摘要
研究背景：
胃癌的发生是由不同时期，不同癌基因的激活和/或抑癌基因的失活导致的分子疾病，作用机制可能涉及到不同染色体上多个基因的变化，其中端粒酶的活化在胃癌的发生发展中就具有重要的作用，端粒酶在85%以上的癌组织中表达，而正常的体细胞中不表达或者低表达。人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)作为端粒酶的限速亚单位直接决定端粒酶的活性。因此，hTERT的活化导致的端粒酶激活是肿瘤发生发展的重要环节。课题组前期研究发现miR-1182、miR-1266、miR-1207-5p可以从转录后水平调控胃癌细胞hTERT的表达，从而抑制胃癌细胞增殖迁移。而占全部ncRNA转录大部分的长链非编码RNA（long non-ccoding RNA，lncRNA）因其丰富的调控手段在基因转录后的调节中日益受到重视，近来提出了一种全新基因表达调控模式，即竞争性内源性RNA模式（competing endogenous RNAs,ceRNA），也就是说lncRNA、假基因转录物以及mRNA转录物可以竞争性地结合miRNA应答元件(miRNA response element，MRE)来影响基因的转录后调控。本研究的目的在于探索是否存在调控胃癌hTERT的lncRNA，以及它的作用机制。
研究方法：
(1)采用lncRNA芯片技术筛选可能参与调控hTERT的lncRNA；(2)采用生物信息学技术预测可能调控hTERT的lncRNA；(3)采用实时荧光定量PCR(Real-time PCR)验证筛选的hTERT调控相关的lncRNA；(4)采用特异的lncRNA siRNA化学片段转染细胞，或负载干扰lncRNA序列的慢病毒感染细胞，下调细胞内lncRNA的表达丰度；(5)采用MTT实验、流式细胞仪检测细胞周期、平板克隆形成实验、平面化痕实验、小室侵袭实验以及裸鼠皮下成瘤实验对hTERT调控相关的lncRNA进行功能分析；(6)采用生物信息学技术预测可能与lncRNA发生作用的miRNA；(7)使用双荧光素酶实验检测lncRNA和miRNA对hTERT的调控机制；(8)采用Western blot检测lncRNA和miRNA对靶基因hTERT蛋白含量的调控作用；(9)采用TRAP实验检测lncRNA和miRNA对靶基因hTERT蛋白活性的调控作用；(10) Real-time PCR检测过表达lncRNA后，miRNA含量的变化；(11)RNA dot blot实验检测lncRNA与hTERT3’UTR都可以直接结合miRNA。
研究结果：
一、调控hTERT的lncRNA的预测及鉴定
1. lncRNA基因芯片筛查共有4620条lncRNA在hTERT阳性的胃癌组织和hTERT阴性的癌旁正常组织比较中存在显著差异，其中有1134条在hTERT阳性的组织中表达上调超过5倍或以上。
2.生物信息学分析预测到了第5对染色体hTERT基因组附近的12条lncRNA，并在5对胃癌及对应癌旁组织中采用Real-time PCR进行验证，共筛选出8条lncRNA在hTERT阳性胃癌组织中表达显著高于hTERT阴性的癌旁胃粘膜组织。将上述8条lncRNA与lncRNA基因芯片的结果进行比对，发现共有3条存在交叉，且差异最为明显的是lncRNA BC032469，表达差异为20.566。
二、lncRNA BC032469在胃癌组织中的表达及其临床意义
1. Real-time PCR结果显示lncRNA BC032469在癌组织中的表达水平明显高于癌旁胃组织，且lncRNA BC032469在癌和癌旁胃组织中相对含量的比值（C/P）与hTERT蛋白在对应的癌和癌旁胃组织中相对含量的比值（C/P)具有相关性，p<0.01。
2.进一步分析lncRNA在58例癌和癌旁胃组织中相对含量C/P比值与肿瘤大小相关（p<0.05）；与肿瘤的分化程度相关（p<0.05）。
3.生存分析表明lncRNA BC032469C/P>C/P平均值组的患者术后生存时间较lncRNA BC032469C/P 三、lncRNA BC032469在胃癌细胞中的功能学研究
通过负载shRNA-BC032469慢病毒成功感染MKN28和SGC-7901细胞后，MTT实验表明敲低lncRNA BC032469可明显抑制MKN28和SGC-7901细胞的增殖能力（p<0.01），流式细胞术表明下调lncRNA BC032469可以将MKN28和SGC-7901细胞周期阻滞在G1期；平板克隆形成实验表明下调lncRNA BC032469可抑制SGC-7901细胞克隆形成能力；划痕实验表明下调lncRNA BC032469可抑制MKN28和SGC-7901细胞体外迁移能力；Transwell侵袭实验表明下调lncRNA BC032469对MKN28和SGC-7901细胞体外侵袭能力无影响。裸鼠皮下成瘤实验发现，干扰lncRNA BC032469组的SGC-7901细胞皮下成瘤能力比阴性对照组的成瘤能力下降。
四、lncRNA BC032469调控胃癌细胞hTERT表达的分子机制研究
1.课题组前期实验寻找到5条与胃癌hTERT调控相关的miRNA（miR-138、miR-491-5p、miR-1182、miR-1207-5p、miR-1266），进一步通过PCR实验验证这5条miRNA在58对胃癌组织和癌旁组织中表达的差异，其中miR-1207-5p、miR-1266在hTERT阴性的的癌旁正常粘膜组织中的丰度显著高于hTERT阳性的的胃癌组织（p<0.05）。
