Egr-1基因沉默对A549细胞放射敏感性的影响

目的:探讨Egr-1基因沉默对人肺腺癌A549细胞放射敏感性的影响。方法:选用A549细胞株作为研究对象,将其分成A、B、C三组,即空白对照组(只加入RPMI-1640培养)、阴性对照组(加入LV3-NC-sh RNA)、实验组(加入EGR1-homo-2294-sh RNA),采用慢病毒介导的sh RNA干扰技术使实验组细胞Egr-1基因沉默表达。利用荧光显微镜、自动化荧光定量细胞成像分析系统分析sh RNA转染,利用FQ-PCR分析Egr-1表达,再利用细胞克隆形成实验检测细胞放射敏感性参数的差异。结果:慢病毒介导的sh RNA成功转染阴性对照组、实验组细胞;空白对照组与阴性对照组Egr-1表达无差异(P0.05),实验组与空白对照组、阴性对照组比较Egr-1均受到明显抑制(P0.05);克隆形成实验中细胞放射敏感性参数D0、SF2实验组细胞与空白对照组、阴性对照组比较均存在明显差异(P0.05)。结论:Egr-1基因沉默使A549细胞放射敏感性降低,Egr-1表达可能与肿瘤细胞对放射的敏感性有关。     

靶向抑制miRNA-21提高白血病K562细胞对阿糖胞苷的敏感性

为研究以miRNA-21为靶标的反义核酸(AMO-miR-21)提高白血病K562细胞对阿糖胞苷(Ara-C)的敏感性及可能的作用机制,在LipofectamineTM2000介导下,将化学合成的AMO-miR-21转染K562细胞,四甲基偶氮唑蓝(MTT)法检测单独使用AMO-miR-21、Ara-C以及两者联合使用对细胞增殖抑制作用,并计算抑制率和IC50;流式细胞仪检测细胞凋亡;实时定量PCR(Real-time PCR)检测miRNA-21及其候选靶基因Pdcd4 mRNA的表达水平;免疫印迹(Western blot)检测Pdcd4蛋白的表达水平.结果显示Ara-C与AMO-miR-21联合使用后,IC50从1.95μmol/L降低到0.84μmol/L,敏感性提高到单用Ara-C的2.3倍.AMO-miR-21联合Ara-C组细胞凋亡明显增加(P<0.05).AMO-miR-21显著抑制K562细胞中miRNA-21的表达水平(P<0.05),同时Pdcd4的表达明显增加(P<0.05).提示AMO-miR-21可以提高K562细胞对Ara-C的敏感性,其作用机制与靶向抑制miRNA-21,进而诱...     

HeLa细胞中miRNA-214靶基因Mek3的确定

目的探索miRNA-214在HeLa细胞中的与其靶基因Mek3相互作用。方法通过miRNA靶基因预测网站寻找可能与miRNA-214相互作用的靶基因,合成miRNA-214和对照序列,将miRNA-214、对照序列、Mek3的3’非翻译区（3’UTR）以及突变的Mek3 3’UTR分别克隆到表达载体上,转染HeLa细胞,转染48h后提取蛋白,检测绿色荧光蛋白的表达水平;HeLa细胞转染miRNA-214后,Trizol抽提RNA,通过荧光定量PCR检测Mek3mRNA的表达水平;Western印迹检Mek3的蛋白表达水平。经过以上实验从mRNA和蛋白水平上验证了在HeLa细胞中miRNA-214对靶基因Mek3的作用效应。结果生物信息学方法显示miRNA-214和Mek3存在可能的结合位点。经过实验验证了miRNA-214可以下调Mek3的mRNA和蛋白水平。结论miRNA-214可以负调节靶基因Mek3的表达。     

