肿瘤药学
腫瘤藥學
종류약학
ANTI-TUMOR PHARMACY
2013年
4期
319-320
,共2页
王瑛%尹继业%李湘平%陈娟%钱晨月%郑艺%刘昭前
王瑛%尹繼業%李湘平%陳娟%錢晨月%鄭藝%劉昭前
왕영%윤계업%리상평%진연%전신월%정예%류소전
肺癌是世界范围内癌症死亡的主要原因，而肺鳞癌是肺癌中最常见的组织病理类型，约占原发性肺癌的40～50％。临床和分子机制研究表明肺鳞癌是多因素复杂疾病。长链非编码 RNA（long noncoding RNA, lncRNA）是长度大于200 nt 的非编码 RNA 的统称。最初的研究认为它是转录组的“噪音”，但近十年来的生命科学研究发现，它们参与了多项生物调控过程。有研究表明 lncRNAs 在许多癌症类型中均异常表达。本研究通过全基因组 lncRNAs 表达研究探索 lncRNAs 是否有可能与肺鳞癌的形成相关。我们从中南大学湘雅医院收集了16例肺鳞癌手术病人的肺癌及癌旁组织并保存于液氮中，采用 Trizol法提取组织总 RNA，反转录成 cDNA，体外转录合成 cRNA，使用 RNA 纯化柱纯化 cRNA，以 cRNA 为模板，用随机引物进行反转录，荧光标记反转录产物，最后进行 Agilent human 4×180 K 芯片杂交。使用 Agilent Feature Extraction（v10.7）软件对杂交图片进行分析并提取数据，然后用 Agilent GeneSpring 软件对数据进行归一化和差异分析，使用 R 语言对归一化后的表达数据进行统计分析。此芯片中设计了总共38361个 lncRNAs 探针，我们的数据结果显示，肺鳞癌组织组与正常肺组织组比较，有28055个 lncRNAs 探针的表达有一定水平的改变，其中3460个显著差异表达（倍数变化绝对值≥2），而我们对所有样本对统计发现，127个在所有肺鳞癌组织中与配对正常组织相比，其表达有显著差异。在这3460个 lncRNAs 中，1559个 lncRNAs 表达下调，1901个 lncRNAs 表达上调，绝大部分 lncRNAs 的功能未知，因此我们试图通过构建共表达网络预测lncRNAs 的功能。我们以相关系数0.99和 P<0.05为基础，构建了 lncRNAs 和 mRNAs 的共表达网络，并在此基础上从两个方面对 lncRNAs 可能的靶基因进行预测，分别是顺式距离在10 Kb 内的 lncRNA-mRNA 对和与 mRNA 的3’UTR 的序列比对相似的 lncRNA-mRNA 对，共得到33个 lncRNA-mRNA 对。对这33个 mRNA 进行分析发现，TERC 很可能是非小细胞肺癌的诊断标志物，因此考虑与之对应的两个 lncRNA：ENST00000363312和 sno-RNA_scaRNA_0_548也可能作为非小细胞肺癌的诊断标志物。因此我们得出结论：肺鳞癌组织中差异表达的 lncRNAs 可能参与到肺癌的发展过程中，也可能作为未来肺鳞癌的潜在治疗靶标。
肺癌是世界範圍內癌癥死亡的主要原因，而肺鱗癌是肺癌中最常見的組織病理類型，約佔原髮性肺癌的40～50％。臨床和分子機製研究錶明肺鱗癌是多因素複雜疾病。長鏈非編碼 RNA（long noncoding RNA, lncRNA）是長度大于200 nt 的非編碼 RNA 的統稱。最初的研究認為它是轉錄組的“譟音”，但近十年來的生命科學研究髮現，它們參與瞭多項生物調控過程。有研究錶明 lncRNAs 在許多癌癥類型中均異常錶達。本研究通過全基因組 lncRNAs 錶達研究探索 lncRNAs 是否有可能與肺鱗癌的形成相關。我們從中南大學湘雅醫院收集瞭16例肺鱗癌手術病人的肺癌及癌徬組織併保存于液氮中，採用 Trizol法提取組織總 RNA，反轉錄成 cDNA，體外轉錄閤成 cRNA，使用 RNA 純化柱純化 cRNA，以 cRNA 為模闆，用隨機引物進行反轉錄，熒光標記反轉錄產物，最後進行 Agilent human 4×180 K 芯片雜交。使用 Agilent Feature Extraction（v10.7）軟件對雜交圖片進行分析併提取數據，然後用 Agilent GeneSpring 軟件對數據進行歸一化和差異分析，使用 R 語言對歸一化後的錶達數據進行統計分析。此芯片中設計瞭總共38361箇 lncRNAs 探針，我們的數據結果顯示，肺鱗癌組織組與正常肺組織組比較，有28055箇 lncRNAs 探針的錶達有一定水平的改變，其中3460箇顯著差異錶達（倍數變化絕對值≥2），而我們對所有樣本對統計髮現，127箇在所有肺鱗癌組織中與配對正常組織相比，其錶達有顯著差異。在這3460箇 lncRNAs 中，1559箇 lncRNAs 錶達下調，1901箇 lncRNAs 錶達上調，絕大部分 lncRNAs 的功能未知，因此我們試圖通過構建共錶達網絡預測lncRNAs 的功能。我們以相關繫數0.99和 P<0.05為基礎，構建瞭 lncRNAs 和 mRNAs 的共錶達網絡，併在此基礎上從兩箇方麵對 lncRNAs 可能的靶基因進行預測，分彆是順式距離在10 Kb 內的 lncRNA-mRNA 對和與 mRNA 的3’UTR 的序列比對相似的 lncRNA-mRNA 對，共得到33箇 lncRNA-mRNA 對。對這33箇 mRNA 進行分析髮現，TERC 很可能是非小細胞肺癌的診斷標誌物，因此攷慮與之對應的兩箇 lncRNA：ENST00000363312和 sno-RNA_scaRNA_0_548也可能作為非小細胞肺癌的診斷標誌物。因此我們得齣結論：肺鱗癌組織中差異錶達的 lncRNAs 可能參與到肺癌的髮展過程中，也可能作為未來肺鱗癌的潛在治療靶標。
