胃癌中microRNA-200家族的表达及其功能的研究
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摘要
胃癌是我国最常见的恶性肿瘤之一，2009年全国恶性肿瘤发病和死亡分析显示：胃癌发病率居恶性肿瘤的第二位，死亡率居第三位，依然是我国恶性肿瘤死亡的主要原因。我国胃癌具有发病率高、早期诊断率低、5年生存率低等特点。胃癌的早期诊断率仍徘徊在10%-20%，这亦是胃癌在我国死亡率居高居不下的主要原因之一。目前手术治疗是唯一经证实的可能会治愈胃癌的手段，但即便是已行胃癌根治术的患者，仍有大部分会出现复发或转移。术后的辅助放化疗对控制胃癌的复发或转移可能有效。化疗在晚期胃癌患者的综合性治疗中亦发挥重要作用，然而耐药一直以来是胃癌化疗遇到一个很大瓶颈。由此得出，胃癌的早期诊断和及时有效的治疗是提高其5年生存率的关键，所以寻找敏感而特异性强的分子标志物、明确治疗的分子靶标以及正确的进行预后评估则是至关重要的任务。miRNA是一类机体内源性表达的、长度在18~25个核苷酸的非编码小分子RNA，这些仅占人类基因1%的miRNA分子，却很可能调控着人类三分之一以上基因的表达、修饰、转录和翻译的过程。miRNA的发现可能成为极有应用价值的分子标志物，为恶性肿瘤的早期诊断和预后评估开启了一条新的道路。其中miRNA-200家族成为目前关注的焦点，miRNA-200家族包括miRNA-200a、miRNA-200b、miRNA-200c、miRNA-141和miRNA-429五个成员。具有高度保守、基因簇集现象、时序性和组织特异性等生物学特性。该家族在某些肿瘤中呈低表达(如肝癌、肾细胞癌)，而在一些肿瘤中则呈高表达(如膀胱癌、宫颈癌)。研究已经证明miRNA-200家族的各个成员单独被动表达可以阻止上皮间质转化(epithelial-mesenchymal transition,EMT)的改变，共同作用则可以调节ZEB1和SIP1(ZEB2)的表达，但该家族在人类恶性肿瘤中所扮演的具体角色尚未明晰。本研究将采用实时定量PCR技术检测miRNA-200家族五个成员在胃癌组织及其配对的癌旁正常组织中的表达差异，并分析其表达与胃癌临床病理参数的相关性。选取该家族具代表性的成员miRNA-200c，利用定量Real-time PCR检测其在4株胃癌细胞中的表达水平，采用瞬时转然的方法将miRNA-200c mimics或阴性对照转入胃癌细胞株以获得高表达miRNA-200c的细胞模型，然后通过MTT法检测肿瘤细胞的增殖情况，用流式细胞仪检测细胞周期变化，以及应用细胞划痕实验检测肿瘤细胞的迁移能力，以了解miRNA-200c对胃癌细胞生物学行为的影响，从而能进一步阐明miRNA-200c在胃癌发生、发展中的作用。并应用生物信息学软件对miRNA-200c的靶基因进行筛选，同时采用双荧光素酶报告基因法验证靶基因，为以后深入研究miRNA-200c的分子机制打下基础。
第一部分胃癌组织中microRNA-200家族的表达及意义
目的：探讨miRNA-200家族五个成员在胃癌组织及其配对的癌旁正常组织中的表达差异，并明确其表达与胃癌临床病理参数的相关性。
方法：收集46例胃癌患者手术切除标本，应用定量Real-time PCR检测miRNA-200家族五个成员在胃癌组织及其配对的癌旁正常组织中的差异表达情况，并应用独立样本t检验和单因素方差分析的统计方法研究其表达水平与胃癌临床病理参数之间的相关性。
结果：
1miRNA-200家族在胃癌组织和配对的癌旁正常组织中的表达水平。采用定量Real-time PCR检测miRNA-200家族五个成员在46例胃癌组织及与其配对的癌旁正常组织中的相对表达情况。结果显示46例胃癌患者miRNA-200a、miRNA-200b、miRNA-200c、miRNA-141和miRNA-429在癌组织中的表达水平均显著低于相应的癌旁正常组织，表达差异有显著的统计学意义(P<0.05)，五者分别在86.96%(40/46)、91.30%(42/46)、93.48%(43/46)、93.48%(43/46)和80.43%(37/46)的癌组织标本中表达水平低于癌旁正常组织。
2miRNA-200家族五个成员的表达与胃癌临床病理参数的相关性。采用t检验或单因素方差分析对miRNA-200a、miRNA-200b、miRNA-200c、miRNA-141和miR-429的表达水平与胃癌临床病理参数的相关性进行分析。结果显示：miRNA-200家族五个成员的低表达水平均与组织学分级、脉管瘤栓呈显著相关(P<0.05)，而与胃癌患者的性别、年龄、肿瘤大小、淋巴结转移、浸润深度、TNM分期、Lauren分型和Borrmann分型无明显相关(P>0.05)。结论：
1miRNA-200家族五个成员(miRNA-200a、 miRNA-200b、miRNA-200c、miRNA-141和miRNA-429)在胃癌患者癌组织中的表达水平均显著低于相应的癌旁正常组织，提示该家族可能不适合作为胃癌早期诊断的分子标志物，但提高该家族5个成员内源性的表达，可能有助于胃癌的治疗和有效地预防，可能成为胃癌治疗和预防的分子靶标。
2miRNA-200家族的五个成员在癌组织中的低表达与组织学分级、脉管瘤栓呈显著相关，提示其可能在胃癌发生及发展阶段起着关键性的作用。
第二部分microRNA-200c对胃癌细胞生物学功能的影响
目的：选取该家族具代表性的成员miRNA-200c，研究其对胃癌SGC7901细胞的增殖、周期和迁移能力等生物学行为的影响。
方法：利用定量Real-time PCR法检测其在4株胃癌细胞中的表达水平，采用瞬时转然的方法将miRNA-200c mimics或阴性对照转入胃癌细胞株以获得高表达miRNA-200c的细胞模型，然后通过MTT法检测肿瘤细胞的增殖情况，用流式细胞仪检测细胞周期变化，以及应用细胞划痕实验检测肿瘤细胞的迁移能力。
结果：
1miRNA-200c在不同胃癌细胞株中的表达水平。应用定量Real-timePCR检测4株胃癌细胞MGC803、 HGC27、 SGC7901和AGS中miRNA-200c的表达情况，以胃癌患者癌旁正常组织作随机对照，结果显示：miRNA-200c在4株胃癌细胞中的表达水平均显著下调，减少倍数分别是9.93倍、9.69倍、7.18倍和2.93倍(P<0.0001)。
2人胃癌细胞株SGC7901转染后miRNA-200c的表达水平。选择SGC7901细胞作为体外细胞模型，利用定量Real-time PCR法检测转染miRNA-200c mimics和阴性对照48h后miRNA-200c的表达水平，结果显示：转染miRNA-200c mimics后胃癌SGC7901细胞中miRNA-200c的表达水平较空白对照组和阴性对照组均显著增高，平均升高约10.2倍(P<0.05)。提示miRNA-200c mimics转染成功，可进行后续实验。
3过表达miRNA-200c对SGC7901细胞增殖的影响。在转染miRNA-200c及阴性对照24、48、72h后，应用MTT方法对SGC7901细胞增殖进行检测，结果显示：细胞转染24、48、72小时后，miRNA-200cmimics实验组的OD值为0.308±0.010、0.360±0.016和0.457±0.029，而阴性对照组的OD值为0.339±0.008、0.505±0.006和0.775±0.010，各时间点两组之间比较均有统计学意义(P<0.05)。通过OD值计算细胞抑制率，miRNA-200c mimics实验组三个时间点的抑制率分别为10.76±2.50%、30.83±2.58%和33.86±15.9%，而阴性对照组的抑制率分别为1.78±2.70%、3.01±2.15%和0.64±0.69%，两组间各时间点比较均有显著差异(P<0.05)。表明胃癌细胞内miRNA-200c表达水平的上调可抑制胃癌细胞的增殖。
