siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

去铁酮对宫颈癌HeLa细胞增殖和凋亡的影响

去铁酮可诱导p53的表达和引发肿瘤细胞凋亡。RRM2B基因在铁螯合剂抑制肿瘤细胞生长中同样起重要作用,且其表达受p53调节。为了探讨去铁酮对宫颈癌细胞的影响,以及p53和RRM2B基因二者的关系,通过MTT法检测不同浓度及不同时间去铁酮处理对HeLa细胞活性的影响,采用流式细胞仪检测细胞凋亡水平。同时,利用Western-blot和荧光定量PCR技术分别在蛋白和mRNA水平检测p53和RRM2B表达变化,并进一步利用p53抑制剂检测p53对RRM2B表达的影响。结果表明,HeLa细胞活性随着去铁酮作用浓度及作用时间的增加而下降,在500μmol/L去铁酮处理48h后出现明显凋亡。进一步研究发现,去铁酮可显著上调p53表达水平(P0.05),而p53可显著下调RRM2B表达水平(P0.05)。综上所述,去铁酮可通过p53下调RRM2B表达水平而参与抑制宫颈癌细胞增殖。     

ASPP2增强结肠癌HCT116细胞对奥沙利铂的化疗敏感性

为研究ASPP2对奥沙利铂诱导的结肠癌细胞系HCT116 p53+/+(野生型)凋亡及周期的影响.利用ASPP2（rAd-ASPP2）及p53腺病毒（rAd-p53）感染HCT116 p53+/+细胞,经奥沙利铂50 μmol/L诱导细胞凋亡及周期改变.Western印迹检测ASPP2及p53的表达水平;MTT法检测ASPP2腺病毒对奥沙利铂诱导的HCT116细胞活性的影响;Calcein/PI吸收试验检测细胞凋亡情况;流式细胞术分析细胞周期分布. 结果显示,ASPP2、p53共同过表达,或者ASPP2单独过表达均能增强奥沙利铂诱导的HCT116 p53+/+细胞增殖抑制,以及S期抑制并伴有细胞凋亡水平的升高;而无奥沙利铂诱导时,ASPP2对HCT116 p53+/+细胞的活性、细胞周期及细胞凋亡水平的影响无统计学意义. 上述结果表明,ASPP2能够增强奥沙利铂诱导HCT116 p53+/+细胞的增殖抑制、细胞周期抑制和细胞凋亡.     

乙型肝炎病毒上调的lnc-HUR1抑制肝癌细胞凋亡北大核心CSCD

Lnc-HUR1是乙型肝炎病毒(hepatitis B virus,HBV)上调的长链非编码RNA,具有促进肝癌细胞增殖和促进肝癌发生发展的功能。为探究lnc-HUR1对肝癌细胞凋亡的影响,通过免疫印迹、实时荧光定量PCR、双荧光素酶报告基因、免疫共沉淀、流式细胞术等实验方法,检测lnc-HUR1对肝癌细胞凋亡的影响。转化生长因子-β(tansforming growth factor-β,TGF-β)、五氟尿嘧啶和星孢菌素诱导肝癌细胞凋亡的实验结果显示,过表达lnc-HUR1能够显著降低caspase3/7的活性以及PARP-1的剪切,而敲低lnc-HUR1则能够显著增加caspase3/7的活性,促进PARP-1的剪切;Annexin-Ⅴ和PI联合染色实验也表明过表达lnc-HUR1能够抑制细胞凋亡,敲低lnc-HUR1能够促进细胞的凋亡。同时过表达lnc-HUR1能够在RNA水平和蛋白水平上调凋亡抑制因子Bcl-2(B cell lymphoma-2)、下调促凋亡因子BAX(B cell lymphoma-2-associated x),从而抑制细胞的凋亡。在CCL4诱导的小鼠急性肝损伤模型中,lnc-HUR1转基因小鼠肝组织中Bcl-2的表达高于对照小鼠。染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)实验也证实lnc-HUR1能够降低p53在Bcl-2和BAX启动子区的富集。以上结果说明,lnc-HUR1通过促进凋亡抑制因子Bcl-2及抑制凋亡促进因子BAX的表达抑制肝癌细胞的凋亡。进一步的实验表明,在HCT116细胞中lnc-HUR1能够调控Bcl-2和BAX的转录,而在HCT116 p53−/−细胞中,lnc-HUR1对Bcl-2和BAX的表达没有影响,说明lnc-HUR1对肝癌细胞凋亡的抑制作用依赖于p53的活性。综上所述,HBV上调的lnc-HUR1具有抑制肝癌细胞凋亡的作用,lnc-HUR1通过抑制p53转录活性,上调凋亡抑制因子Bcl-2、抑制凋亡促进因子BAX的转录,从而抑制细胞凋亡。上述结果提示Lnc-HUR1在HBV相关肝癌发生发展中发挥重要作用。     

