HIF-1α基因沉默对口腔癌CEACAM1和VEGF-C表达及脉管生成影响的研究
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摘要
实验目的：
1.观察研究HIF-1α在口腔鳞状细胞癌中表达及与CEACAM1、VEGF-C表达的关系,分析CEACAM1对血管和淋巴管生成的影响,初步探讨HIF-1α、CEACAM1、VEGF-C在口腔癌血管和淋巴管生成中的作用及可能的机制。
2.观察RNAi基因沉默HIF-1α对舌鳞状细胞癌细胞系Tca8113表达HIF-1α的影响,并观察研究HIF-1α基因沉默对CEACAM1和VEGF-C表达的影响,探讨HIF-1α/CEACAM1/VEGF-C的调控关系及在肿瘤脉管生成中的作用机制。
3.以口腔癌裸鼠移植瘤模型为研究对象,研究HIF-1α基因沉默对肿瘤CEACAM1和VEGF-C表达的影响,验证HIF-1α/CEACAM1/VEGF-C对肿瘤血管和淋巴管生成的作用,以及对肿瘤增殖和进展的影响；探讨HIF-1α/CEACAM1/VEGF-C通路对肿瘤脉管生成及其正常化的影响及作用机制。
研究方法：
1.以人口腔鳞状细胞癌为研究对象,检测人口腔鳞状细胞癌中HIF-1α、CEACAM1和VEGF-C表达
收集2005-2007年山东大学齐鲁医院和山东大学口腔医院口腔癌手术标本91例,其中包括舌鳞状细胞癌标本30例。患者临床资料包括年龄、性别、肿瘤大小、肿瘤临床分期、肿瘤分化程度。应用免疫组化方法检测HIF-1α和CEACAM1在91例口腔鳞状细胞癌中的表达及定位,并对免疫组化结果进行分析,评价蛋白的表达与临床资料之间的关系。检测30例舌鳞状细胞癌中VEGF-C表达,并与HIF-1α和CEACAM1在舌癌中的表达进行对比,统计分析HIF-1α与CEACAM1和VEGF-C在舌癌中表达的相关性,并进一步分析在HIF-1α高表达情况下,CEACAM1表达与肿瘤脉管生成的关系。
2.以人口腔癌为研究对象,检测人口腔癌中MVD和LVD以及脉管内皮细胞表型转化情况
采用免疫组化方法,分别应用血管标记CD31和淋巴管标记LYVE-1检测口腔癌中血管和淋巴管生成情况,统计血管密度和淋巴管密度。免疫组化双标记和免疫荧光双标记检测新生脉管表型转化情况,并分析其与CEACAM1表达的关系。
3、以人舌鳞状细胞癌细胞系Tca8113细胞为研究对象,检测RNAi慢病毒干扰载体对Tca8113细胞HIF-1α基因的干扰效率
Tca8113细胞在37℃、5%CO2孵箱中培养,取生长状态良好的细胞以4×104／孔接种于6孔培养板中,每孔加入2m1DMEM培养基。48小时后,细胞融合率可达到30-50%。将细胞分为空白对照组、阴性载体对照组和干扰组,分别加入ENi.S.、NC-GFP-Lentivirus和HIF-1 a-RNAi-Lentivirus(复感染指数MOI为75),37℃、5%CO2孵箱中继续常规培养。每天倒置荧光显微镜观察细胞生长,通过观察慢病毒上报告基因GFP的表达情况,评估感染效率。RNA干扰后第五天,通过观察GFP评估细胞感染率约为70-80%,收集细胞,提取细胞总RNA,应用Real time RT-PCR方法检测不同实验组中HIF-1α基因水平表达,Realtime RT-PCR数值分析采用标准曲线分析法。RNA干扰后第五天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中HIF-1α的蛋白表达水平。
4.以Tca8113细胞为研究对象,检测HIF-1α沉默对CEACAM1、VEGF-C基因和蛋白水平表达的影响
Tca8113细胞培养并感染慢病毒颗粒,RNA干扰后第五天,通过观察GFP评估细胞感染率约为70-80%,收集细胞,提取细胞总RNA,应用real time RT-PCR方法检测不同实验组中HIF-1α、CEACAM1和VEGF-C基因水平的表达,real time RT-PCR数值分析采用标准曲线分析法。RNA干扰后第五天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中HIF-1α的蛋白表达,再次验证蛋白表达水平干扰效率。同时收集细胞,采用流式细胞术检测CEACAM1的表达。RNA干扰后第五天收集Tca8113细胞培养液上清,采用ELISA方法对分泌至细胞外的VEGF-C蛋白进行检测。分析三个不同实验组中Tca8113细胞HIF-1α、CEACAM1和VEGF-C在基因和蛋白水平表达情况,探讨HIF-1α基因沉默后对CEACAM1和VEGF-C表达的影响。
5.建立HIF-1α基因沉默的口腔癌裸鼠移植瘤模型并观察脉管生成及肿瘤进展
观察口腔癌裸鼠移植瘤的增殖进展和荷瘤鼠生存情况,检测移植瘤增殖活性及CEACAM1和VEGF-C表达情况,观察肿瘤脉管生成情况及其形态结构和内皮细胞表型转化情况,分析HIF-1α基因沉默与CEACAM1和VEGF-C表达及脉管生成的关系。
建立口腔癌裸鼠移植瘤模型：37℃、5%CO2孵箱中培养Tca8113细胞,将生长状态良好的细胞以4×104／孔接种于6孔培养板中,每孔加入2m1DMEM培养基。48小时后,细胞融合率可达到30-50%。将细胞分为空白对照组、阴性对照组和干扰组,分别加入ENi.S.、NC-GFP慢病毒颗粒和HIF-1 a-RNAi慢病毒载体(复感染指数MOI为75),37℃、5%CO2孵箱中继续常规培养。在细胞生长状态良好时配制细胞悬液,细胞浓度为2×106个／ml,备用。5周龄的BALB/C nu/nu雌性裸鼠30只,(中国医学科学院实验动物研究所),分为空白对照组、阴性对照组和干扰组,每组10只,注射0.1ml空白对照组、阴性对照组和干扰组Tca8113细胞悬液(细胞数量为2×105个)于裸鼠背部皮下。接种肿瘤细胞后,将裸鼠置于SPF饲养条件下饲养,每天观察裸鼠背部的成瘤情况并在成瘤后定期对肿瘤大小进行测量。
四周后每组裸鼠中的5只分别乙醚吸入麻醉后进行荧光实时活体成像,然后脱颈处死,收集肿瘤标本。将收集的裸鼠肿瘤标本进行常规处理制备成蜡块,应用免疫组化方法检测三组裸鼠肿瘤标本中CEACAM1、VEGF-C、Ki67、LYVE-1(抗鼠)和CD31(抗鼠)的表达,并计数评估MVD和LVD,观察其形态结构变化,免疫双标记法检测新生脉管内皮细胞表型变化,研究HIF-1α基因沉默后对裸鼠移植瘤血管和淋巴管生成及其形态学改变和正常化的影响。
6.观察口腔癌裸鼠移植瘤模型生存情况
三组裸鼠每组剩余5只继续在SPF饲养条件下饲养,观察研究荷瘤鼠生存情况,并进行生存分析。
结果：
1.HIF-1α在口腔鳞状细胞癌中高表达,并与CEACAM1和VEGF-C表达呈正相关
HIF-1α在肿瘤中高表达(口腔癌阳性率95%,舌癌阳性率100%),细胞核和细胞浆着色；CEACAM1在肿瘤中表达增高(口腔癌中阳性率为83.5%,舌癌阳性率80%),胞膜和胞浆着色；VEGF-C在肿瘤中表达增高(舌癌阳性率为90%),胞膜胞浆着色。HIF-1α表达与CEACAM1和VEGF-C呈正相关(r=1.26, P<0.05; r=1.45, P<0.05)
2. CEACAM1表达模式与口腔癌分化密切相关,并与肿瘤脉管生成和内皮细胞表型转化相关
CEACAM1在口腔高分化鳞状细胞癌中呈胞膜阳性表达,在中分化和低分口腔癌中呈胞浆阳性表达(P＜0.01)。在60例CEACAM1阳性的中分化和低分化癌中,有50例显示胞浆呈中到高表达,染色范围从33%到80%,与此形成鲜明对比的是在20例高分化癌中,16例显示胞膜阳性表达。在CEACAM1阳性病例中,CEACAM1阳性的脉管数量较阴性病例中显著增高(P＜0.001)；同时,CEACAM1阳性病例比CEACAM1阴性病例MVD和LVD都显著增加(P＜0.05)；CEACAM1和LYVE-1免疫荧光双标记显示双标记阳性脉管数量在CEACAM1阳性病例中数量增加(P＜0.001)；LYVE-1和CD31免疫组化双标记阳性脉管在CEACAM1胞浆阳性表达病例中升高(P＜0.001)。
3.RNAi慢病毒干扰载体有效抑制HIF-1α基因水平和蛋白水平表达
