【作者】 陈瑜；

【导师】 吴开春；

【作者基本信息】 第四军医大学 ， 内科学， 2007， 博士

【摘要】 胃癌目前仍是我国死亡人数最多的恶性肿瘤之一,五年生存率仅约20%。转移是肿瘤最具破坏性的本质,也是胃癌患者死亡的主要原因。深刻理解胃癌转移的分子机制对治疗十分重要。Runt结构域转录因子(RUNX1、RUNX2和RUNX3)是一组同源性较高的转录因子,它们与核心结合因子β亚基结合形成异源二聚体,作用于基因的启动子、增强子或沉默子区域,调节基因的表达。RUNX-CBFbeta复合体对基因的调节具有组织和阶段特异性,RUNX蛋白在个体发育中具有重要的作用;在受调节的靶基因中包括许多在肿瘤中发生变化的、与细胞命运相关的基因。RUNX3是Runt结构域转录因子家族成员,最近被确定为抑癌基因,在肝癌、胰腺癌、前列腺癌、肺癌中都发现有不同比例的RUNX3表达缺失。研究发现在60%～90%的胃癌病例中存在RUNX3的缺失,与胃癌患者的预后情况呈负相关。随着胃癌分期的进展,缺失比例增高。RUNX3敲除小鼠的胃粘膜上皮细胞发生恶性转化,激活胃癌细胞的RUNX3表达可以显著抑制胃癌细胞的生长和成瘤性。因此RUNX3表达下降或缺失在胃癌的发生和进展中具有重要作用。文献表明,随着胃癌分期进展RUNX3缺失的比例增加。在胃癌肝转移细胞中RUNX3的两等位基因全部缺失,这些均提示RUNX3在胃癌的转移中可能具有重要作用,但RUNX3的表达下降是否与胃癌转移相关还不明确。本课题将对此进行探讨。【目的】1、明确RUNX3下降与胃癌侵袭、转移是否相关;2、探讨RUNX3抑制胃癌转移的可能分子机制;3、筛选RUNX3调控的下游基因。【方法】1、利用免疫组化观察RUNX3在转移和无转移的胃癌组织中的表达;2、基因重组的方法构建含有RUNX3特异性SiRNA的表达载体;3、用构建的特异性SiRNA的表达载体和RUNX3的真核表达载体(Paul J Farrell教授赠予)转染胃癌细胞MKN28和SGC7901,经G418筛选获得稳定表达的细胞系。3、利用失巢培养模型、损伤刮擦实验、体外侵袭实验、裸鼠成瘤及Ⅷ因子染色、裸鼠体内转移实验研究RUNX3对胃癌细胞MKN28和SGC7901侵袭转移能力的影响;4、凝胶酶谱法检测转染胃癌细胞上清中分泌MMP2,MMP9的活性变化;5、RT-PCR及Western blot检测MMP2,MMP9,TIMP-1在mRNA和蛋白水平的表达变化;ELISA检测TIMP-1、VEGF在转染细胞培养上清中的水平。6、基因芯片技术筛选RUNX3相关的差异表达基因,并用RT-PCR进行个别验证。7、双荧光素酶报告基因实验检测RUNX3对TIMP-1启动子活性的调控作用。通过染色质免疫共沉淀实验(ChIP)和电泳迁移率实验(EMSA)验证细胞内RUNX3与TIMP-1启动子是否相互作用。【结果】1、RUNX3在转移的胃癌组织中表达下降,RUNX3可抑制胃癌细胞的转移能力利用免疫组化技术检测了83例无转移胃癌和40例有转移胃癌中RUNX3的表达,发现RUNX3在无转移的胃癌中表达的阳性率(62.7% (52/83))高于有转移的胃癌(42.5% (17/40)),免疫组化评分结果显示,RUNX3在无转移的胃癌中表达高于有转移的胃癌(1.25±0.48 versus 0.57±0.26, P<0.05),说明RUNX3更多地表达在无转移的胃癌组织。成功构建了含有RUNX3特异性SiRNA的表达质粒pSilencer-RUNX3。选取胃癌细胞SGC7901(表达中等强度RUNX3)和MKN28(无RUNX3的表达),pBK-RUNX3转染细胞后能上调RUNX3的表达,pSilencer- RUNX3转染细胞后能显著抑制RUNX3的表达。pBK-RUNX3转染细胞后,MKN28和SGC7901穿过Transewell小室微孔膜的侵袭能力下降,抑制率分别为40.3%和42.5%,而pSilencer-RUNX3转染细胞后,SGC7901细胞穿过Transewell小室微孔膜的侵袭能力显著增加。损伤刮擦实验表明,RUNX3可明显抑制胃癌细胞的迁移运动能力。MTT实验表明,RUNX3抑制细胞的失巢存活能力,转染pBK-RUNX3的SGC7901细胞和MKN28细胞在失巢培养24小时后,其细胞生存率均明显低于空载体对照组(SGC7901:32.2% vs 55.7%, MKN28:58.8% vs 80.7% P<0.05),而转染pSilencer-RUNX3的SGC7901细胞组的细胞生存率均高于空载体对照组(75.3% vs 53.6% P<0.05)。裸鼠成瘤后瘤体固定切片,Ⅷ因子染色表明转染pBK-RUNX3瘤体中微血管密度(MDV)显著下降(P<0.05),而pSilencer- RUNX3转染细胞后瘤体中MDV增多(P<0.05),说明RUNX3可抑制胃癌新生血管生成。裸鼠体内转移实验结果显示转染pBK-RUNX3后细胞在裸鼠肺部及肝部形成的可见转移结节明显少于对照(SGC7901和MKN28中均为P<0.05)而转染pSilencer-RUNX3后形成的可见转移结节明显多于对照组(P<0.05)。2、RUNX3可以通过调节TIMP-1、VEGF、CRKⅡ等分子的表达抑制胃癌细胞侵袭转移的能力我们通过凝胶酶谱试验发现RUNX3可抑制胃癌细胞中MMP9的活性(SGC7901和MKN28中均为P<0.05),RUNX3特异性siRNA则增强SGC7901细胞MMP9的活性(P<0.05);而对MMP2的活性无显著影响。RT-PCR和Western blot检测发现MMP2和MMP9的表达水平并无明显变化。因此,RUNX3对MMP9的调节可能不在mRNA和蛋白水平,而可能通过其它分子影响MMP9的活性。TIMP-1是MMP9主要的内源抑制因子,进一步RT-PCR、Western blot及ELISA的结果证实,RUNX3能上调TIMP-1在mRNA和蛋白水平的表达以及胃癌细胞培养上清中TIMP-1的水平,因此RUNX3可通过上调TIMP-1的表达水平,从而抑制MMP9的酶活性;ELISA还发现RUNX3可以抑制胃癌细胞培养上清中VEGF的表达水平,提示RUNX3通过抑制VEGF的表达而减少胃癌的血管生成。经过Trizol法抽提SGC7901/ pBK-RUNX3和空载体转染的对照细胞SGC7901/ pBK-CMV两种细胞的总RNA,通过琼脂糖电泳检查及紫外定量分析表明所提取得RNA无明显降解,其质和量均符合芯片检测要求。经荧光探针的制备、纯化及定量,芯片杂交、图像采集和数据分析等步骤得到差异表达的基因。我们以两种细胞信号强度的比值小于0.5或者大于2作为判定标准,结果发现,与对照细胞相比,RUNX3表达上调后,SGC7901/ pBK-RUNX3细胞中有14个基因表达被上调,109个基因表达被下调。在基因芯片筛选出的下游基因中,我们通过RT-PCR对个别差异表达得基因进行验证,证实接头分子CRKⅡ的表达在胃癌细胞MKN28和SGC7901中可被RUNX3抑制。3、RUNX3作为转录因子增强TIMP-1在转录水平的表达双荧光素酶报告基因实验结果显示, pBK-RUNX3质粒与pGL-TIMP-1共转染MKN28细胞后,细胞内的激发荧光强度显著高于质粒与pBK-CMV空载体转染的MKN28细胞。表明RUNX3可以增强TIMP-1启动子的活性ChIP实验发现,设计扩增包含TIMP-1启动子上2个RUNX3结合位点的序列的特异性引物,经RUNX3抗体沉淀后的DNA模版可以扩增出特异性的片段,提示无论是内源性表达的RUNX3还是外源性的RUNX3均可在细胞内与TIMP-1的启动子结合并相互作用。进一步EMSA和Supper Shift试验结果显示:SGC7901细胞核蛋白提取物可以使TIMP-1启动子的双链DNA探针的电泳条带滞后,而RUNX3抗体可以使电泳条带进一步滞后。表明RUNX3可以与TIMP-1启动子上的两个保守结合位点结合。【结论】本研究提供了抑癌基因RUNX3具有抑制胃癌转移作用的证据,同时,我们发现这种作用可能与以下机制有关:RUNX3作为转录因子,增强下游靶基因TIMP-1的转录及表达,抑制MMP9的活性,从而抑制胃癌细胞侵袭能力;RUNX3抑制下游基因CRKⅡ的表达,从而抑制胃癌细胞的运动迁移能力及失巢存活能力;RUNX3抑制下游基因VEGF的表达,从而抑制胃癌血管生成。这些发现有助于RUNX3在胃癌转移的分子治疗和诊断中的应用。更多还原