AG490对甲状腺髓样癌TT细胞放射敏感性的影响

为了研究AG490对甲状腺髓样癌TT细胞放射敏感性,本研究选择甲状腺髓样癌TT细胞作为研究对象,并采用不同浓度AG490 (0, 25μmol/L, 50μmol/L, 100μmol/L)处理细胞,利用MTT法、流式细胞术和克隆形成实验分别对细胞增殖、凋亡及放射敏感性进行检测,并采用Western blotting法检测各组JAK2、p-STAT3、Bcl-2、Bax蛋白表达。结果表明,与A组相比,B、C、D组的抑制增殖作用增强,呈浓度和时间依赖性;随着AG490药物浓度的增加凋亡率逐渐增加;与A组相比,C组细胞存活分数显著降低;JAK2、p-STAT3和Bcl-2的蛋白表达量随着药物浓度的升高而逐渐降低,Bax蛋白表达量随着药物浓度的升高而逐渐增加。AG490对甲状腺髓样癌TT细胞增殖有抑制作用,可促进细胞凋亡,提高放射敏感性。     

miRNA-21对肺癌细胞放疗敏感性调控及肺损伤的影响

[目的]探究miRNA-21对肺癌细胞放疗敏感性的调控及放疗所致肺损伤的影响。[方法]选取人小细胞肺癌细胞系NCI-H446作为肺癌细胞模型、人支气管上皮细胞HBE作为放疗所致肺损伤细胞模型,取对数生长期NCI-H446和HBE细胞,分为空白对照组(不转染细胞)、阴性对照组(转染阴性对照序列细胞)和anti-miRNA-21组(转染anti-miRNA-21序列细胞),检测各组细胞中miRNA-21表达水平细胞活率、细胞周期进程。[结果]NCI-H446中miRNA-21表达量显著高于BEAS-2B,5.0Gy组的NCI-H446和HBE细胞活率均显著低于0.0Gy组,与0.0Gy比较,2.5、5.0Gy组的NCI-H446和HBE细胞的G2/M期细胞百分比显著增高(P<0.05),G1/S期细胞百分比显著降低,5.0Gy γ照射后,在8 h前,NCI-H446细胞中的miRNA-21表达水平显著上调,8 h后逐渐回落;在12 h前,HBE细胞中的miRNA-21表达水平显著上调,12 h后逐渐回落。转染后,anti-miRNA-21组NCI-H446和HBE细胞中的miRNA-...     

Egr-1基因与肿瘤腺癌放射敏感性关系的研究

目的：研究Egr-1基因在2种腺癌细胞A549和Hela放射前后的表达变化及对放射敏感性的影响。方法：培养肺腺癌A549细胞和宫颈腺癌Hela细胞,分别提取4Gyx射线照射前及照射后不同时间点的细胞的总RNA行荧光定量PCR（FQ-PCR）检测Egr-1表达水平;收获4GyX射线照射前及照射后不同时间点的细胞处理行流式细胞术检测其凋亡;对照射不同剂量的细胞继续培养10-14天,进行克隆形成计数,计算克隆形成率及存活分数。结果：FQ-PCR结果显示,放射后Egr．1基因表达水平在2种细胞中均明显升高且于放射后1h达到峰值,A549细胞的峰值明显高于Hela细胞;流式细胞术检测结果显示,A549细胞凋亡明显高于Hela细胞;克隆形成实验结果显示,A549细胞存活分数明显低于Hela细胞。结论：Egr-1基因在不同腺癌细胞表达水平不同并影响其放射敏感性。     

LncRNA LINC02310对非小细胞肺癌细胞上皮-间质转化和放射敏感性的影响

该研究探讨长链非编码RNA(LncRNA)LINC02310对非小细胞肺癌上皮-间质转化(EMT)和放射敏感性的影响.采用实时定量聚合酶链反应(qRT-PCR)法检测非小细胞肺癌组织和细胞(H1299、A549、H1650)内LINC02310相对表达量.体外培养H1299细胞,按照脂质体法将si-NC、si-LINC...     

Hsa-miR-212靶向调控BRCA1对乳腺癌MDA-MB-231细胞放疗敏感性的影响

[目的]探讨Hsa-miR-212靶向调控BRCA1对乳腺癌MDA-MB-231细胞放疗敏感性的影响。[方法]设MDA-MB-231细胞组、RT组(X射线以12Gy/min的剂量率发射,照射孵育时间为48h)、Hsa-miR-212 mimics组、RT+Hsa-miR-212 mimics组,各组设6个平行样,培养72 h。试验结束后,采用细胞计数试剂盒-8和结晶紫测定增殖及单克隆形成数目,流式细胞仪测量细胞凋亡水平,RT-PCR法及蛋白印迹法测定Hsa-miR-212、BRCA1 mRNA和蛋白水平。[结果]与MDA-MB-231细胞组比较,RT组、RT+Hsa-miR-212 mimics组OD值、存活率、细胞克隆形成数目、Hsa-miR-212 mRNA、BRCA1 mRNA与蛋白、穿膜数均降低(P<0.05),Hsa-miR-212 mimics组OD值、存活率、细胞克隆形成数目、Hsa-miR-212 mRNA、BRCA1 mRNA与蛋白、穿膜数升高(P<0.05);与RT组比较,Hsa-miR-212 mimics组、RT+Hsa-miR-212 mimics...     

