Inhibition Of Metastasis And Angiogenesis Of Laryngeal Carcinoma Cell Via Targeting Egf17 Gene In Vivo And Vivo By RNA Interference

PartyⅠThe study on clinical significance of the expression of Egfl7 in laryngeal squamous cell carcinomaObjective:To investigate the expression of Egfl7 in laryngeal squamous cell carcinoma(LSCC)and its significance.Methods:The expression of Egfl7 mRNA and protein were detected by RT-PCR and Western-blot in 33 fresh cases laryngeal cancer tissue; The expression of Egfl7 protein in 116 cases laryngeal carcinoma were detected by immunohistochemical methods.To research the expression of Egfl7 relation to clinicopathologic characteristics and prognosis,the multi-factor COX and the combination of clinical pathology and clinical follow-up data were analysis.Results:1.The expression of Egfl7 mRNA positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 87.9%(29/33) to non-carcinoma laryngeal tissues(NCLT) 33.3%(11/33)(P＜0.01).The expression average level of Egfl7 mRNA in LSCC was significantly higher than NCLT(1.42±0.21/ 0.86±0.11,P=0.008).The expression of Egfl7 protein positive rate is significantly difference between 33 cases fresh laryngeal squamous cell carcinoma(LSCC) tissues 90.9%(30/33) to non-carcinoma laryngeal tissues(NCLT) 27.3%(9/33)(P＜0.01).The expression average level of Egfl7 protein in LSCC was significantly higher than NCLT(0.97±0.21/ 0.41±0.13,P=0.001).Furthermore,the expression of Egfl7 mRNA between protein is significant positive correlation(R=0.786,P＜0.01). Egfl7 mRNA and protein expression were significantly correlated with LSCC clinical stage,tumor size and the presence of lymph node metastasis(P＜0.05).The expression of Egfl7 mRNA and protein is significant positive correlation with MVD in 33 cases LSCC(R=0.842, P=0.231).2.The positive immue staining of Egfl7 protein expression is majority located in the cytoplasm.Among 116 cases LSCC tissues,the expression of Egfl7 protein positive rate is significantly difference between the positive expression 81.03%(94/116) and negative expression rate 18.97%(22/116).3.The expression Egfl7 protein in 116 cases LSCC is significantly related the clinical stage(P=0.003),tumor size in diameter(P=0.001) and lymph node metastasis(P=0.002).However,the expression Egfl7 protein in LSCC is unrelated sex,age and to the primary site(P＞0.05). Among 116 patients with LSCC,67 in Egfl7^++/+++ group,49 in Egfl7^-/+ group,the average survival time was 36.9 months,median survival time was 43.2 months and 55cases of them died in five years.Of which 55 died cases,40 cases was in Egfl7^++/+++ group,15 in Egfl7^-/+ group.Egfl7 high-expression group post-operative survival rate is significant difference lower than Egfl7 low expression group(P=0.007).Cox analysis result is show that Egfl7 expression(RR,1.74;P=0.012),lymph node metastases(RR,1.52;P=0.015) is an independent predictor of the prognosis laryngeal squamous cell carcinoma.Conclusion:1.Egfl7 may have been involved in the occurrence and development of LSCC.2.Egfl7 might be a tumor marker as a predict prognosis of laryngeal cancer. PartyⅡStudy on the effect of biological behavior of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitroObjective:To investiga the mechanisms of the expression of Egfl7 in LSCC invasion and metastasis by interfence targeting Egfl7 silence.Methods:PT67 packaging cells were transferred construction of retroviral pSUPER.Retro.neo small RNA interference plasmid,the virus particles and instantaneous access to double infection Hep-2 laryngeal cancer cell lines,the intervention of silence Egfl7 in Hep-2 expression in vitro Use the scratch test,Transwell invasion experiments,MTT and flow cytometry and other methods to study the laryngeal cancer cell line Hep-2 secretion Egfl7 control the proliferation of laryngeal cancer cells, laryngeal attack the molecular mechanism of movement.To examine the role of the expression of Egfl7 in laryngeal cancer cells is effected the ability of development of LSCC,Hep-2 cell lines were by RNAi interfered and were carry out a subcutaneous injection into node mice body in vivo.Results:1.In our present study,targeting of Egfl7siRNA plasmid and the control group expression vector were successfully constructed.2.We obtained the stability of the expressed sequence 1 and C sequences of Hep-2 cell line,and named Hep-2^Egfl7RNAi+and Hep.2^Egfl7RNAi-.3.The expression of Egfl7 protein in Hep-2^Egfl7RNAi+ cell lines was significantly inhibited by Western blot detection.4.The expression of Egfl7 is inhibited in laryngeal cancer cells can lead to inhibition the invasion of migration is decreased in vitro.