【作者】 李芳；

【导师】 李勇； 刘月平；

【作者基本信息】 河北医科大学 ， 外科学， 2021， 博士

【摘要】 胃癌是世界上最常见的恶性肿瘤之一,是由多种因素导致的疾病。在过去几十年虽然胃癌的发病率有所下降,预后也有所改善,但通常在发生组织浸润和远处转移时被发现,导致世界大多数地区患者的平均5年生存率低于20%,早期发现和治疗是降低胃癌死亡率的关键。在靶向治疗的时代,找到敏感、特异、可预测预后的生物标志物和新的治疗靶点是治疗成功与否的关键。因此,探索胃癌的调控基因有助于胃癌的精准诊断和治疗。长链非编码RNA(Long non-coding RNAs,lncRNAs)是一组主要由RNA聚合酶II转录的非蛋白编码RNA;是长度超过200个核苷酸的转录本,能够在表观、转录及转录后水平调控基因表达和染色质的结构。许多lncRNA可以作为癌基因或抑制基因启动或抑制癌症的发展。新近有学者提出一种全新的基因表达调控模式,即竞争性内源性RNA(competing endogenous RNA,ce RNA)机制,是指lncRNA、m RNA、靶基因等转录后可作为ce RNA,通过miRNA应答元件(m RNA response element,MRE)来竞争性结合具有MRE作用的miRNA,解除miRNA对其相关的靶基因转录后的抑制作用,对miRNA下游基因表达及翻译进行调控。lncRNA CNALPTC1首先是在甲状腺乳头状癌中被发现的,发挥促进甲状腺乳头状癌细胞增殖、迁移,抑制细胞凋亡的作用。但在其他肿瘤中的研究未见报道。本研究以胃癌组织、癌旁组织、不同分化程度的胃癌细胞株及永生化正常胃黏膜细胞株GES-1为研究对象,采用RT-qPCR、Western Blot技术、细胞转染技术、Transwell小室及细胞划痕实验、流式细胞技术、双荧光素酶报告基因等实验技术,检测胃癌及癌旁组织中lncRNA CNALPTC1表达的差异,并分析与胃癌患者临床病理特征的关系。通过检测不同分化胃癌细胞株中lncRNA CNALPTC1表达情况,选择合适的细胞株进行后期实验。通过抑制及过表达lncRNA CNALPTC1观察对胃癌细胞株增殖、侵袭及迁移的影响。通过生物信息学分析预测与lncRNA CNALPTC1靶向结合的micro RNA(miR-6788-5p),并通过预测与miRNA相互作用的靶基因(PAK1),探讨影响胃癌发生发展的分子机制。人类表皮生长因子受体2(HER2),定位于染色体17q21,属于原癌基因,编码产物为185k D的跨膜受体样蛋白p185,具有酪氨酸激酶活性,含有1255个氨基酸,是EGFR家族的成员之一。赫赛汀作为乳腺癌HER2靶点的靶向药物,在早期乳腺癌和晚期(转移性)乳腺癌的治疗中均凸显出可喜的疗效。To GA研究显示HER2阳性型胃癌是一类独特的疾病亚型,需要采取不同的诊疗策略和方法。胃癌HER2阳性率国外的研究为7.3%-20.2%,国内为12%-13%。很多晚期胃癌患者已经从抗HER2靶向治疗中获益。与乳腺癌不同,迄今为止,胃癌在HER2状态与预后相关性方面的研究发现并不一致,HER2状态与胃癌预后相关,但能否作为胃癌的独立预后因素仍存在争议,与其他的临床病理学特征之间的关系如何,也尚不明确。肿瘤新生血管生成受多种因素调控,除传统的血管系统之外,其他非血管内皮细胞,例如肿瘤细胞参与了肿瘤组织内“血管”的构建等因素共同参与,导致肿瘤组织内出现大量的新生血管,肿瘤细胞由于有充足的营养供给,导致快速增生,肿瘤新生血管与肿瘤的发生、侵袭、转移存在显著相关性。基于第一部分研究中显示lncRNA CNALPTC1表达与血管侵犯相关,在本研究中通过回顾性分析胃癌手术患者的临床病理资料,分析原发性胃癌患者中血管侵犯情况、HER2表达情况与临床病理特征的关系,分析血管侵犯、HER2表达与肿瘤间质血管生成的相关性,探索影响预后的独立影响因素,为临床抗肿瘤间质血管生成的治疗提供基础,为lncRNA CNALPTC1表达与胃癌患者预后相关指标的研究提供理论依据,是临床实施精准个体化治疗的前提。第一部分LncRNA CNALPTC1在胃癌中的表达及其与临床病理学特征的关系目的:探讨lncRNA CNALPTC1在胃癌组织、癌旁组织、不同分化程度胃癌细胞株及永生化胃黏膜细胞株中的表达差异,分析其表达与胃癌临床病理特征之间的关系,进一步探讨与胃癌发生发展的关系。方法:1.应用RT-qPCR方法检测lncRNA CNALPTC1在胃癌及癌旁组织中的表达水平。2.分析胃癌组织中lncRNA CNALPTC1的表达情况与临床病理特征的关系。3.应用RT-qPCR方法检测lncRNA CNALPTC1在3株不同分化程度胃癌细胞株及永生化胃黏膜细胞株中的表达差异。结果:1.采用RT-qPCR的方法检测80对胃癌及癌旁组织中lncRNA CNAL-PTC1的表达情况,结果显示:胃癌组织中lncRNA CNALPTC1的表达水平(2.875±0.674)明显高于癌旁组织(1.081±0.196),(t=25.13,P<0.0001)。2.胃癌组织中lncRNA CNALPTC1表达水平与Lauren分型、分化程度、浸润深度、淋巴结转移数量、临床分期、血管侵犯密切相关,(P<0.05);而与性别、年龄、肿瘤位置、肿瘤大小、远处转移状态、HER2扩增状态无关,(P>0.05)。3.lncRNA CNALPTC1在胃癌细胞系MKN28、AGS、MKN45中的表达水平明显高于人永生化胃黏膜上皮细胞系GES-1,(P<0.05);不同分化胃癌细胞株中,低分化胃癌细胞系MKN45中表达水平最高,高分化胃癌细胞系MKN28中表达水平最低,显示细胞分化越差,lncRNA CNALPTC1的表达水平越高,(P<0.05)。小结:1.胃癌组织中lncRNA CNALPTC1的表达水平显著高于癌旁组织。胃癌组织中lncRNA CNALPTC1表达水平与Lauren分型、分化程度、浸润深度、淋巴结转移数量、临床分期、血管侵犯密切相关,而与性别、年龄、肿瘤位置、肿瘤大小、远处转移状态、HER2扩增状态无关。提示lncRNA CNALPTC1的异常表达可能参与了胃癌的发生。2.lncRNA CNALPTC1的表达水平在不同分化胃癌细胞系中均升高;细胞分化越差,表达水平越高。第二部分抑制lncRNA CNALPTC1表达对胃癌MKN45细胞生物学行为的影响目的:探讨抑制lncRNA CNALPTC1对胃癌MKN45细胞生物学行为的影响。方法:1.RT-qPCR技术检测抑制MKN45细胞lncRNA CNALPTC1表达效果。2.MTT、RT-qPCR、Western Blot技术检测抑制MKN45细胞中lncRNA CNALPTC1表达对细胞增殖能力的影响。3.流式细胞技术、RT-qPCR、Western Blot技术检测抑制lncRNA CNALPTC1表达后对胃癌MKN45细胞细胞周期、细胞周期相关基因的影响。4.细胞划痕实验、Transwell小室实验、RT-qPCR技术检测抑制lncRNA CNALPTC1表达后对胃癌MKN45细胞侵袭、迁移及EMT的影响。结果:1.抑制CNALPTC1表达后相对表达量(0.140±0.021)明显低于si-NC组(1.132±0.180)及NC组(1.000±0.127),(t=54.13,P<0.001),CNALPTC1-si RNA能有效抑制胃癌MKN45细胞中内源性lncRNA CNALPTC1的表达,抑制率超过90%。2.MTT检测结果发现抑制CNALPTC1表达后,胃癌细胞MKN45增殖能力明显降低,(t=152.1,P<0.001)。RT-qPCR方法检测结果显示,抑制CNALPTC1表达后,胃癌细胞MKN45增殖标志基因PCNA m RNA相对表达量(0.238±0.043)明显低于si-NC组(1.018±0.129)及NC组(1.000±0.185),(t=34.92,P<0.001)。Western blot检测证实蛋白水平也明显降低。3.流式细胞技术检测结果显示抑制CNALPTC1表达后,胃癌MKN45细胞G0/G1期细胞数量明显增加,S期和G2/M期细胞数量、DNA含量均明显减少,细胞增殖能力减弱。RT-qPCR及Western Blot方法检测结果显示抑制CNALPTC1表达后P16、P21表达水平明显升高,Cyclin D1、Cyclin E表达水平明显降低,(P<0.05)4.抑制CNALPTC1表达后,细胞划痕实验结果显示,胃癌MKN45细胞向划痕中间聚拢的速度减慢,迁移能力降低;Transwell侵袭实验显示,胃癌MKN45细胞通过微孔膜细胞数目明显减少,细胞侵袭能力降低。RT-qPCR及Western Blot检测发现,抑制CNALPTC1表达后,侵袭、迁移相关分子MMP2、MMP9、NM23、ICAM1均明显降低,(P<0.05);TIMP1、TIMP2变化不明显,(P>0.05)。5.RT-qPCR及Western Blot检测结果显示,抑制CNALPTC1表达后E-cadherin表达水平明显升高,N-cadherin、Snail、Twist、ZEB1、Vimentin表达水平明显降低,(P<0.05),提示抑制lncRNA CNALPTC1的表达,抑制了胃癌MKN45细胞的侵袭及迁移能力。小结:1.成功抑制胃癌MKN45细胞中内源性lncRNA CNALPTC1的表达,抑制率超过90%。2.抑制lncRNA CNALPTC1表达后胃癌MKN45细胞增殖能力明显降低,可能与增殖基因PCNA表达降低相关。3.抑制lncRNA CNALPTC1表达后,胃癌MKN45细胞增殖受阻,可能与P16、P21表达明显升高,Cyclin D1、Cyclin E表达明显降低相关。4.抑制lncRNA CNALPTC1表达后,胃癌MKN45细胞的侵袭及迁移能力受到抑制,可能与MMP2、MMP9、NM23、ICAM1表达下调相关。5.抑制lncRNA-CNALPTC1表达后,胃癌MKN45细胞EMT的过程受到抑制,进一步抑制了侵袭及迁移能力,可能与E-cadherin表达明显升高,N-cadherin、Snail、Twist、ZEB1、Vimentin明显降低相关。第三部分过表达lncRNA CNALPTC1对胃癌MKN28细胞生物学行为的影响目的:探讨过表达lncRNA CNALPTC1对胃癌MKN28细胞生物学行为的影响。方法:1.RT-qPCR技术检测过表达lncRNA CNALPTC1后,对胃癌MKN28细胞lncRNA CNALPTC1表达水平的影响。2.MTT、RT-qPCR、Western Blot技术检测过表达lncRNA CNALPTC1后对胃癌MKN28细胞增殖能力的影响。3.流式细胞技术、RT-qPCR、Western Blot技术检测过表达lncRNA CNALPTC1后对胃癌MKN28细胞细胞周期、细胞周期相关基因的影响。4.细胞划痕实验、Transwell小室实验、RT-qPCR技术检测过表达lncRNA CNALPTC1后对胃癌MKN28细胞侵袭、迁移及EMT的影响。结果:1.RT-qPCR技术检测结果显示,胃癌细胞MKN28细胞pc DNA3.1-CNALPTC1转染组相对表达量明显升高,(t=12.108,P<0.001)。证实重组质粒pc DNA3.1-CNALPTC1已经成功转入胃癌细胞MKN28中。2.MTT检测结果发现过表达CNALPTC1后,胃癌MKN28细胞增殖能力明显升高,(t=211.5,P<0.001)。RT-qPCR及Western blot检测结果显示,过表达CNALPTC1后,胃癌MKN28细胞PCNA表达水平明显升高,从而促进肿瘤的生长(P<0.001)。3.流式细胞技术检测结果显示过表达CNALPTC1后,胃癌MKN28细胞G0/G1期细胞数量明显减少,S期和G2/M期细胞数量、DNA含量均明显增多,提示在过表达CNALPTC1后细胞DNA复制加快,加快了细胞分裂的速度,缩短细胞周期,促进G0/G1期进入S期及G2/M期,细胞增殖能力增强。RT-qPCR及Western Blot检测结果显示,过表达CNALPTC1后,P16、P21表达水平明显降低,Cyclin D1、Cyclin E表达水平明显升高,(P<0.05)。4.细胞划痕实验检测结果显示,胃癌MKN28细胞转染lncRNA CNALPTC1过表达质粒后,细胞向划痕中间聚拢的速度变快,促进胃癌细胞迁移速率。Transwell侵袭实验检测结果发现,过表达CNALPTC1后,胃癌MKN28细胞侵袭数量明显增多,促进了胃癌细胞MKN28的侵袭能力。