【作者】 刘丹丹；

【导师】 李连宏；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2019， 硕士

【摘要】 研究背景肾细胞癌(Renal Cell Carcinoma,RCC)是一种起源于肾实质泌尿小管上皮系统的恶性肿瘤,新近研究报道,在欧美地区恶性肿瘤发病率中,RCC在男性中发病率排第六,女性中为第十,RCC已成为威胁人类健康的又一高风险性的恶性肿瘤。初期RCC通常无明显症状,且RCC常以转移快,复发性高为特征,约25%的患者在确诊时已发生远处转移。晚期RCC对于放疗、化疗等临床普遍的治疗手段表现出抵抗效应,因此,探讨其有关生物学特征及其发生机制,寻找有效的药物对于RCC治疗,降低患者死亡率尤为重要。小白菊内酯(Parthenolide,PTL)为天然存在的倍半萜内酯,从草本植物小白菊胚芽中纯化而来,可用于治疗发热、痉挛、眩晕等,另外还具有止痛及抗炎等作用。近年来,PTL已被证明具有调节免疫,抗氧化、抗肿瘤,尤其是杀伤肿瘤干细胞的作用。目前,PTL对肿瘤的抑制作用已在乳腺癌,肺癌和胰腺癌等肿瘤中得到证实。有证据表明,PTL的这些生物学特性归因于其对NF-kB信号通路的抑制,因此,PTL被认为是NF-kB信号通路的天然抑制剂。同时,另有研究表明NF-kB信号通路可与多种信号通路发生相互作用,包括PI3K-Akt信号通路等,PI3K-Akt信号通路与肿瘤细胞的发生发展密切相关,可调控肿瘤细胞增殖、凋亡、迁移、侵袭、上皮-间质转化等多种生物学特征。上皮-间质转化(Epithelial-Mesenchymal Transition,EMT)作为哺乳动物胚胎发育的重要生理过程,也对促进恶性肿瘤发生发展起到重要作用。在EMT过程中,原本呈现上皮样表型的细胞失去其细胞极性,表现出间充质样表型细胞的特点,细胞间黏附力减弱,能够在细胞基质自由移动,同时伴有上皮标志物E-钙粘蛋白(E-cadherin)和间质标志物N-钙粘蛋白(N-cadherin)以及波形蛋白(Vimentin)表达水平的显著变化。另外,EMT过程还涉及多种信号通路和转录因子的参与,其中之一是Snail,作为E-cadherin的阻遏物可触发EMT过程,通过与E-cadherin启动子区域的E-box盒结合,下调E-cadherin表达,诱导EMT发生。近年来研究证实PI3K-Akt信号通路在肿瘤细胞的EMT过程中起重要作用,PI3K-Akt信号通路关键分子Akt可调节Snail表达从而调节肿瘤细胞的EMT过程。同时也有报道称Snail是GSK-3β的下游靶标,GSK-3β可调节Snail的核定位影响肿瘤细胞的EMT过程。研究证实,发生EMT的乳腺癌细胞除获得抗凋亡和侵袭能力外,还产生了自我更新能力,表现出干细胞特点,这也为探究肿瘤发生发展的机制及治疗提供新的方向。越来越多的研究表明识别肿瘤的生物学特征及其发生机制,开发针对性的治疗手段是治疗肿瘤的重要步骤。据报道,PTL可通过抑制NF-kB信号通路抑制肿瘤细胞的增殖、迁移与侵袭、促进凋亡,此外,PTL还可通过PI3K-Akt信号通路调节宫颈癌细胞的凋亡和自噬,PI3K-Akt又可调节EMT的驱动因子Snail,且PI3K-Akt信号通路又可与NF-kB信号通路发生相互作用,因此,PI3K-Akt信号通路可能是PTL的潜在作用通路并极大可能影响肿瘤的EMT过程。当前,PTL是否能够抑制肿瘤细胞的EMT过程以及肿瘤细胞干性的相关研究少见,尤其在RCC中。因此,本研究以人RCC细胞系(786-O和ACHN)为对象,探讨PTL对人RCC细胞EMT过程的影响,同时,验证并探讨PTL对人RCC细胞增殖、RCC细胞干性等生物学特征的影响,并进一步探讨PTL影响人RCC细胞EMT过程可能的作用机制。研究方法CCK-8法检测PTL对RCC细胞(786-O和ACHN)生存活力的影响,同时根据IC50值设置实验分组:对照组(PTL未处理组)、低浓度实验组(4μm/L PTL处理组)、高浓度实验组(8μm/L PTL处理组)。应用划痕实验及Transwell小室实验检测PTL对两种RCC细胞迁移、侵袭能力的影响,并在蛋白水平上通过Western Blot实验观察基质金属蛋白酶MMP2、MMP9的表达变化。应用TUNEL法检测PTL对两种RCC细胞凋亡的影响,并在蛋白水平上通过Western Blot实验观察凋亡相关蛋白BAX、Bcl2及Caspase3的表达变化。应用免疫荧光实验检测PTL对两种RCC细胞EMT过程的抑制作用,同样,通过Western Blot实验检测EMT相关蛋白E-cadherin、N-cadherin、Vimentin及EMT驱动因子Snail的表达变化,并在基因水平通过实时荧光定量PCR对个别指标mRNA表达情况进一步验证。应用干细胞微成球实验检测PTL对两种RCC细胞干性的影响,也在蛋白水平通过Western Blot实验观察干性标志物ALDH1、CD133、Sox2及Oct4的表达变化,最终,我们探讨PTL对RCC上皮-间质转化的可能作用机制,通过Western Blot实验观察PI3K-Akt信号通路关键分子PI3K、Akt、p-PI3K、p-Akt的表达变化。实验结果PTL处理两种RCC细胞,一定时间后,CCK-8实验结果显示,PTL可抑制RCC细胞增殖,且随着药物浓度增加,抑制效率增加。集落形成实验直观上验证显示,实验组较对照组集落形成数量减少,说明PTL可剂量依赖性抑制RCC细胞增殖。划痕实验结果显示,划痕愈合比例实验组较对照组明显减少,同时Transwell小室实验中,实验组细胞穿膜数量较对照组减少,Western Blot实验检测到转移相关蛋白基质金属蛋白酶MMP2、MMP9实验组较对照组表达水平降低,说明PTL可剂量依赖性抑制RCC细胞迁移与侵袭。TUNEL实验显示,实验组较对照组,荧光亮染细胞增多,即被标记的DNA断裂的细胞多,DAPI染色后,细胞核具有变形现象,Western Blot实验检测凋亡相关蛋白,实验组较对照组BAX、Cleaved caspase3表达水平升高,Bcl2表达水平降低,说明PTL可促进RCC细胞凋亡。免疫荧光实验显示,实验组较对照组EMT相关蛋白E-cadherin表达水平升高,可见荧光亮染点增加,N-cadherin、Vimentin及转录因子Snail表达水平表达降低,可见荧光亮染点减少,Western Blot实验中,发现实验组较对照组,EMT相关蛋白E-cadherin表达水平升高,N-cadherin、波形蛋白Vimentin及转录因子Snail表达水平表达降低。实时荧光定量PCR结果显示实验组较对照组,E-cadherin的mRNA表达水平升高,Snail的mRNA表达水平降低,说明PTL可抑制RCC细胞EMT过程。干细胞微成球实验结果显示,实验组较对照组干细胞成球能力弱,球体状态差,高浓度组抑制更明显,细胞膜呈破裂溶解趋势。Western Blot实验同时检测到干性相关标志物:ALDH1、Sox2、Oct4及RCC特异性干性标志物CD133实验组较对照组均低表达,说明PTL可抑制RCC细胞干性。此外,研究中Western Blot实验观察到PI3-Akt信号通路关键分子PI3K、p-PI3K、Akt、p-Akt实验组与对照组相比表达水平均降低。结论1.PTL可抑制RCC细胞增殖。2.PTL可抑制RCC细胞迁移、侵袭。3.PTL可促进RCC细胞凋亡。4.PTL可抑制RCC细胞EMT过程。5.PTL可抑制RCC细胞干性。6.PTL可能通过PI3K-Akt信号通路抑制RCC细胞的EMT过程。更多还原