2.生物信息学预测到lncRNA BC032469序列上有多个高评分的miR-1266、miR-1207-5p种子区的结合位点。 Western blot结果表示在胃癌细胞中干扰lncRNABC032469能下调hTERT的表达，在此基础上强制表达hTERT ORF区（缺乏hTERT3’UTR）能回复hTERT蛋白的下调。双荧光素酶实验发现： miR-1266、miR-1207-5p可以下调hTERT3’UTR荧光素酶的表达。结合western blot回复实验的结果，干扰lncRNA，可能是通过影响hTERT3’UTR发挥作用。推测也许lncRNABC032469与miR-1266、miR-1207-5p存在相互作用来调控hTERT的表达。
3. lncRNABC032469被干扰后，hTERT3’UTR荧光素酶活性降低，同时转染anti-miR-1207-5p对hTERT3’UTR荧光素酶的下调能力显著减弱；同时转染anti-miR-1266未能显著下调hTERT3’UTR荧光素酶的表达。在lncRNA BC032469被干扰的基础上，转染突变了miR-1207-5p结合位点的hTERT3’UTR荧光素酶报告基因，萤火虫荧光素酶表达下调的情况显著减弱；转染突变了miR-1266结合位点的hTERT3’UTR荧光素酶报告基因，萤火虫荧光素酶表达的下调未受影响。
4. Western blot结果表示在干扰lncRNA BC032469的同时，拮抗miR-1207-5p，可以部分地回复hTERT蛋白的下调。而拮抗miR-1266，不能回复hTERT蛋白的表达。
5. TRAP实验结果显示干扰lncRNA BC032469，SGC-7901细胞的hTERT活性降低，在干扰lncRNA BC032469的基础上，外源性地拮抗了miR-1207-5p，可以部分地回复hTERT蛋白的活性。
6. Real-timePCR发现过表达lncRNABC032469后，miR-1207-5p含量降低，提示lncRNA BC032469可能促进miR-1207-5p的降解。
7. RNA dot blot实验发现lncRNA BC032469和hTERT3’UTR都可以直接结合miR-1207-5p，证明lncRNABC032469能竞争结合miR-1207-5p，从而部分解除miR-1207-5p对hTERT的抑制。
研究结论：
1、通过生物芯片及生物信息学技术，首次证实lncRNA BC032469参与了胃癌细胞hTERT表达的调控，并与胃癌患者的不良预后密切相关；
2.功能学研究表明，lncRNA BC032469的敲低可显著抑制胃癌细胞体外增殖、细胞周期、克隆形能力以及迁移能力，抑制胃癌细胞动物体内的增殖能力，在胃癌中起到促癌基因的作用；
3. lncRNA BC032469可能通过对miR-1207-5p的封闭，部分解除对hTERT的负性调控，促进hTERT蛋白含量和活性的增高，来促进胃癌细胞增殖和迁移，从lncRNA层面初步阐明胃癌发生发展的新机制，
以上研究表明lncRNA BC032469参与了胃癌细胞hTERT表达的调控，并与胃癌的预后密切相关，为胃癌的治疗提供了新的靶点。
Background:
Gastric cancer is molecular disease caused by activation of different oncogene and/orinactivation of anti-oncogene in different periods. The mechanisms may involve mutationof multiple genes on different chromosomes, among which the activation of telomeraseplays a significant role in the development of gastric cancer. Telomerase expressed in morethan85%of cancer tissues, whereas not expressed or low expressed in normal somatic cells.Human telomerase reverse transcriptase,(hTERT), as the rate-limiting subunit oftelomerase, directly determines the activity of telomerase. Therefore, the activation oftelomerase caused by hTERT's activation is an important part in tumorigenesis. Ourprevious studies mainly focused on miRNA-related gastric hTERT regulation, whilenon-coding RNA has increasingly got much focus on post-translational modifications ingene by its abundant regulating methods. Now, a significant portion of the noncodingtranscriptome, including long noncoding RNAs and pseudogenes, harbors microRNA(miRNA)-response elements (MRE). A totally new regulation pattern of gene expressionwas put forward recently, namely, competing endogenous RNAs. That is to say, lncRNA,false gene transcripts and mRNA transcripts can competitively combine with miRNAresponse element (MRE) to influence the regulation of gene after transcription. This studywas aimed to explore lncRNA's regulation of gastric cancer hTERT and its function.
