【作者】 苗壮；

【导师】 马虹；

【作者基本信息】 中国医科大学 ， 麻醉学， 2021， 博士

【摘要】 目的:肺癌是全球癌症相关死亡的首位原因,肺癌中超过85%为非小细胞肺癌(non-small cell lung cancer,NSCLC)。NSCLC患者早期临床症状不明显,有60%～70%的患者在首诊时已处于晚期,预后常较差。目前NSCLC治疗方法包括手术治疗、放化疗、分子靶向治疗和免疫治疗等,由于NSCLC具有遗传异质性,个体差异较大,5年生存率改善仍不明显。近年来阿片生长因子(opioid growth factor,OGF)-阿片生长因子受体(OGF receptor,OGFR)轴被发现具有抗肿瘤特性。OGF,即蛋氨酸脑啡肽,是一种重要的内源性阿片肽,几乎在人体所有组织中均有不同水平的表达。OGFR是一种与细胞核相关的完整的膜蛋白,其表达位于外核膜上或其附近,含有核定位信号(nuclear localization signal,NLS),当OGF与OGFR特异性结合后,OGF-OGFR复合物能被转运至细胞核内调节DNA合成,对组织器官发育、细胞更新、肿瘤的发生发展、血管生成、创伤修复和免疫调节等起到重要作用。在人类许多肿瘤细胞系和肿瘤组织中均发现OGF-OGFR轴的表达,其中包括NSCLC细胞系和肿瘤组织。研究发现OGFR在肿瘤细胞和组织中低表达。改变OGFR表达量可影响肿瘤恶性生物学行为,OGFR过表达可抑制肿瘤细胞的增殖,而沉默OGFR可促进肿瘤细胞的增殖。OGFR对人NSCLC肿瘤发生发展过程可能的调节作用未有报道,其表达量与NSCLC肿瘤的预后的相关性也未有报道。近期大量研究发现阿片药物和阿片受体可通过不同的作用机制来影响NSCLC的发生发展及患者预后。阿片受体包括μ受体(μopioid receptor,MOR)、κ受体(κopioid receptor,KOR)、δ受体(δopioid receptor,DOR)和ζ阿片受体(OGFR)。大量研究表明MOR和OGFR参与肿瘤的发生发展,并与阿片类药物相互作用影响肿瘤进程。MOR被发现在NSCLC肿瘤组织中高表达,MOR的活化和过表达可促进NSCLC细胞的增殖、迁移和上皮间质转化(epithelial mesenchymal transition,EMT)。吗啡等可高选择性作用于MOR促进肿瘤细胞生长,而OGFR被发现可与吗啡相互作用抑制NSCLC肿瘤细胞生长。目前尚无OGFR与其他阿片药物相互作用对NSCLC肿瘤细胞生长影响的相关研究,尚无OGFR与MOR阿片受体在相同肿瘤组织中表达相关性的研究。因此,本研究以抑癌因子OGFR作为NSCLC可能的潜在治疗靶点,通过分析OGFR表达量对NSCLC肿瘤细胞恶性生物学行为的调节作用及分子机制,分析OGFR和促癌因子MOR在NSCLC肿瘤组织中的表达量及与临床病理因素及预后之间的关系,分析OGFR与MOR在相同NSCLC肿瘤组织中表达的相关性,探讨OGFR在NSCLC肿瘤进程中可能的调节作用。还研究了不同浓度舒芬太尼(sufentanil,SUF)对NSCLC细胞系生长的影响,以及OGFR表达量对该作用的影响,为临床癌痛药物选择提供新思路。研究方法:1、本研究选取人肺成纤维细胞(human lung fibroblast-like cell,HLF)和5种NSCLC肿瘤细胞系A549、H460、H322、H358和H1299,通过Western Blot实验检测不同细胞系OGFR蛋白表达情况。慢病毒质粒转染在H1299细胞系构建OGFR低表达细胞系,在A549细胞系构建OGFR高表达细胞系,并使用Western Blot实验验证转染效率。通过MTT法、平板克隆形成实验检测转染的H1299和A549细胞系增殖能力的变化。Western Blot实验检测PI3K/Akt信号通路蛋白表达。对转染的H1299细胞系进行AV/PI染色,通过FACS法观察OGFR敲低后细胞凋亡变化;使用免疫荧光法检测H1299细胞线粒体细胞色素C(cytochrome c,Cyt-c)的释放情况;通过Western Blot实验检测凋亡蛋白的表达情况。通过划痕实验、Transwell实验观察转染的H1299细胞迁移和侵袭能力的变化,并通过Western Blot实验检测EMT相关蛋白的表达。通过肿瘤细胞成球实验观察OGFR敲低后H1299细胞成球能力变化,通过Western Blot实验检测细胞干性相关蛋白的表达。2、选取4例行肺癌根治术的NSCLC患者组织标本,采用Western Blot实验检测癌及癌旁正常组织OGFR蛋白表达水平。选择一组含98例NSCLC患者的组织芯片,通过免疫组化染色检测癌及癌旁正常组织OGFR及MOR蛋白表达水平。结合组织芯片患者临床病理参数及预后资料,采用卡方检验比较OGFR及MOR表达量与患者各临床病理参数,如年龄、肺癌组织分化程度和临床分期间的差异。采用Kaplan-Meier法绘制NSCLC生存曲线,Log-rank检验比较不同OGFR及MOR表达量NSCLC患者总生存期(overall survival,OS)的差异。卡方检验比较不同OGFR表达量的NSCLS患者MOR表达量的差异。3、选取A549细胞系,慢病毒质粒构建OGFR高表达细胞系,利用si RNA构建OGFR低表达细胞系,并使用RT-PCR试验验证转染效率。使用不同浓度(0n M、0.5n M、5n M、50n M和500n M)SUF对A549细胞进行处理,在6h、12h、24h和48h后使用CCK-8法观察SUF对不同OGFR表达量A549细胞增殖情况的影响。结果:1、OGFR在NSCLC细胞系中表达量较HLF细胞显著降低(P<0.05)。在NSCLC不同细胞系OGFR蛋白表达量有显著差异,其中H1299和H322细胞系表达水平相对较高,H358、H460和A549细胞系表达水平相对较低。选择H1299细胞系对OGFR表达量进行敲低,A549细胞系对OGFR进行过表达。MTT法和平板克隆形成实验结果表明,OGFR沉默的H1299细胞系增殖能力显著增加(P<0.05),OGFR过表达的A549细胞系增殖能力显著降低(P<0.05)。Western Blot实验结果显示,OGFR沉默的H1299细胞系p100α、p100β和p-Akt表达量显著增高(P<0.05),OGFR过表达的A549细胞系PI3K/Akt信号通路蛋白p100α、p100β和p-Akt表达量显著降低(P<0.05)。将转染的H1299细胞进行AV/PI染色,通过FACS分析,结果显示OGFR沉默的H1299细胞系凋亡率较对照组显著降低(P<0.05)。使用免疫荧光法发现OGFR敲低的H1299细胞线粒体Cyt-c释放较对照组显著减少(P<0.05)。Western Blot实验结果提示下调OGFR表达量可显著降低凋亡相关蛋白cleaved-caspase-7、cleaved-caspase-9和cleaved-聚(腺苷二磷酸-核糖)多聚酶(poly ADP-ribose polymerase,PARP)的表达水平(P<0.05)。划痕实验和Transwell实验结果显示,OGFR沉默的H1299细胞系迁移和侵袭的能力较阴性对照组增加(P<0.05)。Western Blot实验检测EMT相关蛋白表达情况,发现OGFR表达量降低的H1299细胞系E-cadherin表达量下降,N-cadherin和Vimentin表达量显著增加(P<0.05)。肿瘤细胞成球实验结果显示OGFR敲低后H1299细胞系成球能力显著增加,且Western Blot实验检测细胞干性相关蛋白的表达情况,结果显示CD133、CD44和OCT4表达量较对照组显著增加(P<0.05)。