微小核糖核酸-7调控表皮细胞生长因子受体对肝癌细胞增殖和转移的影响

目的 探讨微小核糖核酸(miR)-7是否通过靶向调控表皮细胞生长因子受体(EGFR)对人肝癌细胞侵袭和增殖产生影响.方法 传代培养HepG2人肝癌细胞,脂质体转染miR-7后使其表达上调或下调.荧光定量聚合酶链反应检测转染效率.蛋白印迹法检测miR-7上调或下调后肝癌细胞EGFR表达变化.溴化噻唑蓝四氮唑(MTT)法检...     

地佐辛复合全身麻醉对老年患者趋化因子CXC配体8信使核糖核酸、微小核糖核酸-124-3p、微小核糖核酸-143-3p表达水平影响

目的 探讨地佐辛复合全身麻醉对老年患者趋化因子CXC配体8(CXCL8)信使核糖核酸(mRNA)、微小核糖核酸-124-3p(miR-124-3p)、微小核糖核酸-143-3p(miR-143-3p)表达水平的影响。方法 选取四川大学华西医院自2020年5月至2023年5月收治的498例老年全身麻醉患者为研究对象。将患者随机分入常规组和地佐辛组,每组各249例。地佐辛组麻醉诱导前静脉给予地佐辛,常规组给予氯化钠,随访至术后1 d。比较两组患者麻醉诱导和麻醉苏醒的相关指标;同时,比较两组患者术前和术后1 d的CXCL8 mRNA、miR-124-3p、miR-143-3p表达水平,以及疼痛和认知功能相关指标。结果 两组患者气管插管后1 min的收缩压、心率均高于麻醉诱导前,差异有统计学意义(P<0.05)。与常规组比较,地佐辛组苏醒期躁动和呛咳的发生率更低,苏醒时间和拔管时间更短,差异有统计学意义(P<0.05)。与术前比较,两组患者术后1 d的CXCL8 mRNA、miR-143-3p水平升高,miR-124-3p水平降低,差异有统计学意义(P<0.05);地佐辛组术...     

N6-甲基腺苷甲基化对小鼠脑缺血-再灌注损伤中微小核糖核酸-155表达和脑损伤的调控作用

目的 研究N6-甲基腺苷(m6A)甲基化对小鼠脑缺血-再灌注损伤(CIRI)模型中微小核糖核酸(microRNA,miRNA,miR)-155表达和脑损伤的调控作用.方法 栓线法构建小鼠大脑中动脉闭塞(MCAO)模型,实时定量聚合酶链反应(RT-qPCR)分析MCAO小鼠脑细胞中pri-miR-155、pre-miR-...     

微小核糖核酸-205靶向调控血管内皮生长因子-A对肝癌细胞的影响

【摘要】 目的 探讨微小核糖核酸（miR）- 205在肝细胞癌（HCC）中的作用及其与肝癌进展的关系，以及miR- 205是否通过靶向血管内皮细胞生长因子（VEGF）- A影响肝癌细胞增殖。方法 采用逆转录- 定量聚合酶链反应（RT- qPCR）检测miR- 205在HCC、相邻正常肝组织和肝癌细胞株HepG2、HuH- 7、SMMC- 7721、BEL- 7402、人正常肝细胞株L02中的表达。RT- qPCR检测转染miR- 205激动剂和空白对照的HepG2和HuH- 7细胞中miR- 205表达。Transwell和溴化噻唑蓝四氮唑（MTT）法检测转染后肝癌细胞增殖、迁移和侵袭。双荧光素酶报告基因检测VEGF- A是否为miR- 205直接作用靶标。VEGF- A siRNA敲低分析VEGF- A表达是否为miR- 205调控HCC细胞增殖、迁移和侵袭的关键介质。结果 miR- 205在HCC组织和肝癌细胞株中表达下调。miR- 205过表达可抑制肝癌细胞增殖、迁移和侵袭。VEGF- A是HCC中miR- 205直接作用靶标，miR- 205通过靶向VEGF- A抑制肝癌细胞增殖、迁移和侵袭。 结论 miR- 205可通过下调VEGF- A表达抑制HCC生长、迁移和侵袭，这可能是未来HCC治疗的潜在靶点。     