폐암시세계범위내암증사망적주요원인，이폐린암시폐암중최상견적조직병리류형，약점원발성폐암적40～50％。림상화분자궤제연구표명폐린암시다인소복잡질병。장련비편마 RNA（long noncoding RNA, lncRNA）시장도대우200 nt 적비편마 RNA 적통칭。최초적연구인위타시전록조적“조음”，단근십년래적생명과학연구발현，타문삼여료다항생물조공과정。유연구표명 lncRNAs 재허다암증류형중균이상표체。본연구통과전기인조 lncRNAs 표체연구탐색 lncRNAs 시부유가능여폐린암적형성상관。아문종중남대학상아의원수집료16례폐린암수술병인적폐암급암방조직병보존우액담중，채용 Trizol법제취조직총 RNA，반전록성 cDNA，체외전록합성 cRNA，사용 RNA 순화주순화 cRNA，이 cRNA 위모판，용수궤인물진행반전록，형광표기반전록산물，최후진행 Agilent human 4×180 K 심편잡교。사용 Agilent Feature Extraction（v10.7）연건대잡교도편진행분석병제취수거，연후용 Agilent GeneSpring 연건대수거진행귀일화화차이분석，사용 R 어언대귀일화후적표체수거진행통계분석。차심편중설계료총공38361개 lncRNAs 탐침，아문적수거결과현시，폐린암조직조여정상폐조직조비교，유28055개 lncRNAs 탐침적표체유일정수평적개변，기중3460개현저차이표체（배수변화절대치≥2），이아문대소유양본대통계발현，127개재소유폐린암조직중여배대정상조직상비，기표체유현저차이。재저3460개 lncRNAs 중，1559개 lncRNAs 표체하조，1901개 lncRNAs 표체상조，절대부분 lncRNAs 적공능미지，인차아문시도통과구건공표체망락예측lncRNAs 적공능。아문이상관계수0.99화 P<0.05위기출，구건료 lncRNAs 화 mRNAs 적공표체망락，병재차기출상종량개방면대 lncRNAs 가능적파기인진행예측，분별시순식거리재10 Kb 내적 lncRNA-mRNA 대화여 mRNA 적3’UTR 적서렬비대상사적 lncRNA-mRNA 대，공득도33개 lncRNA-mRNA 대。대저33개 mRNA 진행분석발현，TERC 흔가능시비소세포폐암적진단표지물，인차고필여지대응적량개 lncRNA：ENST00000363312화 sno-RNA_scaRNA_0_548야가능작위비소세포폐암적진단표지물。인차아문득출결론：폐린암조직중차이표체적 lncRNAs 가능삼여도폐암적발전과정중，야가능작위미래폐린암적잠재치료파표。
Lung cancer is the leading cause of cancer death worldwide, and squamous carcinoma is the most common histological subtype. Clinical and molecular evidence indicated that lung squamous carcinoma is heterogeneous disease. Long non-coding RNAs (lncRNAs) were noncoding RNAs with more than 200 nucleotide length. They have been found to be involved in a variety of biological processes. Many studies indicated that they were aberrantly expressed in some types of carcinomas. In this study, we tested the hypothesis that some lncRNAs may correlate with lung cancer tumor genesis by detecting genome-scale lncRNAs expressions. 16 lung squamous cell carci-noma patients’paired normal and lung tumor tissues were obtained after surgery. First, extracted total RNA from frozen tissues by Trizol reagent; next, reverse-transcripted the total RNA to cDNA, got cRNA in vitro transcription synthesis, and then purified cRNA by spin columns, cRNA was transcribed into cDNA utilizing a random priming method and cDNA was