【作者】 梁琨；

【导师】 胡克； 孙德俊；

【作者基本信息】 武汉大学 ， 呼吸内科学， 2018， 博士

【摘要】 研究背景肺癌是临床常见的恶性肿瘤之一,同时是全球死亡率最高的癌症。近年来,肺癌在中国的发生率逐年提高。NSCLC是肺癌的主要形式,大约80～85%的肺癌患者被诊断为NSCLC。尽管临床上治疗NSCLC的手段较为先进,但是NSCLC晚期患者不适合接受手术根治,导致其死亡率仍然居高不下。虽然临床研究多数着重于肺癌的发生和转移的机制,但是对于癌细胞的转移过程,目前尚未有详细而明确的报导。因此,临床急需发现新的生物标记物来预测NSCLC,以及筛选有效的治疗肺癌的方法。在第一部分的实验中,我们通过收集临床40例接受手术治疗的NSCLC患者的肿瘤组织及其癌旁组织,检测CXCL16及CXCR6在组织中的表达水平;同时检测患者血清中炎性相关因子的水平,并分析CXCL16/CXCR6与血清炎性因子及临床特征的关系。在第二部分的实验中,我们通过在肺癌细胞中敲低CXCR6或抑制NF-κB来检测CXCL16/CXCR6与NF-κB之间的相互作用对于肺癌细胞的生物学特性的影响,从而探讨CXCL16/CXCR6对于肺癌的发生和转移的作用。第一部分CXCL16/CXCR6在肺癌中的表达及其与临床特征的关系1.1目的通过检测40例NSCLC患者的肿瘤组织和癌旁组织中的CXCL16/CXCR6表达水平、并检测患者血清中炎性相关因子的水平,从而探讨CXCL16/CXCR6与血清炎性因子及临床特征之间的关系。1.2方法1.2.1临床资料的收集收集40例长期居住在内蒙古地区NSCLC患者的临床资料,包括:男性27例,女性13例;年龄≤65岁者25例,>65岁者15例;平均年龄60.43±10.86岁,抽取患者血样本进行指标检测。1.2.2肿瘤标本的收集40例患者术前未接受过任何化疗或放射治疗。40例患者均接受手术切除肿瘤组织及癌旁组织,统一送至病理科制作组织切片,确定肿瘤组织学分型及临床病理分期,包括:Ⅰ期5例、Ⅱ期7例、Ⅲ期19例、Ⅳ9例;鳞癌(squamous cell carcinoma SC)12 例、腺癌(adenocarcinoma,AC)20 例、腺鳞癌(adenosquamous carcinoma,ASC)8例;淋巴结转移27例,无淋巴结转移13例;胸膜浸润4例,无胸膜浸润36例。1.2.3免疫组化(IHC)使用IHC的方法检测肿瘤组织中CXCL16和CXCR6的阳性率。1.2.4 苏木素伊红(hematoxylin and eosin,HE)染色使用HE染色试剂盒进行组织HE染色。1.2.5采血及血清炎性相关因子检测术前、术后,40例NSCLC患者均接受空腹静脉采血并分离血清,使用酶联免疫法(enzyme-linked immunosorbent assay,ELISA)检测试剂盒进行血清炎性因子水平的检测,包括:高迁移率族蛋白B1(high-mobility group box 1,HMGB1)、白介素-6(interleukin-6,IL-6)、白介素-10(interleukin,IL-10)和白介素-1β(interleukin-1 β,IL-1β)。1.3结果1.3.1 NSCLC患者肿瘤及癌旁组织CXCL16/CXCR6的表达程度40例NSCLC患者肿瘤组织的CXCL16表达情况为:强阳性(+++)20例(50.0%),阳性(++)10 例(25.0%),弱阳性(+)8 例(20.0%),阴性(-)2例(5.0%);癌旁组织中,CXCL16表达情况为:弱阳性(+)7例(17.5%),阴性(-)33例(82.5%)。肿瘤组织CXCL16的阳性率明显高于癌旁组织(95.0%v.s.17.5%),差异具有明显的统计学意义(t=14.859,P<0.05)。40例NSCLC患者肿瘤组织的CXCR6表达情况为:强阳性(+++)14例(35.0%),阳性(++)12 例(30.0%),弱阳性(+)8 例(20.0%),阴性 6例(-)(15.0%);癌旁组织中,CXCR6表达情况为弱阳性(+)4例(10.0%),阴性(-)36例(90.0%)。肿瘤组织CXCR6的阳性率明显高于癌旁组织(85.0%v.s.10.0%),差异具有明显的统计学意义(t=10.729,P<0.05)。40例NSCLC患者肿瘤组织CXCL16的表达强度明显高于CXCR6(t=4.583,P<0.05),且 CXCL16 与 CXCR6 的表达呈现正相关(r=0.894,95%CI=0.843～0.934,P<0.05)。1.3.2肿瘤组 CXCL16/CXCR6与NSCLC患者血清炎性相关因子水平的相关性40 例 NSCLC 患者术后的血清 HMGB1(t=27.189,P<0.05)、IL-1β(t=27.121,P<0.05)、IL-6(t=13.966,P<0.05)和 IL-10(t=12.599,P<0.05)的水平均明显低于术前。肿瘤组织CXCL16强阳性(+++)和阳性(++)NSCLC患者的术前血清HMGB1 水平明显高于 CXCL16 弱阳性(+)NSCLC 患者(F=4.476,P=0.009);肿瘤组织CXCR6强阳性(+++)和阳性(++)NSCLC患者的术前血清HMGB1水平明显高于CXCR6弱阳性(+)和阴性(-)NSCLC患者(F=4.476,P=0.009)。40例NSCLC患者的肿瘤组织CXCL16水平与血清HMGB1水平(r=0.479,95%CI=0.261～0.678,P<0.05)、CXCR6 水平与血清 HMGB1 水平(r=0.539,95%CI=0.308～0.715,P<0.05)均呈现正相关。肿瘤组织CXCL16强阳性(+++)NSCLC患者的术前血清IL-10水平明显低于肿瘤组织CXCL16阳性(++)NSCLC患者(P<0.05)。1.3.3肿瘤组织CXCL16/CXCR6与NSCLC患者临床特征的相关性对于不同年龄的NSCLC患者,>65岁的NSCLC患者,其CXCL16多数呈现强阳性(+++),而≤65岁的NSCLC患者,其CXCL16呈现强阳性(+++)和阳性(++)的数量相当(F=4.775,P=0.035)。CXCL16的表达强度与年龄呈正相关(r=0.334,95%CI=0.036～0.585,P=0.035)。