| Objective:CCR9(chemokine receptor 9)was related with various kinds of malignant tumors and participated in cancer occurrence and development.This study was designed to observe and study the expression of not only CCR9 in non-small cell lung cancer(NSCLC)but VEGF-C,VEGF-D,MMP2,MMP9,TIMP1 and TIMP2 in two kinds of NSCLC cells,and analyze their association with clinicopathologic feature of NSCLC as well as to study how does CCL25/CCR9 axis have effects on cell migration and invasion in NSCLC,and to explore the relationship between CCL25/CCR9 axis and the expression of VEGF-C,VEGF-D,MMP2,MMP9,TIMP 1 and TIMP2,providing the reference data for the research on the metastasis of NSCLC and theoretical basis for clinical targeted therapy of NSCLC.Methods:(1).60 casses of NSCLC specimens and corresponding normal lung specimens were collected from The People'S Hospital Of Guang Xi Zhuang Autonomous Region.The expression of CCR9,VEGF,MMP2,MMP9 in 60 NSCLC patients and the corresponding paraneoplastic normal lung tissues were detected by immunohist-ochemistry(IHC),and the level of CCR9 expression was analyzed whether it was associated with clinicopathologic feature such as gender,age,histopathological classification,TNM stage,lymph node metastasis in NSCLC.(2).the NSCLC cells(A549 and SK-MES-1)which are treated by special drugs and divided into 3 groups(blank control group,CCL25 group,CCL25+anti-CCR9 group)were used to analyse the effection of the NSCLC cells' migration and invasion through the method of transwell.(3).the aim of usage of the Western-Blot?RT-PCR techniques was to analyse the expression of VEGF-C,VEGF-D,MMP2,MMP9,TIMP1,TIMP2 at the level of protein and genetic transcription,and then to study how dose CCL25/CCR9 axis have effects on cell migration,invasion and metastasis in NSCLC.Results:1.The positive expression rate of CCR9,VEGF,MMP2 and MMP9 in lung cancer was significantly higher than that in the corresponding paraneoplastic normal lung tissues(65.00%vs.25.00%,51.67%vs.16.67%,60.00%vs.18.33%,55.00%vs.10.00%,P<0.05).The positive expression rate of CCR9 in lung adenocarcinoma was higher than that in lung squamous cell carcinoma(77.14%vs.48.00%,P<0.05).The positive expression rate of CCR9,VEGF,MMP2,MMP9 in cases with lymph node metastasis was higher than that in cases without lymph node metastasis(77.78%vs.26.67%,68.89%vs.33.33%,66.67%vs.20.00%,P<0.05).The positive expression rate of CCR9 in cases with VEGF or MMP2 or MMP9 positive expression was higher than that in cases without VEGF or MMP2 or MMP9 positive expression(80.65%vs.48.28%,75.00%vs.50.00%,87.88%vs.37.04%,P<0.05).for the patients with lymph node metastasis,the expression of CCR9 is positively associated with the expression of VEGF,MMP2 and MMP9(r=0.339,P<0.05;r=0.257,P<0.05;r=0.530,P<0.05).2.The results of RT-PCR showed that the mRNA expression of VEGF-C,VEGF-D,MMP2,MMP-9,TIMP-1,TIMP-2 in NSCLC cell lines.Real-time PCR analysis showed CCL25 can increase the expression of respective factors in both A549 and SK-MES-1 cell lines but the expression of MMP-9 and TIMP-1 in the SK-MES-1 cell lines(P*<0.05);The promotion can be weakened by the CCR9 antibody(P**<0.05).3.The results of Western-Blot techniques showed that the protein expression of VEGF-C,VEGF-D,MMP2,MMP-9,TIMP-1,TIMP-2 in NSCLC cell lines.Western-Blot technique showed CCL25 can increase the expression of VEGF-C,VEGF-D,MMP2,TIMP-2 factors in both A549 and SK-MES-1 cell lines(P*<0.05);the promotion can be weakened by the CCR9 antibody(P**<0.05).We could not detect the expression of MMP-9 and find out the different expression of TIMP-1 between the controls and the CCL25 treated group or CCL25-anti-CCR9 group in the SK-MES-1 cells.Conversely,for A549 cells,CCL25 can improve the expression of MMP-9 and TIMP-1(P*<0.05)and CCR9 antibody can also abrogate the improvement(P**<0.05).4.The results of Transwell invasion and migration assay showed that the number of cells migrated was significantly altered after the CCL25 treatment(P*<0.05)and the antibody of CCR9 can abrogate the promotion(P**<0.05);The number of cells invasion was significantly increased after the CCL25 treatment(P*<0.05)and the antibody of CCR9 can decrease the promotion(P**<0.05).Conclusion:1.The expression of CCR9,VEGF,MMP2 and MMP9 was detected in the NSCLC tissues and the adjacent normal tissues and the expression in NSCLC tissues was higher.The CCR9 expression was significantly associated with histological types,clinic pathological grading,lymphatic status,which indicate the high invasion ability and poor prognosis.2.Our Western-Blot,RT-PCR and other techniques indicated that the CCL25/CCR9 biological axis can promote the lymphatic metastasis in NSCLC through regulating the expression of VEGF-C,VEGF-D,MMP2,MMP9,TIMP1 and TIMP2. |
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