【Abstract】 Despite the progress in diagnosis and treatment of gastric cancer, only about 20% of patients survive to 5 years. Metastasis, the most fearful aspect of cancer, is one of the major causes of mortality for gastric cancer patients. In order to improve the treatment of this disease, it is of great importance to clearly understand the molecular mechanism of gastric cancer metastasis. All the RUNX family members, namely, RUNX1, RUNX2 and RUNX3, encode DNA bindingαsubunits which form heterodimers with the commonβsubunit CBFβand act as transcription regulators. It has been realized that all three RUNX family members play important roles in normal developmental processes and carcinogenesis. They regulate the expression of cellular genes through binding to promoters, enhancer or silencer elements, with growing list of targes that including many genes relevant to carcinogenesis.Recently, RUNX3, one of Runt-related genes, is taken as a candidate tumor suppressor gene whose deficiency is causally related with gastric cancer. In RUNX3-knock-out mice, the stomach mucosal cells showed characteristics of malignant transformation. Reactivation of RUNX3 in gastric cancer cells inhibited their growth and tumorigenicity. There have emerged numerous reports on gene methylation and silencing of RUNX3 in tumors of many other tissues, such as liver, colon, gallbladder, lung, etc. Furthermore, decrease of RUNX3 protein expression was significantly associated with inferior survival duration of gastric cancer patients. It was reported RUNX3 expression was decreased in about 60% of the analyzed primary human gastric tumors, while the reduced RUNX3 levels rising to nearly 90% among the late stage, representing highly metastatic tumors. These data indicated that RUNX3 deficiency is closely related with gastric cancer metastasis.However, the function of RUNX3 in pathogenesis of gastric cancer metastasis remains not fully understood. So in the present work, we investigated whether RUNX3 might supress the invasive and metastatic abilities of gastric cancer cells and explored the relative molecular mechanism involved.【Objectives】(1) To investigate whether RUNX3 expression level is assocciated with invasion and metastasis of gastric cancer; (2) To examine the possible molecular mechanisms underlying the metastasis-suppressing effect by RUNX3 in gastric cancer; (3) To screen the downstream effectors of RUNX3【Methods】(1) The expression level of RUNX3 in metastatic gastric cancer and non-metastatic gastric cancer were determined by immunohistochemistry assay. (2) The RUNX3-specific siRNA vector was designed and constructed. (3) The plasmids pBK-RUNX3 (kindly provided by Prof. Paul J Farrell) or pSilencer-RUNX3 was transfected into SGC7901 and MNK28 cells using lipofectamine 2000 reagent. Empty vector of pBK-CMV or pSilencer were used as control. The transfected cells were screened with G418 for 2 months, single cell colonies were obtained by limited dilution method. Semi-quantitative RT-PCR and Western blot were used to detect the RUNX3 expression in the transfected cells for confirmation of transfection. (4) The effects of RUNX3 on the motility, invasive, angiogensis and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN28 were respectively investigated by wound-healing assays, invasion assay, tail vein metastatic assay andⅧfactor staining. (5) The activities of MMP2 and MMP9 in the conditioned medium were visualized by performing