Methods：
1. lncRNA gene chip technology was applied to screen lncRNA which may beinvolved in regulation of hTERT;
2. Bioinformatics technique was used to predict the possible lncRNA which might playa role in regulating hTERT;
3. Real-time quantitative PCR (real-time PCR) analysis was performed to validatelncRNA may be regulated by selected hTERT;
4. The specific lncRNA siRNA oligo was transiently transfected into gastric cancercells and the lentivirus loading shRNA-lncRNA was used to infect the cells todown-regulate the lncRNA expression level;
5. MTT assay, flow cytometry, colony formation assay, wound healing assay and theinvasion assay and tumorigenicity in nude mice experiments were applied to analyze thefunction of lncRNA regulating hTERT;
6. Bioinformatics technique was used to predict the possible miRNA which mightinteract with lncRNA;
7. The probable lncRNA and miRNA targeting hTERT was verified by dual luciferasereporter assay;
8. Western blot was applied to validate the regulating role of lncRNA and miRNA ontarget genes of hTERT protein content;
9. TRAP was used to detect the regulating role of lncRNA and miRNA on target genesof hTERT protein activity.
10. Real-timePCR was applied to validate the content of miR-1207-5p after overexpression of lncRNABC032469.
11. RNA dot blot test showed that lncRNA BC032469and hTERT3'UTR which bothcan interact with miR-1207-5p directly.
Results:
1. Prognosis and certification of lncRNA regulating hTERT.
There are4620lncRNAs varied a lot in comparing the hTERT-positive gastric cancertissues and hTERT-negative normal pericarcinous tissues in gene chip screening, and1134of which were up-regulated more than5-fold or more in hTERT-positive tissues.
Bioinformatics predicted that twelve lncRNAs near the fifth genome hTERT, andreal-time PCR was used to verify five pairs of gastric cancer and corresponding adjacenttissues, and8lncRNAs were screened out which expressed significantly higher inhTERT-positive gastric cancer tissues than in hTERT-negative adjacent mucosa. Comparethose8lncRNAs mentioned above with lncRNA gene chip results, and we found that there were3of them overlapped each other, and the most obvious difference is lncRNABC032469with differential expression of20.566.
