【作者】 李萍；

【导师】 张国俊；

【作者基本信息】 郑州大学 ， 内科学（呼吸病学）， 2017， 博士

【摘要】 背景和目的肺癌根据临床治疗方案的不同,通常将其分为两种类型:非小细胞肺癌（non-small cell lung cancer,NSCLC）和小细胞肺癌（SCLC）;前者较为多见,约占肺癌患者比例的80%,近年来,随着吸烟和各种环境因素的影响,肺癌的发病率和死亡率逐年上升,尽管一直以来有关肺癌的诊治水平不断提高,但肺癌细胞生长速度快、易转移的特点使得多数患者总的五年存活率很低。为此,从分子水平探讨肺癌的发生发展的调控机制,早期干预肿瘤的发展进程,有助于提高肺癌患者的生存率,降低其死亡率。非编码RNA（Non-coding RNA,nc RNA）生物形成机制及调控作用也开始引起研究者的关注。mi RNA已证实在转录或翻译水平调控靶基因的表达,进而在细胞的生长、发育、分化、增殖、凋亡和细胞周期等细胞生物学行为都起着十分重要的作用。长链非编码RNA（Long non-coding RNA,lnc RNA）是一种长度大于200个核苷酸的非编码RNA。目前已发现lnc RNAs在多种癌症中差异表达,且参与调控多种分子途径,引起基因表达改变,调控细胞生物学行为的变化。为此,lnc RNAs的功能及相关机制研究将为肿瘤的治疗提供潜在的靶点。Lnc RNA RGMB-AS1定位于5q21.1,目前关于lnc RNA RGMB-AS1的国内外报道非常少,结合预实验结果肺腺癌标本中检测分析lnc RNA RGMB-AS1表达时发现lnc RNA RGMB-AS1在肺腺癌组织中呈高表达,且其表达水平均与肺腺癌的分化程度及淋巴结转移有相关性,但是lnc RNA RGMB-AS1在肺腺癌中的具体生物学作用及相关的分子机制仍需进一步探讨。我们的研究第一次综合分析了lnc RNA RGMB-AS1在肺腺癌中表达和生物学作用,并初步探讨了其肿瘤调节作用的分子机制。本研究包括四部分:第一部分:肺腺癌组织中lnc RNA RGMB-AS1表达及与临床病理学特征相关性分析;第二部分:体外调控lnc RNA RGMB-AS1表达对肺腺癌细胞系A549和SPC-A-1生长、迁移和侵袭的影响;第三部分:体内调控lnc RNA RGMB-AS1表达对肺腺癌对裸鼠移植瘤生长的影响;第四部分:lnc RNA RGMB-AS1作用机制的初步研究。第一部分肺腺癌组织中lnc RNA RGMB-AS1表达及与临床病理学特征相关性分析方法1.收集了110例肺腺癌组织标本,包括110例肺腺癌组织和110例配对的癌旁正常组织标本。2.运用lnc RNA基因芯片检测肺腺癌组织和配对的癌旁组织标本中lnc RNA的表达情况。3.运用q RT-PCR检测110例标本9种lnc RNAs,其中包含5个在肺腺癌中表达上调的lnc RNAs（LOC284801,KIAA1908,XLOC₀08466,LINC00665 and RGMB-AS1）和4个在肺腺癌中表达下调的lnc RNAs（MAGI2-AS3,LOC100505495,LOC400550,XLOC₀01412）验证lnc RNA芯片结果的可靠性。4.依据lnc RNA RGMB-AS1的表达水平,并分析其与肺腺癌患者年龄、性别、TNM分期、分化程度及有无淋巴结转移之间的相关性。结果1.lnc RNA芯片检测分析得到肺腺癌组织标本中差异表达的lnc RNAs有907个,其中272个（约30%）表达上调,635个（70%）表达下调。lnc RNA RGMB-AS1在肺腺癌组织中表达增高。2.q RT-PCR结果显示在9种lnc RNA中,验证结果和基因芯片结果一致,证实了芯片数据的可靠性。3.Lnc RNA RGMB-AS1在肺腺癌组织中表达上调,且相关性分析结果显示lnc RNA RGMB-AS1的表达水平与肺腺癌分化程度、TNM分期及淋巴结转移情况有相关性（P>0.05）,而与病人的年龄和性别无相关性（P<0.05）。第二部分体外调控lnc RNA RGMB-AS1表达对肺腺癌细胞系A549和SPC-A-1生长、迁移和侵袭的影响方法1.设计合成沉默lnc RNA RGMB-AS1表达的si RNA及无关的si RNA序列,分别使用脂质体LipofectamineTM2000进行转染对数生长期A549和SPC-A-1细胞。2.荧光定量PCR检测各实验组细胞中lnc RNA RGMB-AS1的表达变化,筛选沉默lnc RNA RGMB-AS1表达效果最佳的si RNA序列。3.CCK-8试剂和克隆形成实验检测下调lnc RNA RGMB-AS1对A549和SPC-A-1细胞细胞增殖能力的影响。4.流式细胞术检测下调lnc RNARGMB-AS1对A549和SPC-A-1细胞周期的影响。5.划痕实验用来检测下调lnc RNA RGMB-AS1对A549和SPC-A-1细胞迁移能力的影响。6.Transwell实验用来检测下调lnc RNA RGMB-AS1对A549和SPC-A-1细胞侵袭能力的影响。结果1.对比空白对照组和阴性对照组,转染lnc RNA RGMB-AS1 si RNA的四个组中si RNA1（si-lnc RNA组）的沉默作用较为明显,lnc RNA RGMB-AS1表达水平显著下调（P<0.01）。2.CCK8实验结果显示:与对照组比较,si-lnc RNA组A549和SPC-A-1细胞的生长在转染24h后出现显著抑制（P<0.05）,并且随时间抑制效果日益显著。3.克隆形成实验结果显示A549和SPC-A-1细胞中si-lnc RNA组的细胞克隆团形成明显少于NC组和Blank组,差异具有统计学意义（P<0.05）,说明下调lnc RNA RGMB-AS1表达,A549和SPC-A-1细胞的生长受到显著抑制。4.流式细胞术检测细胞周期结果显示相比NC组和Blank组,si-lnc RNA组A549和SPC-A-1细胞处于G0/G1期的细胞比例显著增加,而S期细胞比例显著减少,进一步说明下调lnc RNA RGMB-AS1表达可影响A549和SPC-A-1细胞的生长。5.划痕实验结果显示si-lnc RNA组A549和SPC-A-1细胞划痕的宽度随时间延长愈合较慢,而NC组和Blank组的细胞划痕愈合较快,且具有明显统计学差异（P<0.05）,说明下调lnc RNA RGMB-AS1表达可抑制A549和SPC-A-1细胞的迁移能力。6.Transwell侵袭实验结果显示si-lnc RNA组A549和SPC-A-1细胞侵袭数与Blank组和NC组相比差异有显著性（P<0.05）,说明下调lnc RNA RGMB-AS1表达可抑制A549和SPC-A-1细胞的侵袭能力。第三部分体内调控lnc RNA RGMB-AS1表达对肺腺癌裸鼠移植瘤生长的影响方法1.设计合成针对lnc RNA RGMB-AS1的sh RNA序列及无关阴性对照sh RNA序列,构建慢病毒重组载体后,制备沉默lnc RNA RGMB-AS1表达的重组慢病毒,感染对数生长期A549-Luc和SPC-A-1-Luc细胞,得到下调lnc RNA RGMB-AS1表达的A549-Luc和SPC-A-1-Luc细胞株。