生物钟蛋白TIMELESS对乳腺癌细胞增殖和侵袭转移的差异调控

生物钟蛋白TIMELESS是生物体周期节律相关的核心分子钟的一员。TIMELESS可以调节细胞的增殖和代谢,DNA损伤的识别和修复等。研究发现,TIMELESS的表达与肝癌、肺癌、结直肠癌和鼻咽癌等的发生发展明显关联。在乳腺癌中,TIMELESS的调节作用和机制仍不十分清晰。本文分别研究了TIMELESS在乳腺癌细胞增殖和转移方面的调控作用。通过细胞增殖实验发现,TIMELESS可明显促进乳腺癌细胞的增殖。克隆形成实验发现,在乳腺癌细胞ZR-75-30和T-47D中,TIMELESS过表达分别上调克隆数量约1.6倍和1.8倍。通过报告基因检测对雌激素信号通路的研究发现,TIMELESS和雌激素受体ERα(estrogen receptor)共表达与单转ERα相比,使得雌激素受体ERα的转录活性提高约3倍;而通过对于Basal型乳腺癌患者生存曲线分析发现,Timeless高表达与basal型乳腺癌患者的生存率正相关。基于此,我们进一步研究了TIMELESS对于乳腺癌细胞转移的调控作用。通过细胞划痕实验和F-肌动蛋白组装检测发现,TIMELESS抑制乳腺癌细胞的转移;同时,TIMELESS敲低提高了乳腺癌细胞的转移数量约1.6倍,并下调了上皮标志物E 钙黏着蛋白的表达,上调了间质标志物N-钙黏着蛋白表达。本研究提示,TIMELESS在调控乳腺癌的增殖和转移过程中存在差异调控,即TIMELESS促进乳腺癌细胞增殖,而抑制了乳腺癌细胞的转移。这为生物钟蛋白质调控乳腺癌的发生发展提供了分子基础,同时为乳腺癌的分型治疗提供了一定的理论依据。     

Caveolin-1基因沉默促进乳腺上皮细胞ERα36 介导的PI3K/AKT信号通路的激活

Caveolin-1(窖蛋白-1)单倍不足可以促进正常乳腺细胞的早期转化, 与新型雌激素受体亚型ERα36介导的膜始动雌激素信号通路的激活有关, 但是关于其机制并未被研究清楚. 本文利用siRNA技术建立了Caveolin-1低表达而ERα36高表达的稳定传代细胞模型MCF10ACE, 采用基因芯片技术检测了ERα36高表达时雌激素信号通路基因表达谱, 研究了ERα36在雌激素激活的PI3K/AKT信号通路中的作用及其与乳腺细胞转化的关系. 结果表明: (1) Caveolin-1表达降低时可以以雌激素依赖性方式促进ERα36表达增加; (2) ERα36高表达可介导MCF10ACE细胞雌激素抗凋亡信号通路(PI3K/AKT)的激活, 促进其与增殖信号通路MEK/ERK之间的交叉对话, 从而使人乳腺细胞增殖加快, 逐渐转化. 结果提示, Caveolin-1与ERα36相互作用调节膜起始的雌激素信号通路是控制乳腺细胞转化的重要机制.     