对于是否具有吸烟史的NSCLC患者,CXCL16(F=13.096,P=0.001)和CXCR6(F=17.136,P<0.001)的阴阳性分布均存在明显的差异。CXCL16(r=0.506,95%CI=0.217～0.738,P<0.001)和 CXCR6(r=0.557,95%CI=0.281～0.751,P<0.001)的表达强度均与吸烟史呈正相关。对于不同TNM分期的NSCLC患者,CXCL16(F=9.129,P<0.001)和CXCR6(F=8.187,P<0.001)的阴阳性分布均存在明显的差异。CXCL16(r=0.483,95%CI=0.245～0.679,P=0.002)和 CXCR6(r=0.553,95%CI=0.346～0.733,P<0.001)的表达强度均与TNM分期呈正相关。对于具有不同肿瘤病理分型的NSCLC患者,CXCL16(F=8.735,P<0.001)和CXCR6(F=9.815,P<0.001)的阴阳性分布均存在明显的差异。CXCL16(r=0.531,95%CI=0.293～0.699,P<0.001)和 CXCR6(r=0.552,95%CI=0.309～0.749,P<0.001)的表达强度均与SC、AC、ASC呈正相关。对于是否出现淋巴结转移的NSCLC患者,CXCL16(F=22.531,P<0.001)和CXCR6(F=22.022,P<0.001)的阴阳性分布均存在明显的差异。CXCL16(r=0.610,95%CI=0.355～0.816,P<0.001)和 CXCR6(r=0.606,95%CI=0.338～0.807,P<0.001)的表达强度均与淋巴结转移呈正相关。对于是否出现胸膜浸润的NSCLC患者,CXCR6(F=5.695,P=0.022)的阴阳性分布存在明显的差异。CXCR6的表达强度与胸膜浸润呈正相关(r=0.361,95%CI=0.180～0.530,P=0.022)。1.3.4 NSCLC患者的临床特征之间的相关性NSCLC 患者的性别与吸烟史(r=-0.414,95%CI=-0.701～-0.102,P=0.008)呈负相关,与 TNM 分期(r=0.322,95%CI=0.083～0.535,P=0.043)、肿瘤病理分型(r=0.328,95%CI=0.029～0.582,P=0.039)、淋巴结转移(r=0.368,95%CI=0.097～0.568,P=0.020)呈正相关;TNM分期与肿瘤病理分型(r=0.855,95%CI=0.706～0.954,P<0.001)、淋巴结转移(r=0.771,95%CI=0.564～0.891,P<0.001)、胸膜浸润均(r=0.431,95%CI=0.225～0.594,P=0.005)呈正相关;肿瘤病理分型与淋巴结转移(r=0.704,95%CI=0.581～0.851,P<0.001)、胸膜浸润(r=0.524,95%CI=0.285～0.691,P=0.001)呈正相关。1.3.5 NSCLC患者的临床特征与血清炎性相关因子水平的相关性NSCLC患者的术前血清HMGB1水平与性别(r=0.371,95%CI=-0.004～0.094,P=0.018)、TNM分期(r=0.893,95%CI=0.815～0.936,P<0.001)、肿瘤病理分型(r=0.683,95%Cr=0.465～0.862,P<0.001)、淋巴结转移(r=0.800,95%CI=0.703～0.892,P<0.001)、胸膜浸润(r=0.343,95%CI=0.168～0.501,P=0.030)呈正相关。1.4结论NSCLC患者肿瘤组织中CXCL16/CXCR6呈现高表达,其表达强度与NSCLC患者的年龄、吸烟史、TNM分期、肿瘤病理分型、淋巴结转移、胸膜浸润、以及血清炎性相关因子水平存在一定的相关性。CXCL16/CXCR6的表达强度可以作为NSCLC发展进程及肿瘤转移的生物指标。第二部分 CXCL16/CXCR6与NF-κB之间的相互作用对肺癌细胞生物学特性的影响2.1目的通过在肺癌细胞中敲低CXCR6或抑制NF-κB来检测CXCL16/CXCR6与NF-κB之间的相互作用对于肺癌细胞的生物学特性的影响。2.2方法2.2.1细胞培养使用含有10%胎牛血清(fetal bovine serum,FBS)、100UI//ml青霉素、100μg/ml链霉素的RPMI-1640培养基(完全培养基)培养人肺腺癌A549细胞和人肺鳞癌NCI-H520 细胞。2.2.2 沉默 CXCR6使用 CXCR6 小干扰 RNA(small interfering RNA,siRNA)转染 A549 和NCI-H520 细胞。2.2.3 抑制 NF-κB使用JSH-23阻断A549和NCI-H520细胞中的NF-κB。2.2.4蛋白检测使用蛋白免疫印迹(Western blotting,WB)检测A549和NCI-H520细胞中的 CXCL16、CXCR6、p105、c-Rel 和 Rel-B 的表达水平。2.2.5 CXCL16蛋白定位使用免疫荧光(immunofluorescence,IF)定位A549和NCI-H520细胞中的CXCL16 蛋白。2.2.6 CXCL16蛋白分泌检测使用人CXCL16 ELISA试剂盒对A549和NCI-H520细胞上清的CXCL16分泌量进行检测。设置细胞CXCL16分泌量检测天数为连续的5天。2.2.7细胞增殖检测使用细胞计数试剂盒-8(cell counting kit-8,CCK-8)对 A549 和 NCI-H520细胞的增殖状况进行检测。设置细胞增殖检测天数为连续的5天。2.2.8细胞凋亡和周期检测使用细胞凋亡与细胞周期检测试剂盒对A549和NCI-H520细胞的凋亡状况和细胞周期进行检测。2.2.9细胞侵袭能力检测使用Transwell法对A549和NCI-H520细胞的侵袭能力进行检测。2.3结果2.3.1沉默CXCR6或抑制NF-κB对肺癌细胞CXCL16/CXCR6和NF-κB相关蛋白表达的影响沉默CXCR6不但降低了 A549和NCI-H520细胞中的CXCR6蛋白的表达,同时降低了 CXCL16和NF-κ相关蛋白(p105、c-Rel、Rel-B)的表达;抑制NF-κB不但降低了 A549和NCI-H520细胞中的p105、c-Rel和Rel-B蛋白的表达,同时降低了 CXCL16和CXCR6蛋白的表达。