Real time RT-PCR和western blot检测Tca8113细胞系中HIF-1α干扰效率：RNA干扰基因沉默HIF-1α后,Tca8113细胞基因水平和蛋白水平HIF-1α表达均明显降低(P＜0.05),基因水平表达下降80%,蛋白水平表达降低50%。
4.基因沉默HIF-1α后CEACAM1基因水平和蛋白水平表达下调
Real time RT-PCR和FCM(流式细胞术)分别检测各组Tca8113细胞CEACAM1表达,干扰组较对照组CEACAM1在基因水平和蛋白水平表达均降低。FCM结果显示CEACAM1蛋白在空白对照组、阴性对照组和干扰组表达率分别为10.30%、12.73%和5.49%(P＜0.05)。
5.基因沉默HIF-1α后VEGF-C基因水平和蛋白水平表达下调
Real time RT-PCR和ELISA检测各组Tca8113细胞VEGF-C基因水平和蛋白水平表达,ELISA结果显示干扰组VEGF-C较对照组表达明显降低与空白对照组(P=0.029)和阴性对照组(P=0.032)比较具有显著差异性,表明HIF-1α基因沉默显著下调VEGF-C表达。
6.HIF-1α基因沉默组口腔癌裸鼠移植瘤模型中CEACAM1和VEGF-C表达显著下调,脉管生成明显减弱,形态结构有明显的正常化趋势
免疫组化方法检测裸鼠移植瘤模型中CEACAM1、VEGF-C表达及脉管生成情况：干扰组较空白对照组和阴性对照组CEACAM1表达明显降低(P＜0.05),MVD和LVD明显降低(P＜0.05),并且形态趋于正常化,管壁完整,内皮细胞连续,管腔规则,直径变小。干扰组与对照组相比,管腔直径差异性显著(P＜0.001)。LYVE-1和CD31双标记阳性脉管在干扰组中显著减少(P＜0.05)。结果表明肿瘤HIF-1α基因沉默后,脉管生成受到抑制,并且新生脉管结构形态趋于正常。
7.HIF-1α基因沉默降低口腔癌裸鼠移植瘤增殖活性,降低肿瘤生长速度,延长荷瘤裸鼠生存期
RNA干扰组及阴性载体对照组和空白对照组裸鼠移植瘤生长曲线显示,干扰组移植瘤生长速度明显减慢(P＜0.05),可能与HIF-1α沉默导致受HIF-1α调控的细胞增殖相关基因活性降低,使得肿瘤细胞增殖降低。干扰组移植瘤Ki-67表达明显降低(P＜0.005),说明HIF-1α沉默导致细胞增殖活性减弱。
裸鼠生存曲线分析显示：干扰组裸鼠生存时间较空白对照和阴性对照明显延长(P＜0.05)。
结论：
1.HIF-1α在人口腔鳞状细胞癌中高表达,并且与CEACAM1和VEGF-C表达呈正相关。CEACAM1表达与肿瘤分化程度关系密切,并与肿瘤脉管生成相关。CEACAM1阳性病例中,血管和淋巴管生成显著增高,内皮细胞表型转化也更活跃,这表明在HIF-1α高表达口腔癌中,HIF-1α可能通过调控CEACAM1和VEGF-C的表达,促进肿瘤血管和淋巴管生成,并对肿瘤血管内皮细胞和淋巴管内皮细胞表型转化具有重要作用。
2.体外实验显示HIF-1α基因沉默显著抑制HIF-1α基因水平和蛋白水平表达,同时随着HIF-1α表达降低,CEACAM1和VEGF-C在基因水平和蛋白水平表达均下调。这表明在人口腔癌中HIF-1α可以调控CEACAM1和VEGF-C表达,HIF-1α/CEACAM1/VEGF-C可能在口腔癌脉管生成中具有重要作用。
3.体内实验显示,HIF-1α基因沉默组口腔癌裸鼠移植瘤模型HIF-1α表达明显降低,肿瘤血管和淋巴管生成被显著抑制,并且脉管结构形态明显趋于正常,血管内皮细胞和淋巴管内皮细胞表型转化也更趋稳定,同时,干扰组移植瘤CEACAM1和VEGF-C表达都明显下调,表明RNA干扰基因沉默HIF-1α后,通过下调CEACAM1和VEGF-C表达抑制肿瘤血管和淋巴管生成,并介导内皮细胞的表型转化,使肿瘤脉管系统形态结构趋于正常。HIF-1α基因沉默还显著降低裸鼠移植瘤增殖活性和生长速度,延长荷瘤裸鼠生存期,这表明HIF-1α基因沉默导致受HIF-1α调控的细胞增殖相关基因活性降低,使得处于静止期的肿瘤细胞比例增高,肿瘤增殖活性降低,延迟了肿瘤的异质性进展过程,有利于荷瘤鼠生存期延长。此外,干扰组移植瘤新生脉管系统形态结构趋于正常也会使其功能在一定程度上趋于正常,趋于正常化的脉管系统形成的微环境抑制了肿瘤细胞在乏氧环境下的异质性克隆筛选,也会延缓肿瘤的异质化进程,也有利于荷瘤鼠生存期的延长。
4.HIF-1α沉默通过下调口腔癌CEACAM1和VEGF-C的表达抑制口腔癌血管和淋巴管生成,促进新生血管和淋巴管结构功能的正常化,并能有效抑制肿瘤增殖活性和生长速度,延长荷瘤鼠生存期,因此,HIF-1α/CEACAM1/VEGF-C可能成为口腔癌治疗中的有效联合靶点。6国家自然科学基金资助项日(30572056)
Objective:
1.To investigate the expression and relationsip of HIF-1α, CEACAM1 and VEGF-C in oral squamous cell carcinoma(OSCC). To analyze CEACAM1 expression and its impact on angiogenesis and lymphangiogenesis. Then to discuss and probe into the roles of HIF-la, CEACAM1 and VEGF-C in the angiogenesis and lymphangiogenesis in oral carcinoma.
2. To study the effects of gene silencing of HIF-1αby RNAi on HIF-1αexpression in tongue squamous cell carcinoma cell line-Tca8113. To investigate the impact of HIF-la gene silencing on CEACAM1 and VEGF-C in Tca8113 cell line. Then to discuss and probe into the possible mechanism of HIF-la and CEACAM1 in angiogenesis and lymphangiogenesis.
3. To study angiogenesis and lymphangiogenesis in tumor xenograft of oral carcinoma in nude mice and probe into the impact of HIF-1αgene silencing on angiogenesis and lymphangiogenesis. To explore the mechanism of HIF-la regulating CEACAM1 and VEGF-C expression and its impact and on angiogenesis and lymphangiogenesis
Methods:
1. Evaluation of HIF-1α, CEACAM1 and VEGF-C expression in oral squamous carcinoma
Ninety one cases of oral squanous cell carcinoma were collected from Qilu hospital and Stomatology Hospital of Shandong University, including 30 cases of squamous cell carcinoma in tongue.The clinical data included age, sex, size of tumor, stage and histological classification. Immunohistochemistry was performed to observe the expression and localization of HIF-la, CEACAM1 and VDGF-C in oral squanous cell carcinoma. The results were evaluated and the relationship between the results and clinical data was analyzed.
