【作者】 薛景；

【导师】 吴锦昌；

【作者基本信息】 苏州大学 ， 放射医学， 2014， 博士

【摘要】 目的：目前克服肿瘤细胞放射抗拒性策略较多,但效果欠佳。研究发现SOD1高表达是肿瘤对放化疗抵抗的重要机制之一，但SOD1高表达的机制不明。基因表达的精细调控多与3’UTR的转录后调控有关，但关于肿瘤SOD1的转录后调控报道较少，因此本课题拟探讨肿瘤SOD1转录后调控机制及其对肿瘤反射效应的影响，为以SOD1为靶标的放疗增敏策略提供理论基础。方法：本课题主要以卵巢癌细胞系A2780和胰腺癌PANC1为模型，（1）构建SOD-3’UTR的荧光素酶报告载体，观察SOD1、SOD2-3’UTR在肿瘤细胞对荧光素酶活性的影响，以及在过氧化氢和X射线作用下的变化；并应用RT-PCR和蛋白印迹法验证肿瘤细胞在过氧化氢作用下SOD1mRNA和蛋白表达水平的变化（2）比较SOD1-3’UTR在两种肿瘤细胞中对荧光素酶活性的影响，并应用RT-PCR法检测萤火素酶mRNA的水平；并进一步应用蛋白印迹法观察外源SOD-3’UTR对内源性SOD蛋白表达的影响。构建不同的SOD1-3’UTR荧光素酶报告载体，包括含有不同AREs的、miRNA（miR-224、-377、-621）结合位点缺失的、单个或多个AREs联合缺失的3’UTR载体，或应用miRNA的拟似物或抑制剂等观察RNA元件对3-‘UTR所介导的荧光素酶性的影响，或SOD1蛋白表达的变化。构建SOD1-Promoter-SOD1-3’UTR荧光素酶报告载体，进一步对该载体进行AREs缺失突变，检测X射线处理后荧光素酶活性的变化。（3）RNA干扰技术下调AUF1或外源真核表达上调AUF1，观察其对SOD1蛋白表达的变化；以及SOD1-3’UTR所介导的荧光素酶活性的变化；并观察AUF1下调对肿瘤细胞ROS水平的影响；进一步应用RNA免疫共沉淀检测AUF1与SOD1-3’UTR的直接作用；免疫组化法检测胰腺癌标本中AUF1和SOD1蛋白的表达。结果：（1）SOD1、SOD2-3’UTR正义载体与反义载体相比，荧光素酶活性增加近20倍；过氧化氢100μM明显增加了A2780细胞内SOD1、SOD2-3’UTR所介导的荧光素酶活性；10Gy射线明显增加了A2780细胞内SOD1-3’UTR所介导的荧光素酶活性;过氧化氢100μM增加了A2780细胞SOD1mRNA和蛋白表达水平；（2）在A2780和PANC1细胞内SOD1-3’UTR正义载体相比反义、空载体的荧光素酶活性明显增加；SOD1-3’UTR增加了A2780细胞萤火素酶mRNA的水平；外源SOD-3’UTR降低了A2780细胞内源性SOD蛋白的表达，并且外源SOD1-3’UTR能降低内源性SOD2蛋白的表达；逐步的对SOD1-3’UTR作片段的截断后结果显示荧光素酶活性也逐渐降低；miR-224, miR-377结合位点的缺失导致荧光素酶活性显著下降，而miR-621结合位点的缺失未见明显变化；miR-377或miR-621的拟似物或抑制剂并没有显著影响内源性SOD1的表达，也没有影响SOD1-3’UTR所介导的荧光素酶活性；任一个ARE的缺失均能降低荧光素酶活性，以ARE6的作用最强；但当载体仅包含2个或4个ARE本身的序列时荧光素酶活性几乎测不出。ARE6的缺失逆转了X射线诱导的SOD1-Promoter-SOD1-3’UTR所介导的荧光素酶活性的增加。（3）AUF1的下调明显降低了SOD1蛋白的表达水平，而HuR的下调对SOD1蛋白表达的影响不大，并与荧光素酶活性的结果一致；AUF1的高表达增加了这两种细胞系内的SOD1-3’UTR介导的荧光素酶活性；RT-PCR法仅能从AUF1沉淀物中检测到SOD1-3’UTR的表达；93例胰腺癌组织标本检测的结果显示AUF1和SOD1的蛋白表达明显相关。结论：（1）SOD1-3’UTR与肿瘤细胞基础和应激状态下SOD1的表达相关（2）SOD1-3’UTR能增加肿瘤细胞SOD1mRNA的稳定性，并与ARE特别是ARE6相关，与所检测的miRNA结合位点相关的可能性不大。（3）AUF1通过与SOD1-3’UTR结合影响肿瘤SOD1表达是SOD1转录后调控的机制之一，并能影响肿瘤细胞基础的和放射诱导的ROS水平。更多还原

【Abstract】 Purpose:Known strategies of reversing resistance to radiotherapy in cancer werecommonly unsatisfied. It has been reported that a high expression of SOD1is related tochemo-radiotherapy resistance in cancer, the mechanism underlying an increasedexpression of SOD1are unknown. It is well known that the precise regulation of geneexpression is relative to post-transcriptional regulation through3’UTR. But little wasknown about SOD1post-transcriptional regulation in cancer, which was investigated inthis project, as well as the impact on radiation induced effect in cancer. These studieswill possibly provide new insights for strategies targeted SOD1in increasing cancerradiosensitivity.Methods：The ovarian cancer cell line A280and the pancreatic cancer cell linePANC1were the main cell models in this project.(1) The fragment of SOD-3’UTR wasinserted into pGL3-Promoter vector.The influences of3’UTR on the expression ofSOD1/2in cancer cell were explored,as well as after treatment by the hydrogenperoxide and X-ray radiaiton.Meanwhile,the SOD1mRNA and protein level weremeasured before and after treatment by hydrogen peroxide.