2.荧光定量PCR方法检测稳定下调lnc RNA RGMB-AS1表达的A549-Luc和SPC-A-1-Luc细胞株中lnc RNA RGMB-AS1的表达水平。3.据上述lnc RNA RGMB-AS1下调水平的检测结果将A549-Luc和SPC-A-1-Luc各组处理细胞注射裸鼠右侧腋下后在1周,2周,3周,4周的时间运用小动物成像仪观察并监测裸鼠移植瘤的生长情况。4.免疫组化和Western blot技术检测移植瘤组织中Ki67的表达水平。结果1.成功制备了沉默lnc RNA RGMB-AS1表达的重组慢病毒,到下调lnc RNA RGMB-AS1表达的A549-Luc和SPC-A-1-Luc细胞株。2.在1周,2周,3周,4周的时间观察并监测裸鼠移植瘤的生长情况,结果显示sh RNA1组裸鼠移植瘤的生长较空白对照组和阴性对照组明显减慢,差异有统计学意义（P<0.05）。4周时的移植瘤体积相比空白对照组和阴性对照组明显减小,差异有统计学意义（P<0.05）。3.免疫组化和Western blot技术检测移植瘤组织中Ki67的表达,结果发现显示sh RNA1组裸鼠移植瘤组织中Ki67的表达较空白对照组和阴性对照组明显降低,差异有统计学意义（P<0.05）。第四部分lnc RNA RGMB-AS1作用机制的初步研究方法1.UCSC分析lnc RNA RGMB-AS1序列预测可能的靶基因。2.根据分析结果构建lnc RNA RGMB-AS1第三外显子的表达载体（p Silencer3.1-Lnc-Exon3）和RGMB第一和第二外显子报告基因载体（p EGFP-N3-Exon1和p EGFP-N3-Exon2）,运用脂质体LipofectamineTM2000进行转染对数生长期A549细胞。实验分六组进行,（1）单独转染p EGFP-N3-Exon1;（2）共同转染p EGFP-N3-Exon1和p Silencer 3.1;（3）共同转染p EGFP-N3-Exon1和p Silencer 3.1-Lnc-Exon3;（4）单独转染p EGFP-N3-Exon2（5）共同转染p EGFP-N3-Exon2和p Silencer 3.1;（6）共同转染p EGFP-N3-Exon2和p Silencer3.1-Lnc-Exon3。3.运用倒置荧光显微镜和微量荧光分光光度计观察和检测各组细胞的荧光强度。4.q RT-PCR检测110例肺腺癌标本和对应癌旁正常组织中RGMB m RNA表达情况并与临床病理特征和lnc RNA RGMB-AS1进行相关性分析,Western blot技术检测肺腺癌标本和对应癌旁正常组织中RGMB蛋白的表达水平。5.构建pc DNA3.1-RGMB表达载体,运用脂质体LipofectamineTM2000进行转染A549和SCP-A-1细胞,western blot技术检测RGMB蛋白的的表达变化,同时比较转染lnc RNA RGMB-AS1 si RNA与过表达RGMB两细胞增殖、侵袭和细胞周期的影响。结果1.USCS序列分析结果发现lnc RNA RGMB-AS1序列的第三外显子区域与RGMB的第一和第二外显子区域存在相互作用。2.对比癌旁正常组织,肺腺癌组织中RGMB m RNA和蛋白表达水平均显著降低（P<0.05）,经统计学分析显示肺腺癌组织中RGMB m RNA的表达水平与肺腺癌分化程度、TNM分期及淋巴结转移情况有相关性（P>0.05）,而与病人的年龄和性别无相关性（P<0.05）。Lnc RNA RGMB-AS1在肺腺癌组织中表达与RGMB表达之间呈负相关。3.Western blot技术检测转染后各组细胞RGMB蛋白的表达变化结果显示转染lnc RNA RGMB-AS1相关的si RNA组RGMB蛋白表达明显增加,转染pc DNA3.1-RGMB组细胞,RGMB蛋白的表达量也明显增加,相对于Blank组差异具有统计学意义（P<0.05）4.微量荧光分光光度计检测转染后各组细胞的荧光强度变化,结果发现转染重组载体p EGFP-N3-Exon1和p Silencer 3.1-Lnc-Exon3的细胞的荧光强度相对于单独转染p EGFP-N3-Exon1组和共转染p EGFP-N3-Exon1和p Silencer 3.1组细胞的荧光强度无明显变化（P>0.05）;而转染重组载体p EGFP-N3-Exon2和p Silencer 3.1-Lnc-Exon3的细胞的荧光强度相对于单独转染p EGFP-N3-Exon2组和共转染p EGFP-N3-Exon2和p Silencer 3.1组细胞的荧光强度明显减弱（P<0.05）。5.CCK8结果显示:对比Blank组,转染后1d后si-lnc RNA组和RGMB过表达组细胞的增殖能力均出现明显抑制（P<0.05）,并且随时间的延长抑制作用日益显著。该两组之间细胞的增殖能力变化无显著差异（P>0.05）。6.流式细胞术检测细胞周期结果显示:比Blank组细胞,si-lnc RNA组和RGMB过表达组细胞在G0/G1,S和G2/M期的比例发生变化,处于G0/G1期比例均显著升高,而处于S期的比例显著降低（P<0.05）,该两组之间相比无显著差异（P>0.05）7.Transwell实验结果显示:对比Blank组,si-lnc RNA组和RGMB过表达组细胞的侵袭能力均出现明显抑制（P<0.05）,且比较两组之间细胞的侵袭能力变化结果无显著差异（P>0.05）结论1.lnc RNA芯片检测分析得到肺腺癌组织标本中差异表达的lnc RNAs有907个,其中272个（约30%）表达上调,635个（70%）表达下调。lnc RNARGMB-AS1在肺腺癌组织中表达增高,其表达水平与患者的肿瘤分化程度、TNM分期及淋巴结转移有相关性。2.体内外下调肺腺癌细胞中的lnc RNARGMB-AS1表达可有效抑制肺腺癌细胞的生长、抑制细胞侵袭迁移能力。3.Lnc RNA RGMB-AS1通过负调控RGMB的表达,从而发挥相应的生物学功能。4.Lnc RNA RGMB-AS1发挥促癌作用,有望成为肺腺癌治疗新的靶点。更多还原

