【作者】 朱琳；

【导师】 周光明； 胡文涛；

【作者基本信息】 苏州大学 ， 放射医学， 2020， 硕士

【摘要】 目的:因为太空资源的利用显现出巨大的经济价值和军事价值,航天活动过程中受到的各种制约因素也受到人们的广泛关注,尤其是空间辐射。空间辐射的一个重要特征是低剂量、低剂量率的电离辐射。低剂量辐射诱导的旁观者效应是其介导正常组织细胞恶性转化的重要原因之一。辐射诱导的旁效应指受照射细胞可分泌相关物质作用于未直接受照射细胞,使未直接受照射细胞出现一些生物学反应。受照细胞分泌多种细胞因子作用于其周围细胞产生旁效应,转化生长因子β(TGF-β)便是广受关注的旁效应信号分子之一。前期研究中,我们发现了 TGF-β诱导永生化人肺上皮细胞(BEAS-2B)中LNC HPN-AS1上调,生物信息学分析发现LNC HPN-AS1及其靶基因受到转录因子FOS的调控。FOS作为一个重要的恶性转化因子,与正常细胞的恶性转化过程息息相关,我们推测LNC HPN-AS1可能参与TGF-β诱导正常细胞的恶性转化过程。在本课题中,我们主要着眼于TGF-β诱导永生化人肺上皮细胞BEAS-2B中LNC HPN-AS1表达上调后,细胞发生恶性转化的一系列生物过程。对lncRNA功能及其相关机制的研究,也为空间辐射致癌机理研究和空间辐射防护提供崭新的思路。方法:(1)在肺上皮细胞中通过慢病毒感染敲低、过表达LNC HPN-AS1后,检测永生化人肺上皮细胞BEAS-2B的增殖、迁移和侵袭能力的改变。从而确定LNC HPN-AS1在诱导BEAS-2B细胞发生恶性转化过程中的作用。(2)利用RT-qPCR技术检测加入miR-98-5p mimic后LNC HPN-AS1表达水平的变化,同时检测分别转染miR-98-5p mimic和miR-98-5p inhibitor后TGFBR1表达的变化。之后利用荧光素酶报告载体技术验证LNC HPN-AS1与miR-98-5p之间、miR-98-5p与TGFBR1的相互作用关系。随后在BEAS-2B细胞过表达LNC HPN-AS1细胞系中过表达miR-98-5p,或者加入TGFBR1的抑制剂处理,检测其增殖能力、迁移水平以及侵袭能力的改变,最后分析不同处理对细胞EMT程度的影响。(3)经过碳离子和氦离子照射处理后,利用ELISA技术,检测受照培养基内TGF-β1的浓度。利用RT-qPCR技术,分析经由重离子照射后的条件培养基处理后LNC HPN-AS1、miR-98-5p、TGFBR1表达量的变化。最后在BEAS-2B细胞中过表达LNC HPN-AS1,观察小鼠皮下移植瘤的生长状况。结果:(1)利用转录组测序技术,以永生化人肺上皮细胞BEAS-2B作为研究对象,测定并筛选出TGF-β1处理后差异性表达明显的lncRNA。发现LNC HPN-AS1表达量显著升高(p<0.001),利用RT-qPCR技术进一步验证了 TGF-β1处理增加了LNCHPN-AS1的表达量(p<0.05)。慢病毒感染敲低LNCHPN-AS1的表达,利用RT-qPCR技术验证了 LNC HPN-AS1表达量明显减少(p<0.001)。通过CCK8法检测敲低LNC HPN-AS1表达后细胞增殖能力的改变,发现不同时间点的细胞增殖速度均降低(24 h,p<0.05;48 h,p<0.01;72 h,p<0.05);敲低 LNC HPN-AS1 细胞的划痕愈合速度降低(p<0.01),细胞的侵袭能力也有所下降(p<0.001)。同时利用慢病毒感染过表达LNC HPN-AS1,利用RT-qPCR技术验证了 LNC HPN-AS1表达量明显增加(p<0.001)。通过细胞增殖实验发现,过表达LNC HPN-AS1后,不同时间点的细胞增殖速度明显加快(24h,p<0.01;48h,p<0.05;72h,p<0.05);划痕实验发现,过表达LNC HPN-AS1后,划痕的愈合速度增加(p<0.01)。侵袭实验发现,过表达LNC HPN-AS1后,细胞的侵袭能力也大大增强(p<0.001)。最后利用RT-qPCR技术检测了敲低LNC HPN-AS1、过表达LNC HPN-AS1细胞系中EMT标志物mRNA表达水平,结果显示过表达LNC HPN-AS1细胞系中上皮细胞标志物E-cadherin表达降低(p<0.001),而间充质细胞标志物Fibronectin1、Vimentin和 N-cadherin 的表达上调(p<0.001;p<0.01;p<0.001);敲低 LNC HPN-AS1 细胞系中的 E-cadherin 表达微微上调(p<0.05),Fibronectin1、Vimentin 和 N-cadherin的表达均呈不同程度的降低(p<0.01;p<0.05;p<0.05)。以上结果表明LNC HPN-AS1可以促进BEAS-2B细胞的增殖,迁移,侵袭和EMT发生。(2)首先利用RT-qPCR技术检测加入miR-98-5p mimic后LNC HPN-AS1和TGFBR1表达水平的变化,发现过表达miR-98-5p后LNC HPN-AS1和TGFBR1的表达水平降低(p<0.001;p<0.01),随后我们利用荧光素酶报告载体技术验证LNC HPN-AS1与miR-98-5p之间、miR-98-5p与TGFBR1的靶向关系。结果发现转染miR-98-5p mimic后生物发光强度均明显降低(p<0.001;p<0.001)。我们又通过TargetScan预测的结合位点构建结合位点突变的荧光素酶报告载体,发现在共转染LNC HPN-AS1结合位点突变体和miR-98-5p mimic,TGFBR1结合位点突变体和miR-98-5p mimic后荧光素酶活性并没有明显的改变。以上结果表示,LNC HPN-AS1和miR-98-5p相互作用;TGFBR1是miR-98-5p的直接靶标分子。根据之前的结果,对LNC HPN-AS1通过与miR-98-5p互作,并且miR-98-5p靶向TGFBR1来影响BEAS-2B细胞的恶性转化进行验证,结果发现,转染miR-98-5p mimic后,细胞的增殖速度下降(24 h,p<0.05;48 h,p<0.05;72 h,p<0.05);划痕愈合减缓(p<0.01);侵袭能力也有降低(p<0.001);而且过表达LNC HPN-AS1后促进细胞内EMT转化程度也被部分逆转:转染miR-98-5p mimic后细胞中E-cadherin表达升高(p<0.01),Fibronectin1、Vimentin 和 N-cadherin 的表达下调(p<0.001;p<0.01;p<0.001)。