RT-qPCR及Western Blot检测结果显示,过表达CNALPTC1后,侵袭、迁移相关基因MMP2、MMP9、NM23、ICAM1均明显增高,(P<0.05);TIMP1、TIMP2变化不明显,(P>0.05)。5.RT-qPCR及Western Blot实验检测显示,过表达CNALPTC1后EMT相关基因E-cadherin表达水平明显降低;而N-cadherin、Snail、Twist、ZEB1、Vimentin明显增高,(P<0.05)。提示过表达CNALPTC1促进了胃癌MKN28细胞的侵袭及迁移能力。小结:1.将重组质粒pc DNA3.1-CNALPTC1成功转入胃癌细胞MKN28中。2.过表达lncRNA CNALPTC1后胃癌MKN28细胞增殖能力明显升高可能与增殖基因PCNA表达升高相关。3.过表达lncRNA CNALPTC1后,促进胃癌MKN28细胞增殖,可能与P16及P21细胞增殖及分裂的负性调节功能下降,Cyclin D1、Cyclin E表达明显升高相关。4.过表达lncRNA CNALPTC1后促进胃癌MKN28细胞的侵袭及迁移能力,可能与MMP2、MMP9、NM23、ICAM1表达上调相关。5.过表达lncRNA CNALPTC1后,促进了胃癌MKN28细胞的EMT过程,进一步促进了侵袭及迁移能力,可能与E-cadherin表达明显降低,N-cadherin、Snail、Twist、ZEB1、Vimentin明显升高相关。第四部分LncRNA CNALPTC1作为ce RNA通过调控miR-6788-5p/PAK1轴对胃癌细胞增殖、侵袭及迁移能力的影响目的:通过生物信息学分析预测与lncRNA CNALPTC1靶向结合的micro RNA(miR-6788-5p),并通过预测与miRNA相互作用的靶基因(PAK1),探讨对胃癌细胞增殖、侵袭及迁移能力的影响。方法:1.RT-qPCR技术检测过表达及抑制lncRNA CNALPTC1表达对miR-6788-5p m RNA表达的影响。2.生物信息学及双荧光素酶报告基因技术分析lncRNA CNALPTC1对miR-6788-5p的靶向调控作用。3.RT-qPCR及Western Blot检测过表达或抑制miR-6788-5p表达对PAK1表达水平的影响。4.生物信息学及双荧光素酶报告基因技术分析miR-6788-5p对PAK1的靶向调控作用。5.RT-qPCR及Western Blot检测探讨lncRNA CNALPTC1通过miR-6788-5p调控PAK1的表达。结果:1.RT-qPCR技术检测结果显示,胃癌MKN28细胞过表达CNALPTC1后显著抑制了miR-6788-5p的表达(t=11.829,P<0.001);胃癌MKN45细胞抑制CNALPTC1表达后,miR-6788-5p的表达显著升高(t=9.326,P<0.01),提示lncRNA CNALPTC1的表达水平与miR-6788-5p呈负相关。2.通过RNAhybrid网站预测结果发现miR-6788-5p与lncRNA CNALP-TC1最多有连续8个碱基发生互补配对。通过构建CNALPTC1野生型(WT)、CNALPTC1突变型(MUT)荧光素酶报告载体,发现与miR-6788-5p mimic共同转染至胃癌MKN45细胞后,lncRNA CNALPTC1野生型荧光素酶活性显著降低,(t=18.111,P<0.001);突变型荧光素酶活性没有明显变化,(t=0.197,P=0.851)。提示miR-6788-5p可与lncRNA CNALPTC1靶向结合,CNALPTC1可负向调控miR-6788-5p的表达。3.过表达或抑制miR-6788-5p的表达对PAK1表达水平的影响选用胃癌MKN45细胞转染miR-6788-5p mimic,上调miR-6788-5p的表达,RT-qPCR及Western Blot技术检测结果显示,胃癌MKN45细胞过表达miR-6788-5p后,PAK1表达水平显著降低,(t=9.269,P=0.001)。选用胃癌MKN28细胞经转染miR-6788-5p inhibitor,抑制miR-6788-5p的表达,RT-qPCR及Western Blot检测结果显示,抑制MKN28细胞中miR-6788-5p后,PAK1表达水平显著升高,(t=6.406,P=0.003),提示miR-6788-5p与PAK1表达呈负相关。4.通过生物信息学数据库Target Scan对miR-6788-5p的靶基因进行预测,发现PAK1是miR-6788-5p的候选靶基因,构建PAK1 3’UTR野生型(WT)质粒、PAK1 3’UTR突变型(MUT)的荧光素酶报告载体,与miR-6788-5p mimic共同转染至胃癌MKN45细胞后,PAK1野生型荧光素酶活性显著降低,(t=13.154,P<0.001);PAK1突变型荧光素酶活性没有明显变化,(t=0.130,P=0.901)。提示PAK1可以与miR-6788-5p靶向结合,且miR-6788-5p可负向调控PAK1的表达。5.RT-qPCR及Western Blot检测结果显示:胃癌MKN28细胞过表达CNALPTC1后,PAK1表达水平上调;而共转染CNALPTC1+miR-6788-5p mimic后,PAK1的表达上调消失,(t=16.100,P<0.001)。抑制MKN45细胞中CNALPTC1表达后,PAK1表达降低;共转染si-CNALPTC1+miR-6788-5p inhibitor后,PAK1表达下调消失,(t=6.601,P=0.003)。提示lncRNA CNALPTC1作为通过调控miR-6788-5p/PAK1轴促进胃癌细胞增殖、侵袭及迁移能力。小结:1.过表达lncRNA CNALPTC1后显著抑制了miR-6788-5p的表达,抑制lncRNA CNALPTC1表达后,miR-6788-5p的表达显著升高,两者表达水平呈负相关。2.双荧光素酶报告载体实验证实,与miR-6788-5p mimic共转染后lncRNA CNALPTC1野生型荧光素酶活性显著降低,突变型荧光素酶活性没有明显变化,提示miR-6788-5p可与lncRNA CNALPTC1靶向结合,并呈负向调控。3.过表达miR-6788-5p后,胃癌细胞PAK1表达显著降低,抑制miR-6788-5p后,PAK1表达显著升高,两者表达呈负相关。4.双荧光素酶报告载体实验证实,与miR-6788-5p mimic共转染后,胃癌细胞PAK1野生型荧光素酶活性显著降低,突变型荧光素酶活性没有明显变化,提示PAK1可与miR-6788-5p靶向结合,并呈负向调控。5.证实lncRNA CNALPTC1作为ce RNA通过调控miR-6788-5p/PAK1轴促进胃癌细胞增殖、侵袭及迁移能力。第五部分进展期胃癌血管侵犯的影响因素及预后分析目的:探讨进展期胃癌血管侵犯的影响因素、对预后的影响及血管侵犯与PAK1表达之间的关系。方法:1.采用免疫组化方法对进展期胃癌肿瘤组织内血管侵犯进行识别,并分析血管侵犯与临床病理特征的关系。2.通过定期随访,分析影响进展期胃癌预后的独立影响因素。3.采用免疫组化方法检测进展期胃癌中PAK1的表达情况,分析其与临床病理特征的关系。4.采用统计学分析对进展期胃癌血管侵犯及PAK1表达进行相关性分析。结果:1.622例进展期胃癌中血管侵犯的检出率23.25%(144/622)。血管侵犯与肿瘤组织分化程度、浸润深度、淋巴结转移数量相关,(P<0.05)。而与性别、年龄、肿瘤位置、肿瘤大小、Lauren分型、肿瘤远处转移状态、临床分期无关,(P>0.05)2.622进展期胃癌患者5年生存率为43.89%(273/622)。患者5年生存率与血管侵犯、Lauren分型、浸润深度、淋巴结转移数量、临床分期密切相关,(P<0.05)。而与性别、年龄、肿瘤位置、肿瘤大小、肿瘤组织分化程度、肿瘤远处转移状态无关,(P>0.05)。血管侵犯、临床分期是进展期胃癌根治性术后患者预后的独立预测因子。3.125例进展期胃癌组织PAK1阳性率72.80%(91/125),PAK1蛋白表达与Lauren分型、肿瘤组织分化程度、临床分期密切相关,(P<0.05)。而与性别、年龄、肿瘤位置、肿瘤大小、浸润深度、淋巴结转移数量、远处转移状态无关,(P>0.05)。4.经相关性分析,125例进展期胃癌中,血管侵犯与PAK1两者存在明显相关性,且呈正相关,(P<0.05)。小结:1.进展期胃癌组织中血管侵犯与肿瘤组织分化程度、浸润深度、淋巴结转移数量相关;而与性别、年龄、肿瘤位置、肿瘤大小、Lauren分型、远处转移状态、临床分期无关。2.进展期胃癌患者5年生存率与血管侵犯、Lauren分型、浸润深度、淋巴结转移数量、临床分期密切相关,且血管侵犯、临床分期是进展期胃癌根治性术后患者预后的独立预测因子。3.进展期胃癌组织中PAK1蛋白表达与Lauren分型、肿瘤组织分化程度、临床分期密切相关;而与性别、年龄、肿瘤位置、肿瘤大小、浸润深度、淋巴结转移数量、远处转移状态无关。4.进展期胃癌组织中,血管侵犯与PAK1两者存在明显正相关。第六部分胃癌HER2表达与肿瘤新生血管的关系及对预后的影响目的:探讨1121例胃癌患者中HER2表达情况与临床病理特征的关系,分析HER2表达与肿瘤新生血管的相关性,探索影响预后的独立影响因素。方法:1.采用IHC及FISH相结合的方法检测1121例胃癌患者中HER2阳性率,分析HER2扩增状态与临床病理特征的关系。2.采用免疫组化方法进行MVD计数,分析胃癌HER2表达情况与肿瘤新生血管的相关性分析。3.采用Kaplan-Meier法绘制生存曲线,采用log-rank法进行生存分析,采用Cox比例风险模型进行多因素分析,确定影响胃癌的独立预后因素。结果:1.HER2免疫组化染色结果:0为268例,1+为450例,2+为297例,3+为106例。染色结果为2+的297例标本,均进一步进行FISH检测。FISH检测发现其中9例HER2扩增,其余288例未见扩增。HER2阳性的病例为115例,HER2阴性的病例为906例。胃癌HER2阳性率10.26%(115/1121)。2.1121例胃癌患者HER2扩增状态与肿瘤大小、WHO分型、Lauren分型、浸润深度相关,提示可能与肿瘤进展密切相关,(P<0.05);而与性别、年龄、肿瘤位置、临床分期、淋巴结转移数量、远处转移状态、神经受侵、血管侵犯无关,(P>0.05)。3.HER2阳性型胃癌MVD计数(24.10±4.59)明显高于HER2阴性型(16.74±4.81),(t=12.994,P<0.001)。4.1350例患者,随访成功率为83.04%(1121/1350),中位随访时间62.17个月(1-107个月);总生存率50.76%(569/1121);5年总生存率51.92%(582/1121)。5.1121例胃癌患者5年生存率与肿瘤大小、WHO分型、Lauren分型、浸润深度、淋巴结转移数量、临床分期、HER2扩增状态、MVD计数相关,(P<0.05);与性别、年龄、肿瘤位置、远处转移状态、神经受侵、血管侵犯无关,(P>0.05)。Lauren分型、浸润深度、淋巴结转移数量、临床分期、HER2扩增状态、MVD计数是影响胃癌预后的独立影响因素,(P<0.05)。小结:1.1121例胃癌患者HER2阳性率为10.26%,HER2扩增状态与肿瘤大小、WHO分型、Lauren分型、浸润深度相关,提示可能与肿瘤进展密切相关;而与性别、年龄、肿瘤位置、临床分期、淋巴结转移数量、远处转移状态、神经受侵、血管侵犯无关。2.HER2阳性型胃癌MVD计数明显高于HER2阴性型。3.1121例胃癌患者5年生存率与肿瘤大小、WHO分型、Lauren分型、浸润深度、淋巴结转移数量、临床分期、HER2扩增状态、MVD计数相关;且Lauren分型、浸润深度、淋巴结转移数量、临床分期、HER2扩增状态、MVD计数是影响胃癌预后的独立影响因素。4.HER2表达与MVD计数两者呈正相关,与预后呈负相关。结论:1.胃癌及胃癌细胞株中lncRNA CNALPTC1的表达水平明显升高。lncRNA CNALPTC1表达水平与胃癌组织Lauren分型、分化程度、浸润深度、淋巴结转移数量、临床分期、血管侵犯密切相关。胃癌细胞分化越差,lncRNA CNALPTC1表达水平越高。2.抑制lncRNA CNALPTC1表达后,抑制了胃癌MKN45细胞的增殖、侵袭及迁移能力。3.过表达lncRNA CNALPTC1后,促进了胃癌MKN28细胞增殖、侵袭及迁移能力。4.lncRNA CNALPTC1作为ce RNA通过调控miR-6788-5p/PAK1轴促进胃癌细胞增殖、侵袭及迁移能力。5.进展期胃癌患者血管侵犯与肿瘤组织分化程度、浸润深度、淋巴结转移状态显著相关。血管侵犯状态、临床分期是影响进展期胃癌根治性术后患者预后的独立预测因子。6.进展期胃癌组织中PAK1表达与Lauren分型、肿瘤组织分化程度、临床分期密切相关;PAK1表达与血管侵犯两者存在明显正相关。7.HER2扩增状态与肿瘤大小、WHO分型、Lauren分型、浸润深度、MVD计数密切相关,提示可能与肿瘤进展密切相关。Lauren分型、浸润深度、淋巴结转移数量、临床分期、HER2扩增状态、MVD计数是影响胃癌预后的独立影响因素。更多还原