【Abstract】 Background and objective Lung cancer is one of malignant tumors.Based on the clinic standard treatment,most of lung cancer is non-small cell lung cancer（NSCLC）,about 80%.Another is small cell lung cancer（SCLC）.Environmental pollution and smoking are more serious in recent years.The morbidity and mortality of lung cancer is rising year by year.Although the treatment methods keep in progress,the 5-year survival rate is less than 15% because of the proliferation,invasion and metastasis of lung cancer cell.In order to raise the survival rate and reduce the mortality of lung cancer patients,we must further study its pathogenesis,and intervene the progression of lung cancer in early stage.The biological formation mechanism and function of non-coding RNA（nc RNAs）have been drawn public attention.Mi RNA has been verified to play an important role in cell biological behavior,such as cell growth,differentiation,apoptosis and cell cycle by regulating the target gene at the transcription and post-translation levels.Long non-coding RNA（lnc RNA）is a diverse class of nc RNA with >200 nucleotides in length.So far,expression of several lnc RNAs have been reported in many kinds of tumors,and involved in regulating expression of gene by a series of molecular pathways,which resulted the change of cell biological behavior.Therefore,it is conducive to provide new and potential target for tumor therapy to study the function and mechanism of lnc RNAs in-depth.Lnc RNA RGMB-AS1 is located on human chromosomal band 5q21.1.No report was found about lnc RNA RGMB-AS1 in lung cancer at home and abroad.Combined with the results of preliminary experiment about the high expression of lnc RNA RGMB-AS1 in lung adenocarcinoma tissues and its correlation with cancer tissue differentiation and lymph node metastases,we think it is worthy of exploring the function and mechanism of lnc RNA RGMB-AS1 in the development of lung adenocarcinoma.Therefore,our study synthetically analysed the expression and function of lnc RNA RGMB-AS1 in lung adenocarcinoma and discussed its potential mechanism.Our study included four parts.The first part is expression and analysis of lnc RNA RGMB-AS1 in lung adenocarcinoma tissues;the second part is biological effects of downregulating potential lnc RNA RGMB-AS1 expression in A549 and SPC-A-1 cell;the third part is biological effects of downregulating potential lnc RNA RGMB-AS1 expression;the fourth part is preliminary study of mechanism of lnc RNA RGMB-AS1.Part One: Expression and analysis of lnc RNA RGMB-AS1 in lungadenocarcinoma tissuesMethods: 1.110 paired lung adenocarcinoma tissues and adjacent normal tissues（≥3 cm away from tumor）were obtained from patients who received surgical resection of lung adenocarcinoma.2.The expression of lnc RNAs was detected in 5 cases lung adenocarcinoma tissues and adjacent normal tissues by lnc RNA chip.3.q RT-PCR method was used to detect expressions of 9 lnc RNAs in 110 cases of lung adenocarcinoma tissues,including 5 up-regulated lnc RNAs（LOC284801,KIAA1908,XLOC₀08466,LINC00665 and RGMB-AS1）and 4 down-regulated lnc RNAs（MAGI2-AS3,LOC100505495,LOC400550 and XLOC₀01412）.The aim is to test and verify the reliability of lnc RNA chip.4.Beased on lnc RNA RGMB-AS1 expression,correlation analysis was used to verify the correlation between lnc RNA RGMB-AS1 expression and gender,age,differentiation status,lymph node metastases and TNM stage.Results: 1.Compared with adjacent normal tissues,lung adenocarcinoma