过表达小鼠UNCV蛋白质对HeLa细胞凋亡的影响

目的研究小鼠UNCV蛋白质对HeLa细胞凋亡的影响。方法将BALB/c小鼠Uncv基因重组质粒转染HeLa细胞,筛选出稳定过表达UNCV蛋白质的HeLa细胞株。通过细胞计数法和流式细胞术,检测过表达UNCV蛋白质对血清饥饿和阿霉素诱导的HeLa细胞凋亡的影响。结果获得稳定正确过表达UNCV蛋白质的HeLa细胞株。细胞计数和流式细胞术结果显示过表达UNCV蛋白质对血清饥饿诱导的HeLa细胞凋亡有抑制作用,对于阿霉素诱导的HeLa细胞凋亡没有显著影响。结论过表达UNCV蛋白质对血清饥饿诱导的HeLa细胞凋亡有抑制作用。     

LINC00261/miR-182-5p/PFN1对乙肝病毒相关性肝细胞癌HepG2.2.5细胞放疗抵抗的影响

[目的]探讨LINC00261调控miR-182-5p/PFN1轴对乙型肝炎病毒相关性肝细胞癌HepG2.2.15细胞放疗抵抗的作用机制。[方法]用1 Gy的X射线处理HepG2.2.15细胞得放疗抵抗细胞HepG2.2.15/R,RT-qPCR检测LINC00261和miR-182-5p表达,Western Blot检测PFN1的表达,CCK-8检测细胞增殖活力,流式细胞术检测细胞凋亡率,彗星实验检测DNA损伤情况;双荧光素酶报告基因实验验证miR-182-5p和LINC00261或PFN1的靶向关系。[结果]LINC00261在HepG2.2.15细胞中低表达(P<0.01),且在其放疗抵抗细胞HepG2.2.15/R中表达更低(P<0.01)。过表达LINC00261抑制HepG2.2.15/R细胞增殖(P<0.01),诱导细胞凋亡(P<0.05)和DNA双链断裂(P<0.01);6 Gy X射线处理可上调过表达LINC00261对HepG2.2.15/R细胞凋亡(P<0.05)和DNA损伤的促进作用(P<0.01)。双荧光素酶报告基因实验证实miR-182-5p与LINC00261或PFN1的靶向关系。过表达LINC00261或过表达PFN1可下调过表达miR-182-5p对HepG2.2.15/R细胞增殖的促进作用(P<0.05)以及对凋亡和DNA损伤的抑制作用(P<0.05)。[结论]过表达LINC00261靶向下调miR-182-5p,促进PFN1表达,促进HepG2.2.15细胞放疗抵抗。     

siRNA介导的NFBD1表达沉默对HeLa细胞增殖与凋亡的影响

NFBD1,也称MDC1,是1个参与细胞内DNA损伤后细胞应答反应的重要分子．为探讨NFBD1在细胞增殖和凋亡中的作用及其作为分子靶点用于肿瘤治疗的潜在价值,本研究采用siRNA技术抑制NFBD1的表达,并观察了其对人宫颈癌细胞HeLa细胞增殖和细胞凋亡的影响．半定量RT-PCR和蛋白质印迹分析结果表明,筛选到的短链NFBD1 siRNA能有效抑制内源NFBD1的表达,抑制程度约100％．细胞生长曲线分析结果表明,siRNA介导的NFBD1表达沉默导致HeLa细胞生长增殖的显著抑制．FACS分析结果表明,NFBD1表达抑制导致sub-G₁峰的出现,同时蛋白质印迹分析观察到了caspase 3和PARP（poly-ADP-ribose polymerase）的剪接激活,表明NFBD1表达抑制诱发了细胞凋亡．在该凋亡过程中,P-53及其下游靶分子Bax和Puma的表达水平均没有发生明显变化,但Noxa的表达在mRNA和蛋白质水平上均显著上调,强烈提示该凋亡过程很可能是1个不依赖P53的凋亡途径,且Noxa的转录激活在该凋亡过程中可能起着重要作用．这些结果表明,NFBD1参与细胞生长增殖和凋亡的调节,是一个潜在的肿瘤治疗新靶点．     

维生素C诱导人宫颈癌Caski细胞凋亡及其分子机制的研究

为了研究维生素C对人宫颈癌Caski细胞体外抑制、诱导凋亡的作用及其分子机制,使用不同剂量维生素C处理人宫颈癌Caski细胞,采用噻唑蓝(MTT)法检测药物对细胞增殖的抑制作用;流式细胞仪检测Cas-ki细胞周期变化;琼脂糖电泳法观察凋亡细胞DNA Ladder现象;Western blot检测凋亡相关蛋白Bcl-2、Bax和E6的表达以及Caspase 3的激活;荧光染色观察细胞线粒体膜电位的改变.分析发现,维生素C可显著抑制人宫颈癌Caski细胞增殖,呈现明显的时间和剂量依赖性;将细胞阻滞于S期;诱导细胞凋亡,下调Bcl-2和E6、上调Bax蛋白表达,促进Caspase3活化,降低线粒体膜电位.表明维生素C在体外可有效抑制人宫颈癌Caski细胞增殖,诱导细胞凋亡.     