4过表达miRNA-200c对SGC7901细胞周期的影响。应用流式细胞仪对miRNA-200c转染48h后的SGC7901细胞进行细胞周期的检测，结果显示：细胞转染48小时后，miRNA-200c mimics实验组的G0/G1期、S期和G2/M期的比例分别为72.77±0.85%、8.87±0.45%和18.36±1.29%，而阴性对照组三期的比例分别为42.10±0.80%、43.57±1.27%和14.33±2.06%，两组之间比较G0/G1期和S期存在显著差异(P<0.05)，呈现G0/G1期阻滞。表明胃癌细胞内miRNA-200c表达水平的上调可阻滞G0/G1期细胞向S期进展。
5过表达miRNA-200c对SGC7901细胞迁移能力的影响。采用细胞划痕实验检测过表达miRNA-200c对SGC7901细胞迁移能力的影响，结果显示：转染24小时后，miRNA-200c mimics实验组镜下细胞迁移宽度为344.40±17.24μm，而阴性对照组为270.51±18.21μm，两组之间比较有显著差异(P<0.05)。表明过表达miRNA-200c的细胞体外迁移能力明显低于阴性对照组。
结论：
1miRNA-200c在4株胃癌细胞株MGC803、HGC27、SGC7901和AGS中均存在不同程度的表达下调，与胃癌组织结果相一致。
2miRNA-200c mimics能成功转染人胃癌细胞SGC7901，以上调SGC7901细胞中miRNA-200c的表达水平。
3miRNA-200c在胃癌细胞SGC7901的过表达可抑制细胞增殖。提示miRNA-200c可能参与了细胞增殖的分子调控，有望成为胃癌治疗新的靶标。
4miRNA-200c在胃癌细胞SGC7901的过表达可阻滞肿瘤细胞从G0/G1期向S期的进展，从而推测miRNA-200c可能是通过改变细胞周期来抑制细胞增殖。
5miRNA-200c在胃癌细胞SGC7901的过表达可降低细胞体外迁移能力。提示miRNA-200c的表达水平可影响肿瘤细胞的迁移能力，为进一步研究胃癌治疗新的靶标提供了依据。
第三部分microRNA-200c靶基因的筛选和验证
目的：预测并验证RhoE是miRNA-200c的靶基因，为深入研究miRNA-200c的分子机制奠定基础。
方法：应用生物信息学软件对miRNA-200c的靶基因进行预测和筛选，同时采用双荧光素酶报告基因法对预测的靶基因进行验证。
结果：
1miRNA-200c靶基因的生物信息学预测。目前对miRNA的靶基因筛选是应用生物信息学软件进行预测，综合分析TargetScan、PicTar、mirBase和miRanda等多个生物信息学软件预测的结果显示：miRNA-200c属于12号染色体，位置为12：7072862-7072929[+]。TargetScan和PicTar预测到的miRNA-200c潜在靶基因数分别为1057和639。本实验选取了近年来备受关注的RhoE基因(NM_005168)作为进一步研究目标，分析发现其3’UTR含有2段可能与miRNA-200c结合的位点。因此，我们针对此序列构建报告载体，进行双荧光素酶报告基因系统实验验证。
2双荧光素酶报告基因系统验证RhoE是miRNA-200c的靶基因。我们应用验证靶基因与miRNA最经典的方法“双荧光素酶报告基因系统”对RhoE是否为miRNA-200c的靶基因进行验证。本实验先成功构建了RhoE3’UTR和突变型RhoE3’UTR的表达载体，通过在SGC7901细胞中分别共转染RhoE3’UTR+miRNA-200c mimics、RhoE3’UTR+miRNA-200cNC、RhoE mut3’UTR+miRNA-200c mimics和RhoE mut3’UTR+miRNA-200c NC分为四个实验组，48h后的检测结果显示：只有RhoE3’UTR+miRNA-200c mimics组荧光素蛋白的表达水平显著降低(P<0.05)，说明miRNA-200c可靶向抑制RhoE3’UTR报告基因的活性。但miRNA-200c对RhoEmut3’UTR的表达载体没有抑制作用，miRNA-200cNC对RhoE3’UTR和RhoEmut3’UTR的表达载体均没有抑制作用(P>0.05)。因此本实验结果验证了RhoE是miRNA-200c的靶基因，提示miRNA-200c对RhoE存在抑制性功能的作用。
结论：
miRNA-200c可靶向抑制RhoE3’UTR报告基因的活性，验证了RhoE是miRNA-200c的靶基因，提示miRNA-200c对RhoE存在抑制性功能的作用，可能通过抑制RhoE发挥其生物学功能，从而影响着胃癌的发生、发展和侵袭转移的过程。为我们进一步阐明miRNA-200c在胃癌中的具体分子机制奠定了基础。
Gastric cancer is one of the most common malignant tumors in China.The incidence of gastric cancer in malignant tumors is the second place. It isthe third leading cause of cancer-related death in2009. Gastric cancer has thehigh incidence, the low rate of early diagnosis and the5-year survival in China.The early diagnostic rate of gastric cancer is still hovering around10%-20%,which is also the main reason of high mortality rate in China. At present,surgery is the only method to be proven to cure gastric cancer. But even if theyhad accepted the radical operation, most of patients with gastric cancer wouldinduce recurrence or metastasis. Postoperative adjuvant radiation andchemotherapy may be available to control the recurrence or metastasis ofgastric cancer. Chemotherapy plays an important role in comprehensivetreatment of advanced gastric cancer, but the drug resistance of chemotherapyhas become a big bottleneck. Therefore, we concluded that the early diagnosisand timely effective treatment is the key to improve the5-year survival rate ingastric cancer, so it is the most crucial task to look for a sensitive and specificmolecular markers, clarifing molecular targets of treatment and correctingprognosis assessment. MicroRNAs (miRNAs) are a new class of endogenousand non-coding small RNA, the length of which is approximately18-25nucleotides. These miRNAs account for only1%of human genes, but theymay regulate more than a third of gene