①There is significant difference between Hep-2^Egfl7RNAi+ (62%)and Hep-2^Egfl7RNAi-group(77%)(P＜0.05) by wound healing assay.②There is significant difference Hep-2^Egfl7RNAi+(63±11) than Hep-2^Egfl7RNAi-cell invasion(129±13)(P＜0.05) by Transwell invasion experiment:5.The expression of Egfl7 is inhibited in laryngeal cancer cells without affected cell proliferation and apoptosis.①There is no significant difference between Hep-2^Egfl7RNAi+ and Hep-2^Egfl7RNAi- proliferation by MTT detection method(P＞0.05).②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that in Hep-2^Egfl7RNAi+ and Hep-2^Egfl7RNAi- groups apoptosis difference is not significant(P＞0.05).6.To inhibit the expression of Egfl7 in laryngeal cancer cells can inhibit the ability of tumor development.The size of Mice^Egfl7RNAi+group is smaller(3.01±0.22) compare to Mice^Egfl7RNAi- group(3.45±0.23) after 45 days by subcutaneous injection,(P＜0.05) Conclusion:1.Egfl7 may be play a key role in LSCC invasion and metastasis.2.Egfl7 would to be a new target for gene therapy in LSCC invasion and metastasis. PartyⅢStudy on the effect of angiogenesis of laryngeal cancer to interfere with RNAi target to Egfl7 in vivo and in vitroObjective:To investiga the mechanism of the expression of Egfl7 in LSCC angiogenesis by interfering targeted Egfl7 silence.Methods:Silent targert to Egfl7 expression in human microvascular endothelial cells(HMEC-1) was interfered by silencing specific Retrovirus-mediated small interfering RNA;The molecular mechanism that secreted Egfl7 in Hep-2 regulate the cell differentiation,proliferation, invasion and angiogenesis of LSCC was studied by using scratch test, Transwell invasion test,two and three-dimensional vasicular tubulogenesis test,MTT and flow cytometry assay and other detection methods in vitro;The mechanisms which Egfl7 regulate vasicular tubulogenesis leading to tumor biological behavior change in LSCC was confirmed by using three-dimensional co-culture of tumor cells into nude mice experiments in vivo.Results:1.The HMEC-1 infection efficiency is about to 80%to 90%.The expression of Egfl7 in HMEC-1 lines were silencing specific Retrovirus-mediated small interfering RNA.The expression of Egfl7 protein is significantly decreased in HMEC-1^Egfl7RNAi+(0.36±0.07) group than that in the control HMEC-1^{Egfl7RNAi-Egfl7}(0.86±0.02) group by Western blot decation(P＜0.05).2.The expression of Egfl7 is inhibited in HMEC-1 can lead to inhibition the invasion of migration is decreased in vitro.①There is significant difference between HMEC-1^Egfl7RNAi+ (69%)and HMEC-1^Egfl7RNAi-group(92%)(P＜0.05) by wound healing assay.②There is significant difference in HMEC-1^Egfl7RNAi+ invasion(49±6) compared with HMEC-1^Egfl7RNAi- invasion(70±3)(P＜0.05) by Transwell invasion experiment.3.The expression of Egfl7 is inhibited in laryngeal cancer cells cannot be effected cell proliferation and apoptosis.①There is no significant difference between HMEC-1^Egfl7RNAi+ and HMEC-1^Egfl7RNAi- proliferation by MTT detection method(P＞0.05).②AnnexinⅤand PI double staining combined with flow cytometry detection technology show that apoptosis in HMEC-1^Egfl7RNAi+ and HMEC-1^Egfl7RNAi- groups is not significant(P＞0.05).4.Egfl7 inhibition in vitro can inhibit the expression of HMEC-1 angiogenesis.①The HMEC-1^Egfl7RNAi+ endothelial cells can not form a mesh structure on Matrigel.There is a significant difference between the two groups(2397±726 vs 489±215)(P＜0.05). ②The control group HMEC-1^Egfl7RNAi-endothelial cell proliferation in the three-dimensional environment and the formation of threedimensional structure of the tube,while the HMEC-1^Egfl7RNAi+group in the three-dimensional environment and the proliferation took place only a few three-dimensional structure of the tube,In the control group and experimental group of 10 micro-carrier into a pipe a few were 45±9 and 9±7,with the difference was significant(P＜0.05).5.In vivo inhibition Egfl7 can inhibit the expression of VEGF-induced angiogenesis.①To compare Mice^Egfl7RNAi+ with Mice^Egfl7RNAi- two nude models the primary tumor microvessel density(MVD),There is a significant difference MVD values between the two groups(29±2 vs 37±3)(P＜0.05).②Matrigel cultivation of VEGF in nude mice as a bio-induced angiogenesis,the experimental group in the Matrigel neovascularization was less than the control group,experimental and control groups of and, with the difference was significant(P＜0.05).There is a significant difference in Matrigel neovascularization of the total length between the two groups(23634±6421μm vs 32145±4314μm)(P＜0.05).Conclusion:1.Egfl7 may be involved in angiogenesis of LSCC invasion and metastasis; 2.Egfl7 could be as a key anti-angiogenesis target for LSCC gene therapy.