【Abstract】 Gastric cancer is one of the most common malignant tumors in the world,and it is also a multi-factorial disease.Although the incidence of gastric cancer has decreased over the past decades and the prognosis has improved,it is usually detected when tumor tissue infiltration and distant metastasis occur,resulting in an average 5-year survival rate of less than 20%in most regions of the world.Early detection and treatment are the key to reducing the mortality of gastric cancer.In the era of targeted therapy,finding sensitive,specific,prognostic biomarkers and new therapeutic targets are the key to achieve successful treatment.Therefore,to explore regulatory genes of of gastric cancer is helpful for the early diagnosis and precise treatment.Long non-coding RNAs(lncRNAs)are a group of non-protein coding RNAs that are mainly transcribed by RNA polymerase II.LncRNAs are defined as transcripts of more than 200 nucleotides in length that regulate gene expression and chromatin structure at the apparent,transcriptional,and posttranscriptional levels.Many lncRNAs are abnormally expressed in a vari-ety of human diseases,including cancer,and are important regulators of tumor genesis and development.They can act as oncogenes or suppressors to initiate or inhibit cancer development.Recently some scholars put forward a new pattern of gene expression regulation,namely competing endogenous RNAs(ce RNA)mechanism,which is refers to the lncRNA,m RNA and target genes transcription content can be used as original ce RNA through micro RNAs response(m RNA response element,MRE)competitive combining with MRE micro RNAs,remove the miRNA related to its inhibition after transcription of target genes,thereby regulating downstream gene expression and translation of micro RNAs.lncRNA CNALPTC1 was first found in thyroid papillary carcinoma to promote the proliferation and migration of thyroid papillary carcinoma cells and inhibit cell apoptosis.But it has not been reported in other tumors.In this study,gastric cancer tissues,tissues adjacent to carcinoma,gastric cancer cell lines of different differentiation levels and immortified normal gastric mucosa cell line GES-1 were selected as the research objects.RT-qPCR,western Blot,cell transfection,transwell chamber,cell scratch assay,flow cytometry,dual luciferase reporter gene and other experimental techniques were used to detect the expression difference of lncRNA CNALPTC1 in gastric cancer and the tissues adjacent to carcinoma.The relationship between lncRNA CNALPTC1expression and the clinicopathological characteristics of gastric cancer patients was analyzed.By detecting the expression of lncRNA CNALPTC1 in different differentiated gastric cancer cell lines,suitable cell lines were selected for later experiments.The effects of silencing and overexpression of lncRNA CNALPTC1 on proliferation,invasion and migration of gastric cancer cell lines were observed.Bioinformatics analysis was used to predict the micro RNA(miR-6788-5p)targeting to lncRNA CNALPTC1,and the target gene interacting with miRNA(PAK1)was predicted to explore the molecular mechanism affecting the occurrence and development of gastric cancer.Human epidermal growth receptor 2 gene(HER2),a proto-oncogene,is located on chromosome 17q21 and encodes a 185k D transmembrane receptor-like protein p185,which has tyrosine kinase activity and contains 1255 amino acids,and is a member of the EGFR family.Herceptin is a targeted drug for the HER2 target of breast cancer and has shown efficacy in both early and advanced(metastatic)breast cancer.TOGA studies showed that HER2-positive gastric cancer was a unique disease subtype,which required different diagnosis and treatment strategies and methods.The HER2 positive rate in gastric cancer was 7.3%-20.2%in foreign studies and 12%-13%in China.Many patients with advanced gastric cancer had already benefited from anti-HER2-targeted therapies.Different from breast cancer,so far,there has been inconsistent research findings on the correlation between HER2 status and prognosis of gastric cancer.HER2 status is related to prognosis of gastric cancer,but whether it can be used as an independent prognostic factor for gastric cancer is still controversial,and its relationship with other clinico-pathological features is still unclear.Tumor formation of new blood vessels is controlled by many factors,in addition to the traditional cardiovascular system,other non-vascular endothelial cells,such as tumor cells,also participate in the reconstruction of"blood vessel"in tumor tissues.Many factors contribute to the emergence of a large number of new blood vessels in the tumor tissue,and the rapid proliferation of tumor cells due to adequate nutritional supply.Tumor neovascularization is significantly correlated with the occurrence,invasion and metastasis of tumors.Based on the first part result of the study,the expression of lncRNA