测靶基因集合显著富集在与肿瘤发生相关的信号通路中.     

Laminin-332 promotes the invasion of oesophageal squamous cell carcinoma via PI3K activation

Laminin-332 is major component of epithelial basement membrane, and has an important role in cell migration and tumour invasion. Recently, the phosphatidylinositol 3-kinase (PI3K) activation induced by laminin-332 during carcinogenesis or tumour invasion has been highlighted in skin squamous cell carcinoma. The expression of laminin-332 in 126 resected oesophageal squamous cell carcinoma (ESCC) specimens was immunohistochemically examined to determine its associations with the clinicopathological characteristics, and the effect of laminin-332 on the invasiveness and the PI3K activation was assessed by in vitro experiments using ESCC cell lines (ESCCs). Sections with immunostaining signals in >30% cancer cells, which were observed in 55 of 126 cases, were judged to be positive for laminin-332. The positivity was significantly correlated with pTNM stage and poor prognosis. Inactivation of the PI3K pathway by laminin-332 blocking antibody suppressed the invasiveness of TE8 cell line, which secreted laminin-332 at high level and had high PI3K activity. The addition of the purified laminin-332 activated the PI3K pathway and increased the invasiveness of TE11 cell line, which secreted laminin-332 at lower level and had low PI3K activity. The deactivation of PI3K pathway using the PI3K inhibitor decreased the invasiveness of ESCCs and the secretion of laminin-332 in vitro. The expression of laminin-332 was one of the prognostic factors of ESCC. Laminin-332 could provide the autocrine positive-feedback loop through PI3K activation, contributing the invasive ability. Therefore, the inhibitor of PI3K pathway might be useful as the anticancer therapies for ESCC.     

LAMB3 is associated with disease progression and cisplatin cytotoxic sensitivity in head and neck squamous cell carcinoma

ObjectivesLaminin subunit beta-3 (LAMB3) is a major component of the basement membrane zone. In our study, we investigated the role of LAMB3 in head and neck squamous cell carcinoma (HNSCC) progression and its clinical implication as a prognostic biomarker.Materials and methodsA retrospective analysis of 100 patients with HNSCC who had undergone curative surgery from 1999 to 2011 was performed. We evaluated LAMB3 expression by immunohistochemistry and its associations with clinicopathological characteristics and survival. For functional in vitro analyses, cell proliferation, migration, and invasion and western blot assays were performed following LAMB3 suppression. In addition, the role of LAMB3 in cisplatin-induced cytotoxicity was clarified by measuring cell proliferation.ResultsLAMB3 expression was up-regulated in HNSCC cell lines and patient tissues. High LAMB3 expression was significantly associated with positive lymph node metastasis (odds ratio: 6.316; P < 0.001) and poor prognosis in patients with HNSCC. LAMB3 suppression reduced cell migration/invasion via down-regulation of epithelial-to-mesenchymal transition-associated proteins (Vimentin and Slug). Moreover, LAMB3 suppression increased cisplatin cytotoxicity in HNSCC cells.ConclusionOur findings indicate that LAMB3 may be used as a prognostic biomarker in HNSCC and support that LAMB3 silencing could induce the sensitivity of anti-cancer drugs such as cisplatin.     

Tumour-suppressive microRNA-29s inhibit cancer cell migration and invasion by targeting laminin–integrin signalling in head and neck squamous cell carcinoma

Background:

Our recent studies of microRNA (miRNA) expression signatures demonstrated that microRNA-29s (miR-29s; miR-29a/b/c) were significantly downregulated in head and neck squamous cell carcinoma (HNSCC) and were putative tumour-suppressive miRNAs in human cancers. Our aim in this study was to investigate the functional significance of miR-29s in cancer cells and to identify novel miR-29s-mediated cancer pathways and responsible genes in HNSCC oncogenesis and metastasis.

Methods:

Gain-of-function studies using mature miR-29s were performed to investigate cell proliferation, migration and invasion in two HNSCC cell lines (SAS and FaDu). To identify miR-29s-mediated molecular pathways and targets, we utilised gene expression analysis and in silico database analysis. Loss-of-function assays were performed to investigate the functional significance of miR-29s target genes.

Results:

Restoration of miR-29s in SAS and FaDu cell lines revealed significant inhibition of cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that miR-29s modulated the focal adhesion pathway. Moreover, laminin γ2 (LAMC2) and α6 integrin (ITGA6) genes were candidate targets of the regulation of miR-29s. Luciferase reporter assays showed that miR-29s directly regulated LAMC2 and ITGA6. Silencing of LAMC2 and ITGA6 genes significantly inhibited cell migration and invasion in cancer cells.

