【作者】 邓坦；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理与病理生理学， 2009， 博士

【摘要】 鼻咽癌是一种在中国南部和东南亚地区高发的多基因遗传性恶性肿瘤。SPLUNC1基因是我室克隆的鼻咽癌候选抑瘤基因,它在鼻咽正常组织中高表达,而在鼻咽癌细胞系和组织中低表达。我室前期研究发现SPLUNC1蛋白是一种细胞表面的分泌性蛋白,它不仅能够通过结合细菌脂多糖与革兰氏阴性菌相结合,还能够抑制EB病毒。说明SPLUNC1可能是一种天然免疫保护分子。我室还克隆了多个候选鼻咽癌抑瘤基因,比如BRD7,NGX6,UBAP1,LPLUNC1,NAG7等,长期以来,我们都是用单基因转染的方法来研究它们抑制鼻咽癌的机制,但这些抑瘤基因是如何共同作用来阻止多基因遗传性肿瘤发生发展的机制还不为人知。miRNA是近年来生命科学的热点,它是一种内源性调控转录后信使RNA的小分子RNA,由于miRNA对其数以百计的靶基因的调控使miRNA与信使RNA之间组成了复杂的调控网络。正是miRNA调控多基因的这一特点,也许能帮助解决我们多个抑瘤基因共同作用机制的疑问。为了了解SPLUNC1与miRNA在鼻咽癌中调控的分子机制,我们进行了如下研究,并取得了相应的结果:[has-mir-141与has-mir-205在SPLUNC1转染细胞系中表达下调]我们将含有全长的SPLUNC1基因的载体转染至高侵袭,高转移性鼻咽癌细胞系5-8f中,并建立了稳定转染的细胞系。我们利用博奥生物有限公司开发的针对435个人(包含Nature杂志预测的有122个miRNA)、196个大鼠、261个小鼠成熟miRNA的哺乳动物miRNA芯片分析了转染SPLUNC1与其空白对照组的miRNA的差别。结果转基因组和对照组相比,共有25种miRNA表达明显上调,(fold change>3)29种miRNA表达明显下调。(fold change>3).其中两种强烈下调的miRNA(fold change>10),has-mir-141与has-mir-205通过northernblot和Q-PCR验证,确定确实在在SPLUNC1转染细胞系中表达下调。随后通过显微切割10例鼻咽癌组织和10例正常对照鼻咽组织进行Q-PCR验证,我们发现hsa-mir-141在鼻咽癌组织中的表达略强于鼻咽组织对照,两者之间的差异存在统计学意义,而has-mir-205表达则没有差别。[通过细胞生物学实验证实has-mir-141和has-mir-205在鼻咽癌细胞系中起着瘤基因的作用]对特定miRNA的相关细胞生物学功能的研究通常是通过脂质体转染它的成熟体(mimics)与抑制子(inhibitors)进入相应细胞,人为改变细胞内相应miRNA的含量,达到上调和下调细胞内miRNA的表达的作用进而研究这一系列改变对细胞生长,运动功能,穿膜能力,凋亡,细胞周期等功能的影响。MTT实验显示通过转染hsa-mr-141与hsa-mir-205的成熟体(mimics)与抑制子(inhibitors)及其相对应的成熟体对照(NC)抑制子对照(INC),在实验的前三天各转染组之间没有明显差别,第四天以后,hsa-mir-141抑制子组(inhibitors)较其他对照组生长速度减慢了30%左右,而hsa-mir-205成熟体(mimics)组较其他各对照组生长速度提高了40%左右。由于5-8f细胞是一种高侵袭,高转移性肿瘤细胞,我们想知道miRNA对细胞这一特性的影响。通过细胞划痕实验,我们发现hsa-mir-141和hsa-mir-205的成熟体组(mimics)均能显著促进5-8f细胞划痕伤口的愈合,而hsa-mir-141的抑制子组(inhibitors)则明显能减缓5-8f细胞划痕伤口的愈合;而通过transwell实验我们发现hsa-mir-141与hsa-mir-205的抑制子组(inhibitors)能明显降低5-8f细胞穿膜能力。流式细胞术分析发现抑制hsa-mir-141和hsa-mir-205能促进细胞凋亡,并能将5-8f细胞阻滞于G1到S期之间。通过western blot对细胞周期相关的一些分子进行检测,我们发现显示hsa-mir-205与141抑制子(inhibitors)均能促进caspase8与caspase3的表达达到促进凋亡的目的,同时它们同时具有促进JNK2及NF-κB P65表达,并抑制Iκα的表达。这些结果说明hsa-mir-205与141抑制子能够通过影响MAPK通路和TNFR通路的相关分子达到阻滞细胞周期的作用。[has-mir-141与has-mir-205所预测的靶基因与SPLUNC1转染影响的差异mRNA基因多位于相同的肿瘤调控通路]我们对转染SPLUNC1的差异miRNA has-mir-141和has-mir-205进行了靶基因预测,分别通过在线软件pictar,targetscan4.0进行预测。随后我们运用在线基因功能分析软件DAVID 2008 FunctionalAnnotation Bioinformatics Microassay Analysis Tools对hsa-mir-141与hsa-mir-205所预测的靶基因进行功能归类和信号通路分析,发现它们的靶基因涉及到MAPK,JAK-STAT,Cell cycle,wnt,tightjunction,Adherens junction等多条与肿瘤调控细胞凋亡等功能相关通路。由于hsa-mir-141与hsa-mir-205在鼻咽癌细胞相对低表达,所以我们考虑它们在通路中主要影响抑瘤基因,我们发现了其中一些具有研究价值的抑瘤基因:MAP2K4,MAP3K3,DLC1,TP53BP2等。更让我们感兴趣的是,我们在has-mir-141的靶基因中发现了同属我室克隆的抑瘤基因UBAP1。随后通过安捷伦全基因组芯片The WholeHuman Genome Oligo Microarray分析SPLUNC1转染对鼻咽癌细胞系mRNA水平的影响,我们发现转基因组与空白组比较上调明显(FOLDchange>2)的有1698个基因,转基因组与空白组比较下调明显(FOLDchange>2)的有2135个基因。通过对转染SPLUNC1的差异miRNA的预测靶基因和全基因组芯片上下调的差异基因进行比较对接,我们发现它们之间完全意义的一对一重合几乎没有。但运用安捷伦的差异基因通路分析软件,我们对SPLUNC1转染的差异基因对涉及到的细胞通路进行了归类和分析。我们发现SPLUNC1转染差异基因的通路分布状况和hsa-mir-141与hsa-mir-205所预测靶基因的通路分布极为相似,比如其中的MAPK,JAK-STAT,Cell cycle,wnt通路等。提示我们SPLUNC1基因影响这些肿瘤相关通路不仅可以自身直接调控,也可以通过调控miRNA来间接实现。