| 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 lymphangiogenesisMethods:1. Evaluation of HIF-1α, CEACAM1 and VEGF-C expression in oral squamous carcinomaNinety 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 carcinomaThe 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 vectorTca8113 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-CTca8113 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 cellsConstruction 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 miceThe 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 expressionHIF-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 carcinomaCEACAM1 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 efficientlyReal 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 proteinReal 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 proteinReal 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 normalizedIn 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 miceThe 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. |
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