Rho kinase inhibitor fasudil suppresses migration and invasion though down-regulating the expression of VEGF in lung cancer cell line A549

Rho and Rho-associated kinase play an important role in focal adhesion, stress fiber formation and cell motility. Fasudil is a kind of Rho kinase inhibitor. The effect and precise molecular mechanism of fasudil on the biology behavior of lung cancer cell A549 remains unclear. The cytotoxic effect of fasudil on A549 cell was measured by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Wound-healing assay was used to evaluate the effect of fasudil on migration activity of A549 cells. The invasion activity of A549 cells was detected by transwell chamber assay. The expression of MMPs was measured by gelatin zymography. RT-PCR and western blot were used to investigate the molecular change of A549 cells after treated with fasudil. Fasudil-inhibited proliferation of A549 cells in a concentration-dependent manner, decreased the migration and invasion activity. After treated with fasudil, the expression of MMP-2 and MMP-9 was significantly inhibited compared with the control group. Furthermore, the expression of RhoA and VEGF of A549 cell treated with fasudil was significantly down-regulated. Our findings indicate that fasudil might have a therapeutic potential for lung cancer though inhibiting cell proliferation, migration, invasion, MMPs activity and down-regulating the expression of RhoA and VEGF.     

Epigallocatechin gallate sensitizes CAL-27 human oral squamous cell carcinoma cells to the anti-metastatic effects of gefitinib (Iressa) via synergistic suppression of epidermal growth factor receptor and matrix metalloproteinase-2

Human head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related death during the last decade due to its related metastasis and poor treatment outcomes. Gefitinib (Iressa), a tyrosine kinase inhibitor has been reported to reduce the metastatic abilities of oral cancer. Previous studies have shown that epigallocatechin gallate (EGCG), a green tea polyphenol, possesses cancer chemopreventive and anticancer activity. However, the mechanisms involved in the suppression of invasion and metastasis of human oral cancer cells following co-incubation with gefitinib and EGCG remain poorly understood. In the present study, we attempted to investigate the synergistic effects of a combined treatment of gefitinib and EGCG in CAL-27 cells in?vitro and to elucidate the underlying molecular mechanisms associated with the supression of cell migration and invasion. In the present study, we found that the individual treatments or the combined treatment of gefitinib and EGCG synergistically inhibited the invasion and migration of CAL-27 cells using Transwell invasion and wound-healing scratch assays, respectively. Similarly, gefitinib in combination with EGCG synergistically attenuated enzymatic activity and the protein expression of MMP-2 in CAL-27 cells. Furthermore, individual or combined treatment with EGCG and gefitinib suppressed the protein expression of p-EGFR and the phosphorylated protein levels of ERK, JNK, p38 and AKT and displayed inhibitory effects on metastatic ability of CAL-27 cells. Combined effects of EGCG and gefitinib-altered anti-metastatic actions for related gene expression were observed using DNA microarray analysis. Importantly, EGCG sensitizes CAL-27 cells to gefitinib-suppressed phosphorylation of epidermal growth factor receptor (EGFR in?vitro. Taken together, our results suggest that the synergistic suppression of the metastatic ability of CAL-27 cells after EGCG and gefitinib individual or combined treatment are mediated through mitogen-activated protein kinase (MAPK) signaling. Our novel findings provide potential insights into the mechanism involved with synergistic responses of gefitinib and EGCG against the progression of oral cancer.     

基质金属蛋白酶-9反义寡核苷酸对卵巢癌细胞侵袭黏附能力的影响

目的 观察基质金属蛋白酶-9（MMP-9）反义寡核苷酸（ASODN）转染对卵巢癌细胞体外侵袭黏附行为的影响,并探讨其作用机制。方法 以Lipofectinmin介导的MMP-9反义寡核苷酸转染至经纤黏连蛋白诱导MMP-9表达的卵巢癌细胞株HO-8910PM,利用RT—PCR、Western blot及明胶酶谱法检测转染寡核苷酸后HO-8910PM细胞MMPO的mRNA、蛋白表达及酶活性的变化;通过细胞体外侵袭、迁移实验和黏附实验,检测细胞侵袭黏附能力的变化。结果 卵巢癌细胞HO-8910PM转染MMP-9反义寡核苷酸后,MMP-9的mRNA及蛋白的表达受到抑制,抑制率分别为34．8％和42．5％,与对照组比较,差异有统计学意义（P〈0．05）;明胶酶活性也受到了抑制。反义寡核苷酸的转染降低了肿瘤细胞体外侵袭、迁移和黏附能力,侵袭和迁移抑制率分别为22．4％和24．8％,在60min和90min黏附抑制率分别为49．8％和38．3％。结论 MMP-9反义寡核苷酸可抑制卵巢癌细胞的侵袭黏附能力,MMP-9有可能成为抗卵巢癌侵袭转移的分子靶点。     

