| Objective:To investigate the expression of TEM8 and its biological functions and molecular mechanisms in NSCLC.Methods:1.The expression of TEM8 and its clinical significance in NSCLCImmunohistochemical staining was used to compare the expression of TEM8 in tumor and adjacent tissues of 67 NSCLC patients.The relationship between TEM8 expression and clinical characteristics and prognosis was analyzed.The expression of TEM8 in cancer and para cancerous tissues of 21 patients with NSCLC was investigated by qRT-PCR and western blot.Micro-vascular density was identified by IHC staining of CD34 to observe the relationship between TEM8 expression and tumor angiogenesis.Uni-variate survival analysis was used to analyze the relationship between survival and TEM8 expression.Cox regression model was applied to analyze TEM8 expression and prognostic factors in NSCLC patients.2.Studies on the relationship between TEM8 expression and NSCLC cell biological behavior and its mechanism in vitroLentivirus vector was used to construct and screen XWLC-05 cells that silencing or over-expressing TEM8.Proliferation rate of lung cancer cells was detected by MTT,apoptosis and cell cycle changes were inspected by flow cytometry,cell morphology was observed by scanning electron microscope,scratch assay and transwell experiment were performed to discuss migration and invasion ability of cancer cells.qRT-PCR and western blot were conducted to observe ERK(or p-ERK1/2),BCL-2,cyclinD1 expression and the in vitro action mechanism of TEM8.3.Studies on the effects of silencing TEM8 expression on XWLC-05 lung cancer xenografts in nude mice and its mechanism in vivoXWLC-05 cells,which stably silent TEM8,were inoculated subcutaneously in nude mice.General situation,tumor formation rate and tumor growth were observed.All nude mice were sacrificed on the 28th day after successful inoculation.The size and weight of isolated tumors were measured and tumor suppress rate was calculated.The expression of TEM8 and CD34 was detected by HE,IHC and IF staining.CD34 positive vessels were counted,and the relationship between TEM8 expression and tumor angiogenesis was discussed.qRT-PCR and western blot were used to detect the expression of ERK(or p-ERK1/2),BCL-2 and cyclinDl so as to preliminarily explore the mechanism of TEM8 in vivo.Results:1.The expression of TEM8 and its clinical significance in NSCLCThe positive expression rate of TEM8 in NSCLC cancer tissues was 52.23%(35/67),and that in adjacent tissues was 17.91%(12/67).There was a significant difference between them(P<0.05).The expression of TEM8 was significantly correlated with degree of tumor differentiation,clinical stage,pleura invasion and distant metastasis after operation in NSCLC patients(P<0.05).Compared with TEM8 negative patients,CD34 positive micro-vascular density increased significantly in TEM8 positive patients,the difference was statistically significant(P<0.05).The results of single factor survival analysis showed that the survival time of NSCLC patients was related to the level of TEM8 protein expression,clinical stage and distant metastasis(P<0.05).The results of Cox regression analysis indicated that TEM8 protein expression level,clinical stage and distant metastasis were prognostic factors of NSCLC patients(P<0.05).2.Studies on the relationship between TEM8 expression and NSCLC cell biological behavior and its mechanism in vitroAfter silencing TEM8 expression,compared with blank and negative control group,MTT assay indicated that cell proliferation was significantly inhibited(P<0.01);flow cytometry apoptosis detection suggest that apoptosis rate increased significantly(P<0.01);XWLC-05 cells showed morphological changes of apoptosis under scanning electron microscopy;cell cycle analysis suggest that cell cycle arrest in G1 phase(P<0.05);scratch assay indicated that cell migration was significantly inhibited(P<0.01);Transwell experiment showed that the cell invasion ability was significantly inhibited(P<0.01);qRT-PCR and Western blot results showed ERK(or p-ERK1/2),BCL-2 and cyclinDl expression were significantly reduced(P<0.01).After over-expression of TEM8,compared with blank and negative control group,MTT assay showed significant cell proliferation enhancement(P<0.01);Flow cytometry showed no significant change of apoptosis rate(P>0.05);cell cycle analysis showed more G2 phase cells,suggesting that cells acquired stronger proliferative activity(P<0.05);scratch assay indicated that cell migration was significantly increased(P<0.01);Transwell experiment showed that the cell invasion ability was enhanced significantly(P<0.01);qRT-PCR and western blot indicated ERK(or p-ERKl/2),BCL-2 and cyclinDl expression were significantly increased(P<0.01).3.Studies on the effects of silencing TEM8 expression on XWLC-05 lung cancer xenografts in nude mice and its mechanism in vivoVisible tumors were observed as early as 7 days after transplantation,with a tumor vaccination success rate of 100%.All nude mice survived at the experimental endpoint.During observation period,the body weight of each group of mice increased at different degree,but there were no significant difference among all groups(P>0.05).Growth of xenograft tumors were significantly inhibited after silencing TEM8 expression with a tumor inhibition rate of 57.82%,compared with either blank or negative control group(P<0.05).The isolated tumor weight was(0.29±0.04)g in experimental group,which was significantly lower than that of negative control(0.58±0.08)g(P<0.01)and blank control(0.63±0.17)g(P<0.01),whilst with no significant difference between the latter two groups(P>0.05).Tumor volume of nude mice inoculated with TEM8 silenced lung cancer cells(436.64±102.39)mm3 were also significantly less than those of negative control(944.18±178.52)mm3(P<0.01)and blank control group(988.68±128.43)mm3(P<0.01).No significant difference of tumor volume was observed between blank and negative control group(P>0.05).IHC and IF staining showed TEM8 protein was mainly expressed in tumor cell membrane,while the expression of CD34 was mainly in vascular intima of tumor tissues.The expression of CD34 in experimental group was significantly lower than that in blank and negative control group(P<0.05).After silencing TEM8,the micro-vascular density of tumor bearing tissues decreased significantly,and the difference was significant compared with that of the blank and negative control group(P<0.05).qRT-PCR and western blot results showed that compared with control group,expression of ERK(or p-ERK1/2),BCL-2 and cyclinDl were significantly down regulated(P<0.01).Conclusions:1.TEM8 has an abnormal expression on both transcription and translation levels in cancer tissues of NSCLC patients.2.The expression level of TEM8 in cancer tissues,combined with clinical stage and distant metastasis can be used as a reference index for evaluating prognosis of NSCLC patients,which has certain clinical guiding significance.3.TEM8 may regulate the expression of cyclinDl BCL-2 via ERK/BCL-2 signaling pathway,and then modify proliferation and apoptosis of NSCLC cells.4.TEM8 is associated with tumor angiogenesis in NSCLC,and may become target of anti-angiogenesis therapy for NSCLC. |
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