2.3.2沉默CXCR6或抑制NF-κB对肺癌细胞CXCL16蛋白表达和定位的影响Control组中,CXCL16聚集在A549和NCI-H520细胞的细胞膜上,说明CXCL16 为 tCXCL16;si-CXCL16 和 in-NF-κB 组显示,沉默 CXCR6 和抑制 NF-κB均能够降低A549和NCI-H520细胞的tCXCL16的表达。2.3.3沉默CXCR6或抑制NF-κB对肺癌细胞CXCL16蛋白分泌的影响对于 A549 细胞,si-CXCR6(r=0.997,95%CI=0.995～0.999,P<0.05)、in-NF-κB(r＝0.995,95%CI=0.991～0.998,P<0.05)、Control(r=0.992,95%CI=0.986～0.997,P<0.05)三组的CXCL16蛋白分泌量随着时间的增加明显递增,差异具有统计学意义;对比三组间的差异,从24h到120h,Control组A549细胞的CXCL16蛋白分泌量明显高于si-CXCR6和in-NF-KB组(F=128.195,P<0.05)。对于 NCI-H520 细胞,siCXCR6(r=0.997,95%CI=0.995～0.999,P<0.05)、in-NF-κB(r=0.968,95%CI=0.956～0.989,P<0.05)、Control(r=0.991,95%CI=0.985～0.996,P<0.05)三组的CXCL16蛋白分泌量随着时间的增加明显递增,差异具有统计学意义;对比三组间的差异,从24h到120h,Control组NCI-H520细胞的CXCL16蛋白分泌量明显高于si-CXCR6和in-NF-κB组(F=73.201,P<0.05)。2.3.4沉默CXCR6或抑制NF-κB对肺癌细胞增殖的影响对于 A549 细胞,siCXCR6(r=0.978,95%CI=0.964～0.992,P<0.05)、in-NF-κB(r=0.975,95%CI=0.945～0.993,P<0.05)、Control(r=0.976,95%CI=0.969～0.991,P<0.05)三组的细胞光密度(optical density,OD)值随着时间的增加明显递增,差异具有统计学意义;对比三组间的差异,从第一天到第五天,Control组的A549细胞增殖量明显高于si-CXCR6和in-NF-κB组(F=36.993,P<0.05)。对于 NCI-H520 细胞,siCXCR6(r=0.970,95%CI=0.949～0.988,P<0.05)、in-NF-κB(r=0.985,95%CI=0.972～0.995,P<0.05)、Control(r=0.987,95%CI=0.981～0.995,P<0.05)三组的细胞OD值随着时间的增加明显递增,差异具有统计学意义;对比三组间的差异,从第一天到第五天,Control组的NCI-H520 细胞增殖量明显高于 si-CXCR6 和 in-NF-κB 组(F=18.448,P<0.05)。2.3.5沉默CXCR6或抑制NF-κB对肺癌细胞侵袭能力的影响组间对比显示,无论是对于A549(F=241.422,P<0.05)还是NCI-H520(F=74.581,P<0.05)细胞,si-CXCR6、in-NF-κB、Control 三组的侵袭性细胞计数均值之间都存在明显的差异,其中,Control组的侵袭性细胞计数均值都明显高于 si-CXCR6 和 in-NF-κB 组。对比A549和NCI-H520两种细胞之间的侵袭能力,si-CXCR6(F=22.050,P<0.05)和Control(F=70.225,P<0.05)组的侵袭性细胞计数均值都存在明显的差异。2.3.6沉默CXCR6或抑制NF-κB对肺癌细胞凋亡和周期的影响2.3.6.1沉默CXCR6或抑制NF-κB对肺癌细胞凋亡的影响组间对比显示,无论是对于A549(F=59.242,P<0.05)还是NCI-H520(F=40.721,P<0.05)细胞,si-CXCR6、in-NF-κB、Control 三组的细胞凋亡率均值之间均具有明显的差异,且Control组的细胞凋亡率均值明显低于si-CXCR6和 in-NF-κB 组。对比A549和NCI-H520两种细胞之间的凋亡率,Control组(F=8.863,P=0.041)的细胞凋亡率均值存在明显的差异。2.3.6.2沉默CXCR6或抑制NF-κB对肺癌细胞周期的影响A549 细胞中,处于 G1(F=696.087,P<0.05)期和 S(F=230.266,P<0.05)期的细胞数目比例、以及NCI-H520细胞中处于G1(F=427.251,P<0.05)期和S(F=255.513,P<0.05)期的细胞数目比例在 si-CXCR6、in-NF-κB、Control 三组之间存在明显的差异;A549和NCI-H520细胞的Control组中,处于G1期的细胞数目比例明显低于si-CXCR6和in-NF-KB组(P<0.05),而Control组中,处于S期的细胞数目比例明显高于si-CXCR6和in-NF-κB组(P<0.05)。对比 A549 和 NCI-H520 细胞,si-CXCR6(G1:F=23.287,P=0.008;S:F=0.056,P＝0.825)、in-NF-κB(G1:F=11.471,P=0.028;S:F=2.816,P=0.169)、Control(G1:F＝44.518,P=0.003;S:F=15.249,P=0.017)三组中,处于 G1 期的细胞数目比例在两种细胞之间存在明显的差异,而处于S期的细胞数目比例只在Control组中存在明显的差异。2.4结论CXCL16/CXCR6通过与NF-κB相互调节,促进肺癌的发展和转移。更多还原