Conclusion:

Downregulation of miR-29s was a frequent event in HNSCC. The miR-29s acted as tumour suppressors and directly targeted laminin–integrin signalling. Recognition of tumour-suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis and metastasis and suggests novel therapeutic strategies for the disease.     

MiR-218通过抑制Robo1的表达影响肺癌细胞迁移侵袭

背景与目的 本研究旨在探讨肺癌中miR-218的表达,研究miR-218在肺癌细胞中的功能及其可能的分子机制.方法 应用实时荧光定量PCR(qRT-PCR)检测15例肺癌组织和15例癌旁组织中miR-218的表达.在肺癌细胞A549中转染miR-218的抑制物(Anti-miR-218),在肺癌细胞HCC4006中转染miR-218的模拟物后,用Transwell实验检测细胞的迁移侵袭能力的变化.用Targetscan和MiRanda软件预测miR-218的可能靶点,转染miR-218的抑制物及模拟物后用qRT-PCR和Western blot检测Robo1的mRNA和蛋白表达水平.用双荧光素酶报告基因方法鉴定miR-218和Robo1的调控关系.用Anti-miR-218、miR-218模拟物或阴性对照与Si-Robo1或Si-NC同时转染细胞,应用Transwell实验检测转染后细胞的侵袭迁移能力的变化.结果 与癌旁组织比较,肺癌组织中miR-218在肺癌组织中表达水平显著降低(P<0.01).在A549细胞中转染miR-218的抑制物,能够显著降低miR-218的表达,促进了细胞的迁移侵袭.在HCC4006中转染miR-218的模拟物能够显著提高miR-218的表达,同时抑制了细胞的迁移侵袭能力.利用生物信息学预测出在Robo1的3′UTR区有miR-218的结合位点,双荧光素酶报告基因实验进一步证实miR-218能够调控Robo1的转录活性.抑制miR-218能够提高Robo1的表达;过表达miR-218显著降低Robo1的表达,且miR-218能够通过调控Robo1影响细胞的迁移侵袭.结论 MiR-218在肺癌组织中呈现低表达状态,miR-218可能是通过抑制Robo1的表达抑制肺癌细胞侵袭迁移.     

Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that can function as oncogenes or tumor suppressors in human cancer. Down-regulation of the miRNA miR-133a in many type of cancers, and a reduction of cell proliferation, migration, and invasion upon over-expression, suggests that miR-133a is a tumor suppressor. In this study, genome-wide gene expression analysis of HNSCC cells that over-express miR-133a showed that caveolin-1 (CAV1), a multifunctional scaffolding protein, is down-regulated, a result that was confirmed by real-time PCR and Western blot analysis. A luciferase reporter assay revealed that miR-133a is directly bound to CAV1 mRNA. Cancer cell migration and invasion were significantly inhibited in HNSCC cells transfected with si-CAV1. Therefore, CAV1 functions as an oncogene in HNSCC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into potential mechanism of HNSCC carcinogenesis.     

Tumor‐suppressive microRNA‐218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer

Our recent studies of the microRNA (miRNA) expression signature in prostate cancer (PCa) indicated that miRNA‐218 (miR‐218) was significantly downregulated in clinical specimens, suggesting that miR‐218 might act as a tumor‐suppressive miRNA in PCa. The aim of the present study was to investigate the functional significance of miR‐218 in PCa and to identify novel miR‐218‐regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis. Restoration of miR‐218 in PCa cell lines (PC3 and DU145) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that LIM and SH3 protein 1 (LASP1) is a potential target of miR‐218 regulation. LASP1 is a cytoskeletal scaffold protein that plays critical roles in cytoskeletal organization and cell migration. Luciferase reporter assays showed that miR‐218 directly regulated expression of LASP1. Moreover, downregulating the LASP1 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of LASP1 was upregulated in cancer tissues. We conclude that loss of tumor‐suppressive miR‐218 enhanced cancer cell migration and invasion in PCa through direct regulation of LASP1. Our data on pathways regulated by tumor‐suppressive miR‐218 provide new insight into the potential mechanisms of PCa oncogenesis and metastasis.     