labeled and hybridized to the Agi-lent human 4×180 K microarray. Processed signal data was obtained from hybridized images using Agilent Feature Extraction. Quantile normalization and differential expression data were performed using the Agilent GeneSpring. Data analyses were performed using R and Bioconductor. With abundant and varied probes accounting 38,361 lncRNAs in our microarray, the number of lncRNAs that expressed at a certain level could be detected is 28,055. From the results we found that there were 3,460 lncRNAs that differentially expressed (≥2 absolute fold-change) in lung squamous cell carcinoma tissues compared with normal tissue, among which 127 lncRNAs differentially expressed in all 16 lung squamous cell carcinoma samples. Our study is the first one to determine genome-wide lncRNAs expression pat-terns in lung squamous cell carcinoma by using microarray. The results indicated that clusters of lncRNAs were significantly differentially expressed. Of all 3,460 differentially expressed lncRNAs, 1901 lncRNAs were up-regulated and 1559 down-regulated. 9/16 lung sq-uamous cell carcinoma patients’up-regulated lncRNAs were more common than down-regulated ones. 7/16 patients down-regulated lncRNAs were more common than up-regulated ones. To predict the functions of lncRNAs, we constructed coding-non-coding gene co-expression networks of lncRNAs and mRNAs, on the basic of correlation cutoff 0.99 and P-value<0.05. We searched lncRNA-mRNA pairs whose distance of genomic locations was less than 10kb. In addition, we blatted the sequences of lncRNA and 3‘UTR of mRNAs to screen the sequence similarity lncRNA-mRNA pairs. We then got 33 lncRNA-mRNA pairs. The mRNAs of pairs were considered the target genes of lncRNAs. From the mRNAs, we found that TERC was likely a useful biomarker for the diagnosis of the non-small cell carcinoma. As the ENST00000363312 and sno-RNA_scaRNA_0_548 also had the similar function, we considered that they were likely useful biomarkers for the diagnosis of the non-small cell carcinoma. We concluded that lncRNAs differentially expressed in lung tumor tissues and normal tissues may exert a partial or key role in lung tumor development, and may provide potential targets for future treatment of lung squamous cell carcinoma.