小干扰RNA沉默基质金属蛋白酶-2基因对卵巢癌细胞恶性行为的影响

 目的　探讨靶向基质全属蛋白酶-2（MMP-2）基因的RNA干扰（RNAi）技术对卵巢癌OVCAR-3细胞MMP-2的沉默作用,以及沉默MMP-2基因对OVCAR-3细胞生长、黏附、侵袭和迁移能力的影响。方法　合成特异性靶向MMP-2基因的小干扰RNA（siRNA）并转染卵巢癌OVCAR-3细胞,以非特异性序列转染细胞作为阴性对照组,以培养液代替siRNA转染细胞作为空白对照组,采用实时荧光定量PCR和Western blot分别检测转染后24 、48 和72 h MMP-2 mRNA和蛋白的表达水平,通过四甲基偶氮唑蓝（MTT）法绘制细胞生长曲线,利用Transwell、划痕实验检测细胞侵袭和迁移能力。结果　与阴性对照组相比,OVCAR-3细胞在转染siRNA-MMP-2后24 、48 和72 h MMP-2 mRNA表达量分别下降了73.8 %、78.8 %和78.4 %（均P＜0.05）,蛋白表达量分别下降了72.6 %、81.2 %和76.4 %（均P＜0.05）,以48 h作用最强;细胞生长曲线显示细胞生长无明显变化（P＞0.05）;60 min和90 min时的黏附抑制率分别为55.0 %和44.8 %（均P＜0.05）;细胞的侵袭和迁移能力分别下降29.7 %和35.8 %（均P＜0.05）。结论　siRNA介导的MMP-2基因沉默能明显抑制OVCAR-3细胞的黏附、侵袭和迁移能力,而对其生长无明显影响,MMP-2基因有可能成为卵巢癌基因治疗的重要靶点。     

Src酪氨酸激酶在人胃癌细胞转移中的作用和机制

背景与目的:Src酪氨酸激酶的活化在胃癌浸润和转移中发挥了重要的作用。本研究旨在探讨Src酪氨酸激酶对人胃癌细胞E-钙黏着蛋白(E-cadherin)和基质金属蛋白酶(matrix metalloproteinase,MMP)-2、9表达、血管内皮生长因子(vascular endothelial growth factor,VEGF)分泌、转录因子NF-κB和AP-1表达以及胃癌细胞体外侵袭能力的影响。方法:使用Src酪氨酸激酶抑制剂4-苯胺喹唑啉(3和10μmol/L)后,应用Western blot法检测人胃癌SGC7901细胞中E-cadherin、MMP-2和MMP-9蛋白表达变化;应用实时PCR(real-time PCR)检测细胞中E-cadherin、MMP-2和MMP-9的mRNA表达变化;应用ELISA法检测细胞培养上清液中VEGF表达变化;应用双荧光报告基因法检测NF-κB和AP-1转录活性;应用Transwell法检测肿瘤细胞侵袭能力变化。结果:当Src酪氨酸激酶抑制剂4-苯胺喹唑啉浓度为3和10μmol/L时,SGC7901细胞中E-cadherin蛋白表达量显著增加,E-cadherin的mRNA表达是对照组的263.8%(P<0.05)和403.3%(P<0.05);MMP-2和MMP-9的蛋白表达显著降低,MMP-2的mRNA表达是对照组的28.3%(P<0.05)和16.7%(P<0.05),MMP-9的mRNA表达是对照组的23.6%(P<0.05)和13.3%(P<0.05);VEGF含量是对照组的62.6%(P<0.05)和38.7%(P<0.05);AP-1转录活性是对照组的54.4%(P<0.05)和18%(P<0.05),NF-κB转录活性是对照组的38.3%(P<0.05)和11.5%(P<0.05);胃癌细胞侵袭能力是对照组的43.3%(P<0.05)和28.2%(P<0.05),呈现明显的剂量依赖性抑制作用。结论:Src酪氨酸激酶活化与人胃癌SGC7901细胞体外转移能力密切相关,其机制可能与肿瘤转移相关基因表达有关。     