LRP16通过调控E-钙粘合素的表达促进MCF-7细胞的侵袭生长

LRP16在原代乳腺癌组织中表达水平与雌激素受体α（ERα）表达状态以及腋窝淋巴结侵袭数目密切相关.为研究LRP16基因对乳腺癌MCF-7细胞侵袭生长的影响,并探讨涉及的分子机制,采用基质胶黏附实验与Transwell方法,检测内源性LRP16表达抑制MCF-7细胞的体外黏附、侵袭生长与迁移特征.结果表明,抑制LRP16在MCF-7细胞中的表达,降低了细胞的体外黏附、侵袭与迁移能力;采用FVB小鼠进行的实验性转移试验结果显示,抑制LRP16显著降低了MCF-7细胞的肺转移结节数目;为探索可能的分子机制,采用Western印迹方法,检测了LRP16对乳腺癌转移相关分子MMP-2, MMP-9, CD44和E-钙黏着蛋白表达的影响,结果在LRP16抑制的MCF-7细胞中只有E-钙黏着蛋白蛋白表达上调.进一步的Northern印迹与免疫组化实验结果表明,抑制LRP16可上调MCF-7细胞中E 钙黏着蛋白基因的mRNA与蛋白表达水平;共转染与双荧光素酶方法检测LRP16对E-钙黏着蛋白基因启动子的表达调控效应,结果显示,LRP16抑制E 钙黏着蛋白基因基因5′-近端启动子的转录激活,该抑制效应选择性存在于内源性ERα阳性细胞,并且依赖于雌激素的存在;染色质免疫共沉淀方法（ChIP）检测ERα与E-钙黏着蛋白基因启动子的相互作用,结果显示,在LRP16基因表达缺陷的MCF-7细胞中,ERα抗体沉淀到E-钙黏着蛋白基因启动子的DNA序列;上述研究结果表明,抑制LRP16基因表达,削弱了激素依赖型乳腺癌细胞的侵袭生长能力,其分子机制涉及了LRP16通过ERα介导对E-钙黏着蛋白基因基因转录激活的调控.     

高尔基磷蛋白3参与调控乳腺癌的增殖

高尔基体磷蛋白3 (GOLPH3)是一种新的致癌基因,与某些恶性肿瘤的生长和转移密切相关。本研究旨在考察GOLPH3在乳腺癌细胞增殖中的作用。采用MTS和BrdU 2种方法检测GOLPH3对人乳腺癌细胞系MDA-MB-435S增殖的影响,流式细胞仪检测细胞周期,qRT-PCR检测细胞周期相关基因(Cyclin D, Cyclin E和P21)表达。研究发现,GOLPH3在乳腺癌样本中显著上调;GOLPH3的过表达显著促进MDA-MB-435S细胞的增殖;miRNA-590-3p的过表达抑制了细胞的GOLPH3 mRNA表达及增殖;过表达ATF-3通过抑制miR-590-3p来促进MDA-MB-435S细胞增殖。本研究表明抑制ATF-3/miR-590-3p/GOLPH3信号通路可抑制乳腺癌细胞的增殖,GOLPH3可能成为未来临床治疗中乳腺癌早期诊断的潜在生物标志物。     

泛素偶联酶2C促进肺癌细胞增殖和迁移与抑制细胞衰老

泛素偶联酶2C与多种肿瘤细胞的增殖密切相关,但其与肺癌发生和发展的关系尚不明确。 本研究以肺癌A549细胞为材料,通过RT-PCR、Western印迹、免疫荧光、SA-β-Gal细胞衰老染色、细胞划痕和Trans-well实验,阐明UBE2C与肺癌细胞的增殖、衰老和迁移能力的关系。结果显示,UBE2C在肺癌细胞中的表达明显高于正常细胞。利用基因修饰技术瞬时过表达或靶向沉默UBE2C后,在肺癌A549细胞中,UBE2C的mRNA和蛋白质水平显著增加3.5倍或减少0.5倍,显著促进或抑制细胞增殖,进而减少或增加细胞的凋亡率。过表达UBE2C后,显著抑制细胞衰老;但沉默UBE2C后,则增加细胞衰老。此外,过表达UBE2C后,下调转移相关基因E-钙黏着蛋白的mRNA和蛋白质表达水平,且上调波形蛋白基因的表达水平,进而促进肺癌细胞的迁移。但靶向敲除UBE2C后,上调E-钙黏着蛋白,同时下调波形蛋白表达水平,进而抑制肺癌细胞的迁移。本研究的开展将明确UBE2C在肺癌中的作用及其机制,为以UBE2C为靶点,提高病人生存期提供了理论基础。     