2.Evaluation of MVD, LVD and the transforming of endothelial cell in oral carcinoma
The microvessels and lymphatic vessels labelled with CD31 and LYVE-1 respectively were counted with light microscopy, and MVD and LVD were evaluated. Double-labelling immunofluorescence and immunohistochemistry were used to observe the phenotypes of endothelial cell, and the relationship with CEACAMl expression was analyzed.
3. Examining the interference rate of HIF-1αsilenced by RNAi lentivirus expression vector
Tca8113 cells were incubated in 37℃,5%CO2air then 4×104 cells were seeded into 6-well flat-bottom plates with 2ml DMEM medium. Following 48 hours of cultrue, the cells were classified into three groups:control, negative vector control and interference group, and the Eni.S., NC-GPF-lentivirus and HIF-la-RNAi-lentivirus (MOI:75), then the cells were cultured in 37℃and 5%CO2 air. The cell growth and infection were observed by fluorescence microscopy, and the efficiency of infection or transfection was evaluated by GFP and estimated as 70-80%. After 5 days, the cells were collected, and the total RNA was extracted, then the RNA was used for generation of cDNA, and the real time quantitive RT-PCR was performed. After 6 days, the cells were collected and the total protein was extracted, and then the Western blot was performed to examine the HIF-la protein expression.
4. Assessing and evaluating the impact of HIF-la silencing on expression of CEACAMl and VEGF-C
Tca8113 cells were cultured and infected by Lentivirus, after 5 days, the efficiency of infection or transfection was evaluated by observing GFP and estimated as 70-80%, then the cells were collected and the and the total RNA was extracted, which was used for generation of cDNA, and the real time quantitive RT-PCR was performed to evaluate the expression of CEACAM1 and VEGF-C at gene level. After
6 days, the cells were collected and the total protein was extracted, and then the Western blot was performed to examine the expression of CEACAM1 and VEGF-C at protein level after HIF-1αsilencing.
5. Estimating the growth and proliferative activity of xenograft tumor of oral carcinoma, examing the expression of CEACAM1,VEGF-C in carcinoma cells and the phenotype transforming of endothelial cells
Construction of xenograft tumor in nude mice:Tca8113 cells were incubated in 37℃、5%CO2 air then 4×104 cells were seeded into 6-well flat-bottom plates with 2ml DMEM medium. Following 48 hours of cultrue, the cells were classified into three groups:control, negative vector control and experimental group, and the Eni.S., NC-GPF-lentivirus and HIF-1α-RNAi-lentivirus(MOI:75), then the cells were cultured in 37℃and 5%CO2 air. The cell growth and infection were observed by fluorescence microscopy, and the efficiency of infection or transfection was evaluated by GFP and estimated as 70-80%. Then the cell were collected and the cell density was 2×106个/ml. Thirty BALB/C nu/nu nude mice were five weeks old, which were classified into three groups:control, negative vector control and experimental group, evry group involved 10 mice. After anesthetized by ethyl, the three groups of nude mice were injected with corresponding Tca8113 cell (2×105 cells) respectively in the subcutaneous tissue of the back. After subcutaneous injection, the three groups of mice were raised in SPF condition.The tumorigenicity was observed every day, and the size was measured.