低氧对白血病细胞株Raji细胞侵袭转移能力的影响

目的研究低氧对白血病细胞株Raji细胞侵袭转移能力的影响。方法Raji细胞体外常规培养,用体积分数为1%、3%、5%的氧分别处理Raji细胞24h,以常氧培养Raji细胞为对照组,通过细胞黏附实验检测细胞黏附力,细胞迁移实验检测细胞运动力,肿瘤细胞体外侵袭实验检测细胞侵袭力,RT-PCR检测细胞VEGF、MMP-2、MMP-9 mRNA的表达,Western Blot检测细胞HIF 1α蛋白的表达。结果与对照组相比,3%、5%的氧均能增强Raji细胞黏附力、运动力和侵袭力(P<0.05或P<0.01),而且还能上调Raji细胞HIF-1α蛋白、HIF-1α mRNA、VEGF mRNA、MMP 9 mRNA的表达(P<0.01),各组MMP 2 mRNA均无明显表达;而1%氧与对照组相比各检测指标无明显变化(P>0.05)。结论适度低氧可增强白血病细胞株Raji细胞侵袭转移能力,其机制可能是通过HIF-1α上调VEGF、MMP-9表达实现的。     

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-beta1 integrin complex in colon cancer cells

Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer.     

Photodynamic therapy suppresses the migration and invasion of head and neck cancer cells in vitro

Head and neck cancer is highly invasive. It has a tendency to metastasise to regional or distant sites after incomplete treatment. Photodynamic therapy (PDT) is effective in the treatment of head and neck cancers. To investigate the effect of sublethal PDT on the invasiveness of head and neck cancer cells and to elucidate the possible mechanisms, we initiated this study. Two head and neck cancer cell lines, KJ-1 and Ca9-22, were used in this study. Wound healing assay, migration assay, and matrigel invasion assay were used to evaluate the cell migration and invasion. Immunoblotting was performed to investigate the possibly involved signaling pathways. Sublethal PDT significantly suppressed the migration and invasion of both KJ-1 and Ca9-22 cells. Phosphorylation of the focal adhesion kinase (FAK) and its down-stream Src kinase and extracellular signal-regulated kinase (ERK) were also inhibited after sublethal PDT. Sublethal PDT suppresses the migration and invasion of Ca9-22 and KJ-1 cells. Inhibited phosphorylation of the FAK-Src kinase-ERK signaling pathway may be involved in the PDT-induced migration/invasion suppression.     

鞘氨醇激酶-1激活ERK通路介导人结肠癌细胞株LoVo侵袭与迁移的实验

目的研究Sphk1对人结肠癌LoVo细胞侵袭与迁移能力的影响并探讨其作用机制。方法将人结肠癌LoVo细胞分成Sphk1激活组,Sphk1抑制组,空白对照组。以Phorbol 12-myristate 13-acetate（PMA）为Sphk1激活剂（终浓度为100 nM）,N,N-dimethyl-D-eryt-hro-sphingosine（DMS）为Sphk1抑制剂（终浓度为50 μM）,NaCl（终浓度为0.9%）为空白试剂处理LoVo细胞24 h后,用Transwell Boyden小室模型测定LoVo细胞的相对侵袭率与迁移率;用Western　blot方法测定细胞Sphk1、ERK1/2与p-ERK1/2蛋白水平的变化;用ELISA方法检测细胞培养上清中MMP-2、MMP-9及uPA的蛋白含量;用半定量RT-PCR检测细胞中MMP-2、MMP-9和uPA的mRNA水平。结果Sphk1激活剂可促进LoVo细胞侵袭与迁移,同时明显增强LoVo细胞中Sphk1、ERK1/2及p-ERK1/2的蛋白表达,并促进MMP-2、MMP-9及uPA的mRNA表达与蛋白分泌。Sphk1抑制剂则抑制LoVo细胞侵袭与迁移,同时抑制Sphk1、ERK1/2与p-ERK1/2的蛋白表达,并抑制MMP-2、MMP-9及uPA的mRNA表达与蛋白分泌。结论Sphk1可促进人结肠癌细胞株LoVo细胞的侵袭与迁移,其机制可能与激活ERK1/2信号通路从而促进MMP-2、MMP-9及uPA mRNA表达与蛋白分泌有关。     

A novel taspine derivative,HMQ1611, suppresses adhesion,migration and invasion of ZR-75-30 human breast cancer cells

Background

Taspine was screened for the first time from Radix et Rhizoma leonticis (Hong Mao Qi in Chinese) using cell membrane chromatography in our laboratory. Its anticancer and antiangiogenic properties were demonstrated, and it could serve as a lead compound in anticancer agent development. Here, we investigated the role of one of the derivatives, HMQ1611, with increased activity and solubility, on the regulation of breast cancer cell ZR-75-30 adhesion, migration and invasion.