【Abstract】 BackgroundRenal cell carcinoma(RCC)is a common urinary malignancy which originates from the renal tubular epithelium.Worldwide,RCC is the sixth most common cancer in men,and the tenth in women,which seriously threaten the human health.RCC is often characterized by rapid metastasis and high recurrence.Moreover,RCC often exerts resistance to radiotherapy and chemotherapy.Therefore,find out novel,potent anti-tumor agents would be beneficial for RCC treatment.Parthenolide(PTL)is a natural lactone compound purified from the herbal plant Chryanthemum Parthenium,which was used to treat fever,spasm,analgesia,dizziness and inflammation.In recent years,PTL has been shown to possess anti-inflammatory and anti-oxidant effects,it has been also found to exert anti-tumor,especially anti-cancer stem cell effects in many malignant diseases,including breast,lung and pancreatic cancers.Due to its strong inhibition of the nuclear factor NF-kB,PTLis considered as a natural inhibitor of the NF-kB signaling pathway.In the other hand,studies have shown that NF-kB signaling pathway can interact with a variety of signaling pathways,including PI3K-Akt signaling pathway.PI3K-Akt signaling pathway is closely related to the carcinogenesis and tumor progression.Epithelial-Mesenchymal Transition(EMT)is an important process in the development of mammalian embryos,yet it also plays a key role in tumor invasion and metastasis.During EMT process,epithelial-like phenotype cells lose polarity,obtain mobility,can cross the extracellular matrix like mesenchymal cells,and transform into a mesenchymal-like phenotype,accompanied by the alteration of expression levels of epithelial markers E-cadherin and the mesenchyal marker N-cadherin and vimentin.In addition,multiple signaling pathways and transcription factors are involved in the EMT process,for example,Snail,an E-cadherin repressor and EMT inducer,it down-regulates E-cadherin expression by binding to the E-box of the E-cadherin promoter region.In recent years,studies have confirmed that the PI3K-Akt signaling pathway plays an important role in the EMT process of tumor cells.The key molecule Akt of PI3K-Akt signaling pathway regulates Snail and regulates the EMT process.It was also reported that Snail is a downstream target of GSK-3.GSK-3 regulates the nuclear localization of Snail and thus affects the EMT process of tumor cells.At the same time,Weinberg et al have confirmed that in addition to anti-apoptotic and escape ability,Breast cancer cells underwent EMT also exert Stemness.EMT not only causes cancer cells to spread from the primary locus,but also gives them self-renewal ability.This provides us new ideas for further research on the mechanism of tumor progression.Accumulating evidence has shown that identifying the biological characteristics of tumors and their mechanisms of development,and the development of targeted therapies is an important step in the treatment of tumors.It has been reported that parthenolide can inhibit the proliferation,metastasis and invasion of tumor cells and promote apoptosis by inhibiting NF-kB signaling pathway.In addition,parthenolide can regulate the apotosis and autophagy of cervical cancer cells through modulating PI3K-Akt signaling pathway.PI3K-Akt also regulates the EMT driving factor,Snail,and the PI3K-Akt signaling pathway interacts with the NF-kB signaling pathway,thus parthenolide may exert anti-tumor activities at least partly through regulating PI3K-Akt signaling pathway.Currently,studies on the effects on tumor EMT and stemness of parhenolide is rare.Our present study employed human RCC cell lines(786-O and ACHN)as the research object to investigate the effects of parthenolide on EMT process of human RCC cells,and explored the impact of parthenolide on the proliferation and stemness RCC cells.And the possible molecular mechanism of tumor-suppressive effects of parthenolide was also discovered.Material and methodsCell counting kit-8(CCK-8)assay was employed to detect the impact of PTL on the