Ghrelin对胃癌SGC-7901细胞增殖的影响及其作用机制

目的探讨Ghrelin对人胃癌SGC-7901细胞增殖的影响及其作用机制。方法在人胃癌SGC-7901细胞的培养基中添加不同浓度的Ghrelin(400、500、600、700、800 ng/mL),采用MTT法检测细胞增殖情况,实时荧光定量PCR法检测miR-7的表达量,Western blot法检测细胞中Cyclin D1及p27的表达水平。结果 600～800 ng/mL的Ghrelin可显著抑制胃癌SGC-7901细胞的增殖(P 0. 01)。随着600 ng/mL Ghrelin作用时间的延长,miR-7的表达量显著增加(P 0. 01);人胃癌SGC-7901细胞中Cyclin D1的表达显著下调(P 0. 01),p27的表达显著上调(P 0. 05)。结论 Ghrelin可显著抑制胃癌SGC-7901细胞增殖,可能是通过调控Cyclin D1、p27及miR-7的表达来实现的。     

实时荧光定量PCR方法检测人、鼠正常组织及脑癌组织中CD109蛋白的表达

目的分析CD109基因在正常组织及脑癌组织中的分布。方法采用实时荧光定量PCR方法,分析CD109在人、鼠正常组织及脑癌中的表达,以18SrRNA作为内标。结果CD109蛋白的mRNA丰度在睾丸组织中最高,在其它组织中较低,在12份脑癌标本中,检测到9份CD109表达上调。结论CD109基因在多数正常组织中表达很少,有可能是治疗恶性肿瘤的一个潜在的分子靶点。     

神经纤毛蛋白2干扰质粒对胃腺癌SGC7901细胞株生长、侵袭及转移的影响

目的探讨神经纤毛蛋白2(Neuropilin2,NRP2)干扰质粒对胃腺癌SGC7901细胞株生长、侵袭及转移的影响。方法构建NRP2特异的小RNA干扰质粒shRNA-NRP2,采用脂质体介导的方法将质粒转染入人胃腺癌SGC7901细胞株,Western blot法检测NRP2蛋白的表达;Transwell试验检测胃癌细胞体外迁移和侵袭能力的变化;MTT试验检测胃癌细胞的增殖水平。结果 shRNA-NRP2转染SGC7901细胞后,NRP2蛋白表达明显下降,细胞的侵袭、迁移能力明显受抑制,而细胞的增殖水平无显著变化。结论 shRNA-NRP2干扰质粒可下调胃腺癌SGC7901细胞株NRP2蛋白的表达,进而抑制细胞的侵袭、转移能力,提示NRP2可能成为胃癌分子治疗的靶标。     

运用实时荧光定量RT-PCR检测人正常组织及肿瘤组织中BRDT表达

目的探讨BRDT基因在正常组织及癌组织中的分布及其作为肿瘤治疗靶分子的可能性。方法运用实时荧光定量PCR方法分析BRDT在人正常组织及癌组织中的表达,使用18S rRNA作为内对照。结果在所检测的正常组织中该蛋白的mRNA只存在于睾丸组织中,而在其它组织不表达;在检测的10例胃腺癌组织标本中,有6例mRNA的微弱表达;在检测的10例食管鳞状细胞癌组织标本中,有3例mRNA的微弱表达;在检测的12例子宫颈鳞状细胞癌组织标本中均未有表达;在检测9例子宫内膜腺癌组织标本中,有2例微弱表达;在检测12例脑癌组织标本中,只有1例微弱表达。结论BRDT不可能作为子宫颈鳞状细胞癌、脑癌、子宫内膜腺癌及食管鳞状细胞癌治疗的潜在分子靶点,是否能够做胃腺癌的靶点还需进一步探讨。     

Survivin-siRNA真核表达载体的构建及其对胃癌SGC-7901细胞增殖和凋亡的影响

目的构建Survivin-siRNA真核表达载体,并探讨其对胃癌SGC-7901细胞增殖和凋亡的影响。方法化学合成4条能转录出siRNA的模板DNA,各75个碱基,退火形成2条双链DNA,双酶切后插入pSUPER.basic载体。将阳性重组质粒转染SGC-7901细胞,进行细胞计数,并用MTT法检测细胞的增殖活性;半定量RT-PCR检测细胞Survivin基因mRNA的转录水平;流式细胞术检测细胞周期的变化。结果PCR和酶切鉴定表明Survivin-siRNA真核表达载体构建正确,其能下调SGC-7901细胞Survivin基因mRNA的转录水平,抑制SGC-7901细胞生长和增殖,并促进细胞凋亡,使G0/G1和亚G1期细胞增多,S期细胞减少。结论已成功构建了Survivin-siRNA真核表达载体,其能下调SGC-7901细胞中Survivin基因mRNA的转录水平,使细胞增殖减弱,凋亡增加,为RNA干扰技术应用于胃癌的基因治疗提供了一定的实验依据。     