Methods

The effect of HMQ1611 on adhesion, invasion and migration of human breast cancer cells ZR-75-30 was examined. The migration and invasive potential of ZR-75-30 cells were examined by wound-healing assays and matrigel invasion chamber assays. The adhesion to type IV collagen and laminin were evaluated by MTT assay. The expression and proteinase activity of two matrix metalloproteinases (MMPs), matrix metalloproteinases 2 (MMP-2) and matrix metalloproteinases 9 (MMP-9), were analyzed by Western blot analysis and gelatin zymography, respectively.

Results

HMQ1611 effectively inhibited ZR-75-30 cell invasion and significantly suppressed adhesion to type IV collagen and laminin-coated substrate in a dose-dependent manner. Western blot and gelatin zymography analysis showed that HMQ1611 significantly inhibited the expression and secretion of MMP-2 and MMP-9 in ZR-75-30 cells. Additionally, treatment of ZR-75-30 cells with HMQ1611 downregulated the expression of MMP-2 and MMP-9.

Conclusions

HMQ1611 had potential to suppress the adhesion, migration and invasion of ZR-75-30 cancer cells, and it could serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.     

Anthocyanins from the Fruit of Vitis Coignetiae Pulliat Inhibit TNF-Augmented Cancer Proliferation,Migration, and Invasion in A549 Cells

Objective: Anthocyanins belong to a class of flavonoids, exhibiting antioxidant and anti-inflammatory actions have been reported to have anti-cancer effects. Here, we investigated whether anthocyanins can inhibit cancer cell proliferation, invasion, and angiogenesis in human lung cancer A549 cells, which are critically involved in cancer metastasis. Methods: We used anthocyanins from fruits of Vitis coignetiae Pulliat (AIMs) which has been used in Korean folk medicine for the treatment of inflammatory diseases and cancers. We have performed cell proliferation assays, cell invasion assay, gelatin zymography, wound healing assay and western blotting to examine whether anthocyanins can inhibit cancer cell proliferation, invasion, and angiogenesis in A549 cells. Result: AIMs did not inhibit cancer cell proliferation on A549 cells. Also, AIMs suppressed cancer migration, and invasion by supressing MMP-2 and MMP-9 expression. The Immuno-blotting results also revealed that AIMs suppressed the proteins involved in cancer proliferation (COX- 2, C-myc, cyclin D1), migration and invasion (MMP-2, MMP-9), anti-apoptosis (XIAP, and c-IAP2), adhesion and angiogenesis (ICAM-1, VEGF). Conclusion: This study demonstrates that the anthocyanins isolated from fruits of Vitis coignetiae Pulliat inhibit cancer proliferation, cancer migration, and invasion that is involve in cancer-metastasis. This study provides evidence that AIMs might have anti-cancer effects on human lung cancer.     

丹参酮ⅡA对胃癌细胞迁移和侵袭能力的抑制作用及机制

背景与目的：有研究证实,丹参酮ⅡA (tanshinone ⅡA)对肿瘤细胞具有抑制增殖、诱导分化和促凋亡的作用,并可抑制骨肉瘤细胞迁移和侵袭。但丹参酮ⅡA抑制胃癌侵袭和转移的机制尚不明确。本研究主要探讨丹参酮ⅡA对人胃癌SGC7901细胞体外迁移和侵袭的影响。方法：不同浓度(0.5、1、2、4 μg/mL)丹参酮ⅡA分别作用体外培养的胃癌SGC7901细胞24、48、72 h后,MTT比色法检测细胞增殖活力的改变;细胞划痕实验观察细胞的迁移能力的改变;3D侵袭实验观察细胞侵袭能力的改变;Real-time PCR和蛋白质印迹法(Western blot)分别检测细胞间黏附分子1(ICAM-1)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、金属蛋白酶抑制剂-2(TIMP-2)mRNA和蛋白的表达水平改变。结果：1、2、4 μg/mL丹参酮ⅡA对胃癌细胞株SGC7901有明显的抑制作用,且抑制作用存在时间-剂量依赖性(P<0.05);2 μg/mL丹参酮ⅡA呈时间依赖性抑制SGC7901细胞迁移;1、2、4 μg/mL丹参酮ⅡA呈浓度依赖性抑制SGC7901细胞侵袭;丹参酮ⅡA下调SGC7901细胞ICAM-1、MMP-2、MMP-9表达,同时可上调TIMP-2表达(P<0.05)。结论：丹参酮ⅡA可抑制胃癌SGC7901细胞的迁移和侵袭,上调TIMP-2的表达,下调ICAM-1、MMP-2、MMP-9的表达,可能是其作用机制之一。     