expression, modification, transcriptionand translation in human. These miRNAs may have the potential applicationvalue of molecular markers, for the early diagnosis and prognosis assessmentof malignant tumor to open up a new road. Currently, the miRNA-200familyhas become the focus of miRNAs study. The family includes five members,which are miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141andmiRNA-429. They have several biological characteristics, including the highlyconservative, gene cluster phenomenon, timing, and tissue specificity. Some studies have shown that the passive expression alone of each member in themiRNA-200family can prevent epithelial mesenchymal transformation (EMT)change, while they can adjust ZEB1and SIP1(ZEB2) expression if they worktogether, but specific role of the family in human malignant tumor has notbeen clarified. In this study, we detected the expression level of themiRNA-200family in gastric cancer tissue and adjacent normal tissue by thereal-time quantitative PCR technique, and analyzed the correlation betweenthe expression level and clinical pathological parameters of gastric cancer. Weselected the miRNA-200c, which is the representative members of the family,as study subject. We detected the expression level of miRNA-200c in4casesof gastric cancer cells using real-time quantitative PCR, and adopted themethod of transient transfection to transfer the miRNA-200c mimics ornegative control into the gastric cancer cell lines in order to obtain highexpression cell model of the miRNA-200c. In order to understand themiRNA-200c effects on gastric cancer cell biological behavior, and to furtherclarify the role of miRNA-200c in gastric cancer occurrence and development,we explored the proliferation of tumor cells by MTT method, detected the cellcycle change using flow cytometry instrument, and studied the tumor cellmigration ability by wound healing assay. Then we screened the potentialtarget genes by bioinformatics software, and at the same time used dualluciferase report gene method to validate the candidate target gene, forintensive study the molecular mechanism of the miRNA-200c.
Part One The expression level of the miRNA-200family and thecorrelation with clinical pathological parameters in gastric cancer
Objective: To explore the expression level of the miRNA-200family ingastric cancer tissue and adjacent normal tissue, and to analyze the correlationbetween the expression level and clinical pathological parameters of gastriccancer.
Methods: The expression level of the miRNA-200family was detectedby real-time quantitative PCR in46cases of gastric cancer tissues andadjacent normal tissues. The correlation between the expression level of cancer tissues and clinical pathological parameters of gastric cancer wereanalyzed by independent-samples T tests and one-way ANOVA.
Results:
1We detected the expression level of the miRNA-200family in46casesof gastric cancer tissues and adjacent normal tissues by real-time quantitativePCR. Through the analysis of data, the results demonstrated that five membersof miRNA-200family, including miRNA-200a, miRNA-200b, miRNA-200c,miRNA-141and miRNA-429, were all significantly lower in cancerous tissuesthan those in the corresponding normal tissues (P<0.05). Among46patientswith gastric cancer,86.96%(40/46),91.30%(42/46),93.48%(43/46),93.48%(43/46) and80.43%(37/46) cases were showed low expression of themiRNA-200family, respectively.