双内参实时荧光定量PCR法检测肝癌中易洛魁族同源盒2基因信使RNA的水平

目的采用双内参实时荧光定量PCR法检测肝癌中易洛魁族同源盒2(Iroquois homebox 2,IRX2)基因信使RNA(mRNA)的水平,探讨其对肝癌诊断及治疗的潜在意义。方法采用荧光定量PCR法检测21份肝癌组织和21份癌旁组织中IRX2基因的表达水平,并以双内参β-actin和GAPDH为标准对照,计算每个样本IRX2基因的相对表达量,经Pfaffl法进行相对定量,分析基因表达差异。结果 42份肝组织中均存在IRX2基因的表达,85.71%(18/21)的肝癌组织中IRX2基因表达水平较癌旁组织明显降低(P<0.01)。结论肝癌组织中IRX2基因表达水平较低,表明IRX2基因在肝癌中出现异常表达,为从基因水平诊断及治疗肝癌提供了一个潜在靶点。     

miR-153-5p靶向基质金属蛋白酶-16对胃癌细胞增殖、迁移及侵袭的影响

目的 探讨miR-153-5p对胃癌细胞SGC-7901增殖、迁移和侵袭的影响。方法 RT-qPCR法检测胃癌和癌旁组织(河北北方学院附属第一医院2016年1月—2018年12月经病理确诊的胃癌患者手术切除样本)以及正常胃细胞GES-1和胃癌细胞系(GES-1、MHCC97H、MKN-1、SGC-7901、MKN45、MGC803)中miR-153-5p的表达;将SGC-7901细胞分别或联合转染各质粒后分为miR-NC、miR-153-5p mimic、miR-153-5p inhibitor、pc-NC、pc-MMP-16、miR-153-5p mimic+pc-NC和miR-153-5p mimic+pc-MMP-16组,以未处理的SGC-7901细胞为对照组。克隆形成试验检测细胞增殖能力,Transwell试验检测细胞侵袭能力,划痕试验检测细胞迁移能力,Western blot法检测基质金属蛋白酶-16(matrix metalloproteinase-16,MMP-16)表达水平,Targetscan网站和双荧光素酶报告试验分别预测和验证miR-153-5p与MMP-16的靶...     

实时荧光PCR法快速检测乙型肝炎病毒基因YMDD突变株

目的建立一种敏感、特异、快速的实时荧光PCR定量检测乙型肝炎病毒基因YMDD突变株的方法,为临床治疗提供指导。方法设计引物和TaqMan-MGB探针,利用TaqMan-MGB探针技术,建立实时荧光定量PCR法。检测临床HBV-YMDD变异标本36份和野生型HBV标本20份,并将其YMDD变异结果与DNA测序结果进行比较。结果该方法检测灵敏度为101拷贝/μl;特异性为100%;最低检测限度为101DNA拷贝/30μl反应体系;实时荧光PCR方法测得YMDD野生株20份,变异株36份,与DNA测序结果完全一致,符合率为100%。结论应用TaqMan-MGB探针的实时荧光定量PCR方法检测YMDD变异株基因,具有灵敏、特异和精确等优点,对临床监测拉米夫定耐药具有重要意义。     

朊病毒病相关miRNA在不同发病脏器和细胞中表达的变化

目的利用Real-time PCR法检测朊病毒病相关miRNA在不同发病脏器和细胞中的表达变化。方法提取羊痒病因子22L毒株感染的BALB/c小鼠大脑、海马、延髓、脾脏以及羊痒病因子22L毒株感染的鼠神经瘤ScN2a细胞总RNA,通过特殊设计的茎环结构的反转录引物进行反转录,应用ABI step one plus system定量PCR仪进行定量检测;对Real-time PCR进行特异性和重复性验证;采用SYBR Green荧光染料法,以U6 RNA作为内参照,2-△△Ct法进行miRNA表达相对定量分析,检测小鼠发病脏器中和细胞中miR-7a、miR-128、miR-135a、miR-146a、miR-152、miR-24-2*、miR-380-3p、miR-448-5p的表达。结果 Real-time PCR法检测朊病毒病相关miRNA特异性和重复性良好。各组中miRNA改变5倍以上的有发病终末期小鼠大脑中的miR-146a,海马中的miR-128、miR-135a、miR-152,延髓中的miR-448-5p,ScN2a细胞中的miR-146a,脾脏中的miR-152,其中脾脏中的miR-152显著上调,为18.98倍。结论朊病毒病中这些上调或下调的miRNA能调控细胞内多种代谢和信号传导途径发生异常,引起细胞及组织器官发生病理学变化。     