AdipoRon对小鼠骨骼肌细胞胰岛素敏感性的影响及其机制

目的：观察脂联素受体激动剂AdipoRon对小鼠成肌细胞株（C2C12）胰岛素敏感性的影响,并探讨其作用机制。方法：使用马血清将C2C12诱导分化为成肌细胞,分为6组（9个复孔）：空白对照组、AdipoRon （脂联素受体激动剂）高剂量组、AdipoRon低剂量组、胰岛素组以及AdipoRon低剂量+PI3K （磷脂酰肌醇3激酶）抑制剂组和胰岛素+PI3K抑制剂组,作用12 h,收集上清检测葡萄糖消耗量,使用CCK8测定细胞增殖。六孔板中将C2C12诱导分化为肌管细胞,加入药物作用12 h,并用RT-PCR法检测GLUT4的mRNA水平。结果：与空白对照组相比,AdipoRon高剂量组、AdipoRon低剂量组、胰岛素组耗糖量均有所增加,具有统计学意义（P<0.05）。加入PI3K抑制剂组后,耗糖量与空白对照组无统计学意义。与空白对照组相比,AdipoRon高剂量组、AdipoRon低剂量组、胰岛素组细胞均有增殖,但只有胰岛素组具有统计学意义（P<0.05）。与对照空白组相比,AdipoRon高剂量组、AdipoRon低剂量组、胰岛素组GLUT4mRNA水平均有所提高,具有统计学意义（P<0.05）。加入PI3K抑制剂组后,GLUT4mRNA水平与空白对照组无统计学意义。结论：AdipoRon能够不影响细胞增殖的情况下增加葡萄糖的消耗量,这可能是通过提高胰岛素敏感性发挥作用的,但具体机制尚待进一步的研究。     

IER5基因对HeLa细胞辐射敏感性的影响

为寻找放射敏感性基因,采用基因芯片技术筛选出辐射可诱导表达mRNA上调的基因,其中就有IER5．为探索IER5基因的生物学功能及其在宫颈癌放疗中的作用,采用RNA干扰技术构建IER5基因表达抑制的质粒载体并构建IER5-siRNA-HeLa细胞系．对该细胞系与HeLa细胞进行辐射,旨在了解IER5-siRNA-HeLa的细胞生长曲线、细胞周期等实验参数的变化,揭示了IER5基因在辐射诱导中的生物学功能．实验发现,IER5基因表达抑制可提高细胞分裂进入S期与G2-M期的比例,促进细胞分裂,促进细胞生长,提高细胞对辐射的拮抗性,同时发现IER5-siRNA-HeLa细胞在尺寸上大于HeLa细胞．研究表明,IER5基因表达抑制可促使细胞受辐射后发生S期与G2-M期的阻滞,IER5参与辐射细胞周期的调控,对临床宫颈癌放疗有一定的潜在应用价值．     

Let-7e enhances the radiosensitivity of colorectal cancer cells by directly targeting insulin-like growth factor 1 receptor

Abnormal expression of various microRNAs (miRNAs), as regulators of biological signaling pathways, has a strong association with cancer resistance to chemotherapy and radiotherapy. The let-7 family of miRNAs as tumor suppressors have shown to be downregulated in different types of human malignancies including colorectal cancer (CRC). However, the biological function of let-7 members in the processes of resistance to radiation in CRC has not yet been completely elucidated. Insulin-like growth factor 1 receptor (IGF-1R) signaling pathway is amplified in CRC and leads to its progression, development, and also radiation resistance. So, it seems like an attractive target for anticancer therapy. In this study, by using bioinformatics analysis, it has been revealed that IGF-1R is a direct target of the let-7e member. Consistent with this, we identified that increased levels of let-7e in CRC cells reduced IGF-1R protein level and subsequently its downstream signaling pathways, which resulted in the G1 cell cycle arrest and a significant reduction in the proliferation, survival and also resistance to radiation of CRC cells. Altogether, these results suggested that let-7e by targeting the IGF-1R signaling pathway might serve as therapeutics in anticancer therapy.     

