Inhibiting UHRF1 expression enhances radiosensitivity in human esophageal squamous cell carcinoma

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is a novel nuclear protein which is overexpressed in various cancers but not yet examined in esophageal squamous cell carcinoma (ESCC). The correlation between UHRF1 and the radioresistance in ESCC is still unclear. In the present study, the expression of UHRF1 was examined by immunohistochemistry in specimens of ESCC patients treated with radiotherapy. The results showed that UHRF1 was significantly overexpressed in ESCC specimens. Overexpression of UHRF1 correlated significantly with advanced T-stage, positive lymph node metastasis and poor differentiation. In addition, UHRF1 was associated with radiotherapy response, in which overexpression of UHRF1 was observed more frequently in the radioresistant group than in the effective group. At the molecular level, inhibition of UHRF1 by lentivirus-mediated shRNA targeting UHRF1 increased the radiosensitivity and apoptosis, while decreased radiation-induced G2/M phase arrest in TE-1 cells. Moreover, inhibition of UHRF1 resulted in higher residual γH2AX expression after irradiation, but not initial γH2AX. Further study showed that inhibition of UHRF1 down-regulated the endogenous expressions of DNA repair protein Ku70 and Ku80 in TE-1 cells, and significantly inhibited the increase of these proteins after irradiation. Above all, our data suggested that UHRF1 might play an important role in radioresistance of ESCC, and inhibition of UHRF1 can increase the radiosensitivity of TE-1 cells by altering cell cycle progression, enhancing apoptosis, and decreasing DNA damage repair capacity.     

Regenerating gene I regulates interleukin-6 production in squamous esophageal cancer cells

Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and Iβ and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Iα and Iβ led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-γ, tumor necrosis factor-α, granulocyte-colony stimulating factor or transforming growth factor-β1. The elevated IL-6 expression seen in REG Iα transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.     

CXCR4/Akt信号通路对食管癌细胞侵袭和肿瘤转移的调控作用

多项研究发现CXCR4在各种类型的癌症中高表达,然而尚不清楚CXCR4在食管癌细胞生长和转移中的作用。本研究检测了CXCR4在食管癌组织和细胞系(TE-1)中的表达,并通过转染CXCR4-短发夹RNA(CXCR4-sh RNA)慢病毒来敲低TE-1细胞中CXCR4的表达。应用PI3K/AKT抑制剂LY294002(50μmol/L)处理TE-1细胞12 h来考察AKT信号在食管癌细胞生长和转移中的作用;应用蛋白质印迹分析检测AKT和Rho家族蛋白(RhoA,Rac-1和Cdc42)的表达;应用CCK-8实验检测细胞增殖;Transwell实验检测细胞侵袭;对雄性BALB/c-nu/nu裸鼠皮下注射转染CXCR4-shRNA的TE-1细胞建立肿瘤异种移植模型。研究显示,CXCR4在食管癌组织中的表达水平明显高于癌旁组织,并且与TNM分期和淋巴结转移有关。CXCR4在人食管鳞状细胞癌细胞系(TE-1)中的表达水平明显高于人正常食管上皮细胞系(human normal esophageal epithelial cell line,HEEC)。敲低CXCR4能抑制食管鳞状细胞癌细胞的增殖和侵袭能力,并抑制肿瘤异种移植裸鼠的肿瘤形成。敲低CXCR4抑制了AKT的磷酸化及RhoA、Rac-1和Cdc42的表达。此外,PI3K/AKT抑制剂LY294002处理显著降低了TE-1细胞中AKT的磷酸化,并降低了RhoA、Rac-1和Cdc42的表达。本研究表明,CXCR4在食管癌患者中上调,与不良预后相关。下调CXCR4的表达可在体内和体外抑制食管癌肿瘤的生长和转移。下调CXCR4可通过抑制AKT信号的激活来抑制Rho家族粘附/侵袭相关蛋白的表达,从而抑制肿瘤转移。     