血清微小核糖核酸-599与肝细胞肝癌TACE治疗预后的相关性

【摘要】 目的 探究血清微小核糖核酸-599（miR-599）与肝细胞肝癌（HCC）经导管动脉化疗栓塞术（TACE）治疗预后的关系。方法 选取2016年6月至2018年2月江西省萍乡市人民医院确诊并接受治疗的166例HCC患者作为研究对象,根据3年生存结局将其分为生存组113例和死亡组53例。用实时荧光定量PCR法检测miR-599表达量,用受试者工作特征（ROC）曲线评价miR-599诊断HCC预后的价值,用Cox回归分析miR-599与TACE治疗HCC预后的关系。结果 生存组术前、术后的miR-599相对表达量均高于死亡组,差异均有统计学意义（均P＜0.05）。生存组和死亡组的术后miR-599相对表达量均较术前升高,差异均有统计学意义（均P＜0.05）。术前miR-599诊断HCC预后的ROC曲线下面积高于术后miR-599,差异有统计学意义（P＜0.05）。按1∶1倾向性评分匹配（最邻近原则）后,术前miR-599诊断HCC预后的ROC曲线下面积高于术后miR-599,但差异无统计学意义（P＞0.05）。Cox回归分析结果显示肿瘤直径＞5 cm和肿瘤分期高是HCC预后的独立危险因素（P＜0.05）,接受索拉非尼治疗和术前miR-599表达量高是HCC预后的独立保护因素（P＜0.05）。按1∶1倾向性评分匹配（最邻近原则）后,Cox回归分析结果显示术前miR-599表达量高是HCC预后的独立保护因素（P＜0.05）。结论 术前miR-599与HCC患者TACE治疗预后有关,其表达量低提示预后不良。     

MCF-7乳腺癌细胞摄取188Re-DTPA-DG的实验研究

目的:研究乳腺癌细胞(MCF-7)对188Re标记二乙三胺五乙酸葡糖胺(DTPA-DG)的摄取。方法:采用γ计数法在不同时间段评价乳腺癌细胞(MCF-7)对188Re-DTPA-DG(3.7kBq/10μl)的摄取。结果:乳腺癌肿瘤细胞(MCF-7)对188Re-DTPA-DG的摄取率明显高于对照组188ReO4-(P<0.05);实验组30min与4h细胞摄取率差异存在显著性意义(P<0.05),而对照组不同时间点细胞摄取率差异无显著性意义(P>0.05)。结论:188Re-DTPA-DG容易被乳腺癌细胞(MCF-7)摄取,使188Re在肿瘤细胞内发挥辐射效应成为可能,从而为临床肿瘤治疗提供理论依据。     

小干扰RNA下调乳腺癌扩增基因3表达对乳腺癌 MDA MB 231细胞转化生长因子 β通路的影响

目的观察小干扰RNA(small interfering RNA,siRNA)特异性干扰乳腺癌扩增基因3(amplifiedin breast cancer 3,AIB3)对乳腺癌细胞转化生长因子-β(transforming growth factor-beta,TGF-β)通路关键分子smad2、smad3及转移相关分子基质金属蛋白-9(matrix metalloproteinases-9,MMP-9)表达的影响。方法制备3组靶向AIB3的特异性siRNA分别转染乳腺癌MDA-MB-231细胞,采用逆转录-聚合酶联反应检测siRNA干涉效果以及smad2、smad3与MMP-9的表达水平变化。结果与空白对照组相比,3组合成的靶向特异性siR-NA均可有效抑制AIB3基因表达;干涉AIB3可同时显著下调smad2、smad3及MMP-9的表达水平。结论成功设计有效干扰AIB3的siRNA序列,在乳腺癌细胞中应用siRNA干扰AIB3可有效抑制TGF-β通路和转移相关通路关键分子的表达,干扰AIB3基因表达有可能成为临床治疗乳腺癌转移的有效策略。     