miR-218-1-3p对非小细胞肺癌细胞侵袭和迁移影响

目的 非小细胞肺癌(non-small cell lung cancer,NSCLC)因其侵袭性而导致预后不良,miR-218可对多种肿瘤的进展起到抑制作用.本研究探讨miR-218-1-3p对NSCLC A549细胞侵袭和迁移影响.方法 使用LipofectamineTM 2000 Reagent将miR-218-1-3p mimic转染入NSCLC A549细胞中,实时定量聚合酶链反应(real-time PCR,RT-qPCR)检测各组细胞中miR-218-1-3p的表达,Transwell小室法测定其侵袭及迁移能力,荧光定量PCR检测细胞迁移侵袭相关指标基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)、MMP-7、MMP-9、Rho A、Rho B和Rho C的变化.结果 同对照组相比,上调mir-218-1-3p明显抑制了A549细胞的侵袭(t=4.028,P=0.016)和迁移(t=8.911,P=0.001),其侵袭和迁移的抑制率分别为37.8%和53.6%.MMP-7降低至对照组的(0.68±0.19)倍,t=2.931,P=0.043;MMP-9降低至对照组的(0.58±0.15)倍,t=4.875,P=0.080.结论 miR-218-1-3p能够抑制NSCLC A549细胞的侵袭和迁移.     

Targeting a tumor-specific laminin domain critical for human carcinogenesis

Laminin-332 is critical for squamous cell carcinoma (SCC) tumorigenesis, but targeting it for cancer therapy has been unachievable due to key role of laminin-332 in promoting tissue integrity. Here, we show that a portion of laminin-332, termed G45, which is proteolytically removed and absent in normal tissues, is prominently expressed in most human SCC tumors and plays an important role in human SCC tumorigenesis. Primary human keratinocytes lacking G45 (DeltaG45) showed alterations of basal receptor organization, impaired matrix deposition, and increased migration. After SCC transformation, the absence of G45 domain in DeltaG45 cells was associated with deficient extracellular signal-regulated kinase and phosphotidylinositol 3-kinase (PI3K) pathway activation, impaired invasion, deficient metalloproteinase activity, and absent tumorgenicity in vivo. Expression of G45 or activated PI3K subunit in DeltaG45 cells reversed these abnormalities. G45 antibody treatment induced SCC tumor apoptosis, decreased SCC tumor proliferation, and markedly impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion. These results show a remarkable selectivity of expression and function for laminin-332 G45 in human SCC tumorigenesis and implicate it as a specific target for anticancer therapy.     

MicroRNA-218 inhibits cell invasion and migration of pancreatic cancer via regulating ROBO1

miRNA-218 is a highlighted tumor suppressor and its underlying role in tumor progression is still unknown. Here, we restored the expression of miRNA-218 in pancreatic cancer to clarify the function and potent downstream pathway of miRNA-218. The expressions of both miRNA-218 and its potent target gene ROBO1 were revealed by RT-PCR and western blotting analysis. Transfection of miRNA-218 precursor mimics and luciferase assay were performed to elucidate the regulation mechanism between miRNA-218 and ROBO1. Cells, stably expressing miRNA-218 followed by forced expression of mutant ROBO1, were established through co-transfections of both lentivirus vector and plasmid vector. The cell migration and invasion abilities were evaluated by migration assay and invasion assay respectively. An increased expression of ROBO1 was revealed in cell BxPC-3-LN compared with cell BxPC-3. Elevated expression of miRNA-218 would suppress the expression of ROBO1 via complementary binding to a specific region within 3′UTR of ROBO1 mRNA (sites 971–978) in pancreatic cancer cells. Stably restoring the expression of miRNA-218 in pancreatic cancer significantly downregulated the expression of ROBO1 and effectively inhibited cell migration and invasion. Forced expression of mutant ROBO1 could reverse the repression effects of miRNA-218 on cell migration and invasion. Consequently, miRNA-218 acted as a tumor suppressor in pancreatic cancer by inhibiting cell invasion and migration. ROBO1 was a functional target of miRNA-218’s downstream pathway involving in cell invasion and migration of pancreatic cancer.     

