【作者】 闫鹏；

【导师】 孙玉萍；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2021， 博士

【摘要】 研究背景早期NSCLC的消融治疗肺癌是全世界范围内发病率排在第二位的恶性肿瘤,它的死亡率排在第一位。2020年,全世界有220万的新诊断病例,有180万的病例死于肺癌。肺癌按组织病理学可分为非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)和小细胞肺癌(Small Cell Lung Cancer,SCLC),其中NSCLC约占所有肺癌的85%。早期诊断、早期治疗能够明显地改善肺癌患者的预后。引起广泛关注的美国国家肺癌筛查(NLST)和荷兰-比利时肺癌筛查(NELSON)这两项大型试验,表明了低剂量螺旋CT筛查可降低20%肺癌患者的死亡率。低剂量CT筛查已作为肺癌高危人群筛查的重要手段,越来越多的早期肺癌被发现。早期(Ⅰ期及淋巴结阴性的ⅡA期)NSCLC的治疗主要依赖于手术切除,而对于不适宜或者个人意愿不接受手术的患者,立体定向放射治疗(Stereotactic Body Radiation Therapy,SBRT)是作为首选。图像引导的热消融(Thermal Ablation)自2017年后也被National Comprehensive Cancer Network,NCCN)指南推荐用于不可耐受手术切除的早期NSCLC。但是,目前很多研究证明对于不能手术的早期NSCLC,优先推荐SBRT而不是消融。大量的临床研究结果显示,虽然SBRT和消融的1年和3年生存率没有统计学差异(1 年:68.2-95%vs.81-85.7%;3 年:36-87.5%vs.42.7-56%)。但是消融5年的生存率(20.1-27%)却显著低于SBRT(47%)。因此,相对于SBRT,消融的复发率高还是影响其在临床应用的瓶颈。消融相对于SBRT具有可重复性好,无辐射,对肺功能影响小,住院时间短,花费小的优势。如果能降低消融的复发率,提高5年的生存率,可使许多早期NSCLC患者获益。前期研究显示,对于直径小于3cm的NSCLC,热消融治疗与SBRT临床治疗效果相当。但对于直径大于3cm或者形态不规则的早期肺癌,热消融复发率显著升高,5年生存期显著降低。这是因为肿瘤未达到完全灭活所致。受到热刺激但是未死亡的肿瘤细胞会发生哪些生物学改变,这些改变对于预后的影响,是提高早期肺癌消融生存期一个值得去探索的问题。不全消融与亚致死热损伤在原发性肝癌的热消融中,学者们提出不全消融所致亚致死热损伤(Sublethal Heat Damage)的概念。亚致死热损伤定义为37℃到60℃可导致部分肿瘤细胞死亡,而部分肿瘤细胞存活下来,存活下来的细胞出现了一系列的生物学改变。在肝细胞癌的细胞及动物实验证实,在这个温度区间的某个或某几个温度热刺激后,存活下来的细胞会发生上皮间质转化(Epithelial-mesenchymal transition,EMT),伴随侵袭迁移能力增强。这可能是肝癌消融后复发的关键因素。一项研究将肝癌细胞系经过热暴露后,发现肿瘤细胞形成纺锤状形态,肿瘤干性和EMT指标CD133,CK7,CK19,前胶原-α1(Ⅰ)和Snail表达升高。动物实验也表明热暴露的肝癌细胞移植入裸鼠所诱发的肿瘤生长更快且侵袭性更强。Tan等也得到了类似的结果,在46℃可以导致肝癌细胞系细胞侵袭迁移能力增强。同时该团队建立了肝癌细胞裸鼠移植瘤不全消融模型,发现残留肿瘤转移能力明显升高。Yamada S等同样证实了亚致死温度48℃热刺激下,诱导了肝癌细胞系中N-钙粘蛋白上调,E-钙粘蛋白表达降低。上述一系列的实验证实了亚致死温度确实能导致肿瘤细胞生物学行为的改变,主要是EMT表型上调,侵袭迁移能力增加。亚致死热损伤与NSCLC而在NSCLC消融中,肿瘤复发和转移是否与不全消融导致残余肿瘤细胞恶性生物学行为增加有关,尚没有文献报道。亚致死热损伤如何导致NSCLC细胞发生恶性转化的机制,也还没有人去探索。研究目的本课题从临床上经常碰到的消融不充分而导致复发这一现象出发,通过临床数据回顾得出消融完整的必要性。然后通过体内外实验探索和明确NSCLC的不全消融(亚致死热损伤)会导致残余肿瘤细胞侵袭迁移能力增强并出现EMT的现象。我们推断受到亚致死热损伤刺激的NSCLC是通过EMT导致生物学行为改变的。然后我们通过转录组测序筛选亚致死热损伤后的NSCLC差异表达基因及信号通路,并验证差异表达分子在恶性转化中的作用。目的是通过发现不全消融导致肿瘤恶性转化的机制,来提示临床医师完整消融是降低复发的关键因素。研究方法1.回顾性分析了 36例接受微波消融(Microwave ablation,MWA)的Ⅰ期NSCLC患者,以5mm为界,将患者分为足够消融边界组和消融边界不足两个组。Kaplan-Meier法计算两个组的局部肿瘤进展(Local tumor progression,LTP)间期、远处转移(Distantmetastasis,DM)间期和无病生存期(Disease-free survival,DFS,并通过log-rank法检验。2.采用Cox比例风险模型评价性别、年龄、ECOG评分、病理分型、T分期、肿瘤直径、最小消融边界共7个可能影响患者预后的因素。3.建立小鼠肺癌异种移植瘤模型,成瘤到一定体积后,行不全消融。消融后继续培养10天后处死小鼠,解剖瘤体,苏木精-伊红染色法(Hematoxylin-eosin,H-E)染色法观察细胞形态变化,免疫组化染色法(Immunohistochemistry,IHC)检测上皮间质转化相关表型表达。4.体外使用37℃-50℃热浴NSCLC细胞株,使用台盼蓝染色法,确定NSCLC细胞株的体外亚致死温度区间。5.亚致死温度热处理2种NSCLC细胞株后,CCK-8法及克隆形成实验检测细胞的增殖能力的变化;流式细胞术检测细胞的在热刺激后细胞周期及凋亡率的变化。6.亚致死温度热处理2种NSCLC细胞系后,通过细胞划痕实验检测细胞的迁移能力;通过Transwell小室实验检测侵袭、迁移能力变化;选择BALB/C Nude裸鼠,尾静脉注射检测亚致死热刺激后的肿瘤细胞的体内转移能力。7.RT-PCR及Western blot检测亚致死温度热处理2种NSCLC细胞株后EMT相关表型的变化。8.通过转录组测序(RNA-Seq)检测热刺激后NSCLC细胞差异基因的表达,筛选出差异表达基因及信号通路。9.亚致死热刺激2株NSCLC细胞株后,RT-PCR和Western blot检测差异基因(Carboxypeptidase A4,CPA4)随时间的变化。10.收集临床NSCLC患者病例,CPA4在肿瘤组织,以及癌旁组织中的表达水平采用IHC检测;根据得出的免疫组化染色评分结果,统计分析CPA4的表达水平,并分析与NSCLC患者临床病理学特征之间的相关性;对NSCLC患者进行随访,并收集资料,采用Kaplan-Meier法绘制生存曲线,应用log-rank检验方法,分析CPA4的表达水平与患者生存时间之间的相关关系。11.通过转染建立稳定高表达和低表达的NSCLC细胞系。RT-PCR及Western blot检测CPA4在正常肺上皮细胞系BEAS-2B及4株NSCLC细胞系的表达,筛选出内源性高表达CPA4的细胞株和低表达CPA4的细胞株。选择CPA4表达低的细胞株H1299转染CPA4质粒上调CPA4表达,并通过慢病毒感染建立稳定过表达CPA4的细胞株;选择CPA4高表达的细胞株A549,转染CPA4shRNA干扰CPA4表达,并通过慢病毒感染建立稳定干扰CPA4细胞株。12.CCK-8和平板克隆形成实验检测过表达和沉默CPA4后,对NSCLC的增殖能力影响;流式细胞术检测过表达和沉默CPA4后,NSCLC的细胞周期分布情况;进行细胞划痕实验,Transwell小室检测过表达和沉默CPA4后,分析肿瘤细胞的侵袭迁移能力。