[has-mir-141能通过直接作用UBAP1基因3’非编码区调控UBAP1蛋白的表达]我们将构建好的质粒与相应miRNA mimics或inhibitors共同转染至5-8f细胞内,通过荧光素酶检测,我们发现has-mir-141 mimics对UBAP1空白报告质粒组及UBAP1突变体组无明显影响,但对插入了UBAP13‘UTR片段的报告质粒组荧光素酶强度下降了50%,(图3-606)说明UBAP1实受hsa-mir-141直接影响,即UBAP1为hsa-mir-141的靶基因。通过western blot验证,结果表明UBAP1在5-8f及对照中低表达,在抑制hsa-mir-141和SPLUNC1转染细胞系中表达明显增强。这一结果不仅表明has-mir-141能影响UBAP1蛋白,而且说明SPLUNC1能通过下调has-mir-141来达到上调UBAP1共同发挥抑制鼻咽癌肿瘤的作用。[总结]前期研究表明SPLUNC1转染不仅影响MAPK通路中关键分子如JNK2,NF-κB等,也影响TNFR通路中的caspase3和caspase8进而导致细胞的凋亡和细胞周期的改变。我们通过western blot发现抑制hsa-mir-141有着同样的功能和作用,SPLUNC1转染引起hsa-mir-141与hsa-mir-205下调,而hsa-mir-141不仅可以影响MAPK通路的关键分子,也可以通过上调鼻咽癌候选抑瘤基因呢UBAP1,通过其对肿瘤发生的关键蛋白的抑制作用,共同扭转鼻咽癌的恶性表型。在整个假想调控通路中,差异miRNA的作用是最为关键的。SPLUNC1与has-mir-141是否确实存在共同对MAPK通路的调控作用,还需要更多的后期实验来加以证实。更多还原

【Abstract】 Nasopharyngeal carcinoma(NPC) is a polygenetic inherited malignant tumor with a high incidence in South China and Southeast Asia. SPLUNC1 gene,considered as the NPC candidate tumor suppressor gene, is a tissue-specific gene of human nasopharyngeal epithelium with low expression in both NPC cell lines and tissue.Previous studies in our lab found that SPLUNC1 is a secreted protein which covered on the surface of epithelium and can bind to the Gram negative nanobacteria,indicating that it might function as an innate immune defensive molecule. SPLUNC1 protein can bind to bacterial lipopolysaccharide,inhibit not only P.aeruginosa,but also the EB virus.Meanwhile,our lab also have cloned some other NPC candidate tumor suppressor gene,such as BRD7, NGX6,UBAP1,LPLUNC1,NAG7,but the mechanism how those genes interact with each others which cause the tumorigenesis of NPC is far from full known.Since miRNAs are short RNAs that post transcriptionally regulate messenger RNAs,and the formation of miR-Gene network depended on the hundreds of miRNAs and their numerous target genes,which might be an answer to the puzzle of NPC candidate tumor suppressor genes above mentioned.To uncover the molecular mechanisms between SPLUNC1 and miRNA in NPC,we did the research below,and also acquired some interesting results [Hsa-mir-141 and hsa-mir-205 were downregulated in SPLUNC1 overexpressed NPC ceLll lines]We transfected the full length of SPLUNC1 into the highly invasive and matastatic NPC cell line 5-8F,and constructed a stable cell line.Via microRNAarray analysis from CapitalBio Corporation(Beijing,China), we found that 29 miRNAs were upregulated and 25 miRNAs were downregulated in SPLUNC1 overexpressed cell line.(Fold change＞3) Two of the downregulated miRNA.(Fold change＞10),Hsa-mir-141 and hsa-mir-205 were confirmed by northern blot and qRT-PCR in the same cell lines.We further confirmed that expression of hsa-mir-141 was upregulated in 10 laser capture microdessection NPC tissues compared to the normal nasopharyngeal epithelium tissues,while has-mir-205 showed no difference between them.