【作者】 卜丽佳；

【导师】 孙国平；

【作者基本信息】 安徽医科大学 ， 肿瘤学， 2016， 博士

【摘要】 目的:明确褪黑素在内质网应激(endoplasmic reticulum stress,ERS)条件下通过抑制环氧化酶-2(cyclooxygenase-2,COX-2)对Hep G2肝癌细胞凋亡水平的影响,及其分子机制进行初步探讨。方法:通过免疫组化法检测COX-2、活化转录因子6(activating transcription factor6,ATF-6)、肌醇需求激酶1(inositol-requiring enzyme 1,IRE-1)、双链RNA激活的蛋白激酶样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)在肝癌组织中的表达并分析未折叠蛋白反应(unfolded protein response,UPR)信号通路与COX-2表达的相关性。用Western blot检测不同浓度褪黑素对Hep G2肝癌细胞中三条UPR通路蛋白表达变化情况,使用小干扰RNA(Small interfering RNA,si RNA)分别干扰UPR三条通路的表达,使用Western blot技术检测COX-2蛋白以及RT-q-PCR法检测COX-2 m RNA的表达,从而了解UPR通路与COX-2表达的相关性。Western blot法检测COX-2、CCAAT/增强子结合蛋白的同源蛋白(C/EBP homologous protein 10,CHOP)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)以及Bcl-2相关X蛋白(Bcl-2-associaed X protein,Bax)表达的变化,进而了解褪黑素对Hep G2肝癌细胞凋亡的影响。TUNEL法、Annexin V+PI双染流式细胞术观察褪黑素转染ATF-6 si RNA对Hep G2肝癌细胞凋亡的相关机制。结果:1.肝癌组织中UPR信号通路与COX-2的相关性:采用免疫组化法检测COX2、ATF-6、IRE-1、PERK的表达并分析其相关性,结果显示:100例肝癌患者中,研究发现COX-2阳性组,ATF-6的阳性率为87%,IRE-1的阳性率为60%,PERK的阳性率为60%,COX-2阴性组中ATF-6的阴性率为29%,IRE-1的阴性率为55%,PERK为53%。相关性分析显示,COX-2和ATF-6的表达呈正相关(P=0.011),与IRE-1(P=0.086)及PERK(P=0.134)无相关性。2.不同浓度褪黑素对Hep G-2肝癌细胞UPR信号通路的影响:浓度为3μmol/L的衣霉素预处理Hep G-2肝癌细胞8小时后,预计设4个褪黑素浓度组(10-9、10-7、10-5、10-3mmol/L),以不加褪黑素为空白对照。褪黑素作用于Hep G-2肝癌细胞24小时,使用Western blot检测三条UPR通路蛋白表达变化,最后结果证明10-5mmol/L及10-3mmol/L浓度的褪黑素可有效抑制ATF-6、IRE-1及PERK三条UPR通路的表达,有统计学意义,其中10-5mmol/L考虑系褪黑素药理浓度,故选用该浓度作为后续处理细胞的浓度。3.Western blot检测si RNA干扰效率:分别将PERK、IRE-1和ATF-6通路中的三条si RNA同源序列转染Hep G-2中,24小时后提取肝癌细胞总蛋白,分别检测各组同源序列阻断后PERK、IRE-1和ATF-6蛋白的表达情况。结果显示:si-PERK和si-IRE-1各有两条同源序列明显抑制PERK和IRE-1的表达,si-ATF6-1的抑制作用最明显;后期实验选择抑制作用较强si-PERK-3和si-IRE1-3进行特异性阻断UPR通路。4.内质网应激后Hep G2肝癌细胞UPR通路与COX-2表达的相关性:为了解三个UPR通路与COX-2表达的相关性,使用浓度为3μmol/L的衣霉素预处理Hep G2肝癌细胞8小时,分别转染ATF-6、IRE-1及PERK三条通路的si-RNA进行干扰,24小时后提取各组COX-2蛋白进行Western blot检测和定量RT-PCR检测m RNA含量,结果均显示,TM组COX-2表达较空白对照组明显增加,而si-ATF-6组COX-2表达较TM组明显降低,有统计学意义(P<0.01),而si-IRE-1及si-PERK表达无明显变化。5.转染ATF-6 si RNA对内质网应激后Hep G-2肝癌细胞的影响;3μmol/L衣霉素预处理Hep G-2肝癌细胞用8小时后转染ATF-6 si RNA作用24小时,使用Annexin V-PI双染流式细胞术及TUNEL法技术观察Hep G-2肝癌细胞凋亡情况。结果均显示ATF-6 si RNA转染组与空白对照组和只加衣霉素诱导内质网应激组相比,凋亡比例均明显增加,有统计学意义(P<0.01)。6.褪黑素诱导内质网应激后HepG2肝癌细胞凋亡的通路:3μmol/L衣霉素预处理Hep G2肝癌细胞8小时后,分别加入10-5mmol/L浓度的褪黑素作用24小时或转染ATF-6 si RNA作用6小时,设置空白对照组、单独衣霉素组、加褪黑素组、及加ATF-6 si RNA转染组,Western blot检测四组中COX-2和CHOP、Bcl-2及Bax凋亡相关蛋白水平变化。结果显示,在内质网应激后的Hep G2肝癌细胞中,加褪黑素组和加ATF-6 si RNA转染组CHOP、Bax表达均显著上升,Bcl-2的表达下降,Bcl-2/Bax比值显著降低,有统计学差异(P<0.01)。7.比较褪黑素和转染ATF-6 si RNA对内质网应激后Hep G-2肝癌细胞凋亡的影响:3μmol/L衣霉素预处理Hep G2肝癌细胞8小时后,分别加入10-5mmol/L浓度的褪黑素作用24小时或转染ATF-6 si RNA作用6小时,设置空白对照组、单独衣霉素组、加褪黑素组、及加ATF-6 si RNA转染组,使用Annexin V-PI双染流式细胞术及TUNEL法观察四组Hep G-2肝癌细胞凋亡情况。与空白对照组相比,内质网应激后可轻度诱导肿瘤细胞凋亡(P<0.05)。而加褪黑素组和加ATF-6 si RNA转染组,与单独衣霉素组比较,细胞凋亡率均显著增加(P<0.01)。结论:1.COX-2和ATF-6的表达呈正相关,抑制ATF-6可以抑制COX-2的表达,从而表明过表达的COX-2可能与ATF-6通路的激活相关。2.抑制ATF-6可增加细胞凋亡,ATF-6可能参与了内质网应激后Hep G2肝癌细胞的凋亡抵抗。3.褪黑素可能通过抑制ATF-6通路下调COX-2表达,进而增加Hep G2肝癌细胞凋亡。更多还原