诺福素对MCF-7人乳腺癌细胞的体内外抗癌药效学研究

目的 研究评价诺福素在体内体外对MCF-7人乳腺癌细胞的生长抑制作用.方法 按照抗癌药物临床前药效学评价原则,体外评价试验用MTT法,体内评价通过观察受试药物对MCF-7裸鼠移植瘤生长的影响,并与阳性药紫杉醇进行比较.结果 两批体外MTT法显示,诺福素抑制MCF-7细胞生长的半效抑制浓度分别为1.7762 nmol/L...     

微小核糖核酸-590与急性冠状动脉综合征介入术后1年内支架内再狭窄的关系

目的探讨微小核糖核酸-590(miR-590)与急性冠状动脉综合征(ACS)患者经皮冠状动脉介入治疗(PCI)术后1年内支架内再狭窄(ISR)的关系。方法选取2018年2月至2020年1月在重庆市人民医院接受PCI治疗的452例ACS患者作为研究对象。根据术后1年内是否发生ISR,分为ISR组(n=51)和非ISR组(n=401)。实时荧光定量聚合酶链反应(PCR)检测miR-590水平,分析其与ISR的关系。结果 ISR组miR-590相对表达量低于非ISR组,差异有统计学意义(P<0.05)。多因素logistic回归分析结果显示舒张压(DBP)、空腹血糖(FBG)、低密度脂蛋白胆固醇(LDL-C)和血管病变长度是PCI术后1年内ISR的独立危险因素(P<0.05),miR-590是PCI术后1年内ISR的独立保护因素(P<0.05)。模型Y(由DBP、FBG、LDL-C、血管病变长度和miR-590构成)预测ACS患者PCI术后1年内ISR的ROC曲线下面积高于模型X(由DBP、FBG、LDL-C和血管病变长度构成),差异有统计学意义(P<0.05)。风险...     

电离辐射对小鼠EL-4淋巴瘤细胞P21蛋白表达的影响

目的阐明电离辐射对小鼠EL-4淋巴瘤细胞P21蛋白表达的影响。方法采用免疫细胞化学方法和流式细胞术检测X射线照射对P21蛋白表达的时程和量效变化的影响。结果时程实验结果显示，离体培养的EL-4细胞经4．0Gy X射线照射后，P21蛋白表达在2．72h（免疫细胞化学方法检测）和8—72h（流式细胞术检测）明显增高（分别为P〈0．01、P〈0．001）。量效实验结果显示，X射线照射后24h，P21蛋白表达在0．5—6．0Gy（免疫细胞化学方法检测）和1．0～4．0Gy（流式细胞术检测）明显增高（分别为P〈0．05、P〈0．01、P〈0．001）。结论X射线能诱导P21蛋白表达增高，在一定范围内存在时间和剂量依赖关系。     

Radiation-induced gene expression in MCF-7 cells

PURPOSE: Detection of gene targets applicable to biological dosimetry and early detection of radiation-induced cell damage. MATERIALS AND METHODS: MCF-7 human mammary carcinoma cells were exposed to 2 and 6 Gy X-rays. Expression of 1176 genes was traced with the Atlas Human 1.2 Array (Clontech). RESULTS: (1) Based on data reproducibility (about 98%), a new and improved analysis was employed. (2) Normalization of data using ubiquitin or total gene expression led to the same results. (3) Dose-dependent gene expression was shown for six genes, which were constantly expressed over 1 day (three genes), 2 days (two) and 3 days (one). Differential gene expression was confirmed by RTQ-PCR. Three of the six genes were novel radiation-induced gene targets. (4) When analysing the expression of all genes, the processes of cell degradation (e.g. cell death and protein catabolism) were activated. Simultaneously, inhibition not only of cell proliferation, but also of other cellular functions (e.g. cell repair) was found. Hence, cell death appears a process of 'active silencing'. CONCLUSIONS: With the screening method applied, six radiation-induced gene targets were found, three of which were novel genes. Their applicability in other cell models remains to be tested. Different sets of genes have to be considered for dose assessment, owing to their changes in gene expression with time after irradiation. Furthermore, it has to be investigated whether 'active silencing' represents a general principle of radiation-induced cell death.     