2We analyzed the correlation between the expression level of cancertissues and clinical pathological parameters of gastric cancer byindependent-samples T tests and one-way ANOVA. Medians of the relativeexpression values and clinicopathological factors were presented in Table. Thestatistical analysis showed that the expression of five members wereassociated with histologic grade and intravascular cancer embolus (P<0.05).However, no association was observed about gender, age, tumor size, lymphnode metastasis, invasive depth, TNM stage, Lauren type and Borrmanntype(P>0.05).
Conclusion:
1The expression level of the miRNA-200family was down-regulated ingastric cancer tissues compared with adjacent normal tissues, suggesting thatthe down-regulated of the miRNA-200family might become a therapeutic andpreventive tool in patients with gastric cancer, but might not be a promisingbiomarker in the early diagnosis of gastric cancer.
2Lower levels of five members were associated with histologic gradeand intravascular cancer embolus, suggesting that the five members of themiRNA-200family might play an important role in the occurrence anddevelopment of gastric cancer.
Part Two The effects of the miRNA-200c on biological behaviors inhuman gastric cancer cell line
Objective: The miRNA-200c, which is the representative members of thefamily, was as the study subject. To estimate the effects of the miRNA-200family on biological behaviors in human gastric cancer cell line, including thecell proliferation, the cell cycle change and the tumor cell migration ability.
Methods: The expression level in4cases of gastric cancer cell lines weredetected by real-time quantitative PCR, and the transient transfection wasadopt to transfer the miRNA-200c mimics or negative control into the gastriccancer cell lines in order to obtain high expression cell model of themiRNA-200c. The proliferation of tumor cells was explored by MTT, the cellcycle was detected using flow cytometry, and the tumor cell migration abilitywas studied by wound healing assay.
Results:
1We detected the expression level of miRNA-200c in4cases of gastriccancer cell lines, including MGC803, HGC27, SGC7901and AGS. Comparedwith adjacent normal tissue, the results showed that the miRNA-200c wassignificantly lower in MGC803, HGC27, SGC7901and AGS. The mediandecreased about9.93,9.69,7.18and2.93folds (P<0.0001).
2The SGC7901cell was found to be a suitable cell model in vitro. Wedetected the expression level of miRNA-200c in SGC7901cell transferred themiRNA-200c mimics or negative control after48hours. The results displayedthat the expression level of miRNA-200c transferred the miRNA-200c mimicswas significantly higher than those in the negative control and blank controlgroup, with the change of median about10.2folds (P<0.05). These suggestedthat the transient transfection of miRNA-200c was successful and could beused in the next studies.
3The MTT assays showed that24h,48h and72h after transfection ofthe miRNA-200c mimics or negative control in SGC7901cell, the cellproliferation in miRNA-200c mimics group was significantly inhibitedcompared to the negative control group(P<0.05). The OD values of miRNA-200c mimics group were0.308±0.010,0.360±0.016and0.457±0.029,while the OD values of negative control group were0.339±0.008,0.505±0.006and0.775±0.010. In three time points after transfection, the rateof inhibition in miRNA-200c mimics group were10.76±2.50%,30.83±2.58%and33.86±15.9%, while the rate of inhibition in negative control group were1.78±2.70%,3.01±2.15%and0.64±0.69%. They had significant difference ineach time point (P<0.05).
4The flow cytometry showed that48h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the cell cycle ofmiRNA-200c mimics group was significantly changed compared to thenegative control group(P<0.05). The ratio in G0/G1phase, S phase and G2/Mphase of miRNA-200c mimics group were72.77±0.85%,8.87±0.45%and18.36±1.29%, while the ratio of negative control group were42.10±0.80%,43.57±1.27%and14.33±2.06%, respectively. There was significant differencein G0/G1phase and S phase (P<0.05). The G0/G1phase arrest suggested thatover-expression of miRNA-200c in SGC7901cell could inhibit the cell cyclefrom G0/G1phase to S phase.
5The wound healing assays showed that24h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the tumor cellmigration ability of miRNA-200c mimics group was significantly decreasedcompared to the negative control group(P<0.05). The width in miRNA-200cmimics group was344.40±17.24μm, while the width in negative control groupwas270.51±18.21μm. There was significant difference between miRNA-200cmimics group and negative control group (P<0.05).
Conclusion:
1The expression level of miRNA-200c in4cases of gastric cancer celllines, was significant down-regulated. The results were consistent with theexpression in gastric cancer tissue.
2The transient transfection of miRNA-200c was successful andup-regulated the expression level of miRNA-200c in SGC7901cell.
3The over-expression of miRNA-200c in SGC7901cell inhibited the cell proliferation, suggesting that the down-regulation of miRNA-200c might playa role in molecule regulation of cell proliferation.
4The over-expression of miRNA-200c in SGC7901cell inhibited the cellcycle from G0/G1phase to S phase, suggesting that the down-regulation ofmiRNA-200c might inhibit the cell proliferation through cell cycle change.
5The over-expression of miRNA-200c in SGC7901cell decreased thetumor cell migration ability, suggesting that the down-regulation ofmiRNA-200c might affect the cell migration ability and could provide theproof for searching the molecular targets of treatment in gastric cancer.
Part Three The prediction and verification of miRNA-200c target genes
Objective: To predict and verify that RhoE is a target gene ofmiRNA-200c. To make a foundation for further research on moleculemechanism of miRNA-200c.
Methods: The potential target gene was predicted and screenedaccording to the bioinformatics software, and at the same time the candidatetarget gene was verified by dual luciferase report gene assay.
Results:
1We used the bioinformatics software (TargetScan, PicTar, mirBase andmiRanda) to predict and screen the potential target gene of miRNA-200c. Theresults showed that miRNA-200c belongs to the chromosome12and islocated to12:7072862-7072929[+]. There were1057and639target genes ofmiRNA-200c by TargetScan and PicTar bioinformatics software.Comprehensive analysis we chose RhoE (NM_005168) as the research subject.We found that RhoE3’UTR has two binding sites of miRNA-200c, so we willbuild the report carrier of RhoE3’UTR and verify the target gene by dualluciferase report gene assay.