tissues exhibited 907 differentially expressed ln RNAs,including 272 lnc RNAs upregulated（30%）and 635 lnc RNAs downregulated（70%）.Lnc RNA RGMB-AS1 is upregulated in lung adenocarcinoma tissue（P < 0.05）.2.q RT-PCR results showed that expression level of 9 lnc RNAs were consistent with the results of ln RNA chip.3.Lnc RNA RGMB-AS1 has a significant upregulation in lung adenocarcinoma tissues（P < 0.05）.And its expression in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.There was no statistically significant correlation between lnc RNA RGMB-AS1 expression and either gender or age（P<0.05）.Part Two Effect of down-regulated lnc RNA RGMB-AS1 on growth,migration and invasion of A549 and SPC-A-1 cellsMethods: 1.Small interferring RNA（si RNA）against lnc RNA RGMB-AS1,and control oligonucleotides（negative control,NC）were synthesized and transfected into A549 and SPC-A-1 cells using LipofectamineTM2000.2.q RT-PCR was used to detect the change of lnc RNA RGMB-AS1 expression in every group to select the best si RNA oligonucleotides against lnc RNA RGMB-AS1.3.CCK8 and colony formation assay experiment were used to detect proliferation and growth ability of A549 and SPC-A-1 cells in each group.4.Flow-cytometric analyses were used to assess cell cycle progression of A549 and SPC-A-1 cells in each group.5.Wound healing assay was performed to assess migration ability of A549 and SPC-A-1 cells in each group.6.Transwell invasion assay was performed to assess invasion ability of A549 and SPC-A-1 cells in each group.7.SPSS 21.0 was used to analyze all the dates.Results: 1.Compared with Blank and NC groups,lnc RNA RGMB-AS1 expression was significantly decreased in si RNA1 group（P < 0.05）.2.Results of CCK8 showed that the proliferation of A549 and SPC-A-1 transfected with si RNA was markedly decreased with time passing when compared to the control groups（NC and Blank group）（P<0.05）.3.Results of the colony formation number of si-lnc RNA group had a significant reduce in A549 and SPC-A-1 cells when compared to the control groups（NC and Blank group）（P<0.05）,which revealed that down-regulation of could affect the proliferation capacity of A549 and SPC-A-1 cells 4.Results of flow cytometry assay showed a marked increase in the percentage of cells in the G0/G1 phase and a reduction in the percentage of cells in the S phase in the si-lnc RNA group in A549 and SPC-A-1 cells when compared to the control groups（NC and Blank group）（P<0.05）.5.Results of wound healing assay showed that the migratory ability of A549 and SPC-A-1 cells in the si-lnc RNA group was lower at 24 h post-wounding compared with both NC and Blank groups（P<0.05）.6.Results of transwell invasion assay showed that the number of invading cells was significantly lower in the si RNA group than in the two control groups in both A549 and SPC-A-1 cells（P<0.05）.Part Three Effect of down-regulated lnc RNA RGMB-AS1 on the growth of xenograft tumor in nude miceMethods: 1.sh RNA against lnc RNA RGMB-AS1,and negative control sh RNA were synthesized.And construct