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

Background and purpose

Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation.

Materials and methods

A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p.

Results

A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo.

Conclusion

miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.     

siRNA表达载体对MCF-7细胞生长及其hTERT基因表达抑制的研究

目的构建人端粒酶反转录酶(hTERT)基因的RNA干扰(RNAi)表达载体,探讨该载体对人乳腺癌MCF-7细胞生长及hTERT基因表达的影响。方法设计针对hTERT基因的干扰靶序列,构建siRNA重组表达质粒pGenesil-hTERT,将该质粒酶切测序鉴定后,以脂质体转染MCF-7细胞,RT-PCR和Western blot技术分别检测hTERT基因及其蛋白的表达,MTT法观察转染后MCF-7细胞生长情况。结果酶切电泳和测序分析表明插入序列正确,重组质粒构建成功。该质粒转染MCF-7细胞后,hTERT基因mRNA和蛋白质的表达水平均显著降低,MCF-7细胞生长抑制。结论内源性短发夹状siRNA能有效抑制hTERT基因的表达,进而抑制乳腺癌MCF-7细胞的增殖生长,这也为肿瘤的基因治疗提供了实验依据。     

Enhanced expression of adenovirus-mediated sodium iodide symporter gene in MCF-7 breast cancer cells with retinoic acid treatment.

Increased expression of the sodium iodide symporter (NIS) is required for effective radioiodine treatment and reporter gene imaging of breast cancer. We investigated the effect of retinoic acid on adenovirus-mediated expression of the human NIS gene in the MCF-7 breast cancer cell line. METHODS: The MCF-7 cell line was infected with recombinant adenovirus carrying the human NIS gene (Rad-NIS). Levels of NIS messenger RNA (mRNA) and protein expression and radioiodine ((125)I) uptake were measured to evaluate adenovirus-mediated NIS gene expression in wild-type and Rad-NIS-infected MCF-7 cells after treatment with all-trans-retinoic acid (ATRA; 10(-8)-10(-6) mol/L). RESULTS: The transduction efficiency of adenovirus in MCF-7 cells at a multiplicity of infection (MOI) of 50 was >60%. After incubation with 10(-6) mol/L ATRA, the mRNA level in Rad-NIS-infected MCF-7 cells increased to 118.5 times that of wild-type MCF-7 cells, whereas the mRNA level in wild-type MCF-7 cells showed only a 2.1-fold increase. Western blot, immunocytochemical staining, and flow cytometry analyses showed that NIS protein expression in MCF-7 cells infected with Rad-NIS increased after ATRA treatment. With ATRA treatment, the amount of (125)I uptake increased in a dose-dependent manner (P < 0.001). The (125)I uptake in wild-type MCF-7 cells increased 3.1-, 5.5-, and 7.6-fold with treatment with 10(-8), 10(-7), and 10(-6) mol/L ATRA, respectively. Rad-NIS-infected cells showed a 4.0-fold increase in (125)I uptake. Treatment of Rad-NIS-infected cells with 10(-8), 10(-7), and 10(-6) mol/L ATRA increased (125)I uptake by 4.9-, 8.2-, and 27.6-fold, respectively, compared with wild-type MCF-7 cells. The level of NIS expression in Rad-NIS-infected MCF-7 cells treated with 10(-6) mol/L ATRA (245.0 +/- 13.7 pmol/10(6) cells) was much greater than the sum of the expression levels seen in ATRA-treated wild-type cells and Rad-NIS-infected wild-type cells. CONCLUSION: Retinoic acid increases adenovirus-mediated NIS expression in MCF-7 cells. Our results indicate that improved efficiency of NIS gene therapy or reporter imaging in breast cancer may be possible with retinoic acid treatment.     