CNALPTC1 was associated with blood vessel invasion.In this part,the clinical and pathological data of patients with gastric cancer after surgery were retrospectively analyzed to analyze the relationship between blood vessel invasion,HER2 expression and clinicopathological characteristics in patients with primary gastric cancer.By analyzing the correlation between blood vessel invasion,HER2 expression and tumor interstitial angiogenesis,the independent influencing factors affecting prognosis were explored,so as to provide a basis for clinical anti-tumor angiogenesis therapy,and provide theoretical basis for the study of lncRNA CNALPTC1 expression and progn-osis of gastric cancer patients,and that lay a foundation for the implem-entation of clinical individualized therapy.Part One Expression of lncRNA CNALPTC1 in gastric cancer and its relationship with clinicopathological featuresObjective:To investigate the expression differences of lncRNA CNAL-PTC1 in gastric cancer tissues,tissue adjacent to carcinoma,gastric cancer cell lines with different differentiation levels and immortalized gastric mucosa cell lines,and to analyze the relationship between its expression and the clinicopa-thological characteristics of gastric cancer,so as to further explore the relationship between its expression and the occurrence and development of gastric cancer.Methods:1.The expression level of lncRNA CNALPTC1 in gastric cancer and adjacent tissues was detected by RT-qPCR.2.The correlation between the expression of lncRNA CNALPTC1 and the clinicopathological features in gastric cancer tissues was analyzed statistically.3.The expression differences of lncRNA CNALPTC1 in three gastric cancer cell lines with different differentiation degrees and immortalized gastric mucosa cell lines were detected by RT-qPCR.Results:1.RT-qPCR was used to detect the expression of lncRNA CNALPTC1 in80 pairs of gastric cancer and adjacent tissues.The results showed that the expression level of lncRNA CNALPTC1 in gastric cancer tissues(2.875±0.674)was significantly higher than that in adjacent tissues(1.081±0.196)(t=25.13,P<0.0001).2.The expression level of lncRNA CNALPTC1 in gastric cancer tissues was closely correlated with Lauren’s classification,degree of differentiation,depth of invasion,number of lymph node metastasis,clinical stage and blood vessel invasion,(P<0.05).It was not related to gender,age,tumor location,tumor size,distant metastasis,and HER2 amplification,(P>0.05).3.The expression levels of lncRNA CNALPTC1 in gastric cancer cell lines MKN28,AGS and MKN45 were significantly higher than those in human immortalized gastric mucosa epithelial cell line GES-1,(P<0.05).Among different differentiated gastric cancer cell lines,the expression of poorly differentiated gastric cancer cell line MKN45 was the highest,and the expression of highly differentiated gastric cancer cell line MKN28 was the lowest,indicating that the worse the cell differentiation was,the higher the expression level of lncRNA CNALPTC1 was(P<0.05).Summaries:1.The expression level of lncRNA CNALPTC1 in gastric cancer tissues was significantly higher than that in adjacent tissues.The expression level of lncRNA CNALPTC1 in gastric cancer tissues was closely related to Lauren’s classification,degree of differentiation,depth of invasion,number of lymph node metastasis,clinical stage,and blood vessel invasion,but was not related to gender,age,tumor location,tumor size,distant metastasis,and HER2amplification.The abnormal expression of lncRNA CNALPTC1 may be involved in the occurrence of gastric cancer.2.The expression level of lncRNA CNALPTC1 was increased in different differentiated gastric cancer cell lines.The worse the cell differentiation was,the higher the expression level of lncRNA CNALPTC1was.Part Two Inhibition of lncRNA CNALPTC1 expression on the biological behavior of gastric cancer MKN45 cellsObjective:To investigate the effect of lncRNA CNALPTC1 inhibition on the biological behavior of gastric cancer MKN45 cells.Methods:1.The interference efficiency of gastric cancer MKN45 cells after inhibiting lncRNA CNALPTC1 expression was detected by RT-qPCR.2.MTT,RT-qPCR and Western Blot methods were used to detect the effect of inhibiting lncRNA CNALPTC1 expression on the proliferation ability of gastric cancer MKN45 cells.3.Flow cytometry,RT-qPCR and Western Blot were used to detect the effects of inhibiting lncRNA CNALPTC1 expression on cell cycle and cell cycle-related molecules of gastric cancer MKN45 cells.4.Cell scratch assay,Transwell chamber assay and RT-qPCR were used to detect the effects of inhibiting lncRNA CNALPTC1 expression on invasion,migration and EMT of gastric cancer MKN45 cells.Results:1.The relative expression level of CNALPTC1 expression after inhibition(0.140±0.021)was significantly lower than that of the si-NC group(1.132±0.180)and the NC group(1.000±0.127)(t=54.13,P<0.001).CNALPTC1-si RNA could effectively inhibit the expression of lncRNA CNALPTC1 in gastric cancer MKN45 cells,with an inhibition rate of over90%.2.MTT assay results showed that proliferation ability of MKN45 gastric cancer cells was significantly decreased after inhibition of CNALPTC1expression(t=152.1,P<0.001).The results of RT-qPCR showed that the relative expression level of MKN45 proliferation