| Objective:This study aims to investigate the correlation of expression level of MACC1, c-Met and microvessel density (MVD) combining with clinicopathological features in non-small cell lung cancer (NSCLC), and furtherly discuss the effect and molecule mechanism of MACC1 promoting angiogenesis in NSCLC in vivo and vitro.Methods:1. Study on expression and correlation of MACC1, c-Met and MVD in NSCLC, to explore the relationship and their clinical significance:Protein of MACC1 and c-Met in 50 NSCLC tissues and corresponding adjacent normal tissues were detected by immunohistochemical method, and then analyzed the relationship with clinical pathological characteristics. Additionally, microvessel density (MVD) labeled by CD34 were detected in NSCLC tissues and analyzed the correlation with expression of MACC1 and c-Met.2. Determination of MACC1 mRNA level in NSCLC cell lines:The relative expression level of MACC 1 mRNA was detected by RT-qPCR method in five different NSCLC cell lines.3. The effect and mechanism of silencing MACC1 on angiogenesis in NSCLC:XWLC-05 cell line highly expressed MACC1 was selected to be transient transfected MACC1-siRNA, which was harvested by chemical synthesis. Following confirmed silence of MACC1, the supernatant of cultured XWLC-05 cells were gathered respectively at Oh,24h,48 and 72h after MACC1-siRNA transfection. Secretion level of angiogenesis related factors VEGF, bFGF and IL8 were detected by ELISA in above-mentioned supernatants. In addition, chick embryo chorioallantoic membrane (CAM) experiments were performed to test angiogenesis in several groups. Total proteins in different groups were isolated to detect the relative expression of key molecules in downstream signal pathway of MACC1, known as Met, p-MEK/MEK. p-Erk/Erk and p-Akt/Akt.4. The effect and mechanism of MACC 1 over-expression on angiogenesis in NSCLC:Human MACC1 sequence was harvested from Genebank and amplified by using cDNA cloning vector (Thermo Scientific, USA). Following restricted by double enzyme cleavage, MACC1 sequence was cloned into expression plasmid PIRES2-EGFP with green fluorescent protein report gene. After that, confirmed PIRES2-EGFP-MACC1 expression vector was instantaneously transfected into SPC-A1 cells, in which MACC1 low-expressed. As above-mentioned, angiogenesis related factors VEGF, bFGF and IL8 were detected by ELISA in the cultured cell supernatants which were collected from transfected SPC-A1 cells respectively at Oh,24h,48 and 72h. Status of angiogenesis were tested by chick embryo chorioallantoic membrane (CAM) experiments and the relative expression of key molecules in downstream signal pathway of MACC1, known as Met, p-MEK/MEK、p-Erk/Erk and p-Akt/Akt, were detected in different groups.Results:1. Correlation analysis and clinical significance of expression of MACC 1, c-Met and MVD:The positive expression of MACC 1, c-Met between NSCLC and adjacent tissues have statistical significant with P value<0.05. In addition, positive expression of MACC1 and c-Met were equal related with clinical stage and lymph node migration. Correlative analysis showed a positive correlation in NSCLC tissues. MVD values in NSCLC patients with positive expression of MACC 1 and c-Met were significant difference with that of MACC 1 and c-Met negative patients (P<0.05). Correlation analysis showed that expression of MACC1, c-Met and MVD value exist significant correlation (P<0.05).2. Expression level of MACC 1 mRNA using RT-qPCR was highest in XuanWei lung cancer celllines XWLC-05 and lowest in lung adenocarcinoma cell lines SPC-A1.3. All the three MACC1-siRNAs could efficiently inhibit the expression of MACC1. The best one was selected for further experiments. In CAM experiment, the area of neovascularization was much lower in MACC1-siRNA group than other groups. The expression of Met, p-MEK and p-ERK significantly decreased and MEK, ERK, AKT and p-AKT had no change by Western-blot. In addition, the expression level of VEGF, bFGF and IL8 were significantly suppressed in 48h and 72h in MACC1-siRNA group by ELISA.4. The recombinant plasmid PIRES2-EGFP-MACC1 structure and gene sequence was successfully confirmed by sequencing. Green fluorescence could be seen clearly in SPC-A1 cells after transfection the recombinant plasmid PIRES2-EGFP-MACC1. MACC1 mRNA was stably expressed by PIRES2-EGFP-MACC1 vector in the transfected SPC-A1 cells. CAM experiments showed that vascular branches and area of neovascularization in transfected PIRES2-EGFP-MACC1 group were much higher than PIRES2-EGFP (Vector) group and blank control group (P<0.05). In PIRES2-EGFP-MACC1 transfected group, Western-blot showed that, expression level of Met increased significantly, p-MEK and p-ERK which were key factors of MAPK signal pathway were up-regulated slightly (P<0.05). However, there was no significant change of MEK and ERK’s total protein expression. Compared to that of the blank control group and empty vector group, the secretion level of VEGF, bFGF and IL8 were consistent within 24h after transfection. However, expression of VEGF, bFGF and IL8 protein in PIRES2-EGFP-MACC1 group were significantly higher than that of the blank control group and empty vector group respectively at 48h and 72h after transfection.Conclusions:1. MACC1 and c-Met may play important roles during the process of generation and migration in NSCLC, and can be used as an assessment and prognosis-estimation reference for NSCLC patients combining with clinical stage.2. Expression level of MACC1 and c-Met in lung tissues of NSCLC exist correlation with MVD value, which suggest that expression of MACC1 and c-Met may be related with angiogenesis in NSCLC.3. MACC1 could regulate the ability of angiogenesis induced by NSCLC cells in CAM experiment, which may be associated with regulation of HGF/c-Met and MEK/ERK signal pathway and angiogenesis related factors VEGF, bFGF and IL8. |
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