Elevation of long non-coding RNA GAS5 and knockdown of microRNA-21 up-regulate RECK expression to enhance esophageal squamous cell carcinoma cell radio-sensitivity after radiotherapy

ObjectiveLately, lncRNAs have been proposed to function in the radio-sensitivity of tumor cells, yet the role of lncRNA GAS5 in that of esophageal squamous cell carcinoma (ESCC) has scarcely been studied. This study aims to examine GAS5's effects on ESCC cell radio-sensitivity.MethodsGAS5, miR-21 and RECK expression in radiation-sensitive and radiation-resistant ESCC tissues, and TE-1 and TE-1-R cells was determined. TE-1 and TE-1-R cells were treated with pcDNA-GAS5 or miR-21 inhibitors to figure out their roles in ESCC cell proliferation, radio-sensitivity, and apoptosis via gain- and loss-of-function experiments.ResultsWe found underexpressed GAS5 and RECK, and overexpressed miR-21 in ESCC. GAS5 elevation and miR-21 inhibition reduced viability and the colony formation ability, and enhanced the apoptosis of ESCC cells under radiation.ConclusionOur study reveals that GAS5 elevation up-regulates RECK expression by down-regulating miR-21 to increase ESCC cell apoptosis after radiation therapy, thus enhancing cell radio-sensitivity.     

miR-29a-3p enhances the radiosensitivity of oral squamous cell carcinoma cells by inhibiting ADAM12

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, and radiotherapy is the main approach for this disease, while irradiation resistance is a huge challenge that influences radiosensitivity. This study aims to determine the role and function of miR-29a-3p and ADAM12 in the radiosensitivity of OSCC cells. The expression pattern of ADAM12 in OSCC cells was searched in TCGA database. The binding of miR-29a-3p and ADAM12 was predicted by Starbase and verified using dual luciferase reporter gene assay. The RNA or protein expressions of miR-29a-3p and ADAM12 were measured by RT-qPCR or western blot. OSCC cell lines were treated by various γ-ray irradiation dosages before the alteration on miR-29a-3p expression and on the cell viability, proliferation, migration and cell apoptosis was detected. ADAM12 was highly expressed in OSCC cells, whose expression in resistant cells was positively correlated with irradiation dosage. Overexpression of ADAM12 in OSCC cells lead to increased cell proliferation and migration ability as well as inhibited cell apoptosis. miRNAs potentially binding ADAM12 in PITA, microT, miRmap and targetscan were screened, among which miR-29a-3p had the maximum differential expression levels in OSCC cells determined by RT-qPCR. Overexpression of miR-29a-3p resulted in suppressed cell viability, proliferation, migration ability and increased cell apoptosis, while this expression pattern can be partially counteracted by ADAM12 overexpression in OSCC cells. miR-29a-3p through targeting and inhibiting AMDM12 enhances the radiosensitivity of OSCC cells.Key words: miR-29a, ADAM12, oral squamous cell carcinoma, radio-resistance, cell viability     

Ku80 is differentially expressed in human lung carcinomas and upregulated in response to irradiation in mice

Based on the role of Ku80 in mediating radiation-induced DNA repair, we investigated Ku80 expression in human lung cancers of different pathological types and evaluated the effect of radiotherapy on Ku80 expression levels in a mouse model. We used immunohistochemistry and real-time PCR to determine Ku80 protein and mRNA levels, respectively. We inoculated nude mice with A549 cells and subjected the tumor-bearing mice to varying doses of irradiation. Lung carcinoma tissue exhibited higher Ku80 mRNA and protein levels when compared with normal tissue. Among the tumor subtypes, lung adenocarcinoma and lung squamous carcinoma showed higher levels of Ku80 protein and mRNA, compared with small-cell lung carcinoma. There was a dose-dependent and time-dependent increase in Ku80 mRNA levels in nude mice that were inoculated with A549 cells and exposed to varying doses of irradiation. Ku80 may play an important role in the DNA damage response pathway. Higher Ku80 levels in lung squamous carcinoma and adenocarcinoma may explain their lower radiosensitivity when compared with small-cell lung carcinoma. Ku80 expression levels could be useful in predicting radiosensitivity of lung tumors and inhibition of Ku80 may be an interesting target to improve radiosensitivity in lung cancer patients.     