marker gene PCNA m RNA(0.238±0.043)was significantly lower than that in si-NC group(1.018±0.129)and NC group(1.000±0.185)after inhibiting CNALPTC1 expression,(t=34.92,P<0.001).Western blot method confirmed that the protein levels were also significantly reduced.3.Flow cytometry results showed that after inhibiting the expression of CNALPTC1,the number of G0/G1 phase cells was significantly increased;the number of S phase and G2/M phase cells and the DNA content were signifi-cantly reduced;and the proliferation ability of cells was weakened in MKN45cells of gastric cancer.The results of RT-qPCR and Western Blot showed that the expression levels of P16 and P21 were significantly increased,and Cyclin D1&Cyclin E expression level were significantly decreased after inhibiting lncRNA CNALPTC1 expression,(P<0.05).4.After inhibition of CNALPTC1,the scratch test results showed that the aggregation rate of gastric cancer MKN45 cells to the center of the scratch was slowed down,and the migration ability was reduced.Transwell invasion assay showed that the number of MKN45 cells passing through the microporous membrane was significantly reduced,and the cell invasion ability was reduced.RT-qPCR and Western Blot analysis showed that MMP2,MMP9,NM23,and ICAM1 were significantly decreased after inhibiting CNALPTC1 expression,(P<0.05).TIMP1 and TIMP2 didn’t change signific-antly,(P>0.05).5.RT-qPCR and Western Blot results showed that the expression levels of E-cadherin wa’s significantly increased after inhibition of CNALPTC1expression,while the expression levels of N-cadherin,Snail,TWIST,ZEB1and Vimentin were significantly decreased,(P<0.05),suggesting that the inhibition of lncRNA CNALPTC1 expression inhibited the invasion and migration of gastric cancer MKN45 cells.Summaries:1.The expression of endogenous lncRNA CNALPTC1 in gastric cancer MKN45 cells was successfully inhibited with an inhibition rate of over 90%.2.After inhibiting the expression of lncRNA CNALPTC1,the proliferation ability of gastric cancer MKN45 cells was significantly reduced,which may be related to the decreased expression of proliferation gene PCNA.3.After inhibiting the expression of lncRNA CNALPTC1,the proliferation of gastric cancer MKN45 cells was blocked,which may be related to the significantly increased expression of P16 and P21,and the significantly decreased expression of Cyclin D1 and Cyclin E.4.Inhibition of the expression of lncRNA CNALPTC1 inhibited the invasion and migration of gastric cancer MKN45 cells,which may be related to the down-regulation of the expressions of MMP2,MMP9,NM23 and ICAM1.5.Inhibition of lncRNA-CNALPTC1 inhibited the EMT process of gastric cancer MKN45 cells,and further inhibited the invasion and migration ability,which may be related to the significantly increased expression of E-cadherin,and the significantly decreased expression of and N-cadherin,Snail,Twist,ZEB1 and Vimentin.Part Three effect of lncRNA CNALPTC1 overexpression on biological behavior of gastric cancer MKN28 cellsObjective:To investigate the effect of lncRNA CNALPTC1 overexpre-ssion on the biological behavior of gastric cancer MKN28 cells.Methods:1.The influence of overexpressed lncRNA CNALPTC1 on the express-ion level of lncRNA CNALPTC1 in gastric cancer MKN28 cells was detected by RT-qPCR.2.The effect of overexpressed lncRNA CNALPTC1 on the proliferation ability of gastric cancer MKN28 cells was detected by MTT,RT-qPCR and Western Blot.3.Flow cytometry,RT-qPCR and Western Blot methods were used to detect the effects of overexpressed lncRNA CNALPTC1 on cell cycle and cell cycle-related genes of gastric cancer MKN28 cells.4.Cell scratch assay,Transwell chamber assay and RT-qPCR methods were used to detect the effects of overexpression of lncRNA CNALPTC1 on invasion,migration and EMT of gastric cancer MKN28 cells.Results:1.The results of RT-qPCR showed that the relative expression of PCDNA3.1-CNALPTC1 in gastric cancer MKN28 cells was significantly increased in the transfected group(t=12.108,P<0.001).The recombinant plasmid pc DNA3.1-CNALPTC1 was successfully transfected into gastric cancer cell line MKN28.2.MTT assay showed that the proliferation ability of gastric cancer MKN28 cells was significantly increased after overexpression of CNALPTC1(t=211.5,P<0.001).The results of RT-qPCR and Western blot showed that the expression level of PCNA in MKN28 cells was significantly increased after overexpression of CNALPTC1,thus promoting the growth of tumor,(P<0.001).3.The results of flow cytometry showed that the number of gastric cancer MKN28 cells in G0/G1 phase decreased significantly,and the number&DNA content in S and G2/M phases increased significantly after overexpression of lncRNA CNALPTC1.It was suggested that the overexpression of CNALPTC1 accelerated DNA replication and cell division,shortened the cell cycle,promoted G0/G1 phase into S and G2/M phases,and enhanced cell proliferation ability.RT qPCR and Western blot results showed that overexp-ression of CNALPTC1 significantly decreased the expression levels of P16and P21,and increased the expression levels of Cyclin D1 and Cyclin E,(P<0.05).4.The results of cell scratch test showed that MKN28 cells gathered faster to the middle of scratch and promoted the migration rate of gastric cancer cells after overexpression