13.选择BALB/c Nude裸鼠皮下注射过表达CPA4的NSCLC细胞及其对照组细胞,检测NSCLC成瘤能力;尾静脉注射过表达/干扰CPA4细胞检测肿瘤细胞的体内转移能力。14.通过RT-PCR及Western Blot检测稳定过表达和稳定干扰CPA4的NSCLC细胞系EMT相关表型的变化。15.干扰CPA4后,再通过热刺激NSCLC细胞,Transwell小室侵袭迁移实验检测NSCLC的侵袭、迁移能力变化,通过Annexin V-FITC/PI流式细胞术,检测热刺激对NSCLC凋亡的影响,通过Western blot检测干扰CPA4后热刺激对EMT相关分子表型的影响。研究结果1.最小消融边界和早期NSCLC消融的预后密切相关最小消融边界≥5mm患者的无局部进展间期(LTP interval)及无病生存期(DFS)显著长于最小消融边界<5mm的患者。单因素和多因素分析显示肿瘤直径(风险比[HR]=1.91,P<0.01;HR=2.41,P=0.01)和最小消融边界(HR=0.13,P<0.01;HR=0.11,P<0.01)为LTP间期的独立预后因素。同时,被认为是影响DFS的独立预后因素:肿瘤直径(HR=1.96,P<0.01;HR=2.35,P<0.01)、最小消融边界(HR=0.17,P<0.01;HR=0.13,P<0.01)这两个因素。也是。在我们的回顾性分析中,有50%(18/36)的患者消融边界不到5mm。以上研究的这些患者,被认为是肺癌复发的高危人群。2.动物实验中,不全消融可以导致残余肿瘤病灶EMT相关表型表达升高不全消融组肿瘤体积明显小于对照组。HE染色观察到细胞出现坏死,是发生在进行消融的核心区,坏死的细胞其中大部分细胞结构破坏,仅见有组织轮廓,细胞核碎裂,核固缩。而核心区周围细胞呈梭形变化,同时细胞之间变得松散,可能导致EMT转化。免疫组化检测了 EMT相关的分子表型变化,与对照组相比,残留肿瘤中上皮相关分子E-cadherin表达明显降低,间质相关分子N-cadherin、Vimentin表达显著上调。3.热刺激后的存活率随着温度的升高而降低根据台盼蓝染色的结果,我们定义体外的亚致死温度区间在37℃-48℃,热暴露时间为10min。4.46℃和48℃亚致死热刺激可抑制体外NSCLC细胞增殖,增加细胞凋亡在体外,通过CCK-8及平板克隆实验,46℃和48℃亚致死热刺激可抑制NSCLC细胞增殖,而低于46℃对增殖没有显著影响。Annexin V-FITC/PI流式细胞术显示,46℃和48℃增加了细胞凋亡,而42℃和44℃对NSCLC凋亡没有显著影响。5.46℃和48℃亚致死热刺激会导致G0/G1期阻滞细胞周期检测显示,42℃和44℃对肺癌细胞细胞周期没有显著影响。而46℃和48℃导致NSCLC细胞在G0/G1周期阻滞。6.44℃和46℃亚致死热刺激可增强体外NSCLC细胞的侵袭和迁移能力细胞划痕实验显示,在44℃和46℃热刺激后72h的NSCLC,其迁移能力显著增强。Transwell小室侵袭迁移实验也证实了 44℃和46℃热刺激后72h的NSCLC细胞的侵袭迁移能力显著增强。7.44℃和46℃热刺激的后肺癌细胞株体内转移能力增强尾静脉注射实验验证了 44℃和46℃热刺激的肺癌细胞小鼠的肝肺转移灶数量显著高度对照组。8.在体外,44℃和46℃热刺激诱导了非小细胞肺癌EMT相关表型表达升高RT-PCR显示在44℃和46℃热刺激后,48h EMT的转录因子表达开始上升,72h表达最高。其中44℃组升高最为显著。44℃和46℃热刺激后,western blot结果显示E-cadherin蛋白表达显著下调,N-cadherin和Vimentin表达显著上调。44℃热刺激后72h时N-cadherin和Vimentin表达升高最为显著。9.RNA-Seq发现热刺激后CPA4表达升高转录组测序发现亚致死热损伤后CPA4显著差异表达(P<0.01),EMT相关基因显著富集。RT-PCR和Western blot验证了44℃热刺激可以升高CPA4,且呈时间依赖性。热刺激后,CPA4从8h开始表达上调,到48h达到最高,72h开始下降。10.CPA4在NSCLC中高表达,且与预后差密切相关CPA4在NSCLC组织内表达高于癌旁组织,且高表达CPA4的NSCLC患者预后更差。CPA4表达阳性的非小细胞肺癌患者生存时间较CPA4表达阴性的患者其生存时间明显缩短(14.4月VS.22.2月)。此外,CPA4的表达水平还与三个因素密切相关,分别是肿瘤分化程度(P=0.03)、临床分期(P=0.02)和淋巴结转移(P=0.01)。11.CPA4促进NSCLC细胞体外增殖及侵袭迁移RT-PCR及Western blot检测结果显示:在4株NSCLC细胞株(A549、H1299、H1975及PC9)中CPA4均有所表达,这4株细胞株中,H1299最低,A549最高。过表达CPA4后,出现明显增强的是H1299细胞的增殖及侵袭迁移能力;干扰CPA4后,受到明显抑制的是A549细胞的增殖及侵袭迁移能力。12.CPA4促进NSCLC细胞体内成瘤能力及体内转移能力我们建立了皮下移植瘤模型,从中发现过表达CPA4后,肿瘤生长的速度显著快于对照组,且肿瘤的体积大于对照组。尾静脉注射模型发现,过表达CPA4后,裸鼠肺组织转移灶数目显著多于对照组;干扰CPA4后,裸鼠肺组织转移灶数目少于对照组。13.CPA4过表达诱导NSCLC细胞的EMT通过RT-PCR和Western blot检测了过表达CPA4的H1299细胞,上皮细胞黏附分子E-cadherin表达显著下降,间质的表型N-cadherin和Vimentin表达显著提高;敲低CPA4后的A549细胞,E-cadherin表达显著上升,而间质表型N-cadherin和Vimentin表达下降。免疫组化检测过表达CPA4的H1299细胞的皮下瘤组织,相对于对照组,E-cadherin表达显著下降,N-cadherin和Vimentin表达上升。14.敲低CPA4可部分逆转亚致死热刺激引起的NSCLC的恶性生物学行为和EMT敲低CPA4后,再使用44℃热刺激肺癌细胞株,结果发现,相对于空白的NSCLC细胞株,敲低CPA4后接受热刺激的肺癌细胞株,侵袭迁移能力仍出现增加,但是增加的程度相对于没有敲低CPA4但接受热刺激的肺癌细胞显著降低。提示沉默CPA4可以部分逆转亚致死热损伤导致的侵袭、迁移能力增强。敲低CPA4后,再使用44℃热刺激肺癌细胞株,结果发现,干扰CPA4后,相对于无热刺激的空白组,E-cadherin表达显著下降,N-cadherin和Vimentin表达上升,但相对于热刺激组E-cadherin表达显著上升,N-cadherin和Vimentin表达下降。说明沉默CPA4可以部分逆转亚致死热损伤导致的EMT。15.沉默CPA4可增加A549和H1975热刺激后细胞凋亡干扰CPA4后,A549和H1975细胞对热损伤更加敏感,凋亡率显著增加。结论1.消融的边界与早期NSCLC的预后密切相关,早期NSCLC的消融必须要达到安全的消融边界,最小边界不应小于5mm。2.消融不足引起的亚致死热刺激可增强NSCLC细胞侵袭、迁移能力,其机制为亚致死热损伤诱导了 NSCLC的EMT。体外研究结果显示44℃和46℃这两个亚致死温度是出现恶性转化的扳机温度(Trigger temperature),其中44℃诱导出的表型更为显著。3.消融不足可以诱导CPA4表达上调,CPA4作为一种癌基因,与NSCLC患者预后密切相关。高表达CPA4肿瘤患者生存期更短,过表达CPA4的NSCLC细胞株侵袭、迁移能力更强。4.CPA4在亚致死热损伤导致肺癌细胞出现恶性转化中起到关键作用。干扰CPA4可以部分逆转亚致死热刺激导致的EMT和侵袭迁移能力增强。因此,无论是将CPA4作为一种肿瘤标记物判断消融是否完全或者作为消融后随访的监测指标,亦或者靶向CPA4处理消融不全导致的复发,都为我们以后的工作开展了思路。更多还原

