| BackgroudLung adenocarcinoma is one of the most common malignant carcinomas that seriously endanger human health. As high degree of malignancy, it is prone to recurrence and metastasis. As the treatments of recurrence and metastatic lung adenocarcinoma are less efficacious, the mortality of this cancer is very high. So, researchers and clinicians are constantly trying to find the changes of lung adenocarcinoma invasion and metastasis-related genes to explore the molecular mechanism of the recurrence and metastasis of lung adenocarcinoma, and to conquer it completely.Neural precursor cell expressed developmentally down-regulated9（NEDD9） is an invasion and metastasis-related gene and belongs to CAS （Crk-associated substrate） family. Similar to other members of the CAS family, NEDD9has been proven to be highly expressed in a variety of metastatic tumors. The abundance of NEDD9varies significantly in different cell types and tissues, and under different growth conditions. In normal human tissues, highest levels of NEDD9mRNA and protein are found in the lung and kidney, in tissues rich in immature lymphoid cells, and in the fetal brain （although downregulated in adult brain）. NEDD9is abundant in tumor cell lines derived from epithelial lineages, such as lymphoma cell lines, glioblastomas and melanoma cell lines. However, there is little information on the expression of NEDD9in lung adenocarcinoma cells, and the relationship between NEDD9expression and the clinicopathological features of lung adenocarcinoma requires further investigation.ObjectiveTo detect the expressions of NEDD9in lung adenocarcinoma and adjacent noncancerous tissues, analyse and evaluate the relations and clinical significances of overexpression of NEDD9and the clinical pathological features of lung adenocarcinoma. To investigate the effect of silencing on the expression of NEDD9on the growth, apoptosis, migration and invasion of lung adenocarcinoma cells, investigate the relationships between overexpression of NEDD9and the biological behaviors of lung adenocarcinoma cells and study the underlying mechanism of NEDD9involving in the invasion and metastasis of lung adenocarcinoma by RNA interference technique. The effect of NEDD9on the growth of xenograft tumor in vivo was investigated using xenograft transplantation model. This study is divided into the following four parts:Part I Expression and Significant of NEDD9in lung adenocarcinomaMethods（1）10fresh lung adenocarcinoma and adjacent noncancerous tissues were taken by the Henan Tumor Hospital Tissue Bank. Fresh surgical lung adenocarcinoma and adjacent noncancerous tissues were obtained from26patients who underwent surgery in the Department of Thoracal Surgery in The First Affiliated Hospital of Zhengzhou University.（2） The expression and localization of NEDD9were investigated by immunohistochemical staining in36formalin-fixed and paraffin-embedded （FFPE） lung adencarcinoma tissues with that of surrounding nonneoplastic tissues. The scores of36lung adenocarcinoma cancer tissues were evaluated. NEDD9mRNA levels were quantitatively analyzed by fluorescent quantitative reverse transcription polymerase chain reaction （qRT-PCR） in fresh lung adencarcinoma tissues and matched nonneoplastic lung parenchymas to analyze the correlation between NEDD9expression and clinicopathological.（3） NEDD9mRNA expression levels were quantitated by qRT-PCR in the three lung adenocarcinoma cell lines A549,95D and SPC-A-1with different invasive potential.Results（1） From the result of immunohistochemistry, I observed high levels of positive NEDD9staining localizd in the nucleus and/or cytoplasm of lung adenocarcinoma. The immunostaining scores showed statistically significant difference between lung adenocarcinoma and adjacent noncancerous tissues （P＜0.01）. The immunostaining scores also have statistical significance between the metastatic and nonmetastatic lung adenocarcinomas （P<0.01）.（2） qRT-PCR showed the expression of NEDD9mRNA in lung adenocarcinoma tissues is higer than the adjacent noncancerous tissues, overexpression of NEDD9mRNA correlated positively with lymph node metastasis, TNM stage and differentiation in the36cases of lung adenocarcinomas （All P<0.05）.（3） I examined the mRNA expression of NEDD9of three lung adenocarcinomas cell lines （A549,95D and SPC-A-1） by qRT-PCR. The results showed NEDD9was overexpressed in the three lung adenocarcinomas cell lines, the highest expression of NEDD9was detected in hyper-invasive potential cell line （A549）.SummariesOur results indicated that the overexpression of NEDD9may be involved in the development of lung adenocacinoma, the process of invasion and metastasis. Part Ⅱ Construction and characterization of the NEDD9specific shRNA eukaryotic fluorescence expression expression vectorMethods（1） Three NEDD9-specific siRNA sequences and one unrelated control sequence were designed according to NEDD9gene （NM182966） in GenBank, using RNAi Designer Software of Clontech Corporation.（2） The eukaryotic fluorescence expression vector pRNAT-CMV3.2was used to construct NEDD9-specific siRNA recombinant eukaryotic expression vectors: the above sequences were annealed under certain conditions, and then the annealed oligos were cloned into the plasmid pRNAT-CMV3.2/neo by T4DNA ligase. The three pRNAT-CMV3.2-NEDD9-siRNAs and the negative pRNAT-CMV3.2-siNC recombinant plasmids were confirmed by DNA sequencing.（3） The recombinant plasmids were transfected into A549cells according to the manufacturer’s instruction, the expression of NEDD9were exa mined by qRT-PCR and western blot analysis to select the most effieient NEDD9-specific siRNA sequence for further study.Results（1） NEDD9-specific shRNA vectors were constructed successfully.