of lncRNA CNALPTC1.Transwell invasion assay showed that overexpression of lncRNA CNALPTC1 significantly increased the invasion of gastric cancer MKN28 cells,which promoted the invasion of gastric cancer MKN28 cells.RT-qPCR and Western blot results showed that overexpression of CNALPTC1 significantly increased the expression of invasion and migration related genes MMP2,MMP9,NM23,ICAM1,(P<0.05).TIMP1 and TIMP2 did not change significantly,(P>0.05).5.RT-qPCR and Western Blot analysis showed that the expression levels of EMT-related genes,E-cadherin was significantly decreased after the overexpression of CNALPTC1;N-cadherin,Snail,Twist,ZEB1 and Vimentin increased significantly,(P<0.05).It was suggested that the overexpression of CNALPTC1 promoted the invasion and migration of gastric cancer MKN28cells.Summaries:1.The recombinant plasmid pc DNA3.1-CNALPTC1 was successfully transfected into gastric cancer cell line MKN28.2.The proliferation ability of gastric cancer MKN28 cells was significantly increased after overexpression of lncRNA CNALPTC1,which may be related to the increased expression of proliferation gene PCNA.3.Overexpression of lncRNA CNALPTC1 promoted the proliferation of gastric cancer MKN28 cells,which may be related to the decreased negative regulatory function of the proliferation and division of P16 and P21 cells,and the significantly increased expression of Cyclin D1 and Cyclin E.4.Overexpression of lncRNA CNALPTC1 promoted the invasion and migration ability of gastric cancer MKN28 cells,which may be related to the up-regulation of MMP2,MMP9,NM23,ICAM1.5.Overexpression of lncRNA CNALPTC1 promoted the EMT process of gastric cancer MKN28 cells,and further promoted the invasion and migration ability,which may be related to the significantly decreased expression of E-cadherin,and the significantly increased expression of and N-cadherin,Snail,TWIST,ZEB1 and Vimentin.Part Four LncRNA CNALPTC1,as a ce RNA,regulates the effects of miR-6888-5p/PAK1 axis on the proliferation,invasion and migration of gastric cancer cellsObjective:To investigate the effects of micro RNA(miR-6788-5p)targeting to lncRNA CNALPTC1 and target gene(PAK1)interacting with miRNA on proliferation,invasion and migration of gastric cancer cells by bioinformatics analysis and prediction.Methods:1.The effect of lncRNA CNALPTC1 overexpression or inhibition on miR-6788-5p m RNA expression was detected by RT-qPCR.2.The targeted regulation effect of lncRNA CNALPTC1 on miR-6788-5p was analyzed by bioinformatics and dual luciferase reporter gene technique.3.The effect of overexpression or inhibition of miR-6788-5p expression on the expression level of PAK1 was detected by RT-qPCR and Western Blot.4.Bioinformatics and dual luciferase reporter gene techniques were used to analyze the targeted regulation of miR-6788-5p on PAK1.5.lncRNA CNALPTC1 regulated the expression of PAK1 through miR-6788-5p by RT-qPCR and Western Blot analysis.Results:1.The results of RT-qPCR showed that the overexpression of CNAL-PTC1 in gastric cancer MKN28 cells significantly inhibited the expression of miR-6788-5p,(t=11.829,P<0.001).After inhibiting the expression of CNAL-PTC1 in gastric cancer MKN45 cells,the expression of miR-6788-5p was significantly increased,(t=9.326,P<0.01),suggesting that the expression level of lncRNA CNALPTC1 was negatively correlated with miR-6788-5p.2.It was found that miR-6788-5p and lncRNA CNALPTC1 had at most 8consecutive complementary pairing bases by using the prediction results of RNAHybrid website.By constructing CNALPTC1 wild-type(WT)and CNALPTC1 mutant(MUT)luciferase reporter vectors,it was found that the activity of lncRNA CNALPTC1 wild-type luciferase was significantly decre-ased after transfected into gastric cancer MKN45 cells with miR-6788-5P mimic,(t=18.111,P<0.001).Mutant luciferase activity did not change significantly,(t=0.197,P=0.851).It suggested that miR-6788-5p could be targeted to lncRNA CNALPTC1,and lncRNA CNALPTC1 could negatively regulate the expression of miR-6788-5p.3.Gastric cancer MKN45 cells were transfected with miR-6788-5p mimic to upregulate the expression of miR-6788-5p.The results of RT-qPCR and Western Blot showed that the expression level of PAK1 was significantly decreased after the overexpression of miR-6788-5p in gastric cancer MKN45cells,(t=9.269,P=0.001).Gastric cancer MKN28 cells were transfected with miR-6788-5p inhibitor to inhibit the expression of miR-6788-5p.RT-qPCR and Western Blot results showed that the expression level of PAK1 was significantly increased after the inhibition of miR-6788-5p in MKN28 cells,(t=6.406,P=0.003),suggesting a negative correlation between miR-6788-5p and the expression of PAK1.4.The target genes of miR-6788-5p were predicted using the bioinfo-rmatics database Target Scan.It was found that PAK1 was a candidate target gene of miR-6788-5p.Constructed PAK1 3’UTR wild-type(WT)plasmid and the luciferase reporter vector of PAK1 3’UTR mutant(MUT),and transfected into gastric cancer MKN45 cells with miR-6788-5p mimic,the luciferase activity of PAK1 wild-type was significantly decreased,(t=13.154,P<0.001).There was no significant change in luciferase activity of mutant PAK1,(t=0.130,P=0.901).These results suggested that PAK1 can target miR-6788-5p,and miR-6788-5p can negatively