(2)SOD1-3’UTR mediatedrelative luciferase activity was conpared between the two cancer cell line,and therelative firely luciferase mRNA level was measured by RT-PCR.The influence of foredexpression of SOD-3’UTR on endogenous SOD protein expression was explored bywesturnblot assay.Using pGL3-Promoter-SOD1-3’UTR sense vector as a template,thecloning of serially deleted SOD-13’UTR reporter vectors and that including differentdeletions of AREs or miRNA binding site was finished.By using these vectors andmiRNA mimics or inhibitors,the impact of different RNA elements(AREs and miRNAbinding site)on SOD1-3’UTR mediated relative luciferase acitivty or SOD1proteinexpression were examined.The SOD1-3’UTR fragment was also inserted intopGL3-SOD1-Promoter reporter vector,and the vector including different deletions of AREs were cloned again,then the impact of AREs on SOD1-Promoter-SOD1-3’UTRsense mediated relative luciferase activity with or without X-ray radiation wereinvestigated.(3)The SOD1protein expression and SOD1-3’UTR mediated luciferaseactivity was measured by westurnblot assay after forced expression or knockdown ofAUF1and HuR in cancer cell. The influence of AUF1knockdown on radiation inducedROS was measured by confocal microscope.In further,we immunoprecipitated AUF1from cell lysates prepared from PANC1cells and analyzed SOD1-3’UTR expression inthe precipitates.The expression of AUF1and SOD1in pancreatic cancer tissue sampleswere detected by immunohistochemistry.In the preliminary studies, we first found an increased stability of SOD1mRNA bySOD1-3’UTR in cancer cells, six AREs in the SOD13’UTR which interacted with theRNA-binding protein, and several miRNAs binding sites. The expression level of SOD1was significantly correlated to the RNA-binding protein AUF1expression in cancertissues examined. Furthermore, we also found knock-down of AUF1significantlyincreased radiation-induced effect in cancer cells.Results：（1）To investigate whether SOD1/23’UTR influences SOD1/2expression,A2780cell line was transfected with the pGL3-promoter-SOD1/23’UTR sense orpGL3-promoter-SOD1/23’UTR antisense vector, respectively. Inclusion of the SOD1/23’UTR dramatically enhanced the reporter gene activity by20fold in A2780cells,respectively. Hydrogen peroxide at100μM significantly increased the SOD1/2mediatedluciferase activtiy in A2780,the similar results was got by treatment with10Gy X-rayradiation.Both were consistent with the increase of SOD1mRNA and protein level withtreatment of hydrogen peroxide.(2) A2780and PANC1cell lines were transfected withthe pGL3-Promoter vector, pGL3-promoter-SOD13’UTR sense orpGL3-promoter-SOD13’UTR antisense vector, respectively. Inclusion of the SOD13’UTR dramatically enhanced the reporter gene activity by109-,219-fold in PANC1,A2780cells, respectively. Inclusion of the SOD13’UTR remarkably increased firelyluciferase mRNA levels, indicating that the high luciferase activity is attributed toincreased mRNA stability.Transfection of SOD13’UTR sense vector, which sharesidentical sequence with endogenous SOD13’UTR, decreased endogenous SOD1expression in a concentration-dependent manner, suggesting that the exogenousintroduced SOD13’UTR competes with endogenous SOD13’UTR for the trans-acting factors that are required for maintaining SOD1gene expression. Interestingly, when theSOD13’UTR construct was introduced into A2780cells, endogenous SOD2proteinexpression was also significantly