同样的,在过表达LNCHPN-AS1细胞系中加入TGFBR1的抑制剂后,细胞的增殖、迁移、侵袭能力也均是减弱的(24h,p<0.01;48 h,p<0.05;72h,p<0.05;p<0.01;p<0.001),EMT转化也被部分逆转。软琼脂克隆实验结果显示,进行转染miR-98-5pmimic或者TGFBR1抑制剂处理后,细胞的恶性生长程度被部分抑制。以上结果说明,TGF-β1诱导的LNC HPN-AS1通过竞争性结合miR-98-5p,上调miR-98-5p靶向的TGFBR1,从而形成对TGF-β1通路的放大作用来促进肺正常上皮细胞恶性转化的过程。(3)我们以上的实验都是建立在X射线照射诱导细胞分泌TGF-β1产生旁效应而导致的一系列生物过程。众所周知空间辐射的主要组成是重离子,所以我们进一步利用重离子照射验证了我们的实验模型。首先,我们利用ELISA实验检测了不同剂量(0.5 Gy,2 Gy)碳离子和氦离子照射2 h后受照培养基中的TGF-β1的浓度,发现 TGF-β1 的浓度都明显升高(0.5 Gy,C:p<0.001;0.5 Gy,He:p<0.01;2 Gy,C:p<0.001;2 Gy,He:p<0.01)。这与X射线照射处理的结果是一致的。然后,我们选用0.5 Gy碳离子照射的培养基(CM1:条件培养基1)、0.5 Gy氦离子照射的培养基(CM2:条件培养基2)以及TGF-β1处理正常的BEAS-2B细胞,利用RT-qPCR技术检测了 LNC HPN-AS1、miR-98-5p、TGFBR1的表达量,结果发现 LNC HPN-AS1 的表达均明显提高(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001);miR-98-5p 表达降低(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001);TGFBR1 表达升高(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001)。当在CM里加入TGF-β1抑制剂后LNC HPN-AS1、miR-98-5p、TGFBR1表达量下降,结果说明了重离子辐射诱导的旁观者效应介导的LNC HPN-AS1-miR-98-5p-TGFBR1通路的表达变化是由TGF-β1引起的。以上与X射线照射处理的结果也是一致的,说明我们的实验模型是适用于空间辐射的。最后,根据之前的实验结果选取LNC HPN-AS1-NC、LNC HPN-AS1-OE两组BEAS-2B细胞,进行免疫缺陷小鼠(NOD/SCID)的皮下移植瘤实验并进行成瘤率分析。结果显示LNC HPN-AS1-OE组细胞具有成瘤能力,且病理学结果显示该组细胞形成的肿瘤为肺腺癌,对照组并无致瘤能力。结论:(1)TGF-β1可以促进LNC HPN-AS1表达。细胞增殖、划痕以及侵袭实验结果表明LNC HPN-AS1是一个原癌基因,可以促进肺正常上皮细胞的增殖、迁移、侵袭和EMT发生;(2)miR-98-5p可抑制癌细胞恶性程度,而TGFBR1则是肿瘤增殖和转移的关键刺激因子;LNC HPN-AS1以ceRNA方式抑制miR-98-5p表达,从而激活miR-98-5p下游靶向的TGFBR1来促进肺上皮细胞恶性转化的过程。(3)LNC HPN-AS1-miR-98-5p-TGFBR1促进肺上皮细胞恶性转化的过程可能是低剂量空间辐射致癌的原因。过表达LNC HPN-AS1细胞系在小鼠体内实验被证实具有成瘤能力。更多还原

【Abstract】 Objective:With the increasingly important military and economic value of space utilization,people paying more attention to space activities.However,various space environmental factors,especially space radiation,poses the most serious threat to the astronauts.An important feature of space radiation is low dose/low dose rate ionizing radiation.The bystander effects induced by low dose radiation is one of the most important reasons for the malignant transformation of normal tissue cells.Radiation-induced bystander effect(RIBE)refers to the fact that the irradiated cells can secrete related factors to act on the non-irradiated cells,then resulting in some biological reactions of the non-irradiated cells.Many factors can be secreted by the irradiated cells to act on their surrounding cells to produce bystander effects,among which TGF-β is the most important.In previous studies,we found LNC HPN-AS1,upregulated by TGF-β1 in BEAS-2B cells.Bioinformatics analysis showed that LNC HPN-AS1 and its target genes were regulated by FOS.As an important malignant transformation factor,FOS plays an important role in the malignant transformation of normal cells.We speculated that LNC