过表达ER恢复雌激素对子宫内膜癌KLE细胞中Notch信号通路的调控

目的:通过观察雌激素对子宫内膜癌KLE细胞中Notch信号通路的影响,探讨过表达雌激素核受体（estrogen receptor,ER）是否可以恢复雌激素对Notch信号通路的调控作用,继而调节细胞增殖活性。方法:MTT检测雌激素及Notch信号通路对细胞增殖活性的影响;RT-PCR及Western-blotting检测雌激素及Notch通路抑制剂DAPT对Notch表达的影响;质粒的抽提及转染使KLE细胞中的雌激素核受体ER过表达。结果:雌激素呈剂量依赖效应促进KLE细胞的增殖活性,其中以雌激素浓度为1.0×10^-9M时最明显（相对于对照组为1.25±0.026,P<0.05）;抑制Notch信号通路的表达可以明显下调KLE细胞的增殖活性（0.76±0.02,P<0.05）;在KLE细胞中,雌激素对Notch的表达没有明显的调控作用,但是将其雌激素核受体过表达后,雌激素可明显上调Notch的表达,并显著促进细胞的增殖活性（1.24±0.02,P<0.05）。结论:在ER阴性的子宫内膜癌细胞中过表达ER,可以恢复雌激素对Notch信号通路的调控,从而进一步的调控细胞增殖活性。     

LRP16对乳腺癌MCF-7细胞增殖的影响

用Northern印迹方法检测雌二醇 (17β E2 )对LRP16mRNA表达的时间及剂量依赖性调控作用 .构建LRP16基因启动子序列调控的萤光素酶报告子 (pS0 ) ,并与雌激素受体α和 β(ERα和ERβ)表达载体共转染COS 7和MCF 7细胞后测定萤光素酶活性 .将LRP16基因的表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性的影响 .17β E2 使MCF 7细胞中LRP16mRNA表达水平增加 ,增加幅度未显示出 17β E2 培养时间和剂量的依赖性 .pS0 与ERα表达载体共转染细胞的相对萤光素酶活性较非共转染组 (对照组 )及pS0 ERβ表载体共转染组升高 5～ 10倍 .LRP16基因过表达促进MCF 7细胞的增殖 .研究表明 ,雌激素可能通过ERα上调乳腺癌MCF 7细胞LRP16基因的表达并促进细胞增殖     

过表达ER恢复雌激素对子宫内膜癌KLE细胞中Notch信号通路的调控

目的：通过观察雌激素对子宫内膜癌KLE细胞中Notch信号通路的影响,探讨过表达雌激素核受体（estrogenreceptor,ER）是否可以恢复雌激素对Notch信号通路的调控作用,继而调节细胞增殖活性。方法：MTT检测雌激素及Notch信号通路对细胞增殖活性的影响;RT．PCR及Westem．blotting检测雌激素及Notch通路抑制剂DAPT对Notch表达的影响;质粒的抽提及转染使KLE细胞中的雌激素核受体ER过表达。结果：雌激素呈剂量依赖效应促进KLE细胞的增殖活性,其中以雌激素浓度为1．0×10-9M时最明显（相对于对照组为1．25±0．026,P〈0．05）;抑制Notch信号通路的表达可以明显下调KLE细胞的增殖活性（0．76±0．02,P〈0．05）;在KLE细胞中,雌激素对Notch的表达没有明显的调控作用,但是将其雌激素核受体过表达后,雌激素可明显上调Notch的表达,并显著促进细胞的增殖活性（1．24±0．02,P〈0．05）。结论：在ER阴性的子宫内膜癌细胞中过表达ER,可以恢复雌激素对Notch信号通路的调控,从而进一步的调控细胞增殖活性。     