gelatin zymography (6) The mRNA and protein expression of MMP2, MMP9 and TIMP-1 were determined by RT-PCR and Western blotting. (7) The TIMP-1 and VEGF protein levels in the culture supernatants were determined using ELISA. (8) The TIMP-1 promoter was amplified by PCR using the genomic DNA from SGC7901 cells as template. The effect on TIMP-1 promoter activity by RUNX3 was evaluated by dual luciferase reporter assay. (9)The direct interaction between RUNX3 and the TIMP-1 promoter in vivo was determined by performing ChIP assay. (10) EMSA and supper shift assay were performed to visualize the binding of RUNX3 to TIMP-1 promoter via the two putative binding sites. (11) The downstream effectors of RUNX3 were screened cDNA microarray.【Results】1.RUNX3 expression is down-regulated in metastatic gastric cancer. To investigate the relationship between the expression of RUNX3 and metastasis of gastric cancer, we compared expression of RUNX3 in primary sites from 83 patients enduring non-metastatic gastric cancer with those from 40 patients enduring metastatic gastric cancer. The positive rate of RUNX3 expression in non-metastatic gastric cancer was 62.7% (52/83), lower than 42.5% (17/40) in metastatic gastric cancer. When considered staining scores, the average staining score in metastatic gastric cancer was significantly higher than that in non-metastatic gastric cancer (1.25±0.48 versus 0.57±0.26, P<0.05). These results suggested that RUNX3 expression level was negatively related to gastric cancer metastasis.2.Reduced RUNX3 expression is associated with increased metastatic potential of gastric cancer cells in vitro and in vivoRT-PCR and Western blot suggested that transfecting SGC7901 and MKN28 cells with pBK/RUNX3 increased the RUNX3 expression in the cells. And transfecting pSilencer-RUNX3 decreased the RUNX3 expression in the SGC7901 cells.Upregulation of RUNX3 in SGC7901 and MKN28 cells decreased their abilities to migrate on matrigel, and reduced RUNX3 in SGC7901 cells inceased its mobility. The RUNX3-transfected cells also showed significantly lower invasive potency than that of the control. The RUNX3 transfection reduced the number of invasive cells by 42.5% and 40.3% in SGC7901 cells and MKN28 cells, respectively. The difference in invasiveness was found to be statistically significant (P<0.05 in both cell lines). Similarly, inhibition of RUNX3 expression in SGC7901 cells by RUNX3 siRNA constructs promoted the cell invasive potency significantly compared with the control (P<0.05). MTT assay also showed that the survival rates of the detached SGC7901 and MKN28 cells transfected with pBK-RUNX3 were markedly decrease as compared with control group ( P < 0.05) . Survival rate of SGC7901 cells transfected with pSilencer-RUNX3 were significantly increase as compared with control (P < 0.05). The above results suggested that RUNX3 play a negative role in the gastric cancer cell resistant to anoilkis.RUNX3 inhibited angiogenesis in xenograft tumor in nude mice (p<0.05). We performed IHC analysis on various tumor sections to assess angiogenesis in mice. The MVD (miro-vessel density) of tumors in mice subcutaneously inoculated with gastric cancer cells trasfected with pBK-RUNX3 were lower than that trasfected with pBK-CMV (6.50±2.13 vs 17.30±8.32, 23.72±9.13 vs 50.25±15.46 respectively in SGC7901 and MKN28, P < 0.05 in both cell lines). And similarly, the MVD in tumor of mice subcutaneously inoculated with SGC7901/pSilencer-RUNX3 were higher than that of SGC7901/pSilencer (28.50±12.73 vs 12.83±4.92 P < 0.05). Tail vein metastatic assay in nude mice was further adopted to examine the suppressing effect on metastatic abilities of gastric cancer cells by RUNX3. Compared with control cells transfected with empty vectors, the injection of the cells transfected with pBK-RUNX3 via a tail vein of athymic nude mice led to significantly less visible tumors in the liver and lung surface (P< 0.05). Conformably, the injection of the SGC7901 cells transfected with pSilencer-RUNX3 led to much more visible tumors in the liver and lung surface than that of control cells (P<0.05). The results above suggest that RUNX3 could suppress the moltility, invasiveness, angiogenesis and metastatic potential of gastric cancer cells.3. RUNX3 regulates the expression level of downstream genes that are relevant to tumor metastasis.To study the possible role of MMPs in RUNX3-induced inhibition of cell invasion, we first analyzed the regulation on the expression and activity of MMP2 ,9 by RUNX3. The RT-PCR and Western blot were used to examine the mRNA and protein expression of MMP2 ,9 in the gastric cancer cells. We found no significant difference in MMP2 or MMP9 expression between cells with up-regulated RUNX3 (or down- regulated RUNX3) and their respective controlsAt the same time, the activities of MMP2, 9 in the serum-free conditioned medium were determined by gelatin zymography. MMP9 that exhibited collagen-degrading activity was detected in the culture supernatant of the SGC7901 and MKN28 cells. Compared with the control cells, the activity of the MMP9 enzyme in pBK-RUNX3 transfectants was significantly lower (both P<0.05 in SGC7901 and MKN28). And the activity of the MMP9 enzyme in pSilencer-RUNX3 transfectants was significantly higher than that of the control cells (P<0.05). While the activity of MMP2 is relatively low compared with that of MMP9 and showed no significant difference between cells with up-regulated RUNX3 (or down- regulated RUNX3) and their respective controls. Above results indicated that instead of regulating the transcription of MMP9, the enhanced expression of RUNX3 attenuate the matrix degrading activity of MMP9 in gastric cancer cells.TIMP1 expression is up-regulated by RUNX3 at transcriptional level. To examine the expression level of the TIMP1 which is considered the specific inhibitor of MMP-9 activity, we performed RT-PCR to detect the mRNA expression level and Western blot to detect the protein expression. We found that transfection of pBK-RUNX3 induced significantly stronger expression of TIMP-1 in SGC7901/pBK-RUNX3 and MKN28/pBK-RUNX3 cells. However, weaker expression of TIMP-1 was observed in cells transfected with pSilencer-RUNX3 as comparing with the control cells.The TIMP-1 levels secreted in the conditioned culture medium of gastric cancer cells were detected by performing ELISA We found that TIMP-1 production in the medium of the gastric cells transfected with pBK-RUNX3 was signifiantly higher than that of the respective control cells (16.5 ng/ml vs10.6 ng/ml in SGC7901, P<0.05; 14.9 ng/ml vs 7.2ng/ml in MKN28, P<0.05). Conformably, TIMP-1 level in the medium of SGC7901 /pSilencer -RUNX3 deceased compared with the control cells SGC7901/pSilencer (5.7 ng/ml vs 12.5 ng/ml, P<0.05).The VEGF levels secreted in the conditioned culture medium of gastric cancer cells were detected by performing ELISA. The VEGF production in the medium of the gastric cells transfected with pBK-RUNX3 was signifiantly lower than that of the