Gallic acid suppresses the migration and invasion of PC-3 human prostate cancer cells via inhibition of matrix metalloproteinase-2 and -9 signaling pathways

Epidemiological studies have demonstrated that a natural diet or consumption of fruits or vegetables can decrease the risk of cancer development. Cancer cells can migrate to and invade other organs or tissues that cause more difficulty to treat them and this also results in the need for treatments targeting multiple cellular pathways. Gallic acid (GA) has been demonstrated to possess multiple biological activities including anticancer function. However, no report exist on GA inhibited invasion and migration of human prostate cancer cells. We investigated the effects of migration and invasion in GA-treated PC-3 human prostate cancer cells with a series of in?vitro experiments. Boyden chamber transwell assay was used to examine the migration and invasion of PC-3 cells. Western blotting, real-time PCR and gelatin zymography were used for determining the protein levels, gene expression and enzyme activities of matrix metalloproteinase-2 (MMP-2) and -9 in?vitro. Results indicated that GA inhibited the invasion and migration of PC-3 cells and these effects are dose-dependent. GA inhibited the protein levels of MMP-2 and -9, son of sevenless homolog?1 (SOS1), growth factor receptor-bound protein 2 (GRB2), protein kinase C (PKC) and nuclear factor-κ B (NF-κB) p65, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, p-AKT (Thr308) and p-AKT (Ser473), but it promoted the levels of phosphatidylinositol 3-kinase (PI3K) and AKT in PC-3 cells. GA also reduced the enzyme activities of MMP-2 and -9 in the examined cells. Moreover, the down-regulation of focal adhesion kinase (FAK) and Ras homolog gene family, member A (Rho A) mRNA expression levels, and up-regulation of the tissue inhibitor of metalloproteinase-1 (TIMP1) gene levels occurred in GA-treated PC-3 cells after 24?h treatment. Based on these observations, we suggest that GA might modulate through blocking the p38, JNK, PKC and PI3K/AKT signaling pathways and reducing the NF-κB protein level, resulting in the inhibition of MMP-2 and -9 of PC-3 human prostate cancer cells.     

Nm23‐H1 promotes adhesion of CAL 27 cells in vitro

nm23‐H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23‐H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23‐H1. In our experiments, cells overexpressing nm23‐H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23‐H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23‐H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23‐H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma. © 2009 Wiley‐Liss, Inc.     

Steroidal Saponins from Paris polyphylla Suppress Adhesion,Migration and Invasion of Human Lung Cancer A549 Cells Via Down-Regulating MMP-2 and MMP-9

Background: Tumor metastases are the main reasons for oncotherapy failure. Paris polyphylla (Chinese name:Chonglou) has traditionally been used for its anti-cancer actions. In this article, we focus on the regulation ofhuman lung cancer A549 cell metastases and invasion by Paris polyphylla steroidal saponins (PPSS). Materialsand Methods: Cell viability was evaluated in A549 cells by MTT assay. Effects of PPSS on invasion and migrationwere investigated by wound-healing and matrigel invasion chamber assays. Adhesion to type IV collagen andlaminin was evaluated by MTT assay. Expression and protease activity of two matrix metalloproteinases (MMPs),MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. Results: PPSSexerted growth inhibitory effects on A549 cells, and effectively inhibited A549 cell adhesion, migration andinvasion in a concentration-dependent manner. Western blotting and gelatin zymography analysis revealed thatPPSS inhibited the expression and secretion of MMP-2 and MMP-9 in A549 cells. Conclusions: PPSS has thepotential to suppress the migration, adhesion and invasion of A549 cells. PPSS could be a potential candidatefor interventions against lung cancer metastases.     