小鼠微RNA miR-21真核表达质粒的构建及其在小鼠肾小球系膜细胞中的表达

目的构建小鼠微RNA miR-21真核表达质粒,并在小鼠肾小球系膜细胞中表达。方法人工合成小鼠miR-21基因序列,构建miR-21真核表达质粒pGenesil-miR-21。使用脂质体Lipofectamine2000转染小鼠肾小球系膜细胞后,G418筛选,获得稳定转染克隆,提取总RNA,通过实时荧光定量RT-PCR技术检测miR-21的表达。结果经酶切鉴定和测序证实,合成的miR-21基因序列完全正确,并已成功克隆到真核表达质粒pGenesil-1上。重组真核表达质粒转染小鼠肾小球系膜细胞后,筛选出的阳性克隆可稳定高表达miR-21。结论已成功构建miR-21真核表达质粒,并在小鼠肾小球系膜细胞中高效表达,为进一步探讨miR-21的生物学功能奠定了基础。     

Ewing’s Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue

The molecular mechanism responsible for Ewing’s Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.     

The Tumor Suppressor Roles of miR-433 and miR-127 in Gastric Cancer

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, the methylation epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is emerging as a common hallmark of different tumors. Here we showed that miR-433 and miR-127 were significantly down-regulated in gastric cancer (GC) tissues compared with the adjacent normal regions in 86 paired samples. Moreover, the lower level of miR-433 and miR-127 was associated with pM or pTNM stage in clinical gastric cancer patients. The restored expression of miR-433 and miR-127 in GC cells upon 5-Aza-CdR and TSA treatment suggested the loss of miR-433 and miR-127 was at least partly regulated by epigenetic modification in GC. Furthermore, the ectopic expression of miR-433 and miR-127 in gastric cancer cell lines HGC-27 inhibits cell proliferation, cell cycle progression, cell migration and invasion by directly interacting with the mRNA encoding oncogenic factors KRAS and MAPK4 respectively. Taken together, our results showed that miR-433 and miR-127 might act as tumor suppressors in GC, and it may provide novel diagnostic and therapeutic options for human GC clinical operation in the near future.     

Extracellular Disposal of Tumor-Suppressor miRs-145 and -34a via Microvesicles and 5-FU Resistance of Human Colon Cancer Cells

The dysregulation of microRNA (miRNA) expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU)-resistant human colon cancer DLD-1 cells (DLD-1/5FU) from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs) before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.     

microRNA与甲状腺乳头状癌相关性分析

目的 筛选甲状腺乳头状癌(papillary thyroid carcinoma,PTC)显著差异表达的microRNA(miRNA),分析其与PTC 发病机制的相关性.方法 选取确诊的PTC 癌组织及癌旁正常组织样本各6 份,提取总RNA,采用转录组测序技术,筛选显著差异表达的miRNA,预测其靶基因并进行基因功能(...     

MMP-2反义寡核苷酸对人胃癌SGC7901细胞增殖和侵袭能力的影响

目的研究基质金属蛋白酶-2(MMP-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖及侵袭能力的影响。方法将不同浓度的硫代磷酸化修饰的MMP-2ASODN转染入人中低分化胃腺癌SGC7901细胞株,RT-PCR法检测细胞MMP-2基因mRNA的转录水平;Transwell试验检测细胞体外迁移和侵袭能力变化;MTT法检测细胞的增殖活性。结果转染MMP-2ASODN后,SGC7901细胞中MMP-2基因mRNA的转录水平显著降低,细胞的体外迁移和侵袭能力受到抑制,且抑制作用随ASODN浓度的增加而增强;而细胞的增殖活性无明显变化。结论MMP-2ASODN可抑制胃腺癌SGC7901细胞MMP-2基因mRNA的转录水平及细胞的体外迁移和侵袭能力,但与胃癌细胞的增殖活性无明显相关性。     

The Role of E3 Ubiquitin Ligase Cbl Proteins in β-Elemene Reversing Multi-Drug Resistance of Human Gastric Adenocarcinoma Cells

Recent studies indicate that β-elemene, a compound isolated from the Chinese herbal medicine Curcuma wenyujin, is capable of reversing tumor MDR, although the mechanism remains elusive. In this study, β-Elemene treatment markedly increased the intracellular accumulation of doxorubicin (DOX) and rhodamine 123 in both K562/DNR and SGC7901/ADR cells and significantly inhibited the expression of P-gp. Treatment of SGC7901/ADR cells with β-elemene led to downregulation of Akt phosphorylation and significant upregulation of the E3 ubiquitin ligases, c-Cbl and Cbl-b. Importantly, β-elemene significantly enhanced the anti-tumor activity of DOX in nude mice bearing SGC7901/ADR xenografts. Taken together, our results suggest that β-elemene may target P-gp-overexpressing leukemia and gastric cancer cells to enhance the efficacy of DOX treatment.