【Abstract】 BackgroundAblation treatment for early stage NSCLCWorldwide,lung cancer is the malignant tumor with the second incidence rate and the first mortality rate.In 2020,there will be 2.2 million new cases and 1.8 million deaths worldwide.According to histopathological classification,lung cancer can be divided into Non-Small Cell Lung Cancer,NSCLC)and Small Cell Lung Cancer,SCLC),of which NSCLC accounts for about 85%of all lung cancer cases.Early diagnosis of lung cancer,Early treatment can greatly improve the prognosis of patients.Two large-scale experiments,National Lung Cancer Screening(NLST)and Dutch-Belgian Lung Cancer Screening(NELSON),show that low-dose spiral CT screening can reduce the mortality of lung cancer by 20%.Low-dose CT screening has become an important means for screening high-risk groups of lung cancer,and more and more early lung cancer has been found.The treatment of early stage(stage I and stage IIA with negative lymph nodes)NSCLC mainly depends on surgical resection.For patients who are unsuitable or unwilling to undergo surgery,stereotactic body radiation therapy(SBRT)is the first choice.Image-guided Thermal Ablation has also been recommended by the national comprehensive cancer network(nccn)for early NSCLC that cannot tolerate surgical resection since 2017.But,For inoperable early NSCLC,SBRT recommended priority over ablation.The results of a large number of clinical studies show that although there is no statistical difference in 1-year and 3-year survival rates between SBRT and RFA(68.2-95%vs.81-85.7%;36-87.5%vs.42.7-56%)。However,the 5-year survival rate of ablation(20.1-27%)was significantly lower than that of SBRT(47%).Therefore,compared with SBRT,the high recurrence rate of ablation is still the bottleneck affecting its clinical application.Compared with SBRT,ablation has the advantages of good repeatability,no radiation,little influence on lung funion,short hospitalization time and low cost.If we can reduce the recurrence rate of ablation and improve the 5-year survival rate,many patients with early NSCLC will benefit.Previous studies have shown that for NSCLC with a diameter less than 3cm,the therapeutic effect of thermal ablation is equivalent to that of SBRT.However,for early stage lung cancer with diameter larger than 3cm or irregular shape,the recurrence rate of thermal ablation increased significantly,and the 5-year survival time decreased significantly.This is because the tumor has not been completely inactivated.What biological changes will happen to tumor cells that are stimulated by heat but not dead?The influence of these changes on prognosis is a problem worth exploring in prolonging ablation of early lung cancer and improving survival time.Total ablation and sublethal thermal injury.In the thermal ablation of primary liver cancer,scholars put forward the concept of Sublethal Heat Damage caused by incomplete ablation.Sublethal thermal injury is defined as 37℃ to 60℃,which can cause some tumor cells to die,while some tumor cells survive,and the surviving cells have undergone a series of biological changes.Cell and animal experiments in hepatocellular carcinoma proved that,After thermal stimulation at one or several temperatures in this temperature range,the surviving cells will undergo epithelial-mesenchymal transition(EMT),with