HPN-AS1 may be involved in the malignant transformation of normal cells induced by TGF-β.In this study,we focused on the biological process mediated by LNC HPN-AS1,which was upregulated by TGF-β and involved in malignant transformation of normal human lung epithelial cells.We believe the study on lncRNA function and its related mechanism will provide a new idea for the study of space radiation-induced tumorigenesis as well as the the protection of astronauts from space radiation.METHODS:(1)The proliferation,migration and invasion of BEAS-2B cells were detected to determine the role of LNC HPN-AS1 in the induction of malignant transformation of normal lung epithelial cells.(2)LNC HPN-AS1 expression was detected after adding miR-98-5p mimic,TGFBR1 expression was detected after adding miR-98-5p mimic or miR-98-5p inhibitor.Luciferase reporter assay was used to verify the targeting relationship between LNC HPN-AS1 and miR-98-5p,miR-98-5p and TGFBR1.Then we overexpressed miR-98-5p in LNC HPN-AS1 overexpression cell line,or treated the cells with TGFBR1 inhibitor,and observed the change of cell proliferation,migration and invasion ability,at last real time quantitative PCR were used to detect the expression of EMT markers.(3)After the treatment of carbon and helium ions,ELISA was used to detect the concentration of TGF-β1.The expression of LNC HPN-AS1,miR-98-5p and TGFBR1 was analyzed by RT-qPCR.Finally,LNC HPN-AS1 overexpression cells were injected into NOD/SCID mice to observe the formation of subcutaneous transplanted tumors.RESULTS:(1)Using transcriptomic sequencing technology to screen the lncRNA which are differentially expressed after TGF-β 1 treatment.LNC HPN-AS1 expression was significantly increased(p<0.001),RT-qPCR was used to further verify that(p<0.05).Then we detected the change of cell proliferation ability after LNC HPN-AS1 knockdown,the cell proliferation rate decreased at different time points(24 h,p<0.05;48 h,p<0.01;72 h,p<0.05).The wound healing rate also decreased after knockdown of LNC HPN-AS1((p<0.01),so was the invasion ability(p<0.001).At the same time,we overexpressed the LNC HPN-AS1 by lentivirus transfectionand found its expression was obviously incresead(p<0.001).The proliferation rate,migration and invasion ability all increased significantly after overexpression of LNC HPN-AS1(24 h,p<0.01;48 h,p<0.05;72 h,p<0.05;p<0.01;p<0.001).Finally,the expression of EMT markers in the LNC HPN-AS1 overexpression cell line and LNC HPN-AS1 knockdown cell line was detected by RT-qPCR.E-cadherin expression was decreased(p<0.001),while the expressions of Fibronectinl,Vimentin and N-cadherin were increased(P<0.001;P<0.01;P<0.001)in the LNC HPN-AS1 overexpression cell line.In LNC HPN-AS1 knockdown cell line,E-cadherin was slightly increased(p<0.05),the expressions of Fibronectin 1,Vimentin and N-cadherin were decreased(p<0.01;p<0.05;p<0.05).These results indicate that LNC HPN-AS1 can promote the malignant growth of lung epithelial cells.