recombined lentiviral vector about down-regulated lnc RNA RGMB-AS1 and negative control,which were infected Luciferase-tagged A549 and SPC-A-1 cells（A549-Luc and SPC-A-1-Luc cells）.2.q RT-PCR was used to test the expreesion of lnc RNA RGMB-AS1 in infected cells.3.Infected A549-Luc and SPC-A-1-Luc cells were implanted in BALB/c nude mice by subcutaneous injection and tumor xenograft model were constructed.The growth of tumor xenograft models were observed at 1,2,3 and 4 week.4.Immunohistochemistry and western blot were used to detect Ki67 expression.Results: 1.Lentiviruses with lnc RNA RGMB-AS1 sh RNA and NC were successfully constructed.Stably infected A549-Luc and SPC-A-1-Luc cells were constructed.2.The growth of xenograft tumors was assessed to further examine the tumor-suppressing effect of si RNA against lnc RNA RGMB-AS1 on lung adenocarcinoma.In A549 and SPC-A-1 cell tumors,the luciferase signal was clearly lower in sh RNA1 mice than in the control groups at 2,3,and 4 weeks.The signal intensity in the sh RNA1 group decreased with time（P < 0.05）.3.Results from immunohistochemistry and western blot showed that significant differences in Ki67 expression were observed between the sh RNA1 group and the control groups（P < 0.05）.There were no differences in Ki67 expression between NC group and Blank group（P > 0.05）.Part Four Preliminary study about mechanism of lnc RNA RGMB-AS1Methods: 1.UCSC database transcription information was used to predict the potential target gene of lnc RNA RGMB-AS1.2.Based on the analysis results,p EGFP-N3-Exon1,p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3 were constructed respectively.And then they were transfected into A549 cells using LipofectamineTM2000 respectively.Cells were subdivided into six groups as follows: For Exon1,Exon1-I: cells transfected with p EGFP-N3-Exon1 only;Exon1-II: cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1;Exon1-III: cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1-Lnc-Exon3;for Exon2,Exon2-I: cells transfected with p EGFP-N3-Exon2 only;Exon2-II: cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1;Exon2-III: cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3.3.Inverted fluorescence microscope and fluorescence photometer were used to observe and test the fluorescence in every group.4.q RT-PCR method was used to detect expressions of RGMB in 110 cases of lung adenocarcinoma tissues,and correlation analysis was used to verify the correlation between RGMB expression and gender,age,differentiation status,lymph node metastases and TNM stage.Western blot was used to detect expression of RGMB in protein level.And the relation of lnc RGMB-AS1 and RGMB was analyzed by the Spearman correlation analysis.5.pc DNA3.1（+）-RGMB recombinant vector was constructed and transfected into A549 and SPC-A-1 cells by lipofectamineTM2000 to further verify the potential relationship between lnc RGMB-AS1 and RGMB in lung adenocarcinoma cells.Results: 1.The UCSC database transcription information indicated that lnc RNA RGMB-AS1 is