8-氯腺苷通过ADAR1调控miR-335-5p抑制乳腺癌细胞的增殖、迁移和侵袭

该文探讨了8-氯腺苷(8-chloro-adenosine, 8-Cl-Ado)调控RNA编辑酶1(adenosine deaminases acting on RNA1, ADAR1)对乳腺癌细胞增殖、迁移和侵袭的影响,确定了ADAR1与miR-335-5p表达的相关性。10μmol/L的8-Cl-Ado作用于乳腺癌细胞后(不同时间点),采用CCK-8检测细胞的增殖情况; Transwell检测细胞的迁移和侵袭情况; Western blot检测ADAR1蛋白的表达水平;CCK-8检测ADAR1过表达对8-Cl-Ado抑制乳腺癌细胞增殖的影响; Transwell检测ADAR1过表达对8-Cl-Ado抑制乳腺癌细胞迁移和侵袭的影响; miRNA芯片筛选8-Cl-Ado处理后乳腺癌细胞中上调的miRNA, qRT-PCR验证其与ADAR1的相关性。结果显示, 8-Cl-Ado能抑制乳腺癌细胞的增殖、迁移和侵袭; ADAR1蛋白的表达量随8-Cl-Ado处理时间的增加而逐渐降低; ADAR1的过表达能减弱8-Cl-Ado对乳腺癌细胞增殖、迁移和侵袭的抑制作用; miR-335-5p经8-Cl-Ado处理后表达水平上调,与ADAR1呈负向调控关系。以上结果表明, 8-Cl-Ado下调ADAR1抑制乳腺癌细胞增殖、迁移和侵袭,其作用机制可能与ADAR1下调mi R-335-5p有关。     

Prohibitin 1 的上调参与肝癌细胞的增殖和迁移

尽管已知prohibitin 1（PHB）与肿瘤有关,但很少有关于PHB在肝癌中的作用的报道．前期的糖蛋白质组学研究显示,PHB在高转移肝癌细胞株MHCC97-H细胞中被上调,且它可以被麦胚凝集素WGA所吸附．在本研究中,通过Western blotting和逆转录PCR实验证实,PHB在MHCC97．H细胞中的表达被上调约2倍．当PHB在MHCC97-H细胞中被过表达时,细胞增殖被抑制35％,细胞迁移率提高约2倍．本研究结果表明,PHB在MHCC97-H细胞中被上调,而且这种上调与肝癌细胞的增殖和迁移有关．     

蛋白激酶AKT 不同亚型影响乳腺癌恶性表型的研究进展

细胞的增殖、转移、存活等细胞生物学过程的异常对人类众多疾病尤其是恶性肿瘤的发生发展至关重要。大量研究表明,PI3K/AKT信号通路的异常激活在肿瘤的恶性转化过程中发挥重要作用并具有普遍意义。但是,目前的研究多集中于探讨AKT总的激酶活性,而往往忽视了AKT不同亚型的特异性功能。近年来在乳腺癌中的研究发现,AKT家族不同亚型的激酶分子在调控肿瘤细胞的存活、生长、增殖、代谢、转移等众多恶性表型方面发挥独特而关键的作用:与Akt1促进肿瘤细胞增殖、抑制肿瘤细胞转移的作用相反,Akt2在促进肿瘤细胞转移、抑制肿瘤细胞增殖方面发挥重要功能;此外,随着对AKT家族研究的深入,人们对Akt3的特异性生物学功能也有了新的认识。本文在此对AKT不同亚型与乳腺癌恶性表型之间关系的研究进展做一总结。     

Delta/notch-like epidermal growth factor-related receptor promotes stemness to facilitate breast cancer progression

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The function of DNER in breast cancer has not been evaluated. The present study demonstrates that the expression of DNER in breast cancer tissue is significantly higher than its expression in breast benign disease and is associated with poor recurrence-free survival (RFS) of breast cancer patients. It demonstrated that DNER could enhance the proliferation and metastasis of breast cancer cells in vitro and significantly increases tumor growth in vivo. Our study uncovered that DNER can promote breast cancer cells proliferation and metastasis by activating Girdin/PI3K/AKT signaling and subsequently regulating several key genes involving the characters of cancer stem cells. Taken together, DNER promotes breast cancer growth and metastasis, which provided a theoretical basis for future applications of DNER inhibitors in the treatment of breast cancer.     