(2)First,RT-qPCR was used to detect LNC HPN-AS1 and TGFBR1 expression after transfection of miR-98-5p mimic.And the LNC HPN-AS1 and TGFBR1 expression decreased after overexpression of miR-98-5p(p<0.001;p<0.01),Then we used luciferase reporter assay to verify the targeting relationship between LNC HPN-AS1 and miR-98-5p,miR-98-5p and TGFBR1.The results showed that the bioluminescence intensity was significantly decreased after miR-98-5p mimics was transfected(p<0.001;p<0.001).We also constructed a mutant of the luciferase reporter vector for the binding site predicted by TargetScan,and found that there was no significant change in luciferase expression after co-transfection of LNC HPN-AS1 binding site mutants and miR-98-5p mimic,as well as TGFBR1 binding site mutants and miR-98-5p mimic.The above results show that LNC HPN-AS1 interacts with miR-98-5p directly and TGFBR1 is the direct target molecule of miR-98-5p.According to the previous results,LNC HPN-AS1 interacts with miR-98-5p,and miR-98-5p targets TGFBR1 to affect the malignant transformation of lung epithelial cells.The results show that after overexpression of LNC HPN-AS1,the proliferation,migration and invasion of lung epithelial cells are significantly enhanced.However,transfection of miR-98-5p mimic abolished the increased proliferation rate of the cells(24 h,p<0.05;48 h,p<0.05;72 h,p<0.05),the healing of scratches slowed down(p<0.01),and the invasion ability also decreased(p<0.01).In addition,miR-98-5p mimic treatment partly reversed the EMT process which has been promoted after overexpressing LNC HPN-AS1:the expression of E-cadherin in the epithelial cells increased(p<0.001),while Fibronectin1,Vimentin and N-cadherin in the mesenchymal cells decreased(p<0.001;p<0.01;p<0.001).Similarly,the proliferation,migration and invasion were inhibited by treating LNC HPN-AS1 overexpression cell lines with TGFBR1 inhibitor.the EMT process was also partly reversed.The results of soft agar cloning also showed that after miR-98-5p mimic and TGFBR1 inhibitors were administrated,the malignant growth of the cells was partially inhibited.These results suggested that LNC HPN-AS1 can promote malignant transformation of lung epithelial cells by reducing miR-98-5p expression and increasing TGFBR1,which is targeted by miR-98-5p.