respective control cells (1270±580 pg/millioncell vs 2950±870pg/millioncell in SGC7901, P<0.05; 2060±790 pg/millioncell vs 4300±1130 pg/millioncell in MKN28, P<0.05). VEGF level in the medium of SGC7901/pSilencer-RUNX3 increased when compared with the control cells SGC7901/pSilencer (6500±1730 pg/millioncell vs 3100±1066 pg/millioncell P<0.05).The total RNA of SGC7901/pBK-RUNX3 and SGC7901/pBK-CMV were extracted. The quality of extracted RNA was evaluated by agarose electrophoresis and UV absorbance spectroscopy. After microarray hybridization and data normalization, 14 genes were showed up-regulated while 109 genes were down-regulated by RUNX3. Among those genes, the expression level of CRKⅡ was evaluated by RT-PCR. It was confirmed that CRKⅡexpression was reduced by RUNX3, which indicated that inhibiting effect of RUNX3 on gastric cancer cell mobility was at least partially mediated by CRKⅡdownregulation.4. RUNX3 regulated TIMP-1 expression in gastric cancer cells by directly interacting with TIMP-1 promoterTo investigate the possibility that RUNX3 increased TIMP-1 expression by stimulating the TIMP-1 promoter activity, the dual luciferase reporter assay was performed. The transfection of different doses of pBK-RUNX3 plasmid led to a significantly increase of TIMP-1 promoter activity compared with the empty vector transfection. The result indicated that RUNX3 caused transactivation of TIMP-1 promoter and then up-regulated the mRNA and protein expression of TIMP-1.Further analysis of TIMP-1 revealed two putative RUNX binding sites was contained in the TIMP-1 promoter (-327 to -321; -66 to -61). Therefore, we tried to determine whether RUNX3 binds to the regions of the TIMP-1 promoter in vivo by performing ChIP assay. The chromatin fragments from SGC7901 cells and MKN28/ pBK-RUNX3 were immunoprecipitated with specific anti-RUNX3 antibody or with normal rabbit serum as a negative control. We found that both exogenously expressed RUNX3 in MKN28/ pBK-RUNX3 cells and endogenous RUNX3 in SGC7901 cells could be recruited to the two sites.Moreover, EMSA was conducted to demonstrate whether RUNX3 could bind to the two putative binding sites. The nuclear extracts of SGC7901 cells were prepared. Biotin-labeled probes including the RUNX3-binding site were incubated with the nuclear extracts, and DNA-protein complexes were analyzed with PAGE. For both sites, shifted bands of labeled probes were detected with nuclear extracts from SGC7901 cells. These bands were dramatically inhibited when competitive cold probes were included in the reaction, while the mutated cold probes could not inhibit the specific binding between labeled probes and nuclear protein. The shifted band was supershifted by addition of the anti-RUNX3 antibody. These observations suggest that the band was a specific complex formed by RUNX3 and the probes including the RUNX3-binding sites and RUNX3 did bind to the putative RUNX binding sites inTIMP-1gene promoter.【Conclusion】We provide evidence that RUNX3 expression was down-regulated in metastatic gastric cancer tissues than metastatic gastric cancer tissues. Our study presented evidence for RUNX3-mediated suppression of gastric cancer metastasis by inhibting the invasiveness, mobility and angiogenesis of cancer cells. We also provided novel molecular mechanism that at least partly contributes to the metastasis inhibiting activity of RUNX3. These data may be helpful for beneficial application of RUNX3 to diagnostics and therapeutics of gastric cancer metastasis.更多还原