2. Expression and clinical significance of lncRNA BC032469in gastric cancer tissue.
Real-time PCR results showed lncRNA BC032469in cancer tissues was significantlyhigher than in adjacent gastric tissue and the ratio of lncRNA BC032469's relative amountin gastric cancer and adjacent tissues(C/P) had a relation with hTERT protein's relativeamount in corresponding gastric cancer and adjacent tissues (C/P), p <0.01
Further analysis was made of the ratio of lncRNA's relative amount in58cases ofgastric cancer and adjacent tissues(C/P) was related the size of tumor (p <0.05); and it'salso associated with the degree of tumor's differentiation (p <0.05).
Survival analysis showed survival time of patients after surgery from lncRNABC032469C/P> C/P group decreased comparing with the patients from lncRNABC032469(C/P 3. Functional study of lncRNA BC032469in gastric cancer cell.
By loading lentiviral shRNA-BC032469to infect MKN28and SGC-7901cellssuccessfully, MTT experiments show that decreased lncRNA BC03249can inhibit MKN28and SGC-7901cell proliferation (p <0.01), and flow cytometry showed down-regulatinglncRNA BC032469can block MKN28and SGC-7901cell cycle in G1phase; colonyformation assay showed that down-regulating lncRNA BC032469can inhibit SGC-7901cells' coloning ability; scratch experiments showed down-regulating lncRNA BC032469can inhibit MKN28and SGC-7901cells in vitro migration; Transwell invasion assayshowed down-regulating lncRNA BC032469had no influence on MKN28and SGC-7901cells in vitro invasion. Tumor formation in nude mice of interference group of lncRNABC032469was lower than the negative controlled group.
4. Molecular mechanism of lncRNA BC032469regulating hTERT expression ofgastric cancer
In our previous study, we found five miRNAs（miR-138, miR-491-5p, miR-1182,miR-1207-5p, miR-1266）related to regulation of gastric cancer hTERT. It was furtherverified in PCR experiments that the differences of these five miRNAs in58pairs of gastriccarcinoma and adjacent tissue expression, among these miRNAs, the abundance of miR-1207-5p, miR-1266in hTERT-negative cancer adjacent normal mucosa weresignificantly higher than that in hTERT-positive gastric cancer.（p<0.05）
Bioinformatics predicted that in lncRNA BC032469sequence, there were multiplemiR-1266and miR-1207-5p seed region binding sites with high score. Western blot resultsshowed in gastric cancer cells interfering lncRNA BC032469could down-regulateexpression of hTERT, on this basis, forced expression of hTERT ORF region (lackinghTERT3'UTR) can recover the downregulation of hTERT protein. Dual-Luciferase foundthat miR-1266, miR-1207-5p can down-regulate the expression of hTERT3'UTR luciferase.Combining the results from western blot experiments, interferring lncRNA work byaffecting hTERT3'UTR. It's speculated that perhaps lncRNA BC032469interact withmiR-1266and miR-1207-5p to regulate the expression of hTERT.
After lncRNA BC032469was disturbed, the activity of hTERT3'UTR luciferasedecreased, meanwhile, transfecting anti-miR-1207-5p on hTERT3'UTR luciferase wasweakened; and transfected with anti-miR-1266did not significantly down-regulate theexpression of hTERT3'UTR fluorescence luciferase. Based on the disturbance of lncRNABC032469, transfection of mutated miR-1207-5p binding sites in hTERT3'UTR luciferasereporter gene, luciferase activty significantly increased; transfected with mutated miR-1266binding sites of hTERT3'UTR luciferase reporter gene, down-regulating of luciferase wasnot weakened.
Western blot results showed when interfering lncRNA BC032469, antagonizingmiR-1207-5p can partially recover the expression of hTERT protein. However,antagonizing miR-1266can not recover the expression of hTERT protein. TRAP resultsshowed that the interference of lncRNA BC032469could decrease the activity ofSGC-7901cell hTERT, on the basis of interfering lncRNA BC032469, exogenouslyantagonize the miR-1207-5p, may be partially recover the activity of hTERT protein.
Real-timePCR found that over expression of lncRNABC032469, decreased the contentof miR-1207-5p, suggesting that lncRNA BC032469may promote the degradation ofmiR-1207-5p.