the enhancement of invasion and migration ability.This may be the key factor for recurrence of liver cancer after ablation.In a study,liver cancer cell lines were exposed to heat,and it was found that tumor cells formed spindle shape,tumor dryness and EMT indexes cd133,ck7 and ck19.The expression of procollagen-α1(Ⅰ)and Snail increased.Animal experiments also showed that the tumor growth induced by transplantation of heat-exposed liver cancer cells into nude mice was faster and more invasive.Tan et al.also got similar results,and at 46℃,it can enhance the invasion and migration ability of hepatoma cell lines.At the same time,the team established an incomplete ablation model of transplanted hepatocellular carcinoma cells in nude mice.It was found that the metastatic ability of residual tumor increased significantly.Yamada S et al.also confirmed that the sub-lethal temperature of 48℃ induced the up-regulation of Ncadherin and the down-regulation of E-cadherin expression in hepatoma cell lines.The above-mentioned series of experiments confirmed that the sublethal temperature can indeed lead to changes in the biological behavior of tumor cells,mainly the up-regulation of EMT phenotype and the increase of invasion and migration ability.Sublethal thermal damage and NSCLC.However,inNSCLC ablation,whether tumor recurrence and metastasis and incomplete ablation lead to increased malignant biological behavior of residual tumor cells has not been reported in the literature.No one has explored the mechanism of how lethal thermal injury leads to malignant transformation of NSCLC cells.Research purposesIn this paper,the necessity of complete ablation is put forward by reviewing clinical data,and then the incomplete ablation of NSCLC(sub-lethal thermal injury)will lead to enhanced invasion and migration ability of residual tumor cells and EMT.Transcriptome sequencing was used to screen differentially expressed genes and signal pathways in NSCLC after sublethal heat injury,and to verify the role of differentially expressed molecules in malignant transformation.Objective To discover the mechanism of malignant transformation caused by incomplete ablation,and to suggest that complete ablation is the key factor to reduce recurrence.Research method1.Thirty-six patients with stage I NSCLC undergoing Microwave ablation,MWA)were analyzed retrospectively,and the relationship between the minimum ablation boundary and local tumor progression,LTP)interval,distant metastasis,DM)interval and disease-free survival,DFS)was analyzed.Patients were divided into two groups:sufficient ablation boundary group and insufficient ablation boundary group.Kaplan-Meier method was used to calculate the survival time of the two groups and log-rank method was used to test.2.Cox proportional hazards model was used to screen 7 factors that may affect the local progression interval and disease-free survival of patients,including gender,age,ECOG scores,pathological classification,T stage,tumor diameter and minimum ablation boundary.The mouse lung cancer xenograft model was established,and the tumor was formed to a certain volume,and then incomplete ablation was performed.Ten days after ablation,the mice were killed,and the tumor was dissected.Hematoxylin-eosin(H-E)staining was used to observe the morphological changes of the cells,and immunohistochemistry(IHC)was