viabililty of RCC cells(786-O and ACHN).According to the results of CCK-8 assay,the IC50 was calculated,and the concentrations 4μm/L and 8μm/L were selected.Wound healing and Transwell assays were applied to detect the effects of PTL on the migration and invasion of RCC cells,the expression of MMP2 and MMP9 was also monitored via western blotting.TUNEL assay was used to reveal the effect of PTL on the cell apoptosis of RCC cells,and the apoptosis related biomarkers,BAX,Bcl2,and Caspase3 was also detected by western blotting.Furthermore,the expression of EMT related biomarkers E-cadherin,N-cadherin,and Vimentin,and EMT transcription factor Snail was also detected via western blotting and RT-qPCR.Sphere formation assay was employed to discover the influence of PTLon the stemness of RCC cells,the stemness biomarkers ALDH2,CD133,Sox2,and Oct4 were further detected via western blotting.Finally,though western blotting,we evaluated the activity of PI3K-Akt signaling pathway by detecting the expression of PI3 K,Akt,P-PI3 K,and P-Akt.ResultsThe results of CCK-8 showed that PTL inhibited the vialibity of RCC cells dose-dependently.The colony formation assay visually verified that the number of colonies decreased in a dose-dependent manner.These results indicate that PTL could inhibit the proliferation of RCC cells.At the same time,the proportion of scratch healing was significantly lower than the control group.Transwell assay also showed that the number of transmembrane cells in the experimental group was decreased compared with the control group in a concentration-dependent manner.Western Blotting assay was used to detect matrix metalloproteinases MMP2 and MMP9.The results showed that the expression levels of MMP2 and MMP9 in the experimental group were lower than the control group,suggesting that PTL could inhibit the invasion and migration of RCC cells.TUNEL assay showed that the fluorescence intensity and the number bright cells increased with the PTL concentration,and DAPI staining also showed the deformation phenomenon of nucleus.In addition,western blotting assay showed the expression levels of BAX and cleaved Cleaved caspase3 in the experimental group were increased dose-dependently,while the expression of Bcl2 was decreased,indicating that PTL could induce the apoptosis of RCC cells.On the other hand,Immunofluorescence and western blotting assays showed that the expression of epithelial biomarker E-cadherin was increased dose-dependently while the mesenchymal biomarkers N-cadherin,Vimentin,and EMT transcriptional factor Snail were decreased.The results of RT-qPCR showed that the mRNA level of E-cadherin was increased and the mRNA level of Snail was decreased in the experimental group compared with the control group,indicating that PTL can inhibit the EMT process of RCC cells.PTL treatment significantly inhibited the microsphere formation ability of RCC cells in a concentrate-dependent manner.Consistently,via western blotting assay,we the expression of cancer stemness biomarkers ALDH1,Sox2,Oct4 and rRCC-specific stemness biomarker CD133 was decreased concentrate-dependently and In our study,the results of western blotting assay showed the expression levels of PI3 K,P-PIK,AKT and P-AKT in the PI3-AKT signaling pathway were in comparison with the control group.Conclusion 1.PTL inhibits the proliferation of RCC cells.2.PTL inhibits migration and metastasis of RCC cells.3.PTL induces apoptosis of RCC cells.4.PTL inhibits the EMT process of RCC cells.5.PTL inhibits the Stemness of RCC.6.PTL may inhibit EMT through regulation of the PI3K-Akt signaling pathway in RCC cells.更多还原