Three weeks after subcutaneous injection, the 5 mice of every groups were anesthetized by ethyl, which were placed in the real time fluorescence imaging equipment to reveal the xenograft tumor size by GFP. Then the mice were sacrificed, the xenograft tumors were collected and were paraffin-embedded, then 4μm routine slide were made.Immunohistochemistry was performed to detect the expression of CEACAM1, VEGF-C, CD31 and LYVE-1, and the MVD and LVD were calculated based on CD31 and LYVE-1 staining. The histological difference and phenotype transforming of microvessels were also estimated and analyzed. The impact of HIF-1 silencing on angiogenesis, lymphangiogenesis and histological normalization of microvessels in xenograft tumor was studied.
6. Survival analysis of tumor-bearing mice
The remaining 5 mice of every group were raised in SPF condition continuously, and their lifetimes were recorded and analyzed.
Results:
1.Overexpression of HIF-la is positively correlated with CEACAM1 and VEGF-C expression
HIF-1αwas overexpressed in oral squamous cell carcinoma(95%,100%in tongue squamous cell carcinoma), the cytoplasm and nuclear were stained. CEACAM1 expression was upregulated in oral carcinoma(83.5%), and in tongue carcinoma(80%), cytoplasm and membrane were stained. VEGF-C expression was also upregulated in tongue carcinoma(90%),cytoplasm and membrane were stained. In addition, HIF-la expression was positively correlated with CEACAM1 and VEGF-C expression in tongue carcinoma(r=1.26, P<0.05; r=1.45, P<0.05 respectively).
2.The expression patterns of CEACAM1 showed significantly different in well, intermediately and poorly diffrentiated carcinoma, and CEACAM1 expression impacted on angiogenesis and lymphangiogenesis in oral carcinoma
CEACAM1 mainly showed membranous staining in well differentiated carcinoma, and cytoplasmic staining in intermediately and poorly differentiated carcinoma(P0.01):Out of 60 CEACAM1-positive cases of intermediately and poorly differentiated, fifty cases showed intermediate to high expression, and the staining range of positive carcinoma cells was 33%to 80%, in contrast, sixteen CEACAM1-positive cases of 20 well differentiated carcinoma showed membranous staining. In the CEACAMI-positive and CEACAMI-negative cases, the number of CEACAM1-positive vessels showed significant difference(P<0.001), as well as the MVD and LVD(p<0.05), Double-labelling immunofluorescence of CEACAM1 and LYVE-1 showed that coexpression-positive vessels was significant difference in CEAC AMI-positive and negative cases(P<0.001, and double-labelling immunohistochemistry of LYVE-1 and CD31 showed similar result(P<0.001).
3.Lentivirus expression vector of RNAi inhibited the expression of HIF-la at gene and protein level efficiently
Real time quantitive RT-PCR and Western blot showed that HIF-la expression was blocked by RNAi efficiently in Tca8113 cells. 4. HIF-la silencing down regulated CEACAM1 expression at gene and protein
Real time quantitive RT-PCR and FCM showed that the expression of CEACAM1 was downregulated at gene and protein level in experimental group.
5. HIF-la silencing downregulated VEGF-C expression at gene and protein
Real time quantitive RT-PCR and ELISA showed that the expression of VEGF-C was downregulated at gene and protein level in the experimental group.
6. HIF-la silencing dowmregulated the expression of CEACAM1 and VEGF-C in xenograft tumor, angiogenesis and lymphangiogenesis were declined, the histological structure had a tendence to be normalized
In the experimental group, the xenograft tumors showed CEACAM1 and VEGF-C expression downregulated(P<0.05), the amount of CEACAM1-positive vessels decreased(P<0.05). MVD and LVD showed a decrease(P<0.05), and the histological structure tended to be normalized:regular lumen with lessening diametere continuous endothelial cells and basal membrane. The vessels with coexpression of LYVE and CD31 significantly decreased(P<0.05).
7. HIF-la gene silencing declined the proliferative activity of xenograft tumors and slowed the growth speed and prolonged the lifetime of tumor-bearing mice
The growth curve analysis showed that the growth of xenograft tumor in experimental group was slower than control groups, and the immunostaining of Ki-67 revealed the proliferative activity was decreased.
The survival analysis showed that the lifetime of tumor-bearing mice in the experimental group were prolonged significantly.
Conclusion:
1.HIF-1αwas overexpression in oral squamous cell carcinoma and positively correlated with CEACAM1 and VEGF-C expression. CEACAM1 expression patterns showed significant difference in well, intermediately and poorly differentiated carcinoma. Angiogenesis and lymphangiogenesis increased in CEACAM1-positive cases, which revealed that overexpression of HIF-1αmight regulated CEACAM1 and VEGF-C expression to promote the angiogenesisn and lymphangiogenesis, and which played a important role in transforming of endothelial cell from vascular phenotype to lymphatic phenotype.
2.HIF-1αgene silencing downregulated HIF-1αexpression at gene and protein level, with the decrease of HIF-1αexpression, CEACAM1 and VEGF-C expression was declined at gene and protein level. In the experimental group, xenograft tumors with HIF-1αgene silencing showed declined angiogenesis and lymphangiogenesis, and the newborn microvessels tended to be normalized in histological structure, the transforming of endothelial cells from vascular phenotype to lymphatic phenotype tended to be stable and normalized. These all implicatedthat, HIF-la gene silencing inhibited angiogenesin and lymphangiogenesis via downregulating CEACAM1 and VEGF-C expression, and even promote the normalization of vessel system. In addition, HIF-la gene silencing decreased the proliferative activity and growth of the xenograft tumor, but prolonged the survival lifetime of the tumor-bearing mice. These indicated that HIF-la silencing decreased the ratio of silent stage cells to generated stage cells, which resulted in the declining of cell proliferation. The newborn vessel system tended to be normalized in histological structure, which led to the normalization of function. All these inhibited the heterogeneity and cloning selection, this maybe prolong the tumor progression.
3.HIF-1αgene silencing inhibited angiogenesis and lymphangiogenesis via downregulating the CEACAM1 and VEGF-C expression in tumor, and led to the normalization of newborn vessels in histologically and functionally, these implicated that HFI-la, CEACAM1 and VEGF-C might be therapy targets in oral carcinoma.