located on human chromosome 5 and has two interaction sites with the first exon and second exon of RGMB in the third exon region.2.RGMB has a significant downregulation in lung adenocarcinoma tissues（P < 0.05）.And its expression in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.There was no statistically significant correlation between lnc RNA RGMB-AS1 expression and either gender or age（P<0.05）.3.Western blot analysis showed that the expression of RGMB was higher in the RGMB and si-lnc RNA groups than in the Blank groups（P < 0.05）.4.The changes of fluorescence in the different groups were analyzed at 48 h after transfection.The fluorescence intensity of the p EGFP-N3-Exon1 group was used as a control to analyze the relative fluorescence intensity in the other groups.For Exon1,the results of fluorescence microscopy and fluorescence microplate reader showed that the fluorescence intensity of cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1-Lnc-Exon3 did not differ from that of cells transfected with p EGFP-N3-Exon1 only or that of cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1（P>0.05）.For Exon2,the fluorescence intensity of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3 was weaker than that of cells transfected with p EGFP-N3-Exon2 only or that of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1（P < 0.05）.There was no difference between the fluorescence intensity of cells transfected with p EGFP-N3-Exon2 only and that of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1（P > 0.05）.5.Results of CCK8 showed that the OD450 values in RGMB cells on days 2,3 and 4 were significantly decreased compared with those in Blank cells,and the decreasing trend was more obvious in si-lnc RNA cells（P < 0.05）.6.Flow cytometric analysis of cell cycle progression indicated that the proportion of cells in S phase was reduced in the si-lnc RNA and RGMB groups,and thereduction was greater in the si-lnc RNA group than in the RGMB group（P < 0.05）.7.Transwell invasion assays demonstrated that the invasive ability was higher in the si-lnc RNA group than that in the RGMB group（P < 0.05）.Conclusions 1.907 lnc RNAs in lung adenocarcinoma tissues have abnormal expression,including 272（30%）up-regulation and 635（70%）down-regulation.Lnc RNA RGMB-AS1 expression level increased significantly when compared to corresponding adjacent normal lung tissues.Lnc RNA RGMB-AS1 expression level in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.2.Down-regulation of lnc RNA RGMB-AS1 in lung adenocarcinoma cells in vitro can significantly inhibit cells growth,reduce the migration and invasion ability of cells promote cells apoptosis.The animal experiment indicated that the down-regulation of lnc RNA RGMB-AS1 can significantly inhibit the transplant tumor growth in nude mouse models.3.Lnc RNA RGMB-AS1 negatively regulated RGMB expression,thus exert its biological function.4.Lnc RNA RGMB-AS1 can play a role in cancer promotion and is expected to become new targets for lung adenocarcinoma gene therapy.更多还原