2We verified that the RhoE is a target gene of miRNA-200c by theclassical method. Firstly, the RhoE3’UTR vector and RhoE mut3’UTRvector were successfully constructed. We divided into four groups throughtransferring the RhoE3’UTR/RhoE mut3’UTR and miRNA-200c mimics/miRNA-200c NC in SGC7901cell. They were RhoE3’UTR+miRNA-200c mimics group, RhoE3’UTR+miRNA-200c NC group, RhoE mut3’UTR+miRNA-200c mimics group and RhoE mut3’UTR+miRNA-200c NC group.The dual luciferase report gene assays showed that48h after transfection, theluciferin protein expression level of RhoE3’UTR+miRNA-200c mimicsgroup was reduced compared to other groups (P<0.05). The results showedthat miRNA-200c could inhibit the activity of RhoE3’UTR, suggesting thatRhoE was the target gene of miRNA-200c.
Conclusion:
The miRNA-200c could inhibit the activity of RhoE3’UTR, verifyingthat RhoE was the target gene of miRNA-200c. This suggested thatmiRNA-200c has the inhibitory function to RhoE, and may exert its biologicalbehaviors by RhoE. Thus they may affect the occurrence, development,invasion and metastasis of gastric cancer. This provide a foundation for furtherclarify the molecule mechanism of miRNA-200c in gastric cancer.
第一部分胃癌组织中microRNA-200家族的表达及意义
目的：探讨miRNA-200家族五个成员在胃癌组织及其配对的癌旁正常组织中的表达差异，并明确其表达与胃癌临床病理参数的相关性。
方法：收集46例胃癌患者手术切除标本，应用定量Real-time PCR检测miRNA-200家族五个成员在胃癌组织及其配对的癌旁正常组织中的差异表达情况，并应用独立样本t检验和单因素方差分析的统计方法研究其表达水平与胃癌临床病理参数之间的相关性。
结果：
1miRNA-200家族在胃癌组织和配对的癌旁正常组织中的表达水平。采用定量Real-time PCR检测miRNA-200家族五个成员在46例胃癌组织及与其配对的癌旁正常组织中的相对表达情况。结果显示46例胃癌患者miRNA-200a、miRNA-200b、miRNA-200c、miRNA-141和miRNA-429在癌组织中的表达水平均显著低于相应的癌旁正常组织，表达差异有显著的统计学意义(P<0.05)，五者分别在86.96%(40/46)、91.30%(42/46)、93.48%(43/46)、93.48%(43/46)和80.43%(37/46)的癌组织标本中表达水平低于癌旁正常组织。
2miRNA-200家族五个成员的表达与胃癌临床病理参数的相关性。采用t检验或单因素方差分析对miRNA-200a、miRNA-200b、miRNA-200c、miRNA-141和miR-429的表达水平与胃癌临床病理参数的相关性进行分析。结果显示：miRNA-200家族五个成员的低表达水平均与组织学分级、脉管瘤栓呈显著相关(P<0.05)，而与胃癌患者的性别、年龄、肿瘤大小、淋巴结转移、浸润深度、TNM分期、Lauren分型和Borrmann分型无明显相关(P>0.05)。结论：
1miRNA-200家族五个成员(miRNA-200a、 miRNA-200b、miRNA-200c、miRNA-141和miRNA-429)在胃癌患者癌组织中的表达水平均显著低于相应的癌旁正常组织，提示该家族可能不适合作为胃癌早期诊断的分子标志物，但提高该家族5个成员内源性的表达，可能有助于胃癌的治疗和有效地预防，可能成为胃癌治疗和预防的分子靶标。
2miRNA-200家族的五个成员在癌组织中的低表达与组织学分级、脉管瘤栓呈显著相关，提示其可能在胃癌发生及发展阶段起着关键性的作用。
第二部分microRNA-200c对胃癌细胞生物学功能的影响
目的：选取该家族具代表性的成员miRNA-200c，研究其对胃癌SGC7901细胞的增殖、周期和迁移能力等生物学行为的影响。
方法：利用定量Real-time PCR法检测其在4株胃癌细胞中的表达水平，采用瞬时转然的方法将miRNA-200c mimics或阴性对照转入胃癌细胞株以获得高表达miRNA-200c的细胞模型，然后通过MTT法检测肿瘤细胞的增殖情况，用流式细胞仪检测细胞周期变化，以及应用细胞划痕实验检测肿瘤细胞的迁移能力。
结果：
1miRNA-200c在不同胃癌细胞株中的表达水平。应用定量Real-timePCR检测4株胃癌细胞MGC803、 HGC27、 SGC7901和AGS中miRNA-200c的表达情况，以胃癌患者癌旁正常组织作随机对照，结果显示：miRNA-200c在4株胃癌细胞中的表达水平均显著下调，减少倍数分别是9.93倍、9.69倍、7.18倍和2.93倍(P<0.0001)。
2人胃癌细胞株SGC7901转染后miRNA-200c的表达水平。选择SGC7901细胞作为体外细胞模型，利用定量Real-time PCR法检测转染miRNA-200c mimics和阴性对照48h后miRNA-200c的表达水平，结果显示：转染miRNA-200c mimics后胃癌SGC7901细胞中miRNA-200c的表达水平较空白对照组和阴性对照组均显著增高，平均升高约10.2倍(P<0.05)。提示miRNA-200c mimics转染成功，可进行后续实验。
3过表达miRNA-200c对SGC7901细胞增殖的影响。在转染miRNA-200c及阴性对照24、48、72h后，应用MTT方法对SGC7901细胞增殖进行检测，结果显示：细胞转染24、48、72小时后，miRNA-200cmimics实验组的OD值为0.308±0.010、0.360±0.016和0.457±0.029，而阴性对照组的OD值为0.339±0.008、0.505±0.006和0.775±0.010，各时间点两组之间比较均有统计学意义(P<0.05)。通过OD值计算细胞抑制率，miRNA-200c mimics实验组三个时间点的抑制率分别为10.76±2.50%、30.83±2.58%和33.86±15.9%，而阴性对照组的抑制率分别为1.78±2.70%、3.01±2.15%和0.64±0.69%，两组间各时间点比较均有显著差异(P<0.05)。表明胃癌细胞内miRNA-200c表达水平的上调可抑制胃癌细胞的增殖。
4过表达miRNA-200c对SGC7901细胞周期的影响。应用流式细胞仪对miRNA-200c转染48h后的SGC7901细胞进行细胞周期的检测，结果显示：细胞转染48小时后，miRNA-200c mimics实验组的G0/G1期、S期和G2/M期的比例分别为72.77±0.85%、8.87±0.45%和18.36±1.29%，而阴性对照组三期的比例分别为42.10±0.80%、43.57±1.27%和14.33±2.06%，两组之间比较G0/G1期和S期存在显著差异(P<0.05)，呈现G0/G1期阻滞。表明胃癌细胞内miRNA-200c表达水平的上调可阻滞G0/G1期细胞向S期进展。
5过表达miRNA-200c对SGC7901细胞迁移能力的影响。采用细胞划痕实验检测过表达miRNA-200c对SGC7901细胞迁移能力的影响，结果显示：转染24小时后，miRNA-200c mimics实验组镜下细胞迁移宽度为344.40±17.24μm，而阴性对照组为270.51±18.21μm，两组之间比较有显著差异(P<0.05)。表明过表达miRNA-200c的细胞体外迁移能力明显低于阴性对照组。
结论：
1miRNA-200c在4株胃癌细胞株MGC803、HGC27、SGC7901和AGS中均存在不同程度的表达下调，与胃癌组织结果相一致。
2miRNA-200c mimics能成功转染人胃癌细胞SGC7901，以上调SGC7901细胞中miRNA-200c的表达水平。
3miRNA-200c在胃癌细胞SGC7901的过表达可抑制细胞增殖。提示miRNA-200c可能参与了细胞增殖的分子调控，有望成为胃癌治疗新的靶标。
4miRNA-200c在胃癌细胞SGC7901的过表达可阻滞肿瘤细胞从G0/G1期向S期的进展，从而推测miRNA-200c可能是通过改变细胞周期来抑制细胞增殖。