down-regulated, indicating the two SODs likely sharea common post-transcriptional mechanism. Truncated deletion of SOD13’UTRgradually decreased luciferase activity and deletion of any part of SOD13’UTRdramatically affects the reporter activity. Deletion of the binding sites for miR-224andmiR-377, but not miR-621in SOD13’UTR, significantly down-regulated luciferasereporter activity, which was not in accordance with the general belief that miRNAsnegatively regulate gene expression. In addition, application of miR-377or miR-621mimics/inhibitors did not affect endogenous SOD1expression or the SOD13’UTR-driven reporter gene activity, indicating that the SOD13’UTR’s function insustaining SOD1expression is less likely attributed to these miRNA species.Deletion ofany of the four AREs remarkably reduces luciferase activity with the ARE6being themost important one, indicating that SOD1expression is significantly sustained by theAREs in its3’UTR. However, when the vectors, containing only two or four of theputative AREs, were transfected to A2780or PANC1cells, luciferase activity wasalmost undetectable, further suggesting that the importance of the AREs is related totheir contribution to the integrity of the3’UTR, which is essential for sustaining SOD1expression. Deletion of ARE6reversed the increased SOD1-Promoter-SOD1-3’ UTR-driven luciferase activity induced by X-ray radiation in A2780.(3)The knockdown ofAUF1and HuR was confirmed by Western blot analysis. We found that knockdown ofAUF1but not HuR significantly reduces SOD1protein expression levels.Co-transfection of the SOD13’UTR reporter constructs with the AUF1siRNAsignificantly down-regulated the SOD13’UTR-driven reporter gene activity. In contrast,the reporter gene activity in cells transfected with control vector or the SOD13’UTRantisense vector was unaffected by AUF1knockdown. Forced expression of AUF1increases the SOD13’UTR-driven reporter gene activity in a concentration-dependentmanner in both cell lines. We then immunoprecipitated AUF1using cell lysatesprepared from PANC1cells and analyzed SOD13’UTR expression in the precipitates.In contrast to IgG-precipitated sample, expression of SOD13’UTR was only detectedby RT-PCR in AUF1-precipitated sample, indicating a direct interaction of AUF1withSOD13’UTR in these cells. Transfection of shRNA-AUF1to PANC1cells was foundto promote basal ROS generation compared with that of shRNA-NC transfection. Furthermore, transfection of shRNA-AUF1significantly enhanced radiation-inducedROS levels. Similar results were obtained in A2780cells with or without irradiation.Immunohistochemistry analysis showed that, in all93pancreatic cancer tissuesexamined, expression of SOD1is significantly correlated to AUF1expression levels(rho=0.558, P <0.001).Conclusion:(1)SOD1-3’UTR maintains the SOD1expression of cancer cell inbasic and oxidative conditions.（2）SOD1-3’UTR increased SOD1mRNA stability incancer cells,which was attributed to AREs(ARE6) more than miRNA binding site in3’UTR.(3)Post transcriptional regulation of SOD1by3’UTR was attributed to theinteract between AUF1and SOD1-3’UTR,which in further modulated the cellular andradiation induced ROS in cancer cells.更多还原