[Hsa-mir-141 and hsa-mir-205 play a role as oncogenes in NPC cell Iine5-8f.]Since SPLUNC1 gene was a tumor suppressor gene that could inhibit the cell growth of NPC cell lines-HNE1,we then want to know whether the change of hsa-mir-141 level could affect cell growth.Via MTT assay, Although there were no difference among these groups during the first three days after transfection,by day 5 and 6 we detected inhibiting hsa-mir-141 group grew more slowly than other groups with about 30% inhibition,and upregulated hsa-mir-205 group grew more quickly than other groups with about 40%promotion.Since 5-8f is a highly invasive and matastatic cancer cell line,we accomplished cell wound healing assays and transwell assays to identify whether hsa-mir-141 and hsa-mir-205 could influence cell migration and invasive ability.The result showed a significant reduction in both cell migration and invasion in the hsa-mir-141 and hsa-mir-205 inhibitor groups.Via flow cytometry analysis,we further found inhibition of hsa-mir-141 and hsa-mir-205 could slightly increase the cells apoptosis rate,and arrested cells in G0-G1 with a concurrent reduction in S phase cells compared to other control groups.These data suggested the inhibition of hsa-mir-141 and hsa-mir-205 may promote the NPC cell apoptosis and delay cell cycle progression from G1 to S phase.To explore the possible mechanism involved in a delay of G1 to S phase progression by 141 and 205 inhibitor groups in 5-8F NPC cells,cell cycle-related effectors were examined by western blot.And we found an obvious change in the expression of caspase 3,caspase8,NF-κB,I-κα,JNK2,P-ERK in 141 and 205 inhibitor groups.This suggests that inhibition of 141 and 205 could inhibit cell growth and cell cycle progression in 5-8F cells by regulating MAPK/ERK and TNFR pathway. [The Whole Human Genome Oligo Microarray Shows the Differences in genes expression from the transfection of SPLUNC1 also involve in the same signaling pathway as hsa-mir-141 and has-mir-205’s prediction target genes do]Firstly,we used both pictar and targetscan to search the possible targets of hsa-mir-141 and has-mir-205.Secondly,we used DAVID 2008 Functional Annotation Bioinformatics Microassay Analysis Tools to classify the function of the target gene which were predicted both from TargetScanS 4.2 and pictar.The result revealed that mostly(＞50%) target genes of hsa-mir-141 and has-mir-205 involved in the signaling pathways as MAPK,Wnt,JAK-STAT,Tight junction,Cell cycle,Adherens junction,which are important for tumorigenesis and tumor development Since the celluar function showed mir-141 and has-mir-205 may play a role as oncogene in NPC cell lines,we were interested to know whether tumor suppressor genes were potential regulated by hsa-mir-141.From Tumor Suppressor Gene Database,we found some hsa-mir-141 targeted genes,such as MAP2K4,MAP3K3,DLC1,TP53BP2 were considered as classical tumor suppressor genes.More interestingly,we found UBAP1 gene,one of the NPC candidate tumor suppressor gene previously cloned from our lab,was predicted as a hsa-mir-141 targeted gene