(3)Our above experiments are all based on a series of biological processes caused by X-ray induced bystander effect.As we all know,the most important component of space radiation is heavy ion.So in order to verify whether our experimental model is suitable for space radiation,we carried out the following experiments.First of all,we measured the concentration of TGF-β1 in the medium of BEAS-2B cells exposed to different doses(0.5 Gy,2 Gy)of carbon ions and helium ions irradiation,and found that the concentration of TGF-β1 increased significantly with heavy ions irradiation(0.5 Gy,C:p<0.001;0.5 Gy,He:p<0.01;2 Gy,C:p<0.001;2 Gy,He:p<0.01).This is consistent with the result obtained with X-ray treatment.Then,we chose the medium conditioned with 0.5 Gy carbon ion irradiation(CM1:conditioned medium 1),and the medium conditioned with 0.5 Gy helium ion irradiation(CM2:conditioned medium 2)and TGF-β1 treament(concentration:2.5 ng/mL)to culture the cells and detected the expressions of LNC HPN-AS1,miR-98-5p and TGFBR1 by RT-qPCR.The results showed that the expression of LNC HPN-AS1 increased significantly(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001);the expression of miR-98-5p decreased(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001);the expression of TGFBR1 increased(TGF-β1:p<0.001;CM1:p<0.001;CM2:p<0.001).At the same time,the change of LNC HPN-AS1,miR-98-5p and TGFBR1 expression disappeared after TGF-β neutralizing antibody was added into CM1 and CM2,which confirmed that it was a series of biological processes induced by TGF-β1.The above results indicated that our experimental model is suitable for space radiation research.Finally,according to the previous experimental results,LNC HPN-AS1-NC and LNC HPN-AS1-OE cells were selected to carry out the subcutaneous transplantation experiment with immunodeficient mice(NOD/SCID)and the tumor formation rate was analyzed.The results of tumorigenesis experiment in NOD/SCID mice showed that the cells with LNC HPN-AS1 overexpression had tumorigenic ability,and the pathological results showed it was lung adenocarcinoma,while the cells in control group formed no tumors.CONCLUSIONS:(1)TGF-β1 can promote the expression of LNC HPN-AS1.The enhanced cell proliferation,scratch and invasion ability caused by LNC HPN-AS1 overexpression showed that LNC HPN-AS1 is an oncogene,which can promote the malignant growth of lung epithelial cells;(2)miR-98-5p can suppress the malignant degree of cancer cells,while TGFBR1 is the key mediator of tumor proliferation and metastasis induced by TGF-β1;LNC HPN-AS1 can inhibit the expression of miR-98-5p,thus activating the downstream targeted TGFBR1 of miR-98-5p to promote the malignant transformation of lung epithelial cells.(3)The model of LNC HPN-AS1-miR-98-5p-TGFBR1 promoting malignant transformation of lung epithelial cells is also verified with heavy ion radiation.The LNC HPN-AS1 overexpression cell lines can form tumors in mice.更多还原