regulate the expression of PAK1.5.The results of RT-qPCR and Western Blot showed that the expression level of PAK1 was up-regulated after CNALPTC1 overexpression in MKN28cells;the upregulated expression of PAK1 was disappeared after co-transfe-ction of CNALPTC1&miR-6788-5P mimic,(t=16.100,P<0.001).After silencing the expression of Cn ALPTC1 in MKN45 cells,the expression of PAK1 decreased;the down-regulated expression of PAK1 disappeared,and the expression recovered after co-transfection with si-CNALPTC1&miR-6788-5p inhibitor,(t=6.601,P=0.003).It was suggested that lncRNA CNA-LPTC1 promotes the proliferation,invasion and migration of gastric cancer cells by regulating the miR-6788-5p/PAK1 axis.Summaries:1.The overexpression of lncRNA CNALPTC1 significantly inhibited the expression of miR-6788-5p,and the inhibiting of lncRNA CNALPTC1significantly increased the expression of miR-6788-5p,showing a negative correlation between them.2.The dual luciferase reporter vector assay confirmed that the activity of lncRNA CNALPTC1 wild-type luciferase was significantly reduced after co-transfection with miR-6788-5p mimic,while the activity of mutant luciferase was not significantly changed,suggesting that miR-6788-5p could target and negatively regulate lncRNA CNALPTC1 expression.3.The expression of PAK1 in gastric cancer cells was significantly decreased after the overexpression of miR-6788-5p,and significantly increased after inhibition of miR-6788-5p,showing a negative correlation.4.The dual luciferase reporter vector assay confirmed that the activity of PAK1 wild-type luciferase in gastric cancer cells was significantly reduced after co-transfection with miR-6788-5p mimic,while the activity of mutant luciferase was not significantly changed,suggesting that PAK1 could bind to miR-6788-5p in a targeted and negative manner.5.It was confirmed that lncRNA CNALPTC1,as a ce RNA,promoted the proliferation,invasion and migration of gastric cancer cells by regulating miR-6788-5p/PAK1 axis.Part Five Influencing factors and prognosis analysis of blood vessel invasion in advanced gastric cancerObjective:To investigate the influencing factors of blood vessel invasion(BVI)in advanced gastric cancer,the influence on prognosis,and the relationship between blood vessel invasion and PAK1 expression.Methods:1.Immunohistochemical method was used to to identify BVI in advanced gastric cancer tissue and analyze the relationship between BVI and clinico-pathological features.2.Through regular follow-up,the independent factors affecting the prognosis of advanced gastric cancer were analyzed.3.The expression of PAK1 in advanced gastric cancer detected by immunohistochemistry,and the relationship between PAK1 expression&clinicopathological features was analyzed.4.Statistical analysis was used to analyze the correlation between BVI and PAK1 expression in advanced gastric cancer.Results:1.The detection rate of BVI in 622 cases with advanced gastric cancer was 23.25%(144/622).BVI was correlated with the degree of tumor differen-tiation,the depth of invasion and the number of lymph node metastasis,(P<0.05).It was not related to gender,age,tumor location,tumor size,Lauren classification,distant metastatic status,and clinical stage,(P>0.05).2.The 5-year survival rate of 622 patients with advanced gastric cancer was 43.89%(273/622).The 5-year survival rate of the patients was closely related to BVI,Lauren classification,depth of invasion,number of lymph node metastasis and clinical stage,(P<0.05).It was not related to gender,age,tumor location,tumor size,degree of tumor differentiation and distant metastasis,(P>0.05).BVI and clinical staging were independent predictors of prognosis in patients with advanced gastric cancer after radical surgery.3.The positive rate of PAK1 in 125 advanced gastric cancer tissues was72.80%(91/125).The expression of PAK1 protein was closely related to Lauren type,tumor tissue differentiation degree and clinical stage(P<0.05).However,it was not related to gender,age,tumor location,tumor size,depth of invasion,number of lymph node metastasis,and distant metastasis status(P>0.05).4.Correlation analysis showed that there was a significant and positive correlation between BVI and PAK1 in 125 cases with advanced gastric cancer(P<0.05).Summaries:1.BVI in advanced gastric cancer was correlated with the degree of differentiation,depth of invasion and the number of lymph node metastasis.It was not related to gender,age,tumor location,tumor size,Lauren classif-ication,tumor distant metastasis status,and clinical stage.2.The 5-year survival rate of patients with advanced gastric cancer was closely related to BVI,Lauren classification,depth of invasion,number of lymph node metastasis and clinical stage.BVI and clinical stage were independent predictors of prognosis of patients with advanced gastric cancer after radical surgery.3.The expression of PAK1 protein in advanced gastric cancer tissues was closely related to Lauren classification,tumor tissue differentiation degree and clinical stage.It was not related to gender,age,tumor location,tumor size,depth of invasion,number of lymph node metastasis,and distant metastasis status.4.In advanced gastric cancer,there was a significant positive correlation between BVI and PAK1.Part Six Relationship