1.观察研究HIF-1α在口腔鳞状细胞癌中表达及与CEACAM1、VEGF-C表达的关系,分析CEACAM1对血管和淋巴管生成的影响,初步探讨HIF-1α、CEACAM1、VEGF-C在口腔癌血管和淋巴管生成中的作用及可能的机制。
2.观察RNAi基因沉默HIF-1α对舌鳞状细胞癌细胞系Tca8113表达HIF-1α的影响,并观察研究HIF-1α基因沉默对CEACAM1和VEGF-C表达的影响,探讨HIF-1α/CEACAM1/VEGF-C的调控关系及在肿瘤脉管生成中的作用机制。
3.以口腔癌裸鼠移植瘤模型为研究对象,研究HIF-1α基因沉默对肿瘤CEACAM1和VEGF-C表达的影响,验证HIF-1α/CEACAM1/VEGF-C对肿瘤血管和淋巴管生成的作用,以及对肿瘤增殖和进展的影响；探讨HIF-1α/CEACAM1/VEGF-C通路对肿瘤脉管生成及其正常化的影响及作用机制。
研究方法：
1.以人口腔鳞状细胞癌为研究对象,检测人口腔鳞状细胞癌中HIF-1α、CEACAM1和VEGF-C表达
收集2005-2007年山东大学齐鲁医院和山东大学口腔医院口腔癌手术标本91例,其中包括舌鳞状细胞癌标本30例。患者临床资料包括年龄、性别、肿瘤大小、肿瘤临床分期、肿瘤分化程度。应用免疫组化方法检测HIF-1α和CEACAM1在91例口腔鳞状细胞癌中的表达及定位,并对免疫组化结果进行分析,评价蛋白的表达与临床资料之间的关系。检测30例舌鳞状细胞癌中VEGF-C表达,并与HIF-1α和CEACAM1在舌癌中的表达进行对比,统计分析HIF-1α与CEACAM1和VEGF-C在舌癌中表达的相关性,并进一步分析在HIF-1α高表达情况下,CEACAM1表达与肿瘤脉管生成的关系。
2.以人口腔癌为研究对象,检测人口腔癌中MVD和LVD以及脉管内皮细胞表型转化情况
采用免疫组化方法,分别应用血管标记CD31和淋巴管标记LYVE-1检测口腔癌中血管和淋巴管生成情况,统计血管密度和淋巴管密度。免疫组化双标记和免疫荧光双标记检测新生脉管表型转化情况,并分析其与CEACAM1表达的关系。
3、以人舌鳞状细胞癌细胞系Tca8113细胞为研究对象,检测RNAi慢病毒干扰载体对Tca8113细胞HIF-1α基因的干扰效率
Tca8113细胞在37℃、5%CO2孵箱中培养,取生长状态良好的细胞以4×104／孔接种于6孔培养板中,每孔加入2m1DMEM培养基。48小时后,细胞融合率可达到30-50%。将细胞分为空白对照组、阴性载体对照组和干扰组,分别加入ENi.S.、NC-GFP-Lentivirus和HIF-1 a-RNAi-Lentivirus(复感染指数MOI为75),37℃、5%CO2孵箱中继续常规培养。每天倒置荧光显微镜观察细胞生长,通过观察慢病毒上报告基因GFP的表达情况,评估感染效率。RNA干扰后第五天,通过观察GFP评估细胞感染率约为70-80%,收集细胞,提取细胞总RNA,应用Real time RT-PCR方法检测不同实验组中HIF-1α基因水平表达,Realtime RT-PCR数值分析采用标准曲线分析法。RNA干扰后第五天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中HIF-1α的蛋白表达水平。
4.以Tca8113细胞为研究对象,检测HIF-1α沉默对CEACAM1、VEGF-C基因和蛋白水平表达的影响
Tca8113细胞培养并感染慢病毒颗粒,RNA干扰后第五天,通过观察GFP评估细胞感染率约为70-80%,收集细胞,提取细胞总RNA,应用real time RT-PCR方法检测不同实验组中HIF-1α、CEACAM1和VEGF-C基因水平的表达,real time RT-PCR数值分析采用标准曲线分析法。RNA干扰后第五天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中HIF-1α的蛋白表达,再次验证蛋白表达水平干扰效率。同时收集细胞,采用流式细胞术检测CEACAM1的表达。RNA干扰后第五天收集Tca8113细胞培养液上清,采用ELISA方法对分泌至细胞外的VEGF-C蛋白进行检测。分析三个不同实验组中Tca8113细胞HIF-1α、CEACAM1和VEGF-C在基因和蛋白水平表达情况,探讨HIF-1α基因沉默后对CEACAM1和VEGF-C表达的影响。
5.建立HIF-1α基因沉默的口腔癌裸鼠移植瘤模型并观察脉管生成及肿瘤进展
观察口腔癌裸鼠移植瘤的增殖进展和荷瘤鼠生存情况,检测移植瘤增殖活性及CEACAM1和VEGF-C表达情况,观察肿瘤脉管生成情况及其形态结构和内皮细胞表型转化情况,分析HIF-1α基因沉默与CEACAM1和VEGF-C表达及脉管生成的关系。
建立口腔癌裸鼠移植瘤模型：37℃、5%CO2孵箱中培养Tca8113细胞,将生长状态良好的细胞以4×104／孔接种于6孔培养板中,每孔加入2m1DMEM培养基。48小时后,细胞融合率可达到30-50%。将细胞分为空白对照组、阴性对照组和干扰组,分别加入ENi.S.、NC-GFP慢病毒颗粒和HIF-1 a-RNAi慢病毒载体(复感染指数MOI为75),37℃、5%CO2孵箱中继续常规培养。在细胞生长状态良好时配制细胞悬液,细胞浓度为2×106个／ml,备用。5周龄的BALB/C nu/nu雌性裸鼠30只,(中国医学科学院实验动物研究所),分为空白对照组、阴性对照组和干扰组,每组10只,注射0.1ml空白对照组、阴性对照组和干扰组Tca8113细胞悬液(细胞数量为2×105个)于裸鼠背部皮下。接种肿瘤细胞后,将裸鼠置于SPF饲养条件下饲养,每天观察裸鼠背部的成瘤情况并在成瘤后定期对肿瘤大小进行测量。
四周后每组裸鼠中的5只分别乙醚吸入麻醉后进行荧光实时活体成像,然后脱颈处死,收集肿瘤标本。将收集的裸鼠肿瘤标本进行常规处理制备成蜡块,应用免疫组化方法检测三组裸鼠肿瘤标本中CEACAM1、VEGF-C、Ki67、LYVE-1(抗鼠)和CD31(抗鼠)的表达,并计数评估MVD和LVD,观察其形态结构变化,免疫双标记法检测新生脉管内皮细胞表型变化,研究HIF-1α基因沉默后对裸鼠移植瘤血管和淋巴管生成及其形态学改变和正常化的影响。
6.观察口腔癌裸鼠移植瘤模型生存情况
三组裸鼠每组剩余5只继续在SPF饲养条件下饲养,观察研究荷瘤鼠生存情况,并进行生存分析。
结果：
1.HIF-1α在口腔鳞状细胞癌中高表达,并与CEACAM1和VEGF-C表达呈正相关
HIF-1α在肿瘤中高表达(口腔癌阳性率95%,舌癌阳性率100%),细胞核和细胞浆着色；CEACAM1在肿瘤中表达增高(口腔癌中阳性率为83.5%,舌癌阳性率80%),胞膜和胞浆着色；VEGF-C在肿瘤中表达增高(舌癌阳性率为90%),胞膜胞浆着色。HIF-1α表达与CEACAM1和VEGF-C呈正相关(r=1.26, P<0.05; r=1.45, P<0.05)
2. CEACAM1表达模式与口腔癌分化密切相关,并与肿瘤脉管生成和内皮细胞表型转化相关
CEACAM1在口腔高分化鳞状细胞癌中呈胞膜阳性表达,在中分化和低分口腔癌中呈胞浆阳性表达(P＜0.01)。在60例CEACAM1阳性的中分化和低分化癌中,有50例显示胞浆呈中到高表达,染色范围从33%到80%,与此形成鲜明对比的是在20例高分化癌中,16例显示胞膜阳性表达。在CEACAM1阳性病例中,CEACAM1阳性的脉管数量较阴性病例中显著增高(P＜0.001)；同时,CEACAM1阳性病例比CEACAM1阴性病例MVD和LVD都显著增加(P＜0.05)；CEACAM1和LYVE-1免疫荧光双标记显示双标记阳性脉管数量在CEACAM1阳性病例中数量增加(P＜0.001)；LYVE-1和CD31免疫组化双标记阳性脉管在CEACAM1胞浆阳性表达病例中升高(P＜0.001)。
3.RNAi慢病毒干扰载体有效抑制HIF-1α基因水平和蛋白水平表达
Real time RT-PCR和western blot检测Tca8113细胞系中HIF-1α干扰效率：RNA干扰基因沉默HIF-1α后,Tca8113细胞基因水平和蛋白水平HIF-1α表达均明显降低(P＜0.05),基因水平表达下降80%,蛋白水平表达降低50%。