2、采用Western Blot实验对4例行肺癌根治术的NSCLC患者病理标本OGFR和MOR表达量进行检测,结果显示OGFR在癌组织中蛋白表达情况较正常癌旁组织显著降低(P<0.05)。免疫组化染色对含有98例NSCLC患者组织芯片(82例患者同时具有癌和癌旁组织)的组织切片进行OGFR和MOR蛋白定量检测,结果显示OGFR在72例患者肺癌组织中的表达量较癌旁正常组织显著降低(P<0.05)。组织切片中癌组织OGFR表达与NSCLC临床病理参数,如病理分化程度(P=0.046)、T分期(P=0.049)和临床分期(P=0.05)呈负相关,与年龄无相关性(P>0.05)。此外,通过Kaplan-Meier分析生存曲线表明OGFR低表达的患者生存率显著降低(P<0.05)。MOR在75例患者肺癌组织中的表达量较癌旁正常组织显著增加(P<0.05)。肺癌组织MOR表达与NSCLC患者病理分化程度(P=0.032)、T分期(P=0.043)、N分期(P<0.0001)和临床分期(P<0.0001)呈正相关,与年龄无相关性(P>0.05)。MOR表达较高的患者生存率显著降低(P=0.00011)。不同OGFR表达水平的癌组织中MOR表达量差异无统计学意义(P>0.05)。3、使用不同浓度(0n M、0.5n M、5n M、50n M和500n M)SUF对A549细胞进行处理,在6h、12h、24h和48h后观察细胞增殖能力,结果显示5n M及以上浓度的SUF对A549细胞增殖活性的抑制程度随药物处理时间的延长以及SUF浓度增高而显著增强。高表达OGFR的A549细胞系中,0.5～500n M所有浓度SUF均可抑制A549细胞的增殖能力,过表达OGFR可显著增强SUF对A549细胞增殖活性的抑制作用。OGFR沉默的A549细胞系中,在SUF干预12h后细胞增殖活性反而增加,但随着SUF的作用时间和作用浓度的增加,A549细胞增殖活性逐渐减低。其中给予500n M SUF 6h、24h和48 h后,50n M SUF 24h和48h后,5n M SUF 24h和48h后,和0.5n M SUF 48h后,A549细胞增殖活性显著下降。结论:1、OGFR在NSCLC细胞系中表达量较HLF细胞显著降低,在不同NSCLC细胞系表达也存在差异。OGFR抑制NSCLC细胞系的增殖、迁移、侵袭和细胞干性,促进凋亡,抑制PI3K/Akt信号通路活性。2、OGFR在NSCLC肿瘤组织中低表达,OGFR表达量与患者病理分化程度、T分期和临床分期呈负相关,OGFR低表达提示NSCLC预后不良。MOR在NSCLC肿瘤组织中高表达,MOR表达量与患者病理分化程度、T分期、N分期正相关,MOR高表达提示NSCLC预后不良。OGFR与MOR表达量无相关性。3、SUF对A549细胞增殖抑制作用随时间延长和剂量的增加而增强。OGFR促进SUF对A549细胞生长的抑制作用。更多还原

【Abstract】 Objective: Lung cancer is still the leading cause of cancer related death.Non-small cell lung cancer(NSCLC)accounts for more than 85% of the total lung cancer population.As the early clinical symptoms of NSCLC were not obvious,60%-70% of NSCLC patients have advanced disease at diagnosis,and followed with a poor prognosis.Therapeutic strategies for NSCLC,such as surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy show promising outcome.However,because of the genetic heterogeneity of NSCLC,the 5-year overall survival(OS)rate still remains poor.Opioid growth factor(OGF)-opioid growth factor receptor(OGFR)axis has recently received extensive attention due to its physiological anti-tumor properties in diverse human cancers.OGF,chemically termed [Met5]-enkephalin,is an important endogenous opioid peptide widely expressed in almost all human tissues.OGFR,an integral membrane protein associated with the nucleus,is expressed on or near the outer nuclear membrane.It contains specific nuclear localization signals(NLS).When OGF specifically binds to OGFR,the complex can be transported into the nucleus to regulate DNA synthesis,and plays an important role in tissue organization,cellular renewal,tumorigenesis and development,angiogenesis,wound healing and immunoregulation.OGF-OGFR axis has been widely identified in many human tumor cell lines and tumor tissues,including NSCLC.Studies suggested that the functional OGFR expression was significantly down-regulated in tumor cells or tissues.Moreover,the OGFR expression can regulate the malignant biological behaviors of tumors.Overexpression of OGFR can inhibit the proliferation of tumor cells,while OGFR silencing can promote the proliferation of tumor cells.There are limited studies focused on the effect of OGFR on NSCLC tumor progression and clinical prognosis.A large scale of studies have shown that opioids and opioid receptors might affect the occurrence,development