【Abstract】 Aim: To clarify how melatonin sensitized human hepatocellular carcinoma(HCC)Hep G2 cells via inhibition of cyclooxygenase-2(COX-2)expression to endoplasmic reticulum(ER)stress-induced apoptosis and indentify the molecular mechanisms.Methods:We detected the expression of COX-2,activating transcription factor 6(ATF-6),inositol-requiring enzyme 1(IRE-1),protein kinase RNA-like endoplasmic reticulum kinase(PERK)in HCC tissues by using immunohistochemical staining.We next evaluated the effect of four concentrations of melatonin on the three unfolded protein response(UPR)pathways by Western blot.To investigate the underlying mechanisms of which pathway is associated with the expression of COX-2 under the condition of ER stress,we used RNA interference to knockdown the m RNA in all three UPR pathways.Then we observed the changes in COX-2 m RNA and protein levels by q RT-PCR and Western blot.We also evaluated the expression of COX-2,C/EBP homologous protein10(CHOP),B-cell lymphoma-2(Bcl-2)and Bcl-2-associaed X protein(Bax)by Western blot.To further determine whether melatonin influences ER stress-induced apoptosis,FACS analysis and TUNEL staining were performed in Hep G2 cells.Results:1.The association of three UPR pathways in HCC with the expression of COX-2: We have evaluated and analyzed the expression of COX-2,ATF-6,IRE-1 and PERK in HCC tissues by using immunohistochemical staining.Among the tissues with positive COX-2 expression,the positive rate of ATF-6,IRE-1 and PERK was 87%,60% and60%,respectively.Among the tissues with negative COX-2 expression,the negative rate of ATF-6,IRE-1 and PERK was 29%,55% and 53%,respectively.In conclusion,the expression of COX-2 was correlated with the expression of ATF-6(P=0.011).There was no association between COX-2 and IRE-1(P=0.086).We also found no association between COX-2 and PERK(P=0.134).2.The effects of different concentrations ofmelatonin on the three UPR pathways in Hep G2 cells: To meet this requirement,Hep G2 cells were first pretreated withtunicamycin for 8 h;then,we treated the cells with four concentrations of melatonin(10-9,10-7,10-5,10-3mmol/L)for 24 h.We evaluated the expression of proteins in the three pathways by Western blot.We found that melatonin at a concentration of both 10-3and 10-5mmol/L could obviously downregulate the expression of ATF-6,IRE-1 and PERK.We next chosen the concentration of 10-5mmol/L for further study.3.We have evaluated the interference efficiency of si RNA using Western blot: We have transfected the three homologous sequences of si RNA for ATF-6,IRE-1 and PERK to Hep G2 cells individually.The total protein was extracted 24 h after transfection.The expression of ATF-6,IRE-1 and PERK was detected using Western blot.We have obersved that the expression of ATF-6 was inhibited obviously by transfected with si-ATF-6 1#.Furthermore,the expression of IRE-1 was Inhibited obviously by transfected with si-IRE13# and the expression of PERK was Inhibited obviously by