食管鳞状细胞癌中MMP-9、CD-147的表达及意义

目的：探讨基质金属蛋白酶9（MMP-9）和CD-147在食管鳞状细胞癌的表达及其与肿瘤浸润转移的关系。方法：应用免疫组化S-P法观察57例食管鳞癌组织中MMP-9、CD-147的表达,并探讨其与食管鳞癌临床病理资料的关系。结果：MMP-9、CD-147在癌组织中的阳性表达率分别为82．46％,64．91％;在癌旁组织中的阳性表达率分别是29．82％,8．77％。在食管鳞癌中,MMP-9及CD-147的表达均与食管鳞癌的浸润深度有关,与分化程度无明显关联;有淋巴结转移的病例阳性表达率明显高于无淋巴结转移组。结论：MMP-9及CD-147的表达均与食管鳞癌的浸润深度及淋巴结转移有关。     

双向凝胶电泳-飞行时间质谱技术鉴定食管上皮细胞癌变时14-3-3 protein ε、S100A9的表达

[摘要] 目的：分析食管鳞癌和正常食管上皮细胞蛋白质的表达差异,获取鉴别两者的分子标志物。方法：通过激光捕获显微切割技术分离ESCC肿瘤细胞和癌旁上皮细胞,通过双向电泳和质谱技术鉴定表达异常的蛋白,并通过蛋白免疫印记证实部分差异蛋白的表达。结果：建立了食管癌组织和正常食管上皮蛋白的双向凝胶电泳图谱,通过质谱技术鉴定出14-3-3 protein ε、S100A9等蛋白在食管癌变时差异表达,蛋白印记结果证实14-3-3 protein ε、S100A9的表达量在食管癌变时分别上调和下调。结论：激光捕获显微切割是蛋白质组研究中的一个突破性的技术,可以有效地解决组织异质性的问题;本实验检测到的差异蛋白例如14-3-3 protein ε、S100A9可能成为鉴别食管癌组织和正常食管上皮特异性的分子标记物。     

BAG3‐positive pancreatic stellate cells promote migration and invasion of pancreatic ductal adenocarcinoma

BAG3 is constitutively expressed in multiple types of cancer cells and its high expression is associated with tumour progression and poor prognosis of PDAC . However, little is known about the role of BAG3 in the regulation of stromal microenvironment of PDAC. The current study demonstrated that beside PDAC tumour cells, BAG3 was also expressed in some activated stroma cells in PDAC tissue, as well as in activated PSCs. In addition, the current study demonstrated that BAG3 expression in PSCs was involved in maintenance of PSCs activation and promotion of PDACs invasion via releasing multiple cytokines. The current study demonstrated that BAG3‐positive PSCs promoted invasion of PDACs via IL‐8, MCP1, TGF‐β2 and IGFBP2 in a paracrine manner. Furthermore, BAG3 sustained PSCs activation through IL‐6, TGF‐β2 and IGFBP2 in an autocrine manner. Thereby, the current study provides a new insight into the involvement of BAG3 in remodelling of stromal microenvironment favourable for malignant progression of PDAC, indicating that BAG3 might serve as a potential target for anti‐fibrosis of PDAC.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma

Radioresistance‐induced residual and recurrent tumours are the main cause of treatment failure in nasopharyngeal carcinoma (NPC). Thus, the mechanisms of NPC radioresistance and predictive markers of NPC prognosis and radioresistance need to be investigated and identified. In this study, we identified RPA3 as a candidate radioresistance marker using RNA‐seq of NPC samples. In vitro studies further confirmed that RPA3 affected the radiosensitivity of NPC cells. Specifically, the overexpression of RPA3 enhanced radioresistance and the capacity for DNA repair of NPC cells, whereas inhibiting RPA3 expression sensitized NPC cells to irradiation and decreased the DNA repair capacity. Furthermore, the overexpression of RPA3 enhanced RAD51 foci formation in NPC cells after irradiation. Immunohistochemical assays in 104 NPC specimens and 21 normal epithelium specimens indicated that RPA3 was significantly up‐regulated in NPC tissues, and a log‐rank test suggested that in patients with NPC, high RPA3 expression was associated with shorter overall survival (OS) and a higher recurrence rate compared with low expression (5‐year OS rates: 67.2% versus 86.2%; 5‐year recurrence rates: 14.8% versus 2.3%). Moreover, TCGA data also indicated that high RPA3 expression correlated with poor OS and a high recurrence rate in patients with head and neck squamous cell carcinoma (HNSC) after radiotherapy. Taken together, the results of our study demonstrated that RPA3 regulated the radiosensitivity and DNA repair capacity of NPC cells. Thus, RPA3 may serve as a new predictive biomarker for NPC prognosis and radioresistance to help guide the diagnosis and individualized treatment of patients with NPC.     

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。     

LINC00473/miR-374a-5p regulates esophageal squamous cell carcinoma via targeting SPIN1 to weaken the effect of radiotherapy

Esophageal squamous cell carcinoma (ESCC) is the most prevalent type in esophageal cancers. Despite accumulating achievements in treatments of ESCC, patients still suffer from recurrence because of the treatment failures, one of the reasons for which is radioresistance. Therefore, it is a necessity to explore the molecular mechanism underlying ESCC radioresistance. Long intergenic noncoding RNA 473 (LINC00473) has been reported to be aberrantly expressed in several human malignancies. However, its biological function in radiosensitivity of ESCC remains to be fully understood. This study explored the role of LINC00473 in radiosensitivity of ESCC cells and whether LINC00473 acted as a competing endogenous RNA to realize its modulation on radioresistance. We found that LINC00473 was markedly upregulated in ESCC tissues and cell lines, and its expression was remarkably related to cellular response to irradiation. In addition, knockdown of LINC00473 could sensitize ESCC cells to radiation in vitro. As for the underlying mechanism, we uncovered that there was a mutual inhibition between LINC00473 and miR-374a-5p. Spindlin1 (SPIN1) was verified as a downstream target of miR-374a-5p, and LINC00473 upregulated SPIN1 expression through negatively modulating miR-374a-5p expression. Furthermore, we revealed that SPIN1 could aggravate the radioresistance of ESCC cells. Finally, overexpression of SPIN1 reversed the LINC00473 silencing-enhanced radiosensitivity in ESCC cells. To sum up, we demonstrated that LINC00473 facilitated radioresistance by regulating the miR-374a-5p/SPIN1 axis in ESCC.     

Survivin特异性SiRNA提高鼻咽癌移植瘤放射敏感性

目的：研究Survivin特异性SiRNA（small interfering RNA）对鼻咽癌移植瘤的放疗增敏作用,探索提高鼻咽癌疗效的新方法。方法：Survivin特异性SiRNA转染鼻咽癌5-8F细胞系,培养48h后,采用RT-PCR、流式细胞仪（flow cytometry,FCM）分别检测Survivin mRNA和蛋白在5-8F细胞中表达。将Survivin基因特异性SiRNA转染5-8F细胞,培养24h后,用剂量为6GY的放射线处理,培养6h后,收集细胞,进行裸鼠皮下接种,50d后处死裸鼠,对移植瘤进行分析。结果：Survivin特异性SiRNA能有效抑制5-8F细胞中Survivin表达。Survivin特异性SiRNA组,Survivin表达阳性率12.37±1.86%,与对照组阳性率91.93±1.3%和阴性对照组阳性率92.43±2.34%比较,差别具有显著性（p〈0.01）。特异性SiRNA加放射组移植瘤（0.03±0.03g）显著小于特异性SiRNA组（0.28±0.02g,p〈0.01）与阴性SiRNA加放射组（0.17±0.02g,p〈0.01）。结论：Survivin特异性SiRNA增强了鼻咽癌5-8F细胞移植瘤的放射敏感性。