by both pictar and targetscan.We then used the same cell line of transfected SPLUNC1 for the use of the Whole Human Genome Oligo Microarray.We found 1698 upregulated genes and 2135 downregulated genes between the transgenic group and the blank group.There was almost no perfect one to one genes match between the differences in mRNA and miRNA prediction target genes,but the Differences in genes expression from the transfection of SPLUNC1 involve in the gnaling pathway such as MAPK,Wnt,JAK-STAT,Cell cycle,which was also the same pathway hsa-mir-141 and has-mir-205’s prediction target genes involve in.We may draw a conclution that SPLUNC1 can influence these tumor related pathways not only in direct way,but also can regulate the pathways in indirect way by miRNA.[hsa-mir-141 regulated NPC tumor suppressor gene UBAP1 via directly targeting its 3’UTR]The UBAP1 gene was originally cloned in our lab,which has been found to be down-regulated in NPC tissues.The result showed that when co-transfected with hsa-mir-141 mimic in 5-8f cells,luciferase reporter of UBAP1 3’UTRs was dramatically inhibited(down over 50%),but no obvious changes were found when co-transfected with hsa-mir-141 inhibitor and mimic negative control;whereas mutation of the miR-141 binding sites(RL-UBAP1 M) could diminish the regulating function of miR-141 on UBAP1,which verified that the effect of the miR-141 is due to direct interaction with the binding sites in UBAP1 3’UTRs.The next experiment was to detect whether transfection with hsa-mir-141 inhibitor could affect the UBAP1 protein levels.The cell extracts from 5-8f,141 inhibitor group,inhibitor nagetive control group, 5-8F/pcDNA3.1/his-SPLUNC1 and 5-8F/pcDNA3.1/his were used for western blot.The result demonstrated that UBAP1 were upregulated in both SPLUNC1 overexpressed cells and miR-141 inhibitor group,while hardly or barely detectable in the other groups.The result not only indicated that mir-141 could influence the expression of UBAP1,but also showed that the transfection of SPLUNC1 could upregulate UBAP1 via do wnregulating hsa-mir-141.In our previous studies,SPLUNC1 may affect some key molecules such as JNK2,NF-icB,and also influence the expression of caspase 8 and caspase 3 in TNFR pathway,which led to the DNA fragmentation and finally cell apoptosis.In this study,we confirmed the inhibitor of hsa-mir-141 had the same affect to influence the ERK/MAPK and TNFR pathway by western blotting.With our present study,SPLUNC1 might downregulate hsa-mir-141,while the downregulation of hsa-mir-141 might upregulate its target gene UBAP1 which may inhibit tumor growth by degrading the key proteins of tumorigeness.Hsa-mir-141 played a role as a turning point in this system, the NPC tumor suppressor genes SPLUNC1 may upregulate UBAP1 via do wnregulating hsa-mir-141.Whether these genes in ERK/MAPK pathway also involve in the mechanism of tumor inhibition need to be further studied and verified.更多还原