The RNA dot blot test showed that lncRNA BC032469and hTERT3'UTR can bedirectly combined with miR-1207-5p, prove that lncRNABC032469can compete withmiR-1207-5p, which part of the lifting of the inhibition of miR-1207-5p on hTERT.
Conclusion:
1. By biochip and bioinformatics, it was confirmed for the first time that lncRNABC032469was involved in the regulation of gastric cancer cell hTERT and it was closelyrelated to poor prognosis for patients with gastric cancer.
2. Functional study showed that lowering lncRNA BC032469can obviously inhibitgastric cancer cell proliferation, cell cycle, cloning and migration, and restrain cancer cellproliferation in vivo, and play a carcinogenic role in gastric cancer.
3. lncRNA BC032469could possibly remove the negative regulation of hTERTthrough blocking miR-1207-5p, to promote the increase of hTERT protein content andactivity, and then promote gastric cancer cell proliferation and migration. Illustrate newmechanism in the development of gastric cancer from the angle of lncRNA.
Above mentioned showed that lncRNA BC032469was involved regulating theexpression of gastric cancer cell hTERT, and it was closely related to prognosis for gastriccancer. It provided a new target for treatment of gastric cancer.
引文
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2Kantharidis, P., A. El-Osta, M. deSilva, et al., Altered methylation of the human MDR1promoter is associated withacquired multidrug resistance. Clin Cancer Res,1997.3(11):2025-2032.
1Sun, M., R. Xia, F. Jin, et al., Downregulated long noncoding RNA MEG3is associated with poor prognosis andpromotes cell proliferation in gastric cancer. Tumour Biol,2013.
2Yang, F., X.C. Xue, J.W. Bi, et al., Long noncoding RNA CCAT1, which could be activated by c-Myc, promotes theprogression of gastric carcinoma. J Cancer Res Clin Oncol,2013.139(3):437-445.
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3Arita, T., D. Ichikawa, H. Konishi, et al., Circulating long non-coding RNAs in plasma of patients with gastric cancer.Anticancer Res,2013.33(8):3185-3193.
4Chung, S., H. Nakagawa, M. Uemura, et al., Association of a novel long non-coding RNA in8q24with prostate cancersusceptibility. Cancer Sci,2011.102(1):245-252.
5Kotake, Y., T. Nakagawa, K. Kitagawa, et al., Long non-coding RNA ANRIL is required for the PRC2recruitment toand silencing of p15(INK4B) tumor suppressor gene. Oncogene,2011.30(16):1956-1962.
6Wang, Y., W. Chen, C. Yang, et al., Long non-coding RNA UCA1a(CUDR) promotes proliferation and tumorigenesis ofbladder cancer. Int J Oncol,2012.41(1):276-284.
1Sulentic, C.E. and N.E. Kaminski, The long winding road toward understanding the molecular mechanisms for B-cellsuppression by2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci,2011.120Suppl1: S171-91.
2Gutschner, T. and S. Diederichs, The hallmarks of cancer A long non-coding RNA point of view. RNA Biol,2012.9(6):
3703-719.Clemson, C.M., J.A. McNeil, H.F. Willard, et al., XIST RNA paints the inactive X chromosome at interphase: evidencefor a novel RNA involved in nuclear/chromosome structure. J Cell Biol,1996.132(3):259-275.
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2Gutschner, T. and S. Diederichs, The hallmarks of cancer A long non-coding RNA point of view. RNA Biol,2012.9(6):703-719.
3Clemson, C.M., J.A. McNeil, H.F. Willard, et al., XIST RNA paints the inactive X chromosome at interphase: evidencefor a novel RNA involved in nuclear/chromosome structure. J Cell Biol,1996.132(3):259-275.
4Flynn, R.A. and H.Y. Chang, Active chromatin and noncoding RNAs: an intimate relationship. Curr Opin Genet Dev,2012.22(2): p.172-8.
5Rinn, J.L., M. Kertesz, J.K. Wang, et al., Functional demarcation of active and silent chromatin domains in humanHOX loci by noncoding RNAs. Cell,2007.129(7):1311-1323.
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