5miRNA-200c在胃癌细胞SGC7901的过表达可降低细胞体外迁移能力。提示miRNA-200c的表达水平可影响肿瘤细胞的迁移能力，为进一步研究胃癌治疗新的靶标提供了依据。
第三部分microRNA-200c靶基因的筛选和验证
目的：预测并验证RhoE是miRNA-200c的靶基因，为深入研究miRNA-200c的分子机制奠定基础。
方法：应用生物信息学软件对miRNA-200c的靶基因进行预测和筛选，同时采用双荧光素酶报告基因法对预测的靶基因进行验证。
结果：
1miRNA-200c靶基因的生物信息学预测。目前对miRNA的靶基因筛选是应用生物信息学软件进行预测，综合分析TargetScan、PicTar、mirBase和miRanda等多个生物信息学软件预测的结果显示：miRNA-200c属于12号染色体，位置为12：7072862-7072929[+]。TargetScan和PicTar预测到的miRNA-200c潜在靶基因数分别为1057和639。本实验选取了近年来备受关注的RhoE基因(NM_005168)作为进一步研究目标，分析发现其3’UTR含有2段可能与miRNA-200c结合的位点。因此，我们针对此序列构建报告载体，进行双荧光素酶报告基因系统实验验证。
2双荧光素酶报告基因系统验证RhoE是miRNA-200c的靶基因。我们应用验证靶基因与miRNA最经典的方法“双荧光素酶报告基因系统”对RhoE是否为miRNA-200c的靶基因进行验证。本实验先成功构建了RhoE3’UTR和突变型RhoE3’UTR的表达载体，通过在SGC7901细胞中分别共转染RhoE3’UTR+miRNA-200c mimics、RhoE3’UTR+miRNA-200cNC、RhoE mut3’UTR+miRNA-200c mimics和RhoE mut3’UTR+miRNA-200c NC分为四个实验组，48h后的检测结果显示：只有RhoE3’UTR+miRNA-200c mimics组荧光素蛋白的表达水平显著降低(P<0.05)，说明miRNA-200c可靶向抑制RhoE3’UTR报告基因的活性。但miRNA-200c对RhoEmut3’UTR的表达载体没有抑制作用，miRNA-200cNC对RhoE3’UTR和RhoEmut3’UTR的表达载体均没有抑制作用(P>0.05)。因此本实验结果验证了RhoE是miRNA-200c的靶基因，提示miRNA-200c对RhoE存在抑制性功能的作用。
结论：
miRNA-200c可靶向抑制RhoE3’UTR报告基因的活性，验证了RhoE是miRNA-200c的靶基因，提示miRNA-200c对RhoE存在抑制性功能的作用，可能通过抑制RhoE发挥其生物学功能，从而影响着胃癌的发生、发展和侵袭转移的过程。为我们进一步阐明miRNA-200c在胃癌中的具体分子机制奠定了基础。
Gastric cancer is one of the most common malignant tumors in China.The incidence of gastric cancer in malignant tumors is the second place. It isthe third leading cause of cancer-related death in2009. Gastric cancer has thehigh incidence, the low rate of early diagnosis and the5-year survival in China.The early diagnostic rate of gastric cancer is still hovering around10%-20%,which is also the main reason of high mortality rate in China. At present,surgery is the only method to be proven to cure gastric cancer. But even if theyhad accepted the radical operation, most of patients with gastric cancer wouldinduce recurrence or metastasis. Postoperative adjuvant radiation andchemotherapy may be available to control the recurrence or metastasis ofgastric cancer. Chemotherapy plays an important role in comprehensivetreatment of advanced gastric cancer, but the drug resistance of chemotherapyhas become a big bottleneck. Therefore, we concluded that the early diagnosisand timely effective treatment is the key to improve the5-year survival rate ingastric cancer, so it is the most crucial task to look for a sensitive and specificmolecular markers, clarifing molecular targets of treatment and correctingprognosis assessment. MicroRNAs (miRNAs) are a new class of endogenousand non-coding small RNA, the length of which is approximately18-25nucleotides. These miRNAs account for only1%of human genes, but theymay regulate more than a third of gene expression, modification, transcriptionand translation in human. These miRNAs may have the potential applicationvalue of molecular markers, for the early diagnosis and prognosis assessmentof malignant tumor to open up a new road. Currently, the miRNA-200familyhas become the focus of miRNAs study. The family includes five members,which are miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141andmiRNA-429. They have several biological characteristics, including the highlyconservative, gene cluster phenomenon, timing, and tissue specificity. Some studies have shown that the passive expression alone of each member in themiRNA-200family can prevent epithelial mesenchymal transformation (EMT)change, while they can adjust ZEB1and SIP1(ZEB2) expression if they worktogether, but specific role of the family in human malignant tumor has notbeen clarified. In this study, we detected the expression level of themiRNA-200family in gastric cancer tissue and adjacent normal tissue by thereal-time quantitative PCR technique, and analyzed the correlation betweenthe expression level and clinical pathological parameters of gastric cancer. Weselected the miRNA-200c, which is the representative members of the family,as study subject. We detected the expression level of miRNA-200c in4casesof gastric cancer cells using real-time quantitative PCR, and adopted themethod of transient transfection to