【Abstract】 BackgroundsLung cancer is one of the common malignant tumors clinically,with the highest mortality of cancer in the world.In China,lung cancer incidence increased gradually in the last few years.NSCLC is the major type of lung cancer,accounting for 80～85%.Despite the advanced treatments clinically,the patients with advanced lung cancer are not candidates for curative surgery,leading to remaining high mortality.There was not enough and clear evidence on the mechanisms for the progression of lung cancer metastasis,though many investigations focused on the pathogenesis and metastasis of lung cancer.Therefore,new biomarkers are needed to use in prognosis and to treat lung cancer.In the first part,we collected tumorous and adjacent tissues from 40 NSCLC patients.By measuring CXCL16 and CXCR6 expressions and detecting inflammatory factors’ level,we analyzed the association of CXCL16/CXCR6 with inflammatory factors and clinical characteristics.In the second part,we silenced CXCR6 or inhibited NF-κB for confirming the co-interaction between CXCL16/CXCR6 and NF-κB on the biological characteristics of lung cancer cells,in order to investigate the influences of CXCL16/CXCR6 on lung tumor pathogenesis and metastasis.Part Ⅰ.Association of CXCL16/CXCR6 with clinical characteristics1.1 ObjectivesBy detecting CXCL16/CXCR6 expression and serum inflammatory factors’ level,as well as analyzing the clinical data from 40 NSCLC patients,investigated the association of CXCL16/CXCR6 with inflammation and clinical characteristics.1.2 Methods1.2.1 Clinical data collectionThe clinical data of 40 patients who lived in NSCLC in Inner Mongolia were collected.27 males and 13 females were included,including 25 patients lower than 65 years old and 15 patients higher than 65 years old,average age 60.43±10.86 years old.1.2.2 Tumor samples collection40 NSCLC patients were without any radiotherapy or chemotherapy.All the patients received surgical resection,and the tumorous and adjacent tissues were sent to Pathology for confirming pathological stage,including 5 in stage Ⅰ,7 in stage Ⅱ,19 in stage Ⅲ and 9 in stage Ⅳ,as well as 12 squamous cell carcinoma(SC),20 adenocarcinoma(AC)and 8 adenosquamous carcinoma(ASC).27 patients were with lymphatic metastasis,while 13 were without.4 patients were with pleural invasion,while 36 were without.1.2.3 IHCCXCL16 and CXCR6 positive rates were measured by IHC.1.2.4 Hematoxylin and eosin(HE)stainingHE staining was carried on with HE staining kit.1.2.5 Blood samples collection and inflammatory factors detectionBefore and after surgery,venous blood samples were obtained from 40 NSCLC patients.Serum was separated and enzyme-linked immunosorbent assay(ELISA)was for detecting serum inflammatory factors’ level,including high-mobility group box 1(HMGB1),interleukin-6(IL-6),interleukin-10(IL-10)and interleukin-1β(IL-1β).1.3 Results1.3.1 