地锦草乙醇提取物通过circRHOT1/miR-29a-3p轴抑制结直肠癌SW480细胞的恶性生物学行为

目的：探讨地锦草乙醇提取物（EEEH）对人结直肠癌SW480细胞生物学行为的影响及其分子机制。方法：体外培养SW480细胞,实验分为Con组、EEEH-L组、EEEH-M组、EEEH-H组、si-NC组、si-circRHOT1组、EEEH-H+pcDNA组、EEEH-H+pcDNA-circRHOT1组,分别以si-NC、si-circRHOT1、pcDNA、pcDNA-circRHOT1转染SW480细胞,采用CCK-8法、细胞克隆形成实验、Transwell实验分别检测转染后各组细胞的增殖、迁移及侵袭能力,qPCR法检测转染后各组SW480细胞circRHOT1和miR-29a-3p的表达,WB法检测各组细胞中MMP-2、MMP-9蛋白的表达。双荧光素酶报告基因实验检测circRHOT1与miR-29a-3p之间的靶向关系。结果：与Con组比较,EEEH-L组、EEEH-M组、EEEH-H组SW480细胞中MMP-2和MMP-9蛋白表达均明显降低（均P＜0.05）,circRHOT1的表达均降低（均P＜0.05）而miR-29a-3p的表达均升高（均P＜0.05）且呈剂量依赖性;...     

MAPKAPK2 and HSP27 are downstream effectors of p38 MAP kinase-mediated matrix metalloproteinase type 2 activation and cell invasion in human prostate cancer

Although cell invasion is a necessary early step in cancer metastasis, its regulation is not well understood. We have previously shown, in human prostate cancer, that transforming growth factor beta (TGFbeta)-mediated increases in cell invasion are dependent upon activation of the serine/threonine kinase, p38 MAP kinase. In the current study, downstream effectors of p38 MAP kinase were sought by first screening for proteins phosphorylated after TGFbeta treatment, only in the absence of chemical inhibitors of p38 MAP kinase. This led us to investigate mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), a known substrate of p38 MAP kinase, as well as heat-shock protein 27 (HSP27), a known substrate of MAPKAPK2, in both PC3 and PC3-M human prostate cells. After transient transfection, wild-type MAPKAPK2 and HSP27 both increased TGFbeta-mediated matrix metalloproteinase type 2 (MMP-2) activity, as well as cell invasion, which in turn was inhibited by SB203580, an inhibitor of p38 MAP kinase. Conversely, dominant-negative MAPKAPK2 blocked phosphorylation of HSP27, whereas dominant-negative MAPKAPK2 or mutant, non-phosphorylateable, HSP27 each blocked TGFbeta-mediated increases in MMP-2, as well as cell invasion. Similarly, knock down of MAPKAPK2, HSP27 or both together, by siRNA, also blocked TGFbeta-mediated cell invasion. This study demonstrates that both MAPKAPK2 and HSP27 are necessary for TGFbeta-mediated increases in MMP-2 and cell invasion in human prostate cancer.     

重离子辐照对人舌鳞癌CAL27细胞的生物学效应及其分子机制

目的 研究重离子12C6+离子辐照对人舌鳞癌CAL27细胞的生物学效应以及分子机制。方法 应用不同剂量重离子束（12C6+）对体外培养的人舌癌CAL27细胞进行辐照,采用MTT、Transwell、流式细胞术、划痕实验、克隆实验观察重离子束（12C6+）对CAL27细胞迁移和侵袭、凋亡和周期的影响,Western blot法观察重离子束（12C6+）对CAL27细胞增殖的影响。结果 （1）重离子辐照对CAL27细胞的增殖抑制作用与剂量-时间成正相关;（2）和空白对照组比较,在相同时间点,不同剂量的重离子束（12C6+）对细胞中P53、P65和VEGF的表达影响明显,随着重离子束（12C6+）辐照剂量增大,细胞中P53、P65和VEGF的表达明显降低（P<0.05）。结论 重离子辐照能抑制CAL27细胞的增殖,且具有剂量和时间效应。     

STIM1在骨肉瘤细胞增殖和转移中的作用及机制研究

目的:探究STIM1对骨肉瘤细胞143B增殖和转移的作用及其具体机制研究.方法:使用shRNA构建STIM1敲减的143B稳转细胞株;通过实时荧光定量PCR技术和Western Blot技术检测细胞敲减效率,通过克隆形成实验和CCK-8检测STIM1对骨肉瘤细胞143B增殖的影响;通过Transwell实验观察STIM...