蛋氨酸脑啡肽抑制人胃癌细胞BGC823增殖作用及免疫调节机制

采用不同浓度梯度的蛋氨酸脑啡肽(methionine enkephalin,MENK)体外作用于人胃癌细胞BGC823后,探讨对其增殖影响及其作用机制,为胃癌的免疫治疗提供理论依据。体外培养人胃癌细胞株BGC823,PCR检测阿片受体OGFr的表达;用不同浓度(0、1、2、3、4 mg/mL)的MENK体外作用于BGC823细胞24、48、72、96 h后,MTS检测MENK对其增殖影响;流式细胞术和Annexin V-FITC/PI双染法检测4 mg/mL MENK体外处理48、72 h后BGC823细胞凋亡变化。结果显示,人胃癌BGC823细胞有阿片受体OGFr的表达;MENK可抑制BGC823细胞增殖,且随着剂量的增加和时间的延长,其抑制作用逐渐增强(P0.05);4 mg/mL MENK48 h处理组与空白组相比细胞凋亡率增加,72 h处理组与48 h处理组结果一致(P0.05)。结果表明,MENK可抑制BGC823细胞增殖,具有显著的剂量依赖性和时间依赖性,且可通过诱导细胞凋亡抑制BGC823细胞的增殖。     

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF‐β signalling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non‐tumour tissues were examined. Methylation‐specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re‐expression was induced by 5‐Aza‐2′‐deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non‐tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)‐β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1‐expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF‐β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.     

长非编码RNA SNHG18对人胃癌细胞BGC823增殖和凋亡的影响

目的:探究长非编码RNA SNHG18对胃癌细胞增殖和凋亡的影响。方法:采用实时定量PCR(qRT-PCR)技术检测人胃癌组织及癌旁组织和胃癌细胞系中lncRNA SNHG18的表达;采用MTT和克隆形成试验观察转染SNHG18过表达质粒后胃癌细胞BGC823增殖活力的变化;通过流式细胞术检测lncRNA SNHG18对胃癌细胞BGC823凋亡的影响。结果:相较于癌旁组织和胃正常粘膜上皮细胞系GSE-1,胃癌组织及胃癌细胞系中SNHG18的表达水平显著降低(P0.05);胃癌细胞过表达SNHG18增殖活力以及克隆形成的能力均显著降低(P0.05),而细胞凋亡率明显升高(P0.05)。结论:胃癌组织中长非编码RNA SNHG18呈低表达,可促进胃癌细胞增殖并抑制其凋亡,可能在胃癌发生发展过程中发挥重要作用。     

两歧双歧杆菌胞外多糖对胃癌细胞及hTERT的影响

【目的】研究从双歧杆菌属两歧双歧杆菌(Bifidobacterium bifidum)提取的细胞表面成分胞外多糖(Exopolysaccharide, B.EPS)对人胃癌细胞BGC-823的生长抑制作用及对端粒酶限速因子hTERT活性的影响。【方法】将三种不同浓度B.EPS体外作用于胃癌细胞BGC823,MTT法检测细胞生长抑制率并辅以形态学观察;异硫氰酸盐(FITC)联合PI染色,通过流式细胞术检测肿瘤细胞初期调亡情况;肿瘤细胞端粒酶限速因子hTERT mRNA经RT-PCR检测B.EPS对端粒酶活性抑制作用;通过荧光分光光度计显示B.EPS 对胃癌细胞作用后胞内Ca2+含量变化。【结果】经过检测发现,B.EPS对人胃癌细胞BGC823的生长显著抑制(P＜0.05)呈剂量时间反应关系;细胞中hTERT mRNA在B.EPS的作用下表达降低(P＜0.05),有一定剂量效应关系;随着B.EPS对肿瘤细胞的抑制,细胞内Ca2+含量显著增加(P＜0.05)。【结论】B.EPS诱导人胃癌细胞BGC823调亡的机制可能与改变肿瘤细胞端粒酶限速因子hTERT mRNA表达量和细胞内钙离子浓度有关。     

The contrary functions of lncRNA HOTAIR/miR‐17‐5p/PTEN axis and Shenqifuzheng injection on chemosensitivity of gastric cancer cells

