LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5′-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.     

Gene expression profiling in human esophageal cancers using cDNA microarray

Human esophageal cancer cell lines and human esophageal cancer tissues were profiled on cDNA microarrays. In esophageal cancer cell lines, KYAE and OE-33 (adenocarcinomas) were distinguished from KYSE series (squamous cell carcinomas). Although SK-GT-4 and TE7 were derived from adenocarcinomas, they had a comparatively similar expression profile to the KYSE series. A set of genes whose expression commonly either increased or decreased in cancer cell lines was identified. Genes that were characteristically expressed in KYAE and OE-33 were also identified. The gene expression profiles of cancer tissues (CTs) were remarkably different from those of the cancer cell lines (CCLs). Notable differences between CCLs and CTs were observed in matrix metalloproteinases, plasminogen activator, collagens, paxillin, and thrombospondin 2, etc., whose expression was not increased in CCLs but increased in CTs. Twenty-three genes were extracted to categorize patients according to their prognoses, and clustering analyses, using these genes, were performed successfully.     

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.     

MicroRNA-10b Promotes Migration and Invasion through KLF4 in Human Esophageal Cancer Cell Lines

Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis. In this study, we have analyzed the level of miR-10b and cell motility and invasiveness in several human esophageal squamous cell carcinoma cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility. Additionally, we identified KLF4, a known tumor suppressor gene that has been reported to suppress esophageal cancer cell migration and invasion, as a direct target of miR-10b. Furthermore, overexpression of miR-10b in KYSE140 and KYSE450 cells led to a reduction of endogenous KLF4 protein, whereas silencing of miR-10b in EC9706 cells caused up-regulation of KLF4 protein. Coexpression of miR-10b and KLF4 in KYSE140 cells and coexpression of small interfering RNA for KLF4 mRNA and miR-10b-AS in EC9706 cells partially abrogated the effect of miR-10b on cell migration and invasion. Finally, analyses of the miR-10b level in 40 human esophageal cancer samples and their paired normal adjacent tissues revealed an elevated expression of miR-10b in 95% (38 of 40) of cancer tissues, although no significant correlation of the miR-10b level with clinical metastasis status was observed in these samples.     

MiR-935通过其靶基因Notch1调控胃癌SGC7901细胞的发生发展

目的:探究Mi R-935调控胃癌SGC7901细胞的增殖和浸润与Notch1基因表达的关系。方法:分别检测40例正常人胃粘膜组织与40例胃印戒细胞癌的Notch1表达情况,并分析胃印戒细胞癌组织中Notch1表达与患者年龄、性别、组织进展程度、TNM分期、有无淋巴结转移的关系;采用Mi R-935转染体外培养的SGC7901细胞系,检测Notch1的表达情况,其后采用Mi R-935抑制剂处理,通过Transwell实验检测胃癌细胞的侵袭能力,细胞划痕实验检测胃癌细胞迁移能力。结果:正常人胃粘膜组织中Notch1表达呈阴性,而胃印戒细胞癌组织中Notch1表达呈阳性;Notch1的表达与胃印戒细胞癌的TNM分期、有无淋巴结转移存在着显著的相关性;转染Mi R-935的SGC7901细胞Notch1表达明显上调,采用Mi R-935抑制剂处理后,Notch1的表达显著下降。结论:Mi R-935可能通过调控Notch1的表达调控胃癌的扩增和浸润。     

CKS1 表达与食管癌细胞辐射敏感性的关系研究

目的:探讨CKS1表达对食管癌细胞辐射敏感性的影响,初步研究其分子机理.方法:用Western-blotting方法筛选CKS1低表达和高表达的食管癌细胞系;构建CKS1正义表达载体p-pcDNA 3.1/myc-His A-CKS1和RNA干扰载体CKS1 siRNA,分别转染CKS1低表达细胞和高表达细胞,用不同剂量γ-射线照射各组细胞,克隆形成实验检测细胞辐射敏感性的差异.结果:CKS1在四种食管癌细胞中的表达水平依次为EC9706＞ KYSE510＞KYSE450＞ KYSE150.用p-pcDNA 3.1/myc--His A-CKS1表达载体转染KYSE150细胞后CKS12表达升高,不同剂量γ-射线照射后细胞的克隆形成能力显著高于母系对照组(P＜0.01).RNA干扰载体转染KYSE510细胞后CKS1表达水平降低,不同剂量γ-射线照射后细胞的克隆形成能力显著低于母系对照组(P＜0.01).敲降CKS1表达后DNA损伤修复相关蛋白RAD51表达下降,KU70表达没有变化.CKS1过表达后RAD51表达升高,KU70表达没有变化.结论:CKS1表达与食管癌细胞的辐射敏感性密切相关,可能通过影响DNA损伤修复发挥作用.     