transfer the miRNA-200c mimics ornegative control into the gastric cancer cell lines in order to obtain highexpression cell model of the miRNA-200c. In order to understand themiRNA-200c effects on gastric cancer cell biological behavior, and to furtherclarify the role of miRNA-200c in gastric cancer occurrence and development,we explored the proliferation of tumor cells by MTT method, detected the cellcycle change using flow cytometry instrument, and studied the tumor cellmigration ability by wound healing assay. Then we screened the potentialtarget genes by bioinformatics software, and at the same time used dualluciferase report gene method to validate the candidate target gene, forintensive study the molecular mechanism of the miRNA-200c.
Part One The expression level of the miRNA-200family and thecorrelation with clinical pathological parameters in gastric cancer
Objective: To explore the expression level of the miRNA-200family ingastric cancer tissue and adjacent normal tissue, and to analyze the correlationbetween the expression level and clinical pathological parameters of gastriccancer.
Methods: The expression level of the miRNA-200family was detectedby real-time quantitative PCR in46cases of gastric cancer tissues andadjacent normal tissues. The correlation between the expression level of cancer tissues and clinical pathological parameters of gastric cancer wereanalyzed by independent-samples T tests and one-way ANOVA.
Results:
1We detected the expression level of the miRNA-200family in46casesof gastric cancer tissues and adjacent normal tissues by real-time quantitativePCR. Through the analysis of data, the results demonstrated that five membersof miRNA-200family, including miRNA-200a, miRNA-200b, miRNA-200c,miRNA-141and miRNA-429, were all significantly lower in cancerous tissuesthan those in the corresponding normal tissues (P<0.05). Among46patientswith gastric cancer,86.96%(40/46),91.30%(42/46),93.48%(43/46),93.48%(43/46) and80.43%(37/46) cases were showed low expression of themiRNA-200family, respectively.
2We analyzed the correlation between the expression level of cancertissues and clinical pathological parameters of gastric cancer byindependent-samples T tests and one-way ANOVA. Medians of the relativeexpression values and clinicopathological factors were presented in Table. Thestatistical analysis showed that the expression of five members wereassociated with histologic grade and intravascular cancer embolus (P<0.05).However, no association was observed about gender, age, tumor size, lymphnode metastasis, invasive depth, TNM stage, Lauren type and Borrmanntype(P>0.05).
Conclusion:
1The expression level of the miRNA-200family was down-regulated ingastric cancer tissues compared with adjacent normal tissues, suggesting thatthe down-regulated of the miRNA-200family might become a therapeutic andpreventive tool in patients with gastric cancer, but might not be a promisingbiomarker in the early diagnosis of gastric cancer.
2Lower levels of five members were associated with histologic gradeand intravascular cancer embolus, suggesting that the five members of themiRNA-200family might play an important role in the occurrence anddevelopment of gastric cancer.
Part Two The effects of the miRNA-200c on biological behaviors inhuman gastric cancer cell line
Objective: The miRNA-200c, which is the representative members of thefamily, was as the study subject. To estimate the effects of the miRNA-200family on biological behaviors in human gastric cancer cell line, including thecell proliferation, the cell cycle change and the tumor cell migration ability.
Methods: The expression level in4cases of gastric cancer cell lines weredetected by real-time quantitative PCR, and the transient transfection wasadopt to transfer the miRNA-200c mimics or negative control into the gastriccancer cell lines in order to obtain high expression cell model of themiRNA-200c. The proliferation of tumor cells was explored by MTT, the cellcycle was detected using flow cytometry, and the tumor cell migration abilitywas studied by wound healing assay.