Circadian clock genes promote glioma progression by affecting tumour immune infiltration and tumour cell proliferation

ObjectivesCircadian rhythm controls complicated physiological activities in organisms. Circadian clock genes have been related to tumour progression, but its role in glioma is unknown. Therefore, we explored the relationship between dysregulated circadian clock genes and glioma progression.Materials and MethodsSamples were divided into different groups based on circadian clock gene expression in training dataset (n = 672) and we verified the results in other four validating datasets (n = 1570). The GO and GSEA enrichment analysis were conducted to explore potential mechanism of how circadian clock genes affected glioma progression. The single‐cell RNA‐Seq analysis was conducted to verified previous results. The immune landscape was evaluated by the ssGSEA and CIBERSORT algorithm. Cell proliferation and viability were confirmed by the CCK8 assay, colony‐forming assay and flow cytometry.ResultsThe cluster and risk model based on circadian clock gene expression can predict survival outcome. Samples were scoring by the least absolute shrinkage and selection operator regression analysis, and high scoring tumour was associated with worse survival outcome. Samples in high‐risk group manifested higher activation of immune pathway and cell cycle. Tumour immune landscape suggested high‐risk tumour infiltrated more immunocytes and more sensitivity to immunotherapy. Interfering TIMELESS expression affected circadian clock gene expression, inhibited tumour cell proliferation and arrested cell cycle at the G0/G1 phase.ConclusionsDysregulated circadian clock gene expression can affect glioma progression by affecting tumour immune landscape and cell cycle. The risk model can predict glioma survival outcome, and this model can also be applied to pan‐cancer.     

Tumor suppressor function of RUNX3 in breast cancer

Emerging evidence indicates that RUNX3 is a tumor suppressor in breast cancer. RUNX3 is frequently inactivated in human breast cancer cell lines and cancer samples by hemizygous deletion of the Runx3 gene, hypermethylation of the Runx3 promoter, or cytoplasmic sequestration of RUNX3 protein. Inactivation of RUNX3 is associated with the initiation and progression of breast cancer. Female Runx3(+/-) mice spontaneously develop ductal carcinoma, and overexpression of RUNX3 inhibits the proliferation, tumorigenic potential, and invasiveness of breast cancer cells. This review is intended to summarize these findings and discuss the tumor suppressor function of RUNX3 in breast cancer.     

Leukemia inhibitory factor inhibits the proliferation of gastric cancer by inducing G1-phase arrest

Leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family, plays a complex role in cancer. LIF inhibits the proliferation and survival of several myeloid leukemia cells but promotes tumor progression and metastasis in many solid tumors. However, the relationship between LIF and gastric cancer has not been well understood. LIF was downregulated in gastric cancer as detected by western blot analysis and immunohistochemistry (IHC). Notably, LIF was downregulated in approximately 70% (56/80) of primary gastric cancers, in which it was significantly associated with advanced clinical stage, lymph node metastasis, and poor overall survival (median 5-year survival = 26 vs. 43 months for patients with high LIF expression and low LIF expression gastric cancer, respectively). To study the potential function of LIF in the downregulation of gastric cancer, we monitored the behavior using proliferation, cell cycle, and flow cytometry analysis. Overexpression of LIF inhibited the gastric cancer cell cycle in the G1 phase. In our experiment, overexpression of LIF by lentivirus upregulated P21 and downregulated cyclin D1. Recombinant human LIF also downregulated P21 and cyclin D1 at various times. A further in vivo tumor formation study in nude mice indicated that overexpression of LIF in gastric cancer significantly delayed the progress of tumor formation. These findings indicate that LIF may serve as a negative regulator of gastric cancer.