IGHMBP2过表达促进食管鳞癌细胞的侵袭和迁移

IGHMBP2(Immunoglobulin mu binding protein 2)基因编码一种解旋酶,参与DNA的复制和修复,并且作为转录调节因子在基因转录中发挥重要作用。IGHMBP2基因定位于11q13.2,该染色体区段在食管鳞癌中扩增频率较高。为了探讨IGHMBP2基因在食管鳞癌中的扩增情况及其在食管鳞癌中的作用,文章对本实验室前期报道的59例食管鳞癌原发肿瘤array-CGH数据进行分析,结果显示IGHMBP2基因扩增频率为28.9%(17/59)。进一步利用荧光原位杂交(FISH)和Western blot技术,发现食管鳞癌细胞系KYSE30、KYSE180、KYSE510和KYSE150中存在IGHMBP2基因扩增/增益以及蛋白高表达。敲降IGHMBP2后,KYSE30和KYSE150细胞的侵袭迁移能力明显降低(P<0.001),侵袭迁移相关蛋白E-cadherin的表达水平升高;敲降后转染IGHMBP2质粒,回复其蛋白表达后,细胞的侵袭迁移能力又得以恢复(P<0.01)。上述结果表明,IGHMBP2过表达可能通过降低E-cadherin的表达从而增强食管鳞癌细胞的侵袭迁移能力。     

Notch1 and the activated NOTCH1 intracellular domain are expressed in differentiated trophoblast cells

The Notch signalling pathway regulates proliferation, cell death and cell type specification that is critical for organogenesis. Mouse models carrying mutations in the Notch signalling pathway display defects in development of the placenta, suggesting that this pathway is required for placental development. In particular, Notch1 mutant embryos exhibit abnormal placental morphogenesis and arrest early in development. However, expression of Notch1 gene has not been detected during placental development. Trophoblast stem cells are derived from the precursor of the placenta and express Notch1. We report that Notch1 is also expressed in differentiated trophoblast cells. Under standard differentiation conditions, Notch1 expression ceases by day 6. Furthermore, the activated NOTCH1 intracellular domain is enriched at the nucleolus of trophoblast stem cells and differentiated trophoblast cells. Our results suggest that NOTCH1 is active in both trophoblast stem cells and differentiated trophoblast cells.     

Chloride intracellular channel 1 promotes esophageal squamous cell carcinoma proliferation via mTOR signalling

ObjectivesTo investigate the clinical significance of Chloride Intracellular Channel 1 (CLIC1) expression in esophageal squamous cell carcinoma (ESCC) and its functional contribution and molecular mechanisms to the progression of ESCC.MethodsCLIC1 expression was analyzed by immunohistochemistry (IHC) in a cohort of 86 ESCC tissue specimens and paired normal adjacent esophageal tissues. Associations between clinicopathological features of ESCC and CLIC1 expression were determined. In vitro analyses examined CLIC1 expression in the ESCC cell lines KYSE150 and TE1 using RT-PCR and Western blotting. The downstream pathways of CLIC1 were detected by lentiviral shRNA knockdown and subsequent proteomic analyses. CLIC1 siRNA knockdown was performed in ESCC cell lines KYSE150 and TE1 and the functional effects of CLIC1 on the growth and proliferation of ESCC cells were evaluated combined with cell viability and colony formation assays; the mTOR signaling pathway-related proteins were detected by Western blotting based on the previous proteomic data.ResultsCLIC1 expression was significantly increased in ex vivo ESCC tissues compared with corresponding normal tissues, and the up-regulation was associated with clinical tumor node metastasis (TNM) classifications. Knockdown of CLIC1 inhibited in vitro cell proliferation of ESCC cell lines KYSE150 and TE1. CLIC1 knockdown down-regulated the protein expression of p-mTOR and the downstream targets Rictor and p-4EBP1 in both KYSE150 and TE1 cell lines. And the CLIC1 knockdown induced inhibition of cell proliferation on ESCC cells could be rescued by mTOR overexpression.ConclusionsCLIC1 expression increases during esophageal carcinogenesis and it may functionally contribute to the progression of ESCC through growth promotion effects by promoting the mTOR and downstream signaling pathway. CLIC1 therefore constitutes a candidate molecular biomarker of ESCC.     

Esophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

Cytochrome P450 epoxygenases (CYP450) have been recently shown to promote malignant progression. Here we investigated the mRNA and protein expression and potential clinical relevance of CYP2C9 in esophageal cancer. Highest expression was detected in esophageal adenocarcinoma (EAC; n=78) and adjacent esophageal mucosa (NEM; n=79). Levels of CYP2C9 in EAC and NEM were significantly higher compared to esophageal squamous cell carcinoma (ESCC; n=105). Early tumor stages and well-differentiated tumors showed a significantly higher CYP2C9 expression compared to progressed tumors. Moreover, CYP2C9 expression was correlated to high Ki-67 labeling indices in EAC and Ki-67 positive tumor cells in EAC and ESCC. Selective inhibition of CYP2C9 decreased tumor cell proliferation (KYSE30, PT1590 and OE19) in vitro, which was abolished by 11,12-epoxyeicosatrienoic acid (11,12-EET). Cell-cycle analysis using FACS revealed that inhibition of CYP2C9 leads to a G0/G1 phase cell-cycle arrest. CYP2C9 seems to be relevant for early esophageal cancer development by promoting tumor cell proliferation. Pharmacological inhibition of CYP2C9 might contribute to a more efficient therapy in CYP2C9 highly expressing esophageal cancers.     

紫铆因对食管鳞癌细胞增殖和存活影响的初步研究

研究紫铆因对人食管鳞癌细胞增殖和存活的影响。通过MTS和软琼脂集落实验检测紫铆因对食管鳞癌增殖的抑制,生化分析仪检测紫铆因对食管鳞癌糖酵解的影响,并利用免疫印迹检测紫铆因对食管鳞癌细胞增殖和凋亡激活相关蛋白分子的表达。结果发现紫铆因剂量依赖性抑制KYSE150和Eca109细胞增殖,下调EGFR信号通路活化,抑制HK2的表达及糖酵解。一定浓度的紫铆因能诱导食管鳞癌细胞发生凋亡,caspase3和PARP被剪切,Bcl-2和Mcl-1表达下调,但Bcl-XL未见明显改变。结果证明紫铆因抑制食管鳞癌的增殖,可能与EGFR信号通路和糖酵解被抑制,及促存活蛋白Bcl-2和Mcl-1的表达下调有关。     

Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling

Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P<0.01). DACH1 methylation was associated with poor differentiation (P<0.05) and late tumor stage (P<0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-β signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-β signaling.     

Notch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines

In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 intracellular domain (ICD) to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels. In contrast, pharmacologically blocking Notch signaling reversed this situation and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression.     

Discovery of indoline derivatives that inhibit esophageal squamous cell carcinoma growth by Noxa mediated apoptosis

A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent inhibition activity against esophageal cancer cells (Kyse30, Kyse450, Kyse510 and EC109). Cellular mechanism studies in esophageal squamous cell carcinoma (ESCC) cells elucidated compound 20 inhibited cell growths in vitro and in vivo, reduced colony formation, arrested cell cycle at M phase, and induced Noxa-dependent apoptosis in ESCC. Importantly, compound 20 was identified as a novel Noxa mediated apoptosis inducer. These results suggested that compound 20 might be a promising anticancer agent with potential for development of further clinical applications.     

T-type Ca2+ channel expression in human esophageal carcinomas: a functional role in proliferation

In the present study the role of T-type Ca(2+) channels in cancer cell proliferation was examined. Seventeen human esophageal cancer cell lines were screened for T-type channels using RT-PCR and voltage-clamp recordings. mRNAs for all three T-type channel alpha(1)-subunits (alpha(1G), alpha(1H), and alpha(1I)) were detected in all 17 cell lines: either alpha(1H) alone, alpha(1H) and alpha(1G), or all three T-type alpha(1)-subunits. Eleven cell lines were further subjected to voltage-clamp recordings: one, i.e. the TE8 cell line, was found to exhibit a typical T-type current while others exhibited a minimal or no T-type current. Cell proliferation assays were performed in the presence or absence of T-type channel blocker mibefradil in KYSE150, KYSE180 and TE1 cells expressing mRNA for T-type channel alpha(1)-subunits but lacking T-type current, and TE8 cells exhibiting T-type current. Only TE8 cell proliferation was reduced by mibefradil. Silencing the alpha(1G)-gene that encodes functional T-type Ca(2+) channels in TE8 cells with type-specific shRNA transduction also significantly decreased TE8 cell proliferation. The reduction of cell proliferation in TE8 cells was found to be associated with an up-regulation of p21(CIP1). Moreover, p53 silencing nearly abolished the up-regulation of p21(CIP1) resulting from mibefradil T-type channel blockade. Together, these findings suggest a functional role of T-type channels in certain esophageal carcinomas, and that inhibition of T-type channels reduces cell proliferation via a p53-dependent p21(CIP1) pathway.