（2） Western blot and qRT-PCR analysis indicated that the interventional groups had low levels of NEDD9expression while the three control groups had high levels of NEDD9expression （P＜0.05） after recombinant plasmid transfected into A549cell.（3） The most effective interference sequence of NEDD9gene （NM182966） was GTGTCCTATTTCTTAGTGA （648-666）.SummariesNEDD9-specific siRNA recombinant eukaryotic expression vectors were constructed successfully, it can significantly inhibite the expression of NEDD9in lung adenocacinoma A549cells. Part III Effects of silencing the expression of NEDD9on lung adenocarcinoma A549cells biological behaviourMethods（1） Our previous study showed that small interfering RNA （siRNA） downregulated the NEDD9expression in lung adenocarcinoma A549cells. To further explore the molecular mechanisms after NEDD9gene knockoff, I established stable transgenic A549lung adenocarcinoma cell lines through the lentivirus-mediated short hairpin RNA method packaging siN648and control sequence.（2） The inhibition of NEDD9expression was examind by qRT-PCR and western blot in three groups. Bcl-2, Bax, TIMP1, MMP9and Cylin E were detected by western blot（3） The proliferation of A549cells in three groups was measured by soft agar colony formation assay to investigate the effect of NEDD9.（4） The cell cycle and apoptosis of A549cells in three groups was measured by FCM analysis to investigate the effect of NEDD9.（5） The migration and invasion of A549cells in three groups was measured by wound-healing and transwell assays to investigate the effect of NEDD9.Results（1） The stable transgenic cell lines were established. Compared with A549and A549-NC cells, the expressions of NEDD9mRNA and protein were down-regulated in siRNA group.（2） Compared with A549and A549-NC cells, the expressions of MMP9、 Bcl-2and Cyclin E in A549-siRNA cells were decreased significantly, the expressions of TIMP1and Bax in A549-siRNA cells were increased.（3） Compared with A549and A549-NC cells, the proliferation of A549-siRNA cells was suppressed obviously, and the number of colony formation was decreased significantly （F=11.517, P=0.009）.（4） Compared with A549and A549-NC cells, the percentage of cells at S phase of A549-siRNA was decreased obviously （P＜0.01）. Compared with A549and A549-NC cells, the apoptosis rate of A549-siRNA was increased obviously （F=1433.276, P=0.000）.（5） Compared with A549and A549-NC cells, the scratch of A549-siRNA healed more slowly after cultivation48h without FBS; Compared with A549and A549-NC cells, the number of A549-siRNA cell on the polycarbonate membrane of transwell chamber was decreased significantly （F=280.153, P=0.000）.Summaries（1） Downregulation of the expression of NEDD9gene by siRNA could obviously suppress the proliferation, migration and invasion of A549cells, but obviously induce the apoptosis of A549cells.（2） Overexpression of NEDD9might implicate in the invasion and metastasis of lung adenocarcinoma via promoting the expression of MMP9and Cyclin E, decreasing the expression of TIMP1, and inhibiting apoptosis.Part IV Effect of silencing the expression of NEDD9on the growth of xenograft tumor in nude miceMethods（1）18Balb/c male nude mice were randomly divided into control group, scrambled siRNA group, NEDD9-siRNA group. Tumor was implanted by subcutaneous injeetion of5×106cells/mouse in100μl. When tumor nodules were growed, all mices were executed in5weeks after the injection, collected the tumor which to be weighed and then caleulated the tumor volume according to the formula.（2） The cell morphology of xenograft tumors was observed by HE staining, the expression of NEDD9and apoptosis were detected by Immunohistochemistry and TUNEL methods.Results（1） The average tumor weight of A549-siRNA group （0.13±0.06）g was signifieantly decreased compared with that of A549group（0.37±0.13）g and A549-NC group （0.35±0.10）g after five weeks（F=9.745, P=0.002）, while there was no statistieally significance between A549group and A549-NC group（P＞0.05）. The volumes of xenograft tumors of A549-siRNA cells after injection35days were obviously smaller than A549and A549-NC group （F=10.149, P=0.002）.（2） HE staining showed the large tumor cell nuclei, irregular split image, clear nucleoliand fibrous septa in the two control groups, but the large number of tumor necrosis can be observed in A549-siRNA group, necrosis of the tumor cells were replaced by fibrous tissue.（3） Compared with A549and A549-NC cells, the apoptosis of xenograft tumors in A549-siRNA group was increased obviously （F=147.948, P=0.000）.（4） Compared with A549and A549-NC cells, the immunohistochemical staining score of xenograft tumors in A549-siRNA group was significant decreased （F=3.981,P=0.041）.SummariesSilencing on the expression of NEDD9can inhibit the growth of the A549xenografts in nude mice, and promote apoptosis of tumor cells.Conclusions（1） Overexpression of NEDD9was closely related with lung adenocarcinoma, which involves in the malignant transformation of lung adenocarcinoma cells.（2） Overexpression of NEDD9involve in the invasion and metastasis of lung adenocarcinoma via promoting the expression of MMP9and Cyclin E, decreasing the expression of TIMP1, inhibiting apoptosis.（3） Silencing on the expression of NEDD9can inhibit the growth of the A549xenografts in nude mice, and promote apoptosis of tumor cells.（4） NEDD9could serve as a potential gene target for therapy of lung cancer. |
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