This study was implemented to figure out whether lncRNA HOTAIR/miR‐17‐5p/PTEN axis played a role that was opposite to Shenqifuzheng (SQFZ) injection in regulating the chemosensitivity of gastric cancer cells. The gastric cancer tissues were gathered and four gastric cancer cell lines were prepared, including BGC‐823, MGC‐803, SGC‐7901, and MKN28. Moreover, cisplatin, adriamycin, mitomycin, and 5‐fluoroura were managed as the chemo‐therapeutics, and SQFZ was prepared as a Chinese medicine. Striking distinctions of HOTAIR, miR‐17‐5p, and PTEN expressions were observed between gastric cancer tissues and para‐carcinoma normal tissues (P < 0.05). MKN28 was associated with the highest resistance to cisplatin, adriamycin, mitomycin, and 5‐fluoroura among all the cell types, and SQFZ significantly improved the MKN28 cells’ sensitivity to the drugs (P < 0.05). The over‐expressed HOTAIR and miR‐17‐5p, as well as under‐expressed PTEN tended to significantly facilitate the viability, EMT process and proliferation of MKN28 cells that were subject to treatment of chemo‐therapies (P < 0.05). SQFZ could amplify the effects of si‐HOTAIR, miR‐17‐5p inhibitor, and pcDNA‐PTEN on boosting the chemosensitivity of gastric cancer cells (P < 0.05). In addition, HOTAIR was also found to directly target miR‐17‐5p, and PTEN appeared to be subject to the modification of HOTAIR and miR‐17‐5p in its acting on the viability, proliferation, EMT process, and apoptosis of gastric cancer cells. The HOTAIR/miR‐17‐5p/PTEN axis could be regarded as the potential treatment targets for gastric cancer, and adjuvant therapy of SQFZ injection could assist in further improving the treatment efficacy of chemo‐therapies for gastric cancer.     

Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1

Maggot therapy is a simple and highly successful method for healing of infected and necrotic wounds. The increasing evidences indicate that Maggot excretions/secretions (ES) plays important roles in the wounds healing process. But the precise molecular mechanisms remain undefined. Herein, we investigated if ES induced cell migration during wound healing process using microvascular endothelial cells (HMEC-1) as model, and this effect was associated with the activation of AKT1 and ERK1/2. Wound healing and transwell migration assays were performed to study the effects of ES on HMEC-1 cell migration. Our data showed that ES significantly induced HMEC-1 cell migration in both wound healing and transwell assays, and time-dependently (P < 0.05) activated AKT1, but not ERK1/2. Moreover LY294002 (a PI3K inhibitor) partially attenuated (P < 0.05) ES-induced cell migration in wound healing assay while completely inhibited (P < 0.05) ES-induced AKT1 activation. These findings demonstrate that ES directly induces HMEC-1 cell migration and this event is partially mediated by the activation of AKT1.     

生半夏、南星水提物对人胃癌BGC823细胞的侵袭力及HIF-lαmRNA蛋白表达的影响

摘要目的：观察生半夏、南星中药水提物对缺氧环境中人胃癌细胞株BGC823细胞HIF-lα蛋白表达和侵袭力的影响。方法：运用CoCl2（氯化钴）诱导BCG823细胞缺氧,使得细胞中HIF-1α蛋白表达升高．实验组加入生半夏、南星水提物对细胞进行预处理,然后在进行缺氧诱导。通过甲基噻唑基四唑法（MTT）检测细胞活性,使用Transwell检测细胞侵袭能力变化,RT-PCR、Weste-rn blotting分别检测HIF-lαmRNA及蛋白含量及变化。结果：生半夏、南星水提物能抑制人胃癌BGC823细胞的增殖;生半夏、南星水提物均能抑制缺氧诱导胃癌细胞的侵袭力,并且能降低HIF-1αmRNA及蛋白表达。结论：生半夏、南星水提物可抑制人胃癌BGC823细胞的增殖,抑制人胃癌BGC823细胞侵袭力,可能通过降低HIF-1α蛋白表达有关。     

Impact of BTG2 expression on proliferation and invasion of gastric cancer cells in vitro

BTG2 (B cell translocation gene 2) is downregulated in several human tumors and has been known as a tumor suppressor in carcinogenesis of thymus, prostate, kidney, and liver. However, little is known about the role BTG2 plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of BTG2 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer cell lines SGC7901 and MKN45. BTG2 cDNA was insected into a constitutive vector pcDNA3.1 followed by transfection in gastric cancer cell line MKN45 and SGC7901 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these transfectants were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay, respectively. The invasion of these clones was analyzed by using cell migration assay. MKN-BTG2 (MKN45 with stable transfection of BTG2 gene) and SGC-BTG2 (SGC7901 with stable transfection of BTG2 gene) grew slower than their control groups, respectively. The cell counts of MKN-BTG2 in the fourth, fifth, sixth and seventh days were significantly fewer than those of control groups (P < 0.05). Those of SGC-BTG2 in the fourth fifth, sixth and seventh days were significantly fewer than those of control groups too (P < 0.05). Cell cycle analysis showed that proportions of MKN-BTG2 and SGC-BTG2 cells in G0–G1 and S were different significantly with those of their control groups, respectively (P < 0.05). The apoptosis rate of MKN-BTG2 was significantly higher than those of control groups (P < 0.05). Results of colony-forming assay showed that the colon formation rates of MKN-BTG2 and SGC-BTG2 were lower than those of their control groups (P < 0.05). The results of cell migration assay showed that the cell migration rates of MKN-BTG2 and SGC-BTG2 were not significantly different with those of their control groups (P > 0.05). BTG2 can restrain the growth and proliferation of gastric cancer cells powerfully. It can reduce some malignant phenotype of these tumor cells. But it could not impact the ability of invasion of gastric cancer cells, so could not restrain the metastasis of gastric cancer. In gastric cancer, BTG2 could be thought as a tumor-inhibiting gene in some distance, so the gene could be a potential target of gene therapy.     

