MicroRNA‐127 is Downregulated by Tudor‐SN Protein and Contributes to Metastasis and Proliferation in Breast Cancer Cell Line MDA‐MB‐231

Tudor‐SN is a multifunctional protein that is highly expressed in multiple cancers including breast cancer. Tudor‐SN, as a component in RNA‐induced splicing complex, was recently reported to regulate gene expression in a microRNA (miRNA)‐dependent manner, such as let‐7, miR‐34a and miR‐221. However, how Tudor‐SN is associated with cancer development still remains largely elusive. In the present study, we explored the role of Tudor‐SN in breast cancer. Stable knockdown of endogenous Tudor‐SN, performed on the breast cancer cell line MDA‐MB‐231 by small hairpin RNA expression vectors, suppressed the in vitro migration and invasion ability of the metastatic breast cancer cell line. Interestingly, we found Tudor‐SN as a miRNA regulator according to microarray analysis, and further identified that Tudor‐SN negatively regulated the expression of miR‐127, and consequently increased the expression of the proto‐oncogene BCL6 which was a convincing target of miR‐127. Moreover, overexpression of miR‐127 reduced the in vitro migration and proliferation ability of breast cancer cell MDA‐MB‐231. Collectively, our results suggested a novel mechanism that Tudor‐SN promoted metastasis and proliferation of breast cancer cells via downregulating the miR‐127 expression. Anat Rec, 296:1842–1849, 2013. © 2013 Wiley Periodicals, Inc.     

Metformin Induces G1 Cell Cycle Arrest and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma Cells

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP‐activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1–50 mM of metformin in a dose‐ and time‐dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666‐1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time‐dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)‐binding protein 1 (4E‐BP1) (T37/46) and S6 (S235/236) in C666‐1 cells. In a summary, metformin prevents proliferation of C666‐1 cells by down‐regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK‐mediated inhibition of mTORC1 signaling may be involved in this process. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

MiR‐1253 exerts tumor‐suppressive effects in medulloblastoma via inhibition of CDK6 and CD276 (B7‐H3)

Of the four primary subgroups of medulloblastoma, the most frequent cytogenetic abnormality, i17q, distinguishes Groups 3 and 4 which carry the highest mortality; haploinsufficiency of 17p13.3 is a marker for particularly poor prognosis. At the terminal end of this locus lies miR‐1253, a brain‐enriched microRNA that regulates bone morphogenic proteins during cerebellar development. We hypothesized miR‐1253 confers novel tumor‐suppressive properties in medulloblastoma. Using two different cohorts of medulloblastoma samples, we first studied the expression and methylation profiles of miR‐1253. We then explored the anti‐tumorigenic properties of miR‐1253, in parallel with a biochemical analysis of apoptosis and proliferation, and isolated oncogenic targets using high‐throughput screening. Deregulation of miR‐1253 expression was noted, both in medulloblastoma clinical samples and cell lines, by epigenetic silencing via hypermethylation; specific de‐methylation of miR‐1253 not only resulted in rapid recovery of expression but also a sharp decline in tumor cell proliferation and target gene expression. Expression restoration also led to a reduction in tumor cell virulence, concomitant with activation of apoptotic pathways, cell cycle arrest and reduction of markers of proliferation. We identified two oncogenic targets of miR‐1253, CDK6 and CD276, whose silencing replicated the negative trophic effects of miR‐1253. These data reveal novel tumor‐suppressive properties for miR‐1253, i.e., (i) loss of expression via epigenetic silencing; (ii) negative trophic effects on tumor aggressiveness; and (iii) downregulation of oncogenic targets.     

Caveolin‐1 is an Important Factor for the Metastasis and Proliferation of Human Small Cell Lung Cancer NCI‐H446 Cell

Caveolin‐1 (CAV‐1) has been reported to play an important role in the development of a variety of human cancers. CAV‐1 expression is revealed to be reduced or absent in the malignant tumor cells of small cell lung cancers (SCLC). This study was performed to investigate the influences of the stable expression of CAV‐1 on the metastasis and proliferation of SCLC in vitro. The wild‐type CAV‐1 gene was successfully transfected into the NCI‐H446 cells and was stably expressed in the NCI‐H446 cells. The effects of CAV‐1 on the morphology, proliferation, and metastasis potential for NCI‐H446 cell were evaluated by crystal violet staining, MTT analysis, transwell assay, and scratch wound assay, respectively. Western blot and gelatin zymography were used to examine the expression changes of the metastasis‐related MMP‐3 and E‐cadherin. Stable expression of CVA‐1 was observed in the H446‐CAV‐1 cells, which enlarged the cell shape with filopodia. The proliferation of H446‐CAV‐1 was inhibited, while its migration and invasion abilities were promoted in vitro. The re‐expression of CAV‐1 reduced the expression of E‐cadherin, while it increased the protein expression and enzyme activity of MMP‐3. Taken together, the cellular proliferation of the NCI‐H446 could be inhibited by the re‐expression of CAV‐1. CAV‐1 might increase the cell metastasis potential through the interaction with E‐cadherin and MMP‐3 genes. These in vitro findings confirm the involvement of CAV‐1 in the proliferation and metastasis of SCLC. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell Proliferation in Osteoblasts

Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5–200 μM hydrogen peroxide (H₂O₂) dose‐ and time‐dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3‐E1, and in human osteoblast‐like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G₂/M phase increased about 2‐4 fold by 200 μM H₂O₂ treatment for 24–72 hr. A total of 0.5–5 mM of H₂O₂ but not lower concentrations (5–200 μM) of H₂O₂ inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E‐BP1 (T37/46), and S6(S235/236) in MC3T3‐E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H₂O₂ prevents cell proliferation of osteoblasts by down‐regulating cyclin B1 and inducing G₂ cell cycle arrest. Inhibition of mTOR signaling by H₂O₂ may not be involved in this process. Anat Rec, 292:1107–1113, 2009. © 2009 Wiley‐Liss, Inc.     

人CD137单抗诱导不同状态T细胞增殖和凋亡的研究

为了研究一种新的T细胞共刺激分子—CD137对不同状态T细胞增殖和凋亡的双重调节作用。采用3 H TdR掺入法测定T细胞增殖 ,用流式细胞仪测定细胞凋亡。结果显示 :(1)CD137单抗可与T细胞表面的CD137抗原结合 ,明显增强PHA刺激T细胞增殖 ,使T细胞增殖指数较PHA单独作用高 2～ 3倍 ,但CD137单抗单独不能刺激T细胞增殖 ;(2 )对于慢性活化的T细胞 ,CD137单抗可协同PHA诱导T细胞凋亡 ,使T细胞凋亡率从PHA单独作用的 19 2 0 %增加到 36 31% ,CD137单抗单独并不能诱导慢性活化T细胞凋亡。CD137单抗一方面可协同PHA刺激静止状态T细胞的增殖 ,另一方面可协同PHA诱导慢性活化T细胞凋亡 ,对T细胞起双重调节作用。     

PARP-1抑制剂对人肝癌细胞HepG2增殖和迁移的抑制作用

目的 研究证实聚腺苷二磷酸核糖聚合酶-1(poly ADP ribose polymerase,PARP-1)抑制剂对肿瘤细胞的增殖、凋亡和侵袭有影响,但对肝癌细胞生物学特性的影响尚不清楚,此研究的目的是观察3种不同的PARP-1抑制剂对人肝癌细胞株HepG2增殖、凋亡及迁移的影响及可能机制.方法 MTT检测体外不同浓度的PARP-1抑制剂AG014699,BSI-201,AZD-2281处理后HepG2细胞的增殖;随后选择抑制效果明显的抑制剂AG014699和BSI-201处理HepG2;Western印迹法检测HepG2细胞Casepase3、Casepase8、Bax、Bcl-2、PTEN、Timp3、MMP3蛋白的表达水平.Transwell实验检测AG014699和BSI-201对HepG2细胞迁移的影响.结果 AG014699,BSI-201,AZD-2281均具有抑制HepG2细胞增殖的作用,具有时间和浓度依赖性,作用HepG2细胞48 h后的Caspase3、Caspase8、Bax、PTEN、Timp3蛋白的表达水平随药物浓度的增加而增高,而Bcl-2和MMP3蛋白水平随药物浓度的增加而降低且与对照组相比有显著性差异(P<0.01).结论 在体外PARP-1抑制剂AG014699、BSI-201、AZD-2281明显抑制HepG2细胞的增殖,但AG014699和BSI-201展示较好的敏感性,同时二者诱导肝癌细胞凋亡和抑制肝癌细胞的迁移,这其中的机制可能与影响凋亡信号的通路以及迁移相关蛋白的表达有关.     

MiR‐124 Regulates Apoptosis and Autophagy Process in MPTP Model of Parkinson's Disease by Targeting to Bim

Parkinson's disease (PD) is the most prevalent movement disorder characterized by selective loss of midbrain dopaminergic (DA) neurons. MicroRNA‐124 (miR‐124) is abundantly expressed in the DA neurons and its expression level decreases in the 1‐methyl‐4‐pheny‐1, 2, 3, 6‐tetrahydropyridine (MPTP) model of PD. However, whether the upregulation of miR‐124 could attenuate neurodegeneration remains unknown. Here, we employed miR‐124 agomir and miR‐124 mimics to upregulate miR‐124 expression in MPTP‐treated mice and MPP⁺‐intoxicated SH‐SY5Y cells, respectively. We found that loss of DA neurons and striatal dopamine in MPTP‐treated mice was significantly reduced by upregulating miR‐124. In addition, we identified a target of miR‐124, Bim that mediated the neuroprotection of miR‐124. Indeed, treatment of miR‐124 agomir in MPTP‐treated mice inhibited Bim expression, thus suppressing Bax translocation to mitochondria. Moreover, impaired autophagy process in MPTP‐treated mice and MPP⁺‐intoxicated SH‐SY5Y cells characterized as autophagosomes (AP) accumulation and lysosomal depletion were alleviated by the upregulation of miR‐124. Taken together, these results indicate that upregulation of miR‐124 could regulate apoptosis and impaired autophagy process in the MPTP model of PD, thus reducing the loss of DA neurons.