and prognosis of NSCLC.Opioid receptors include μ opioid receptor(MOR),κ opioid receptor(KOR),δ opioid receptor(DOR)and zeta opioid receptor(OGFR).Studies have demonstrated that OGFR and MOR modulated the tumor malignant porgression,and interacted with opioids to affect the tumor outcome.MOR is highly expressed in NSCLC tumor tissues.The activation and overexpression of MOR affect the proliferation,migration and epithelial mesenchymal transition(EMT)of lung cancer cells.Previous study suggested that morphine can selectively combine to MOR to promote the growth of tumor cells.Moreover,morphine was found to interact with OGFR to inhibit the growth of NSCLC cells.At present,there is little relevant research on the effect of interaction of OGFR and other opioids on the growth of NSCLC tumor cells.There is still no research on the possible relationship between OGFR and MOR.Therefore,in this study OGFR was considered as a potential therapeutic target for NSCLC.We studied the regulation and the underlying molecular mechanism of OGFR expression on the malignant physiological behaviors of NSCLC tumor cells.We also detected the expression of OGFR and MOR in NSCLC tumor tissues and analyzed the correlation between OGFR or MOR expression and the clinicopathological factors and OS to illustrate the clinical siginifcance of both opioid receptors in modulation and prediction of the prognosis of NSCLC.We also studied the influence of sufentanil on the growth of NSCLC tumor cells with different expression levels of OGFR.Methods: 1.In this study,human lung fibroblast-like cell line(HLF),human NSCLC cell lines A549,H460,H322,H358 and H1299 were selected.The expressions of OGFR in different cell lines were detected using Western Blot analysis.Lentivirus were used to construct OGFR low-expression cell lines in H1299 and OGFR overexpression cell lines in A549.The transfection efficiency was evaluated using Western Blot analysis.MTT assay and plate clonony formation assay were used to detect transfected cell proliferation ability.The expressions of PI3K/Akt signaling pathway related proteins were detected using Western Blot analysis.AV/PI staining and FACS analysis were used to evaluate the apoptosis status of transfected cells.Immunofluorescence assay was used to detect the release of cytochrome c(Cyt-c)from mitochondria.The expression of apoptosis related proteins were also detected using Western Blot analysis.Wound scratch assay and Transwell assay were used to evaluate the migration and invasion ability of OGFR silencing tumor cells.EMT-related proteins were also detected by Western Blot analysis.Sphere formation assay and detection of cell stemness related proteins using Western Blot were employed to evaluate the tumor cell stemness.2.NSCLC tumor biopsies were obtained from 4 cases undergoing lobectomy at the First Affiliated Hospital of Dalian Medical University between November and December.Proteins were extracted and the OGFR expressions were detected using Western Blot analysis.Then a tissue microarray of 98 human lung adenocarcinoma cases was employed and immunohistochemical(IHC)method was performed to detect the expression level of OGFR and MOR.Chi square test was used to compare the