4.基因沉默HIF-1α后CEACAM1基因水平和蛋白水平表达下调
Real time RT-PCR和FCM(流式细胞术)分别检测各组Tca8113细胞CEACAM1表达,干扰组较对照组CEACAM1在基因水平和蛋白水平表达均降低。FCM结果显示CEACAM1蛋白在空白对照组、阴性对照组和干扰组表达率分别为10.30%、12.73%和5.49%(P＜0.05)。
5.基因沉默HIF-1α后VEGF-C基因水平和蛋白水平表达下调
Real time RT-PCR和ELISA检测各组Tca8113细胞VEGF-C基因水平和蛋白水平表达,ELISA结果显示干扰组VEGF-C较对照组表达明显降低与空白对照组(P=0.029)和阴性对照组(P=0.032)比较具有显著差异性,表明HIF-1α基因沉默显著下调VEGF-C表达。
6.HIF-1α基因沉默组口腔癌裸鼠移植瘤模型中CEACAM1和VEGF-C表达显著下调,脉管生成明显减弱,形态结构有明显的正常化趋势
免疫组化方法检测裸鼠移植瘤模型中CEACAM1、VEGF-C表达及脉管生成情况：干扰组较空白对照组和阴性对照组CEACAM1表达明显降低(P＜0.05),MVD和LVD明显降低(P＜0.05),并且形态趋于正常化,管壁完整,内皮细胞连续,管腔规则,直径变小。干扰组与对照组相比,管腔直径差异性显著(P＜0.001)。LYVE-1和CD31双标记阳性脉管在干扰组中显著减少(P＜0.05)。结果表明肿瘤HIF-1α基因沉默后,脉管生成受到抑制,并且新生脉管结构形态趋于正常。
7.HIF-1α基因沉默降低口腔癌裸鼠移植瘤增殖活性,降低肿瘤生长速度,延长荷瘤裸鼠生存期
RNA干扰组及阴性载体对照组和空白对照组裸鼠移植瘤生长曲线显示,干扰组移植瘤生长速度明显减慢(P＜0.05),可能与HIF-1α沉默导致受HIF-1α调控的细胞增殖相关基因活性降低,使得肿瘤细胞增殖降低。干扰组移植瘤Ki-67表达明显降低(P＜0.005),说明HIF-1α沉默导致细胞增殖活性减弱。
裸鼠生存曲线分析显示：干扰组裸鼠生存时间较空白对照和阴性对照明显延长(P＜0.05)。
结论：
1.HIF-1α在人口腔鳞状细胞癌中高表达,并且与CEACAM1和VEGF-C表达呈正相关。CEACAM1表达与肿瘤分化程度关系密切,并与肿瘤脉管生成相关。CEACAM1阳性病例中,血管和淋巴管生成显著增高,内皮细胞表型转化也更活跃,这表明在HIF-1α高表达口腔癌中,HIF-1α可能通过调控CEACAM1和VEGF-C的表达,促进肿瘤血管和淋巴管生成,并对肿瘤血管内皮细胞和淋巴管内皮细胞表型转化具有重要作用。
2.体外实验显示HIF-1α基因沉默显著抑制HIF-1α基因水平和蛋白水平表达,同时随着HIF-1α表达降低,CEACAM1和VEGF-C在基因水平和蛋白水平表达均下调。这表明在人口腔癌中HIF-1α可以调控CEACAM1和VEGF-C表达,HIF-1α/CEACAM1/VEGF-C可能在口腔癌脉管生成中具有重要作用。
3.体内实验显示,HIF-1α基因沉默组口腔癌裸鼠移植瘤模型HIF-1α表达明显降低,肿瘤血管和淋巴管生成被显著抑制,并且脉管结构形态明显趋于正常,血管内皮细胞和淋巴管内皮细胞表型转化也更趋稳定,同时,干扰组移植瘤CEACAM1和VEGF-C表达都明显下调,表明RNA干扰基因沉默HIF-1α后,通过下调CEACAM1和VEGF-C表达抑制肿瘤血管和淋巴管生成,并介导内皮细胞的表型转化,使肿瘤脉管系统形态结构趋于正常。HIF-1α基因沉默还显著降低裸鼠移植瘤增殖活性和生长速度,延长荷瘤裸鼠生存期,这表明HIF-1α基因沉默导致受HIF-1α调控的细胞增殖相关基因活性降低,使得处于静止期的肿瘤细胞比例增高,肿瘤增殖活性降低,延迟了肿瘤的异质性进展过程,有利于荷瘤鼠生存期延长。此外,干扰组移植瘤新生脉管系统形态结构趋于正常也会使其功能在一定程度上趋于正常,趋于正常化的脉管系统形成的微环境抑制了肿瘤细胞在乏氧环境下的异质性克隆筛选,也会延缓肿瘤的异质化进程,也有利于荷瘤鼠生存期的延长。
4.HIF-1α沉默通过下调口腔癌CEACAM1和VEGF-C的表达抑制口腔癌血管和淋巴管生成,促进新生血管和淋巴管结构功能的正常化,并能有效抑制肿瘤增殖活性和生长速度,延长荷瘤鼠生存期,因此,HIF-1α/CEACAM1/VEGF-C可能成为口腔癌治疗中的有效联合靶点。6国家自然科学基金资助项日(30572056)
Objective:
1.To investigate the expression and relationsip of HIF-1α, CEACAM1 and VEGF-C in oral squamous cell carcinoma(OSCC). To analyze CEACAM1 expression and its impact on angiogenesis and lymphangiogenesis. Then to discuss and probe into the roles of HIF-la, CEACAM1 and VEGF-C in the angiogenesis and lymphangiogenesis in oral carcinoma.
2. To study the effects of gene silencing of HIF-1αby RNAi on HIF-1αexpression in tongue squamous cell carcinoma cell line-Tca8113. To investigate the impact of HIF-la gene silencing on CEACAM1 and VEGF-C in Tca8113 cell line. Then to discuss and probe into the possible mechanism of HIF-la and CEACAM1 in angiogenesis and lymphangiogenesis.
3. To study angiogenesis and lymphangiogenesis in tumor xenograft of oral carcinoma in nude mice and probe into the impact of HIF-1αgene silencing on angiogenesis and lymphangiogenesis. To explore the mechanism of HIF-la regulating CEACAM1 and VEGF-C expression and its impact and on angiogenesis and lymphangiogenesis
Methods:
1. Evaluation of HIF-1α, CEACAM1 and VEGF-C expression in oral squamous carcinoma
Ninety one cases of oral squanous cell carcinoma were collected from Qilu hospital and Stomatology Hospital of Shandong University, including 30 cases of squamous cell carcinoma in tongue.The clinical data included age, sex, size of tumor, stage and histological classification. Immunohistochemistry was performed to observe the expression and localization of HIF-la, CEACAM1 and VDGF-C in oral squanous cell carcinoma. The results were evaluated and the relationship between the results and clinical data was analyzed.