used to detect the phenotype expression related to epithelial-mesenchymal transition.3.In vitro,NSCLC cell lines were heated at 37℃-50℃,and trypan blue staining was used to determine the sublethal temperature range in vitro.4.After heat treatment of two NSCLC cell lines at sub-lethal temperature,CCK-8 method and clone formation experiment were used to detect the changes of cell proliferation ability.The changes of cell cycle and apoptosis rate after heat stimulation were detected by flow cytometry.5.Through cell migration test,the effects of sublethal heat stimulation on the migration ability of two kinds of NSCLC cells in vitro were analyzed.6.After heat treatment of two NSCLC cell lines at sub-lethal temperature,the migration ability of the cells was detected by cell scratch test.Transwell chamber test was used to detect the changes of invasion and migration ability.BALB/C Nude nude mice were selected and injected into tail vein to detect the in vivo metastasis ability of tumor cells after sublethal heat stimulation.7.RT-PCR and Western Blot were used to detect the changes of EMT-related phenotypes after heat treatment at sublethal temperature.8.Transcriptome sequencing(RNA-Seq)was used to detect the expression of differential genes in NSCLC cells after heat stimulation,and the differentially expressed genes(CPA4)and signal pathways were screened out.9.Two NSCLC cell lines were stimulated by sublethal heat,and the changes of CPA4 with time were detected by RT-PCR and Western blot.10.Collect NSCLC cases,and detect the expression of CPA4 in tumor tissues and adjacent tissues by IHC.According to the results of immunohistochemical staining,the correlation between CPA4 expression level and clinicopathological features of NSCLC patients was statistically analyzed.The follow-up data of NSCLC patients were collected,the survival curve was drawn by Kaplan-Meier method,and the relationship between the expression of CPA4 and the survival time of patients was analyzed by log-rank test.11.Establishment of stable NSCLC cell lines with high and low expression by transfection.RT-PCR and Western blot were used to detect the expression of CPA4 in normal lung epithelial cell line BEAS-2B and 4 NSCLC cell lines,and the endogenous cell lines with high CPA4 expression and low CPA4 expression were screened out.Selecting cell line H1299 with low expression of CPA4 to transfect CPA4 plasmid to up-regulate CPA4 expression,and establishing cell line with stable overexpression of CPA4 by lentivirus infection;Selecting A549 cell line with high expression of CPA4,transfecting CPA4shRNA to interfere with CPA4 expression,and establishing stable interfering CPA4 cell line by lentivirus infection.12.CCK-8 and plate clone formation experiments were used to detect the effect of CPA4 overexpression and silencing on the proliferation of NSCLC.The cell cycle distribution of NSCLC was detected by flow cytometry after CPA4 was overexpressed and silenced.Cell scratch test and Transwell chamber were used to detect the invasion and migration ability of tumor cells after CPA4 was overexpressed and silenced.13.Select BALB/c Nude nude mice to subcutaneously inject NSCLC cells that have expressed CPA4 and their control cells,and detect the tumorigenicity of NSCLC.Intravenous injection of overexpressed/interfered CPA4 cells to detect