Results:
1We detected the expression level of miRNA-200c in4cases of gastriccancer cell lines, including MGC803, HGC27, SGC7901and AGS. Comparedwith adjacent normal tissue, the results showed that the miRNA-200c wassignificantly lower in MGC803, HGC27, SGC7901and AGS. The mediandecreased about9.93,9.69,7.18and2.93folds (P<0.0001).
2The SGC7901cell was found to be a suitable cell model in vitro. Wedetected the expression level of miRNA-200c in SGC7901cell transferred themiRNA-200c mimics or negative control after48hours. The results displayedthat the expression level of miRNA-200c transferred the miRNA-200c mimicswas significantly higher than those in the negative control and blank controlgroup, with the change of median about10.2folds (P<0.05). These suggestedthat the transient transfection of miRNA-200c was successful and could beused in the next studies.
3The MTT assays showed that24h,48h and72h after transfection ofthe miRNA-200c mimics or negative control in SGC7901cell, the cellproliferation in miRNA-200c mimics group was significantly inhibitedcompared to the negative control group(P<0.05). The OD values of miRNA-200c mimics group were0.308±0.010,0.360±0.016and0.457±0.029,while the OD values of negative control group were0.339±0.008,0.505±0.006and0.775±0.010. In three time points after transfection, the rateof inhibition in miRNA-200c mimics group were10.76±2.50%,30.83±2.58%and33.86±15.9%, while the rate of inhibition in negative control group were1.78±2.70%,3.01±2.15%and0.64±0.69%. They had significant difference ineach time point (P<0.05).
4The flow cytometry showed that48h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the cell cycle ofmiRNA-200c mimics group was significantly changed compared to thenegative control group(P<0.05). The ratio in G0/G1phase, S phase and G2/Mphase of miRNA-200c mimics group were72.77±0.85%,8.87±0.45%and18.36±1.29%, while the ratio of negative control group were42.10±0.80%,43.57±1.27%and14.33±2.06%, respectively. There was significant differencein G0/G1phase and S phase (P<0.05). The G0/G1phase arrest suggested thatover-expression of miRNA-200c in SGC7901cell could inhibit the cell cyclefrom G0/G1phase to S phase.
5The wound healing assays showed that24h after transfection of themiRNA-200c mimics or negative control in SGC7901cell, the tumor cellmigration ability of miRNA-200c mimics group was significantly decreasedcompared to the negative control group(P<0.05). The width in miRNA-200cmimics group was344.40±17.24μm, while the width in negative control groupwas270.51±18.21μm. There was significant difference between miRNA-200cmimics group and negative control group (P<0.05).
Conclusion:
1The expression level of miRNA-200c in4cases of gastric cancer celllines, was significant down-regulated. The results were consistent with theexpression in gastric cancer tissue.
2The transient transfection of miRNA-200c was successful andup-regulated the expression level of miRNA-200c in SGC7901cell.
3The over-expression of miRNA-200c in SGC7901cell inhibited the cell proliferation, suggesting that the down-regulation of miRNA-200c might playa role in molecule regulation of cell proliferation.
4The over-expression of miRNA-200c in SGC7901cell inhibited the cellcycle from G0/G1phase to S phase, suggesting that the down-regulation ofmiRNA-200c might inhibit the cell proliferation through cell cycle change.
5The over-expression of miRNA-200c in SGC7901cell decreased thetumor cell migration ability, suggesting that the down-regulation ofmiRNA-200c might affect the cell migration ability and could provide theproof for searching the molecular targets of treatment in gastric cancer.
Part Three The prediction and verification of miRNA-200c target genes
Objective: To predict and verify that RhoE is a target gene ofmiRNA-200c. To make a foundation for further research on moleculemechanism of miRNA-200c.
Methods: The potential target gene was predicted and screenedaccording to the bioinformatics software, and at the same time the candidatetarget gene was verified by dual luciferase report gene assay.
Results:
1We used the bioinformatics software (TargetScan, PicTar, mirBase andmiRanda) to predict and screen the potential target gene of miRNA-200c. Theresults showed that miRNA-200c belongs to the chromosome12and islocated to12:7072862-7072929[+]. There were1057and639target genes ofmiRNA-200c by TargetScan and PicTar bioinformatics software.Comprehensive analysis we chose RhoE (NM_005168) as the research subject.We found that RhoE3’UTR has two binding sites of miRNA-200c, so we willbuild the report carrier of RhoE3’UTR and verify the target gene by dualluciferase report gene assay.
2We verified that the RhoE is a target gene of miRNA-200c by theclassical method. Firstly, the RhoE3’UTR vector and RhoE mut3’UTRvector were successfully constructed. We divided into four groups throughtransferring the RhoE3’UTR/RhoE mut3’UTR and miRNA-200c mimics/miRNA-200c NC in SGC7901cell. They were RhoE3’UTR+miRNA-200c mimics group, RhoE3’UTR+miRNA-200c NC group, RhoE mut3’UTR+miRNA-200c mimics group and RhoE mut3’UTR+miRNA-200c NC group.The dual luciferase report gene assays showed that48h after transfection, theluciferin protein expression level of RhoE3’UTR+miRNA-200c mimicsgroup was reduced compared to other groups (P<0.05). The results showedthat miRNA-200c could inhibit the activity of RhoE3’UTR, suggesting thatRhoE was the target gene of miRNA-200c.
Conclusion:
The miRNA-200c could inhibit the activity of RhoE3’UTR, verifyingthat RhoE was the target gene of miRNA-200c. This suggested thatmiRNA-200c has the inhibitory function to RhoE, and may exert its biologicalbehaviors by RhoE. Thus they may affect the occurrence, development,invasion and metastasis of gastric cancer. This provide a foundation for furtherclarify the molecule mechanism of miRNA-200c in gastric cancer.
引文
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