CXCL16/CXCR6 expression in tumorous and adjacent tissues from NSCLC patientsCXCL16 expression in NSCLC tumor tissues:20(50.0%)of strong positive(+++),10(25.0%)of moderate positive,8(20.0%)of weak positive,and 2(5.0%)of negative.CXCL16 expression in adjacent tissues:7(17.5%)of weak positive(+++),and 33(82.5%)of negative.CXCL16 expression in tumor tissues was higher than that in adjacent tissues(95.0%v.s.17.5%),significantly(t=14.859,P<0.05)CXCR6 expression in NSCLC tumor tissues:14(35.0%)of strong positive(+++),12(30.0%)of moderate positive,8(20.0%)of weak positive,and 6(15.0%)of negative.CXCL16 expression in adjacent tissues:4(10.0%)of weak positive(+++),and 36(90.0%)of negative.CXCL16 expression in tumor tissues was higher than that in adjacent tissues(85.0%v.s.10.0%),significantly(t=10.729,P<0.05)CXCL16 expression in NSCLC tumor tissues was higher than CXCR6(t=4.583,P<0.05).CXCL16 expression was positive with CXCR6 in NSCLC tumor tissues(r=0.894,95%CI=0.843～0.934,P<0.05)1.3.2 Correlation of tumor CXCL16/CXCR6 with serum inflammatory factors in NSCLC patientsAfter surgery,serum HMGB1(t=27.189,P<0.05),IL-1β(t=27.121,P<0.05),IL-6(t=13.966,P<0.05)and IL-10(t=12.599,P<0.05)levels fromg 40 NSCLC patients decreased significantly.Serum HMGB1 in NSCLC patients with strong positive(+++)and moderate positive(++)CXCL16 was higher than that with weak positive(+),significantly(F=4.476,P=0.009).Serum HMGB1 in NSCLC patients with strong positive(+++)and moderate positive(++)CXCR6 was higher than that with weak positive(+)and negative(-),significantly(F=4.476,P=0,009).Serum HMGB1 in NSCLC patients was positively correlated with tumor CXCL16(r=0.479,95%CI=0.261～0.678,P<0.05)and CXCR6(r=0.539,95%CI=0.308～0.715,P<0.05)levels.Serum IL-10 in NSCLC patients with strong positive(+++)CXCL16 was lower than that with moderate positive(++),significantly(P<0.05).1.3.3 Correlation of tumor CXCL16/CXCR6 with clinical characteristics from NSCLC patientsFor patients with different age,there was difference on CXCL16(F=4.775,P=0.035)distributions in NSCLC tumor,significantly.Tumor CXCL16 was positive correlated with the age of NSCLC patients(r=0.334,95%CI=0.036～0.585,P=0.035).For patients with or without smocking history,there was difference on CXCL16(F=13.096,P=0.001)and CXCR6(F=17.136,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.506,95%CI=0.217～0.738,P<0.001)and CXCR6(r=0.557,95%CI=0.281～0.751,P<0.001)were positively correlated with smocking history of NSCLC patients.For patients in different TNM stages,there was difference on CXCL16(F=9.129,P=0.001)and CXCR6(F=8.187,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.483,95%CI=0.245～0.679,P=0.002)and