transfected with si-PERK 3#.4.We have investigated the association of different UPR pathways with the expression of COX-2 under the condition of ER stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8h;then,we used si RNA to knockdown the m RNA in all three UPR pathways and observed the changes in COX-2 m RNA and protein levels by q RT-PCR and Western blot.The results revealed that the expression of COX-2 was downregulated in the group transfected with si-ATF-6,compared with the control(P<0.01).However,there was no significant change for the expression of COX-2 when transfected with si-IRE-1 or si-PERK.5.We have further determined the effect of si-ATF6 on Hep G2 cells.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h;then,we transfected si-ATF-6 to Hep G2 cells for 24 h.Apoptosis assays were performed using an Annexin-V FITC apoptosis kit and TUNEL staining.The apoptotic cell ratio was increased in the group transfected with si-ATF-6,as compared with the control group(P<0.01).6.We have identified the pathway of which melatonin induced Hep G2 cell apoptosis under ER stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h.Then,we have evaluated the expression of COX-2,CHOP,Bcl-2 and Bax by Western blot in four groups: blank control group,TM group,melatonin group(treated with melatonin at the concentration of 10-5mmol/L for 24h)and si-ATF-6 group(transfected with si-ATF-6 for 6h).The expression of CHOP and Bax was upregulated in the melatonin group and si-ATF-6 group.Meanwhile,both the expression of Bcl-2 and Bcl-2/Bax ratio were downregulated in the melatonin group and si-ATF-6 group(P<0.01).7.We have compared the effects of melatonin and si-ATF6 on Hep G2 cell apoptosis under ER-stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h.Then,we have determined cell apoptosis by using an Annexin-V FITC apoptosis kit and TUNEL staining in four groups: blank control group,TM group,melatonin group(treated with melatonin at the concentration of 10-5mmol/L for 24h)and si-ATF-6 group(transfected with si-ATF6 for 6h).Compared with blank control group,ER-stress could induce cancer cell apoptosis mildly(P<0.05).Nevertheless,both treated with si-ATF-6and melatonin resulted in a dramatic increase in the number of apoptotic Hep G2 cells compared with TM group(P<0.01).Conclusions:1.The expression of COX-2 was correlated with the expression of ATF-6.Suppression of ATF-6 could downregulate the expression of COX-2,which suggest that overexpression of COX-2 may be associated with the of ATF-6 pathway.2.Supression of ATF6 resulted in a dramatic increase in the number of apoptotic Hep G2 cells.ATF-6 may take part in the process associated with resistance to ER stress–mediated apoptosis.3.Under the tunicamycin-induced ER stress,melatonin can inhibit the expression COX-2 by downregulating one of the UPR pathways,ATF-6,which increases the apoptosis of Hep G2 cells via CHOP and Bcl-2/Bax pathway.更多还原