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis.     

miR-218-5p靶向下调LPAR3抑制宫颈癌细胞增殖、迁移和侵袭

目的:探讨miR-218-5p通过调控LPAR3表达对宫颈癌细胞增殖、迁移和侵袭的影响。方法:qRT-PCR和Western blot检测人正常宫颈细胞H8和4种宫颈癌细胞（SiHa、HeLa、MS751和HT-3）中miR-218-5p和LPAR3的表达。以HeLa细胞为后续研究对象,分别构建过表达miR-218-5p和敲减LPAR3的HeLa细胞株,CCK-8法检测细胞存活情况,Transwell实验检测细胞的迁移和侵袭能力,Western blot检测细胞增殖相关蛋白CyclinD1、迁移侵袭相关蛋白MMP2和MMP9的表达。双荧光素酶报告基因实验和Western blot验证miR-218-5p和LPAR3的靶向调控关系。结果:与人正常宫颈细胞H8相比,4种宫颈癌细胞miR-218-5p的表达显著下调,LPAR3的表达显著上调。过表达miR-218-5p或敲减LPAR3均可抑制HeLa细胞的增殖、迁移和侵袭能力,抑制CyclinD1、MMP2和MMP9蛋白的表达。LPAR3是miR-218-5p的靶基因,miR-218-5p可负性调控LPAR3的表达。过表达LPAR3可逆转miR-218-5p对HeLa细胞增殖、迁移和侵袭的影响。结论:miR-218-5p通过靶向下调LPAR3表达抑制宫颈癌细胞的增殖、迁移和侵袭,miR-218-5p/LPAR3分子轴有望成为宫颈癌的潜在治疗靶点。     

MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer

The Src family of protein kinases (SFK) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration, and survival, and specialized cell signals in various malignancies. The pleiotropic functions of SFKs in cancer make them promising targets for intervention. Here, we sought to investigate the role of microRNA-205 (miR-205) in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a nonmalignant cell line and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR (untranslated region) sequences complementary to either Src, Lyn, or Yes, which was abolished by mutations in these 3'-UTR regions. Overexpression of miR-205 in A498 cells reduced Src, Lyn, and Yes expression, both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phospho-Src-regulated ERK1/2 pathway. Cell motility factor FAK (focal adhesion kinase) and STAT3 activation were also inhibited by miR-205. Transient and stable overexpression of miR-205 in A498 cells resulted in induction of G?/G? cell-cycle arrest and apoptosis, as indicated by decreased levels of cyclin D1 and c-Myc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study showing that miR-205 inhibits proto-oncogenic SFKs, indicating a therapeutic potential of miR-205 in the treatment of renal cancer.     

Tumour-suppressive microRNA-874 contributes to cell proliferation through targeting of histone deacetylase 1 in head and neck squamous cell carcinoma

Background:

Our recent studies of microRNA (miRNA) expression signature demonstrated that microRNA-874 (miR-874) was significantly downregulated in maxillary sinus squamous cell carcinoma (MSSCC), and a putative tumour-suppressive miRNA in human cancers. Our aim of this study was to investigate the functional significance of miR-874 in cancer cells and to identify novel miR-874-mediated cancer pathways and responsible genes in head and neck squamous cell carcinoma (HNSCC).

Methods:

Gain-of-function studies using mature miR-874 were performed to investigate cell proliferation and cell cycle distribution in HNSCC cell lines (SAS and FaDu). To identify miR-874-mediated molecular pathways and targets, we utilised gene expression analysis and in silico database analysis. Loss-of-function assays were performed to investigate the functional significance of miR-874 target genes.

Results:

Expression levels of miR-874 were significantly downregulated in HNSCC tissues (including oral, pharyngeal and laryngeal SCCs) compared with normal counterpart epithelia. Restoration of miR-874 in SAS and FaDu cell lines revealed significant inhibition of cell proliferation and induction of G2/M arrest and cell apoptosis. Our expression data and in silico analysis demonstrated that miR-874 modulated the cell cycle pathway. Moreover, histone deacetylase 1 (HDAC1) was a candidate target of miR-874 regulation. Luciferase reporter assays showed that miR-874 directly regulated HDAC1. Silencing of the HDAC1 gene significantly inhibited cell proliferation and induced G2/M arrest and cell apoptosis in SAS cells.

Conclusions:

Downregulation of miR-874 was a frequent event in HNSCC. miR-874 acted as a tumour suppressor and directly targeted HDAC1. Recognition of tumour-suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis and suggests novel therapeutic strategies for the disease.     

miR-203a-3p在胰腺癌组织与细胞中的表达及其对BxPC-3细胞增殖、迁移和侵袭的影响

目的:探讨miR-203a-3p对胰腺癌BxPC-3细胞增殖、迁移和侵袭能力的影响.方法:运用癌症基因组图谱(TCGA)数据库筛选胰腺癌组织和癌旁组织中差异表达的miRNA,分析miRNA高表达与低表达时胰腺癌患者的生存率和临床分期;利用TarBase数据库分析miRNA与癌症相关的GO功能与KEGG通路,利用DIAN...