miR-22 enhances the radiosensitivity of small-cell lung cancer by targeting the WRNIP1

Small-cell lung cancer (SCLC) is an aggressive malignancy characterized by high cellular proliferation and early distant metastasis. Our study aimed to explore the effect of miR-22-3p (miR-22, for short) on SCLC radiosensitivity and its molecular mechanisms. The expression level of miR-22 was evaluated in a human normal lung epithelial cell line and a human SCLC cell line, and cell apoptosis and migration were detected. The expression of the miR-22 direct target WRNIP1 mRNA and protein were explored. Five differentially expressed genes were detected. The miR-22 expression in NCI-H446 was significantly decreased, and miR-22 overexpression significantly promoted cell apoptosis. miR-22 overexpression could significantly inhibit the cell migration of SCLC cells, and miR-22 had a negative regulatory effect on WRNIP1 mRNA and protein levels. KLK8 was downregulated, and the messenger RNA (mRNA) of four other genes (PC, SCUBE1, STC1, and GPM6A) was upregulated mRNA in cells overexpressing miR-22, which was in accordance with the bioinformatics analysis. miR-22 could enhance the radiosensitivity of SCLC by targeting WRNIP1.     

MK基因对小鼠子宫基质细胞蜕膜化进程的影响

为探索肿瘤坏死因子相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)的死亡受体(mouse killer,MK)对小鼠子宫基质细胞蜕膜化进程的影响,构建MK基因过表达和siRNA干扰重组腺病毒.原代培养的小鼠子宫基质细胞感染MK过表达或者干扰重组腺病毒并诱导蜕膜化,72 h后用免疫细胞化学与流式细胞术分别检测蜕膜细胞的标志物催乳素(prolactin,PRL)与蜕膜细胞凋亡率的变化情况.妊娠d4小鼠子宫角注射MK重组腺病毒,观察胚胎植入点的数量变化.实验结果表明,与对照组相比,在诱导的蜕膜细胞中过表达MK使得催乳素的含量显著降低(P<0.05),同时,蜕膜细胞的凋亡率明显升高(P<0.05),而siRNA干扰之后催乳素的含量显著升高,凋亡率明显下降(P<0.05),但是,宫角注射MK基因过表达和siRNA干扰重组腺病毒之后,胚胎植入数量均显著减少(P<0.01).提示MK基因通过参与小鼠子宫内膜基质细胞的蜕膜化进程,调节蜕膜细胞增殖与凋亡之间的平衡从而影响胚胎的植入.     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors γ used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G₂/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.     

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.     

Effect of resveratrol on the radiosensitivity of 5-FU in human breast cancer MCF-7 cells

The aim of this study was to assess the efficacy of resveratrol (Res) on radiosensitivity of 5-fluorouracil (5-FU) in the spheroid culture of MCF-7 breast cancer cell line using colony formation examination. Spheroids on day 9 with 300 µm diameters were treated with 20 µM resveratrol and/or 1 µM 5-FU for one volume doubling time (VDT) (42 hours) and then irradiated with 2 Gy gamma radiation (⁶⁰Co) in various groups. Then the viability of the cells and clonogenic ability were acquired by blue dye exclusion and colony formation assay, respectively. The population doubling time in the monolayer culture and the VDT of spheroid culture was 22.48 0.23 hours and 42 0.63 hours respectively. None of the drugs and combination of them had any effect on the viability of cells. The combination treatment of 5-FU+Res+ radiation significantly reduced the colony formation ability of spheroid cells in comparison with each treatment alone. Our results indicated that resveratrol can significantly decrease colony number of breast cancer spheroid cells treated with 5-FU in combination with gamma-rays. Thus, resveratrol as a hypoxia-inducible factor-1-alpha inhibitor increased the radiosensitization of breast cancer spheroid cells.