metastasis ability of tumor cells in vivo;14.RT-PCR and Western Blot were used to detect the changes of EMT-related phenotype in NSCLC cell lines with stable high expression and low expression.15.After interfering with CPA4,the invasion and migration ability of NSCLC cells were tested by Transwell chamber invasion and migration test,the influence of thermal stimulation on apoptosis of NSCLC was tested by Annexin V-FITC/PI flow cytometry,and the influence of thermal stimulation on the phenotype of EMT related molecules after interfering with CPA4 was tested by Western blot.Research results:1.The minimum ablation boundary is closely related to the prognosis of early NSCLC ablation.The local recurrence-free survival(LTP)and disease-free survival(DFS)of patients with minimum ablation boundary≥5mm were significantly longer than those with minimum ablation boundary<5mm.Univariate and multivariate analysis showed that tumor size(hazard ratio[HR]=1.91,P<0.01;HR=2.41,P=0.01)and minimum ablation boundary(HR=0.13,P<0.01;HR=0.11,P<0.01)was an independent prognostic factor of LTP interval.Meanwhile,the tumor size(HR=1.96,P<0.01;HR=2.35,P<0.01)and the minimum ablation margin(HR=0.17,P<0.01;HR=0.13,P<0.01)is an independent prognostic factor of DFS.In our retrospective analysis,50%(18/36)patients had ablation boundaries less than 5 mm These patients are considered to be at high risk of recurrence.2.In animal experiments,incomplete ablation can lead to increased expression of EMT-related phenotype in residual tumor lesions.The tumor volume in incomplete ablation group was significantly smaller than that in control group.In tHE core area of ablation,he staining showed that the cells were necrotic,most of the cell structures were destroyed,and only the tissue outline,nucleus fragmentation and nuclear pyknosis were found.However,the cells around the core area are spindle-shaped and loose,which may lead to EMT process.Immunohistochemistry detected the molecular phenotype changes related to EMT,Compared with the control group,the expression of E-cadherin,an epithelial related molecule,was significantly decreased,while the expression of N-cadherin and Vimentin,an interstitial related molecule,was up-regulated.3.The survival rate after thermal stimulation decreases with the increase of temperature.According to the results of trypan blue staining,we defined the sublethal temperature range in vitro as 37℃-4℃.4.Sublethal heat stimulation at 46℃ and 48℃ can inhibit proliferation and increase apoptosis of NSCLC cells in vitro.In vitro,CCK-8 and plate cloning experiments showed that sub-lethal heat stimulation at 46℃ and 48℃ could inhibit the proliferation of NSCLC cells,while below 46℃ had no significant effect on the proliferation.Annexin V-FITC/PIflow cytometry showed that 46℃ and 48℃ increased apoptosis,while 42℃ and 44℃ had no significant effect on apoptosis of NSCLC.5.Sub-lethal heat stimulation at 46℃ and 48 ℃ will lead to G0/G1 arrest.Cell cycle test showed that 42℃ and 44℃ had no significant effect on cell cycle of lung cancer cells.However,46℃ and 48℃ caused cell cycle arrest of NSCLC cells in G0/G1 phase.6.Sublethal heat stimulation at 44℃ and 46℃ can enhance the invasion and migration ability of NSCLC cells in vitro.The cell scratch test showed that the migration ability of NSCLC was significantly enhanced after 72h heat