expression of OGFR or MOR with different clinical pathological factors,such as age,pathological differentiation and clinical stages.Kaplan-Meier method was used to analyze the survival curve of NSCLC.Log rank test was used to compare the difference of OS of NSCLC patients with different levels of OGFR or MOR expression.The correlation between expressions of OGFR and MOR was evaluated as well.3.Lentivirus were used to construct OGFR overexpression cell lines and si RNA was used to construct OGFR low-expression cell lines in A549 cells.The transfection efficiency was evaluated using q RT-PCR.A549 cells were treated with different concentrations of sufentanil(0n M,0.5n M,5n M,50 n M,500 n M).6,12,24 and48 h later the effects of sufentanil on the proliferation of A549 cells were observed using CCK-8 method.Results:1.This study demonstrated that OGFR was down-regulated in NSCLC cell lines compared with HLF cell line(P<0.05),and in NSCLC cell lines the OGFR expressions were at various level.H1299 and H322 cells exhibited a relative higher expression,while H358,H460 and A549 cells showed a lower expression of OGFR.OGFR was knockdown using its specific sh RNAs in H1299 cells and overexpressed with OGFR overexpression vectors in A549 cells.MTT assay and plate colony formation assay were done and found that OGFR silencing enhanced cell proliferation in H1299 cells,while OGFR overexpression inhibited cell proliferation in A549 cells compared to control groups(P<0.05).PI3K/Akt signaling pathway related proteins p110α,p110β and p-Akt were found decreased in OGFR over-expressed A549 cells,and increased in OGFR silencing H1299 cells.AV/PI staining and FACS analysis were used to evaluate the apoptotic status of transfected H1299 cells,and the results suggested that OGFR knockdown significantly inhibited the apoptosis of H1299 cells.Immunofluorescence assay showed decreased release of Cyt-c in the OGFR silencing cells(P<0.05).Consistently the expression of pro-apoptotic protein cleaved caspase-7,cleaved caspase-9 and cleaved poly ADP-ribose polymerase(PARP)were significantly decreased using Western Blot analysis(P<0.05).Wound scratch assay and Transwell assay were done in transfected H1299 cells,and demonstrated that OGFR knockdown significantly enhanced the migration and invasion abilities of NSCLC cells(P<0.05).The expressions of EMT-related markers E-cadherin was down-regulated(P<0.05),while N-cadherin and Vimentin were both up-regulated(P<0.05).The tumor sphere formation assay showed that OGFR silencing significantly increased the sphere formation number and size.OGFR knockdown also increased the expression of the stemness markers CD133,CD44 and OCT4 evaluated by Western Blot analysis(P<0.05).2.We detected OGFR expression in tumor specimens collected from 4 human NSCLC cases undergoing thoracoscopic surgery using Western Blot analysis.The results indicated that OGFR expression was down-regulated in tumor tissues relative to adjacent normal lung tissues(P<0.05).A NSCLC tissue microarray with a cohort of 98 cases was also employed to evaluate OGFR or MOR expression using IHC assay.82 cases owned both tumor tissues and corresponding adjacent normal tissues.The results indicated that OGFR