CXCR6(r=0.553,95%CI=0.346～0.733,P<0.001)were positively correlated with TNM stages of NSCLC patients.For patients with different tumor pathological types,there was difference on CXCL16(F=8.735,P<0.001)and CXCR6(F=9.815,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.531,95%CI=0.293～0.699,P<0.001)and CXCR6(r=0.552,95%CI=0.309～0.749,P<0.001)were positively correlated with SC,AC and ASC.For patients with or without lymphatic metastasis,there was difference on CXCL16(F=22.531,P<0.001)and CXCR6(F=22.022,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.610,95%CI=0.355～0.816,P<0.001)and CXCR6(r=0.606,95%CI=0.338～0.807,P<0.001)were positively correlated with lymphatic metastasis.For patients with or without pleural infiltration,there was difference on CXCR6(F=5.695,P=0.022)distributions in NSCLC tumor,significantly.Tumor CXCR6(r=0.361,95%CI=0.180～0.530,P=0.022)was positively correlated with pleural infiltration.1.3.4 Correlations among clinical characteristics of NSCLC patientsGender of NSCLC patients was negatively correlated with smocking history(r=-0.414,95%CI=-0.701～0.102,P=0.008),but positively correlated with TNM stages(r=0.322,95%CI=0.083～0.535,P=0.043),tumor pathological type(r=0.328,95%CI=0.029-0.582,P=0.039)and lymphatic metastasis(r=0.368,95%CI=0.097～0.568,P=0.020).TNM stage of NSCLC patients was positively correlated with tumor pathological type(r=0.855,95%CI=0.706-0.954,P<0.001),lymphatic metastasis(r=0.771,95%CI=0.564～0.891,P<0.001)and pleural infiltration(r=0.431,95%CI=0.225～0.594,P=0.005).Tumor pathological type of NSCLC patients was positively correlated lymphatic metastasis(r=0.704,95%CI=0.581～0.851,P<0.001)and pleural infiltration(r=0.524,95%CI=0.285～0.691,P=0.001).1.3.5 Correlation of clinical characteristics with serum inflammatory factors in NSCLC patientsSerum HMGB1 of NSCLC patients was positively correlated with age(r=0.371,95%CI=-0.004～0.094,P=0.018),TNM stages(r=0.893,95%CI=0.815～0.936,P<0.001),tumor pathological types(r=0.683,95%CI=0.465-0.862,P<0.001),lymphatic metastasis(r=0.800,95%CI=0.703～0.892,P<0.001)and pleural infiltration(r=0.343,95%CI=0.168～0.501,P=0.030).1.4 ConclusionsCXCL16/CXCR6 expressed highly in tumor tissues,which was correlated with age,smocking history,TNM stages,tumor pathological types,lymphatic metastasis,pleural infiltration and serum inflammatory factors of NSCLC patients.CXCL16/CXCR6 could be the biomarkers in progression and metastasis of NSCLC.Part Ⅱ.Influence of co-interaction between CXCL16/CXCR6 and NF-κB on biological characters of lung cancer cells2.1 ObjectivesInvestigated the Influence of co-interaction between CXCL16/CXCR6 and NF-κB on biological characters of lung cancer cells by silencing