between HER2 expression and tumor angiogen-esis in gastric cancer and its effect on prognosisObjective:To investigate the relationship between HER2 expression and clinicopathological characteristics in 1121 patients with gastric cancer,and to analyze the correlation between HER2 expression&tumor angiogenesis,in order to explore independent influencing factors affecting prognosis.Methods:1.The HER2 positive rate in 1121 patients with gastric cancer was detected by IHC and FISH method,and the relationship between HER2amplification and clinicopathological characteristics was analyzed.2.MVD count was performed by immunohistochemical method to analyze the correlation between HER2 expression and neovascularization in gastric cancer.3.Kaplan-Meier method was used to plot the survival curve.Log-rank method was used for survival analysis.Cox proportional risk model was used for multivariate analysis to determine the independent prognostic factors for primary gastric cancer.Results:1.The results of HER2 immunohistochemical staining were(0)in 268cases,(1+)in 450 cases,(2+)in 297 cases,and(3+)in 106 cases.All 297samples stained with(2+)were further tested by FISH.HER2 amplification was found in 9 cases and no amplification was found in the remaining 288cases by FISH assay.115 cases were HER2-positive and 906 cases were HER2-negative.The HER2 positive rate in gastric cancer was 10.26%(115/1121).2.The HER2 amplification status of 1121 gastric cancer patients were correlated with tumor size,WHO typing,Lauren typing,and invasion depth,suggesting that it may be closely related to tumor progression,(P<0.05).However,there were no correlation with gender,age,tumor location,clinical stage,number of lymph node metastasis,distant metastasis state,nerve invasion,and BVI,(P>0.05).3.The MVD count of HER2-positive gastric cancer(24.10±4.59)was significantly higher than that of HER2-negative gastric cancer(16.74±4.81),(t=12.994,P<0.001).4.The success rate of follow-up was 83.04%(1121/1350),and the median follow-up time was 62.17 months(1-107 months).The overall survival rate was 50.76%(569/1121).The 5-year overall survival rate was51.92%(582/1121).5.The 5-year survival rate of 1121 patients with gastric cancer was correlated with tumor size,WHO type,Lauren type,depth of invasion,number of lymph node metastasis,clinical stage,HER2 amplification status,and MVD count,(P<0.05).There was no correlation with gender,age,tumor location,distant metastatic status,nerve invasion,or BVI,(P>0.05).Summaries:1.The HER2 positive rate in 1121 gastric cancer patients was 10.26%.The HER2 amplification status was correlated with tumor size,WHO typing,Lauren typing,and invasion depth,suggesting that it may be closely related to tumor progression.That was not related to gender,age,tumor location,clinical stage,number of lymph node metastases,distant metastatic status,nerve invasion,or BVI.2.The MVD count of HER2-positive gastric cancer was significantly higher than that of HER2-negative gastric cancer.3.The 5-year survival rate of 1121 patients with gastric cancer was correlated with tumor size,WHO typing,Lauren typing,depth of invasion,number of lymph node metastasis,clinical stage,HER2 amplification status,and MVD count.Lauren classification,depth of invasion,number of lymph node metastases,p TNM stage,HER2 amplification status and MVD count were independent influencing factors for the prognosis of gastric cancer.4.HER2 expression was positively correlated with MVD count and negatively correlated with prognosis.Conclusions:1.The expression level of lncRNA CNALPTC1 was significantly increased in gastric cancer and gastric cancer cell lines.The expression level of lncRNA CNALPTC1 was closely related to Lauren classification,degree of differentiation,depth of invasion,number of lymph node metastasis,clinical stage and BVI of gastric cancer tissues.The more poorly differentiated gastric cancer cells were,the higher the expression level of lncRNA CNALPTC1was.2.The proliferation,invasion and migration of gastric cancer MKN45cells were inhibited after inhibiting the expression of lncRNA CNALPTC1.3.It promoted the proliferation,invasion and migration of gastric cancer MKN28 cells after overexpression of lncRNA CNALPTC1.4.lncRNA CNALPTC1,as a ce RNA,promoted the proliferation,invasion and migration of gastric cancer cells by regulating miR-6788-5p/PAK1 axis.5.BVI in patients with advanced gastric cancer was significantly correlated with the degree of tumor tissue differentiation,invasion depth and lymph node metastasis status.BVI and clinical stage were independent predictors of prognosis in patients with advanced gastric cancer after radical surgery.6.PAK1 expression in advanced gastric cancer tissues was closely related to Lauren classification,tumor tissue differentiation degree and clinical stage;There was a positive correlation between PAK1 expression and BVI.7.HER2 amplification status was closely related to tumor size,WHO type,Lauren classification,depth of invasion,and MVD count,suggesting that HER2 amplification status may be closely related to tumor progression.Lauren classification,depth of invasion,number of lymph node metastases,clinical stage,HER2 amplification status and MVD count were independent influencing factors for the prognosis of gastric cancer.更多还原