was down-expressed in tumor tissues relative to normal lung tissues in 72 cases(P<0.05).The relationship between OGFR expression in tumor tissues and corresponding clinicopathologic characteristics of NSCLC cases were analyzed.The data illustrated that OGFR expression was negative relevant to the pathological differentiation(P=0.046),T stage(P=0.049)and clinical stage(P=0.05),but not age(P>0.05).In addition,the Kaplan-Meier method was used to analyze the life curve and indicated that the patients with lower expression of OGFR owned lower OS rate(P<0.05).75 cases of NSCLC tissue microarray demonstrated higher expression level of MOR in tumor tissues compared to normal tissues(P<0.05).The MOR expression was positively relevant to the pathological differentiation(P=0.032),T stage(P=0.043),N stage(P<0.0001)and clinical stage(P<0.0001),but not age(P>0.05).The patients with higher expression of MOR owned lower OS rate(P=0.00011).There was no significant difference of MOR expression in tumor tissues with different OGFR expression(P=0.105).3.Different concentration of sufentanil(0n M,0.5n M,5n M,50 n M and 500 n M)were used to treat A549 cells,and the proliferation abilities of A549 cells were evalulated 6h,12 h,24h and48 h after medication.The results suggested that sufentanil at the concentration of 5n M or above showed inhibition role on the prolliferation of A549 cells,which was enhanced as the medication duration and concentration increased.In OGFR overexpression A549 cell line,sufentanil at all the concentration from 0.5n M to 500 n M inhibited cell proliferative abilities.Overexpression of OGFR enhanced the inhibition role of sufentanil.In OGFR silencing A549 cell line,the proliferation abilities of transfected cells increased 12 h after the treatment of sufentanil,and gradually decreased as the concentration of sufentanil and treatment time increasing.Sufentanil exhibited the inhibition role on cell proliferation at the concentration of 500 n M 6h,24 h,48h later,50 n M and 5n M under 24 h and 48 h treatment time,and 0.5n M with treatment time of 48 h.Conclusion:1.The expressions of OGFR are low in NSCLC cell lines relative to HLF cell,and varied in different NSCLC cell lines.OGFR can inhibit the proliferation,migration,invasion and cell stemness of NSCLC cell lines,and promote apoptosis.The PI3K/Akt pathway might be involved in the regulation of OGFR on the growth of NSCLC cells.2.The expressions of OGFR are low in NSCLC tumor tissues compared to adjacent normal tissues.The expression of OGFR is negatively correlated with malignant biological characteristics and OS rate.The low expression of OGFR indicates poor prognosis of NSCLC.MOR is highly expressed in NSCLC tumor tissues.The expressions of MOR are positively correlated with malignant biological characteristics and OS rate of NSCLC.The high expression of MOR indicates poor prognosis of NSCLC.There was no correlation between the expression of OGFR and MOR in NSCLC tumor tissues.3.Sufentanil exhibits a suppression role on the proliferation of A549 cells,and the influence is enhanced as the medication duration and concentration increased.OGFR facilitates the supression of sufentanil on the proliferation of A549 cells.更多还原