2.Evaluation of MVD, LVD and the transforming of endothelial cell in oral carcinoma
The microvessels and lymphatic vessels labelled with CD31 and LYVE-1 respectively were counted with light microscopy, and MVD and LVD were evaluated. Double-labelling immunofluorescence and immunohistochemistry were used to observe the phenotypes of endothelial cell, and the relationship with CEACAMl expression was analyzed.
3. Examining the interference rate of HIF-1αsilenced by RNAi lentivirus expression vector
Tca8113 cells were incubated in 37℃,5%CO2air then 4×104 cells were seeded into 6-well flat-bottom plates with 2ml DMEM medium. Following 48 hours of cultrue, the cells were classified into three groups:control, negative vector control and interference group, and the Eni.S., NC-GPF-lentivirus and HIF-la-RNAi-lentivirus (MOI:75), then the cells were cultured in 37℃and 5%CO2 air. The cell growth and infection were observed by fluorescence microscopy, and the efficiency of infection or transfection was evaluated by GFP and estimated as 70-80%. After 5 days, the cells were collected, and the total RNA was extracted, then the RNA was used for generation of cDNA, and the real time quantitive RT-PCR was performed. After 6 days, the cells were collected and the total protein was extracted, and then the Western blot was performed to examine the HIF-la protein expression.
4. Assessing and evaluating the impact of HIF-la silencing on expression of CEACAMl and VEGF-C
Tca8113 cells were cultured and infected by Lentivirus, after 5 days, the efficiency of infection or transfection was evaluated by observing GFP and estimated as 70-80%, then the cells were collected and the and the total RNA was extracted, which was used for generation of cDNA, and the real time quantitive RT-PCR was performed to evaluate the expression of CEACAM1 and VEGF-C at gene level. After
6 days, the cells were collected and the total protein was extracted, and then the Western blot was performed to examine the expression of CEACAM1 and VEGF-C at protein level after HIF-1αsilencing.
5. Estimating the growth and proliferative activity of xenograft tumor of oral carcinoma, examing the expression of CEACAM1,VEGF-C in carcinoma cells and the phenotype transforming of endothelial cells
Construction of xenograft tumor in nude mice:Tca8113 cells were incubated in 37℃、5%CO2 air then 4×104 cells were seeded into 6-well flat-bottom plates with 2ml DMEM medium. Following 48 hours of cultrue, the cells were classified into three groups:control, negative vector control and experimental group, and the Eni.S., NC-GPF-lentivirus and HIF-1α-RNAi-lentivirus(MOI:75), then the cells were cultured in 37℃and 5%CO2 air. The cell growth and infection were observed by fluorescence microscopy, and the efficiency of infection or transfection was evaluated by GFP and estimated as 70-80%. Then the cell were collected and the cell density was 2×106个/ml. Thirty BALB/C nu/nu nude mice were five weeks old, which were classified into three groups:control, negative vector control and experimental group, evry group involved 10 mice. After anesthetized by ethyl, the three groups of nude mice were injected with corresponding Tca8113 cell (2×105 cells) respectively in the subcutaneous tissue of the back. After subcutaneous injection, the three groups of mice were raised in SPF condition.The tumorigenicity was observed every day, and the size was measured.