CXCR6 or inhibiting NF-κB.2.2 Methods2.2.1 Cell culturingLung AC A549 cell and lung SC NCI-H520 cell were cultured with RPMI-1640 containing 10%fetal bovine serum(FBS),100UI/ml penicillin and 100μg/ml streptomycin.2.2.2 Silencing CXCR6Small interfering RNA(siRNA)targeting CXCR6 was used to transfect A549 and NCI-H520 cells.2.2.3 Inhibiting NF-κBJSH-23 was used to inhibit NF-κB in A549 and NCI-H520 cells.2.2.4 Proteins detectionCXCL16,CXCR6,p105,c-Rel and Rel-B levels in A549 and NCI-H520 cells were measured by western blotting(WB).2.2.5 CXCL16 locationImmunofluorescence(IF)was used to locate CXCL16 in A549 and NCI-H520 cells.2.2.6 Secreting CXCL16 detectionCXCL16 secretion in supernatant of A549 and NCI-H520 cells was measured by ELISA kit for 5 days.2.2.7 Cell proliferation detectionProliferation of A549 and NCI-H520 cells was measured by cell counting kit-8(CCK-8)for 5 days.2.2.8 Cell apoptosis and cell cycle detectionsCell apoptosis and cell cycle kit was used to measured A549 and NCI-H520 cells.2.2.9 Cell invasion detectionTranswell assay was used to detected invasion of A549 and NCI-H520 cells.2.3 Results2.3.1 Effect of silencing CXCR6 or inhibiting NF-κB on CXCL16/CXCR6 and related proteins expressions in NF-κBSilencing CXCR6 not only decreased CXCR6 expression in A549 and NCI-H520 cells,but also suppressed CXCL16 and related proteins expressions in NF-κB(p105,c-Rel,Rel-B).Inhibiting NF-κB not only decreased p105,c-Rel and Rel-B in A549 and NCI-H520 cells,but also suppressed CXCL16 and CXCR6 expressions.2.3.2 Effect of silencing CXCR6 or inhibiting NF-κB on CXCL16 expression and locationIn Control group,CXCL16 accumulated on cytomembrane of A549 and NCI-H520 cells,indicating CXCL16 showed as tCXCL16.In si-CXCL16 and in-NF-KB groups,silencing CXCR6 and inhibiting NF-κB both decreased tCXCL16 expression in A549 and NCI-H520 cells.2.3.3 Effect of silencing CXCR6 or inhibiting NF-κB on CXCL16 secretionIn A549 cell,CXCL16 secretion in si-CXCR6(r=0.997,95%CI=0.995～0.999,P<0.05),in-NF-κB(r=0.995,95%CI=0.991～0.998,P<0.05)and Control(r=0.992,95%CI=0.986-0.997,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from 24h to 120h,CXCL 16 secretion in Control group was higher than that in si-CXCR6 and in-NF-κB groups,significantly(F=128.195,P<0.05).In NCI-H520 cell,CXCL16 secretion in si-CXCR6(r=0.997,95%CI=0.995～0.999,P<0.05),in-NF-κB(r=0.968,95%CI=0.956～0.989,P<0.05)and Control(r=0.991,95%CI=0.985-0.996,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from 24h to 120h,CXCL 16 secretion in Control group was higher than that in si-CXCR6 and in-NF-κB groups,significantly(F=73.201,P<0.05).2.3.4 Effect of silencing CXCR6 or inhibiting NF-κB on lung