stimulation at 44℃ and 46℃.Transwell cell invasion and migration experiments also confirmed that the invasion and migration ability of NSCLC cells was significantly enhanced after 72 hours of heat stimulation at 44℃ and 46℃.7.After heat stimulation at 44℃ and 46℃,the metastasis ability of lung cancer cell lines in vivo was enhanced.The tail injection experiment verified that the number of liver and lung metastases in lung cancer cell mice stimulated by heat at 44℃ and 46℃ was significantly higher than that in the control group.8.In vitro,heat stimulation at 44℃ and 46℃ induced an increase in EMT-related phenotype expression in non-small cell lung cancer.RT-PCR showed that after heat stimulation at 44℃ and 46℃,the expression of transcription factors began to rise at 48 hours,and reached the highest level at 72 hours.Among them,the increase in 44℃ group was the most significant.After heat stimulation at 44℃ and 46℃,western blot showed that the expression of E-caherin protein was significantly down-regulated,while the expression of N-cadherin and Vimentin was significantly up-regulated.At 44℃,the expression of E-caherin decreased with time,while the expression of N-cadherin and Vimentin increased with time.The expression increased most significantly at 72h.9.RNA-Seq showed that CPA4 expression increased after heat stimulation.Transcriptome sequencing showed that CPA4 was significantly differentially expressed(P<0.01),and EMT-related genes were significantly enriched.RT-PCR and Western Blot verified that heat stimulation at 44℃ could increase CPA4 in a time-dependent manner.After heat stimulation,the expression of CPA4 was up-regulated from 8h,reached its peak at 48h,and began to decrease at 72 h.10.CPA4 is highly expressed in NSCLC and is closely related to poor prognosis.The expression of CPA4 in NSCLC tissues is higher than that in adjacent tissues,and the prognosis of NSCLC patients with high expression of CPA4 is worse.The survival time of patients with positive CPA4 expression was significantly shorter than that of patients with negative CPA4 expression(14.4 months VS.22.2 months).In addition,the expression of CPA4 was closely related to the degree of tumor differentiation(P=03),lymph node metastasis(P=0.01)and clinical stage(=0.02).11.CPA4 promotes proliferation,invasion and migration of NSCLC cells.RT-PCR and Western blot showed that CPA4 was expressed in four NSCLC cell lines(A549,H1299,H1975 and PC9),among which H1299 was the lowest and A549 was the highest.The ability of proliferation,invasion and migration of H1299 cells was significantly enhanced after overexpression of CPA4.The ability of proliferation,invasion and migration of A549 cells was significantly inhibited after CPA4 interference.12.CPA4 promotes tumor formation and metastasis of NSCLC cells in vivo.The establishment of subcutaneous transplanted tumor model showed that after overexpression of CPA4,the growth rate of tumor was significantly faster than that of the control group,and the tumor volume was larger than that of the control group.The tail injection model showed that after overexpression of CPA4,the number of metastatic foci in lung tissue of nude mice was significantly higher than that of the control group.After interfering with CPA4,the number of lung metastases in nude mice was less than that in control group.13.Overexpression