Three weeks after subcutaneous injection, the 5 mice of every groups were anesthetized by ethyl, which were placed in the real time fluorescence imaging equipment to reveal the xenograft tumor size by GFP. Then the mice were sacrificed, the xenograft tumors were collected and were paraffin-embedded, then 4μm routine slide were made.Immunohistochemistry was performed to detect the expression of CEACAM1, VEGF-C, CD31 and LYVE-1, and the MVD and LVD were calculated based on CD31 and LYVE-1 staining. The histological difference and phenotype transforming of microvessels were also estimated and analyzed. The impact of HIF-1 silencing on angiogenesis, lymphangiogenesis and histological normalization of microvessels in xenograft tumor was studied.
6. Survival analysis of tumor-bearing mice
The remaining 5 mice of every group were raised in SPF condition continuously, and their lifetimes were recorded and analyzed.
Results:
1.Overexpression of HIF-la is positively correlated with CEACAM1 and VEGF-C expression
HIF-1αwas overexpressed in oral squamous cell carcinoma(95%,100%in tongue squamous cell carcinoma), the cytoplasm and nuclear were stained. CEACAM1 expression was upregulated in oral carcinoma(83.5%), and in tongue carcinoma(80%), cytoplasm and membrane were stained. VEGF-C expression was also upregulated in tongue carcinoma(90%),cytoplasm and membrane were stained. In addition, HIF-la expression was positively correlated with CEACAM1 and VEGF-C expression in tongue carcinoma(r=1.26, P<0.05; r=1.45, P<0.05 respectively).
2.The expression patterns of CEACAM1 showed significantly different in well, intermediately and poorly diffrentiated carcinoma, and CEACAM1 expression impacted on angiogenesis and lymphangiogenesis in oral carcinoma
CEACAM1 mainly showed membranous staining in well differentiated carcinoma, and cytoplasmic staining in intermediately and poorly differentiated carcinoma(P0.01):Out of 60 CEACAM1-positive cases of intermediately and poorly differentiated, fifty cases showed intermediate to high expression, and the staining range of positive carcinoma cells was 33%to 80%, in contrast, sixteen CEACAM1-positive cases of 20 well differentiated carcinoma showed membranous staining. In the CEACAMI-positive and CEACAMI-negative cases, the number of CEACAM1-positive vessels showed significant difference(P<0.001), as well as the MVD and LVD(p<0.05), Double-labelling immunofluorescence of CEACAM1 and LYVE-1 showed that coexpression-positive vessels was significant difference in CEAC AMI-positive and negative cases(P<0.001, and double-labelling immunohistochemistry of LYVE-1 and CD31 showed similar result(P<0.001).
3.Lentivirus expression vector of RNAi inhibited the expression of HIF-la at gene and protein level efficiently
Real time quantitive RT-PCR and Western blot showed that HIF-la expression was blocked by RNAi efficiently in Tca8113 cells. 4. HIF-la silencing down regulated CEACAM1 expression at gene and protein
Real time quantitive RT-PCR and FCM showed that the expression of CEACAM1 was downregulated at gene and protein level in experimental group.
5. HIF-la silencing downregulated VEGF-C expression at gene and protein
Real time quantitive RT-PCR and ELISA showed that the expression of VEGF-C was downregulated at gene and protein level in the experimental group.
6. HIF-la silencing dowmregulated the expression of CEACAM1 and VEGF-C in xenograft tumor, angiogenesis and lymphangiogenesis were declined, the histological structure had a tendence to be normalized
In the experimental group, the xenograft tumors showed CEACAM1 and VEGF-C expression downregulated(P<0.05), the amount of CEACAM1-positive vessels decreased(P<0.05). MVD and LVD showed a decrease(P<0.05), and the histological structure tended to be normalized:regular lumen with lessening diametere continuous endothelial cells and basal membrane. The vessels with coexpression of LYVE and CD31 significantly decreased(P<0.05).
7. HIF-la gene silencing declined the proliferative activity of xenograft tumors and slowed the growth speed and prolonged the lifetime of tumor-bearing mice
The growth curve analysis showed that the growth of xenograft tumor in experimental group was slower than control groups, and the immunostaining of Ki-67 revealed the proliferative activity was decreased.
The survival analysis showed that the lifetime of tumor-bearing mice in the experimental group were prolonged significantly.
Conclusion:
1.HIF-1αwas overexpression in oral squamous cell carcinoma and positively correlated with CEACAM1 and VEGF-C expression. CEACAM1 expression patterns showed significant difference in well, intermediately and poorly differentiated carcinoma. Angiogenesis and lymphangiogenesis increased in CEACAM1-positive cases, which revealed that overexpression of HIF-1αmight regulated CEACAM1 and VEGF-C expression to promote the angiogenesisn and lymphangiogenesis, and which played a important role in transforming of endothelial cell from vascular phenotype to lymphatic phenotype.
2.HIF-1αgene silencing downregulated HIF-1αexpression at gene and protein level, with the decrease of HIF-1αexpression, CEACAM1 and VEGF-C expression was declined at gene and protein level. In the experimental group, xenograft tumors with HIF-1αgene silencing showed declined angiogenesis and lymphangiogenesis, and the newborn microvessels tended to be normalized in histological structure, the transforming of endothelial cells from vascular phenotype to lymphatic phenotype tended to be stable and normalized. These all implicatedthat, HIF-la gene silencing inhibited angiogenesin and lymphangiogenesis via downregulating CEACAM1 and VEGF-C expression, and even promote the normalization of vessel system. In addition, HIF-la gene silencing decreased the proliferative activity and growth of the xenograft tumor, but prolonged the survival lifetime of the tumor-bearing mice. These indicated that HIF-la silencing decreased the ratio of silent stage cells to generated stage cells, which resulted in the declining of cell proliferation. The newborn vessel system tended to be normalized in histological structure, which led to the normalization of function. All these inhibited the heterogeneity and cloning selection, this maybe prolong the tumor progression.
3.HIF-1αgene silencing inhibited angiogenesis and lymphangiogenesis via downregulating the CEACAM1 and VEGF-C expression in tumor, and led to the normalization of newborn vessels in histologically and functionally, these implicated that HFI-la, CEACAM1 and VEGF-C might be therapy targets in oral carcinoma.
引文
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