Knockdown of long noncoding RNA urothelial carcinoma associated 1 inhibits colorectal cancer cell proliferation and promotes apoptosis via modulating autophagy

Long noncoding RNA urothelial carcinoma associated 1 (UCA1) has been implicated in the growth and metastasis of colorectal cancer (CRC), and autophagy contributes to tumorigenesis and cancer cell survival. However, the regulatory role of UCA1 in CRC cell viability by modulating autophagy remains unclear. In the present study, a significant positive correlation was observed between UCA1 and microtubule-associated protein 1 light chain 3 (LC3) levels, and the elevated UCA1 was negatively correlated with the PKB/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in 293T cells. Downregulation of UCA1 inhibited autophagy activation and cell proliferation, whereas the apoptosis was increased and the cell cycle was arrested in G2 stage. The next results showed that UCA1 was markedly upregulated in Caco-2 cells. Knockdown of UCA1 significantly decreased the LC3-II and autophagy-related gene 5 (ATG5) protein levels and resulted in an increase in p62 expression. Conversely, the autophagy activator rapamycin (RAPA) reversed the effects. Furthermore, downregulated UCA1 decreased Caco-2 cells population in the G1 phase and increased the cells number in G2 phage. The cell proliferation was inhibited, and apoptosis rate was promoted. More important, RAPA could also abrogate the changes induced by knockdown of UCA1. Collectively, these data demonstrated that downregulated UCA1 induced autophagy inhibition, resulting in suppressing cell proliferation and promoting apoptosis, which suggested that UCA1 might serve as a potential new oncogene to regulate CRC cells viability by modulating autophagy.     

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR‐204 in pediatric AML

The long noncoding RNA urothelial carcinoma‐associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27^kip1. Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR‐204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase‐3 in AML cells. Moreover, UCA1 sponged miR‐204 and suppressed its expression. UCA1 overexpression inversed the miR‐204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR‐204 target, via the sponging interaction. Furthermore, miR‐204 inhibited inducible nitric oxide synthase and cyclooxygenase‐2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR‐204 in pediatric AML.     

circPPP1R12A激活p53信号通路调控骨关节炎中软骨细胞增殖和凋亡

摘要 目的：探讨circPPP1R12A（circ_0000423）调控p53信号通路对骨关节炎（osteoarthritis,OA）中软骨细胞增殖和凋亡的影响。方法：采用qRT-PCR检测circPPP1R12A在OA软骨细胞中的表达水平。在OA软骨细胞中分别转染oe-circPPP1R12A和sh-circPPP1R12A后,采用CCK-8检测细胞增殖情况;免疫荧光检测Ki-67阳性细胞表达率;流式细胞术检测细胞凋亡情况;qRT-PCR检测Ki-67和p53表达水平;Western Blot检测Cleaved-caspase3、P53、BCL-2和BAX的表达水平。结果：OA软骨细胞中circPPP1R12A的表达水平明显高于正常软骨细胞。过表达circPPP1R12A能够抑制OA软骨细胞增殖和促进细胞凋亡,通过上调p53表达激活p53信号通路,低表达circPPP1R12A能够促进OA软骨细胞增殖和抑制细胞凋亡,通过下调p53表达阻滞p53信号通路。在OA软骨细胞中同时低表达circPPP1R12A和过表达p53能够反转单独低表达circPPP1R12A对OA软骨细胞增殖和凋亡的影响。结论：circPPP1R12A在OA软骨细胞中明显高表达,circPPP1R12A能够通过激活p53信号通路抑制骨OA软骨细胞增殖和促进软骨细胞凋亡。circPPP1R12A可能成为OA治疗的干预靶点。     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

siRNA抑制c—myc基因的表达对宫颈癌细胞增殖的影响

目的：利用siRNA（small interference RNA）技术研究C-myc基因的对宫颈癌HeLa细胞增殖的影响。方法：依据Promega公司在网上提供的设计软件,设计针对C-myc基因的siRNA,合成DNA模板,体外转录合成siRNA。通过阳离子聚合物jet—SITM—ENDO将合成的siRNA转染入HeLa细胞,以未转染细胞以及错义序列siRNA—scr转染细胞为对照。用细胞计数法检测siRNA对HeLa细胞增殖的影响。流式细胞法检测细胞周期及蛋白表达的变化,RT—PCR法比较转染前后C-myc mRNA表达水平的变化。结果：细胞计数法结果显示,转染24h后c-myc基因siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。c-myc基因siRNA转染后能有效地抑制HeLa细胞的增殖,阻滞细胞周期于G0／G1期,siRNA转染组c-myc mRNA、蛋白的表达量明显低于空白对照组、错义序列组。结论：体外转录合成的siRNA可有效降低HeLa细胞c-myc基因的表达,抑制细胞增殖。     

Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells

The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.