逆转录病毒载体介导IL－2导入MCF－7细胞基因表达及对LAK细胞杀伤的敏感性研究

用逆转录病毒载体介导人ＩＬ－２基因导入人乳腺癌细胞系ＭＣＦ－７并获得表达。原位杂交结果证实，转染的ＭＣＦ－７细胞中有ＩＬ－２ｍＲＮＡ的表达，用ＩＬ－２依赖细胞系ＣＴＬＬ－２以ＭＴＴ比色法测得ＭＣＦ－７细胞培养上清液中有ＩＬ－２活性，其活性平均为１０ＩＵ／１０￣６细胞。转导后的ＭＣＦ－７细胞对ＬＡＫ细胞的杀伤敏感性降低。     

Low-dose radiation differentially regulates protein acetylation and histone deacetylase expression in human coronary artery endothelial cells

Purpose: Ionizing radiation induces cardiovascular disease, the endothelium being the main target. The exact mechanism of the damage is unclear but the involvement of multiple signaling pathways is probable. Reversible lysine acetylation is a posttranslational protein modification that regulates activity across a broad range of signaling pathways. The aim of this study was to determine if a low radiation dose results in acetylome alteration in endothelial cells.

Materials and methods: Human coronary artery endothelial cell line was irradiated with Cs-137 gamma-rays (0.5?Gy) and proteomics analysis was performed using enriched acetylated peptides and all peptides. Data were validated using immunoblotting, deacetylase activity assay, and RhoA activity assay.

Results: Nearly a hundred proteins were found to have an altered acetylation status 24?h after irradiation, primarily due to an overall decrease in acetylation. The expression of specific deacetylases was significantly increased, coinciding with an enhancement in global deacetylase activity. Proteins changed in their acetylation status belonged to several pathways including protein synthesis, cytoskeleton-related processes, protein folding and calcium signaling. The predicted changes in the RhoA/actin cytoskeleton pathway were validated by immunoassay.

Conclusions: This study shows that protein acetylation is an important mediator of radiation responses in human cardiac coronary endothelial cells. Increased knowledge of the endothelial response to radiation is crucial for the development of normal tissue-sparing modalities during radiation therapy.     

hTERT-siRNA表达载体的构建及对MCF-7细胞生长、端粒酶活性的抑制作用

目的 构建人端粒酶逆转录酶(hTERT)基因的RNA干扰(RNAi)表达载体,并研究该载体对乳腺癌MCF-7细胞端粒酶活性及细胞增殖的影响,为针对端粒酶的乳腺癌基因治疗提供新的途径.方法 设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列TGTTCAGCGTGCTCAACTA,构建重组siRNA表达质粒pGenesil-hTERT,同时构建不针对任何基因的阴性对照重组pGenesiL-HK.两种重组质粒经酶切、电泳分析和测序鉴定后,用脂质体转染法分别转染乳腺癌MCF-7细胞,应用端粒酶重复序列扩增聚合酶链反应(TRAP-PCR)及聚丙烯酰胺凝胶电泳检测端粒酶活性,流式细胞仪测定细胞凋亡率.结果 酶切电泳测序分析表明插入序列正确,重组质粒构建成功.转染pGenesil-hTERT的MCF-7细胞,凝胶电泳见端粒酶特征性条带明显减少,端粒酶活性受到明显抑制pGenesil-hTERT转染细胞后凋亡率较对照组明显升高(P＜0.01),且转染48h凋亡率最高,达54.7%±2.41%.结论 hTERT-siRNA可有效抑制乳腺癌MCF-7细胞端粒酶活性、促进细胞凋亡,此法有望应用于肿瘤基因治疗.