of CPA4 induces EMT in NSCLC cells.H1299 cells overexpressing CPA4 were detected by RT-PCR and Western blot.the expression of E-cadherin in epithelial cells decreased significantly,while the expression of N-cadherin and Vimentin in stroma increased significantly.The expression of E-cadherin in A549 cells after low CPA4 knockdown increased significantly,while the expression of interstitial phenotype N-cadherin and Vimentin decreased.Immunohistochemistry showed that the subcutaneous tumor tissue of H1299 cells expressing CPA4 was significantly lower than that of the control group.The expression of N-cadherin and Vimentin increased.14.Silence of CPA4 can partially reverse the malignant biological behavior and EMT of NSCLC induced by sublethal heat stimulation.Lung cancer cell lines were stimulated by heat at 44℃ after knocking down CPA4.compared with blank NSCLC cell lines,the invasion and migration ability of lung cancer cell lines subjected to heat stimulation after knocking down CPA4 still increased,but the degree of increase was significantly lower than that of lung cancer cells without knocking down CPA4 but subjected to heat stimulation.It is suggested that silencing CPA4 can partially reverse the invasion and migration enhancement caused by sublethal thermal injury.Low CPA4 was knocked out,and then 44 ℃ heat was used to stimulate lung cancer cell lines.the results showed that compared with the blank group without heat stimulation,the expression of E-cadherin decreased significantly,while the expression of N-cadherin and Vimentin increased significantly,while the expression of N-cadherin and Vimentin decreased compared with the heat stimulation group.Therefore,silencing CPA4 can partially reverse EMT caused by sublethal thermal injury.15.Silence of CPA4 can increase apoptosis of A549 and H1975 cells after heat stimulation.With the interference of CPA4,A549 and H1975 cells were more sensitive to heat injury and the apoptosis rate increased.Conclusion1.The ablation boundary is closely related to the prognosis of early NSCLC,and the ablation of early NSCLC must reach a safe ablation boundary,with the minimum boundary not less than 5mm.2.Sublethal thermal stimulation caused by insufficient ablation can promote EMT in NSCLC cells and enhance their metastatic ability.The results of in vitro studies show that two sub-lethal temperatures,44 ℃ and 46℃,are trigger temperature for malignant transformation,and the phenotype induced by 44℃ is more significant.3.Inadequate ablation can induce increased expression of CPA4.CPA4,as an oncogene,plays a key role in malignant transformation of tumor cells caused by sublethal thermal injury.Low CPA4 knockdown can reduce the invasion and migration ability of NSCLC,and overexpression of CPA4 can induce epithelial-mesenchymal transition.4.Patients with tumor overexpressing CPA4 have shorter survival time,and NSCLC cell lines overexpressing CPA4 have stronger proliferation and invasion ability.Silence of CPA4 can partially reverse EMT and enhance invasion and migration ability caused by sublethal heat stimulation.Therefore,whether CPA4 is used as a tumor marker to monitor whether ablation is complete or as an index for follow-up after ablation,Or targeting CPA4 to treat the recurrence caused by incomplete ablation,all of which provide ideas for our future work.更多还原