cell proliferationA549 cell proliferation in si-CXCR6(r=0.978,95%CI=0.964～0.992,P<0.05),in-NF-κB(r=0.975,95%CI=0.945～0.993,P<0.05)and Control(r=0.976,95%CI=0.969-0.991,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from the 1st day to the 5nd day,optical density(OD)value in Control group was higher than that in si-CXCR6 and in-NF-κB groups,significantly(F=36.993,P<0.05).NCI-H520 cell proliferation in si-CXCR6(r=0.970,95%CI=0.949～0.988,P<0.05),in-NF-κB(r=0.985,95%CI=0.972～0.995,P<0.05)and Control(r=0.987,95%CI=0.981～0.995,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from the 1st day to the 5nd day,optical density(OD)value in Control group was higher than that in si-CXCR6 and in-NF-κB groups,significantly(F=18.448,P<0.05).2.3.5 Effect of silencing CXCR6 or inhibiting NF-κB on lung cell invasionComparing between groups,no matter in A549 cell(F=241.422,P<0.05)or in NCI-H520 cell(F=74.581,P<0.05),there were significant differences among si-CXCR6,in-NF-κB and Control groups.Meanwhile,invasive cells in Control group were more than that in si-CXCR6 and in-NF-κB groups,significantly(P<0.05).Comparing between A549 and NCI-H520 cells,there were significant differences on invasive cells in si-CXCR6(F=22.050,P<0.05)and Control(F=70.225,P<0.05)groups.2.3.6 Effect of silencing CXCR6 or inhibiting NF-κB on lung cell apoptosis and cell cycle2.3.6.1 Effect of silencing CXCR6 or inhibiting NF-κB on lung cell apoptosisComparing between,no matter in A549 cell(F=59.242,P<0.05)or in NCI-H520 cell(F=40.721,P<0.05),there were significant differences among si-CXCR6,in-NF-κB and Control groups.Meanwhile,apoptosis rate in Control group was lower than that in si-CXCR6 and in-NF-κB groups,significantly(P<0.05).Comparing between A549 and NCI-H520 cells,there were significant differences on apoptosis rate in Control(F=8.863,P=0.041)group.2.3.6.2 Effect of silencing CXCR6 or inhibiting NF-κB on lung cell cycleA549 cells in G1(F=696.087,P<0.05)and S(F=230.266,P<0.05),and NCI-H520 cells in G1(F=427.251,P<0.05)and S(F=255.513,P<0.05)showed significant differences among si-CXCR6,in-NF-κB and Control groups.In Control group,A549 and NCI-H520 cells in G1 was lower than that in si-CXCR6 and in-NF-κB groups,while that in S was higher than that in si-CXCR6 and in-NF-κB groups,significantly(P<0.05)Comparing between A549 and NCI-H520 cells,there was significant difference on G1 in si-CXCR6(G1:F=23.287,P=0.008;S:F=0.056,P=0.825),in-NF-κB(G1:F-11.471,P=0.028;S:F=2.816,P=0.169)and Control(G1:F=44.518,P=0.003;S:F=15.249,P=0.017)groups,while on S only in Control group.2.4 ConclusionsCXCL16/CXCR6 co-interacted with NF-κB,which promoted progression and metastasis of lung cancer.更多还原