A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.     

ANKRD33 is overexpressed in gastric adenocarcinoma and predictive for poor prognosis

ABSTRACT

The aim of the current study was to investigate and discuss the function of ANKRD33 gene in the pathogenesis of gastric adenocarcinoma. The marked up-regulated expression of ANKRD33 gene in gastric adenocarcinoma tissues compared to normal tissues was found by bioinformatics analysis. Kaplan-Meier analysis revealed that high expression of ANKRD33 is correlated with lower overall survival of gastric adenocarcinoma patients. The results of qPCR revealed that mRNA expression level of ANKRD33 was dramatically higher in AGS, SGC7901, and BGC823 cell lines than that in the GES1 cells. Knockdown of ANKRD33 remarkably inhibited the viability, invasion, and migration of AGS and BGC823 cells. Furthermore, the ratio of p-AKT/AKT and p-mTOR/mTOR was significantly decreased in AGS cells which transfected with si- ANKRD33. All the above results illustrated that ANKRD33 would act as a tumor forwarder in gastric adenocarcinoma development and have a high potential to be a marker molecule in the diagnosis and treatment of gastric tumors.     

A new familial gastric cancer-related gene polymorphism: T1151A in the mismatch repair gene hMLH1

We designed to understand the effects of the T1151A gene polymorphism in the hMLH1 gene on the pathogenesis of familial gastric cancer. Peripheral blood DNA from 113 patients with familial gastric cancer or suspected familial gastric cancer that were newly identified in the same year, along with 180 healthy subjects, was subjected to polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC) and DNA sequencing of exon 12 in the hMLH1 gene. Our results as following, the T1151A detection rate was remarkably higher in patients with familial gastric cancer or suspected familial gastric cancer compared to normal control patients (P < 0.05). Stratified analysis showed that there was a significant difference in the detection rate between the control group and elderly patients whose age of onset was greater than 50 years old (P < 0.05). The detection rate of patients from high-risk families were relatively high (P < 0.05). An especially significant distribution was observed in patients who had suffered precancerous diseases related to gastric cancer (P < 0.01). In conclusion, familial gastric carcinoma families in China carrying the T1151A polymorphism may have a higher risk of suffering from gastric cancer. This gene polymorphism can be used as a candidate screening index for high-risk populations.     

低氧模拟剂氯化钴对胃癌细胞BGC823中S100A4基因表达的影响

摘要: S100A4基因是肿瘤侵袭转移相关的重要基因, 该基因高表达与胃癌浸润、淋巴结转移及胃癌细胞体外侵袭力密切相关。为探讨S100A4基因高表达的机制, 文章应用低氧模拟剂氯化钴处理胃癌细胞BGC823, RT-PCR、免疫组化、免疫荧光及Western blotting方法分别检测BGC823细胞中S100A4 mRNA及蛋白表达情况。结果显示, 氯化钴处理胃癌BGC823细胞后, S100A4 mRNA及蛋白表达明显增加。提示低氧模拟剂氯化钴可促进胃癌细胞BGC823中S100A4 基因表达。     

TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

Cisplatin is a cytotoxic platinum compound that triggers DNA crosslinking induced cell death, and is one of the reference drugs used in the treatment of several types of human cancers including gastric cancer. However, intrinsic or acquired drug resistance to cisplatin is very common, and leading to treatment failure. We have recently shown that reduced expression of base excision repair protein XRCC1 (X-ray repair cross complementing group1) in gastric cancerous tissues correlates with a significant survival benefit from adjuvant first-line platinum-based chemotherapy. In this study, we demonstrated the role of XRCC1 in repair of cisplatin-induced DNA lesions and acquired cisplatin resistance in gastric cancer by using cisplatin-sensitive gastric cancer cell lines BGC823 and the cisplatin-resistant gastric cancer cell lines BGC823/cis-diamminedichloridoplatinum(II) (DDP). Our results indicated that the protein expression of XRCC1 was significantly increased in cisplatin-resistant cells and independently contributed to cisplatin resistance. Irinotecan, another chemotherapeutic agent to induce DNA damaging used to treat patients with advanced gastric cancer that progressed on cisplatin, was found to inhibit the expression of XRCC1 effectively, and leading to an increase in the sensitivity of resistant cells to cisplatin. Our proteomic studies further identified a cofactor of 26S proteasome, the thioredoxin-like protein 1 (TXNL1) that downregulated XRCC1 in BGC823/DDP cells via the ubiquitin-proteasome pathway. In conclusion, the TXNL1-XRCC1 is a novel regulatory pathway that has an independent role in cisplatin resistance, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.     

B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的影响

为探讨B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的增殖和凋亡的影响, 设计并合成B-RAF小分子干扰RNA(B-RAF-siRNA)和阴性对照siRNA, 用TransMessenger介导转染胃癌BGC823细胞, RT-PCR分析检测胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达; MTT检测胃癌BGC823细胞增殖情况; 流式细胞仪检测细胞凋亡情况, 并与对照组进行比较。TransMessenger能够有效介导B-RAF-siRNA和阴性对照siRNA转染胃癌BGC823细胞, TransMessenger介导的B-RAF-siRNA有效地抑制胃癌BGC823细胞B-RAF以及Bcl-2基因的表达, 与对照组相比, 抑制率达90.0%以上, 最高达100%; 同时明显抑制胃癌BGC823细胞增殖; 促进胃癌BGC823细胞的凋亡(P < 0.01)。B-RAF基因特异的siRNA干扰能有效地抑制胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达, 同时促进胃癌细胞凋亡和抑制胃癌细胞增殖。     

CDX2基因高表达对胃癌细胞生物学行为的影响

目的探讨尾侧型同源转录因子-2(CDX2)基因过表达对胃癌BGC-823细胞增殖、迁移、凋亡等生物学特征的影响。方法采用脂质体转染法建立CDX2基因过表达的胃癌BGC-823稳定细胞株,分别采用RT-PCR、Western blotting和免疫细胞化学等方法检测转染重组表达载体pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因及其蛋白的表达。MTT法检测CDX2基因过表达对细胞的增殖能力的影响;划痕实验检测CDX2过表达对细胞迁移能力的影响;流式细胞术检测CDX2过表达对细胞的凋亡的影响;应用基因芯片技术检测转染前后相关基因的差异表达。结果 RT-PCR及Western blotting检测结果显示,与对照组相比,转染pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因和蛋白均呈高表达;CDX2过表达能明显降低转然组BGC-823细胞增殖能力和迁移能力;但对细胞凋亡影响不明显;基因芯片结果提示CDX2基因高表达能影响某些基因的表达。结论 CDX2过表达能明显抑制胃癌细胞增殖、降低迁移能力,提示CDX2在胃癌中可能发挥抑癌基因的作用。     

RNAi引起的Paxillin和p130Cas下调抑制胃癌细胞失巢性生长

P130Cas和paxillin分子是整合素家族下游重要的衔接分子.为了探索这两个分子在肿瘤细胞抗失巢凋亡中的作用,应用RNAi技术分别抑制抗失巢凋亡的胃癌细胞BGC82 3中paxillin和p130cas基因的表达,观察它们对细胞失巢性生长的影响.依据siRNA设计原则,分别设计针对p130cas和paxillin的两条序列;成功的构建了特异性封闭上述两分子的载体pWH1 p130cas和pWH1 paxillin .构建的载体瞬时转染贴壁培养和失巢培养的BGC82 3后,RT PCR和Western印迹检测发现paxillin和p130Cas分子在mRNA及蛋白水平的表达量均明显降低;倒置显微镜下观察发现,贴壁培养的胃癌细胞发生皱缩、脱落;失巢培养的细胞聚集成团的现象受到明显抑制,细胞团比对照组小,且较松散;MTT实验结果表明,失巢培养的BGC82 3pWH1 paxillin 组细胞存活率(32 19%±6 11% )和BGC82 3pWH1 p13 0cas组细胞存活率(2 8 5 2 %±5 0 2 % )与对照组相比显著下降(P <0 0 1vscontrol) ;FCM实验结果发现与失巢培养的对照组相比,BGC82 3pWH1 paxillin和BGC82 3pWH1 p13 0cas组细胞G1期抬高,并出现了凋亡峰.运用RNAi技术分别抑制了BGC82 3细胞中paxillin和p130Cas分子的表达,初步证明paxillin和p130Cas是细胞存活的重要信号分子,在肿瘤细胞抗失巢凋亡过程中具有重要的作用.     

Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.