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part1. Expression of Long Non-coding RNA uc001kfo in Lung AdenocarcinomaObjective:To detect the expression level of long non-coding RNA (lncRNA) uc001kfo in lung adenocarcinoma, and explore the relationship between lncRNA uc001kfo and the clinical signification of lung adenocarcinoma.Methods:Samples of lung adenocarcinoma and the paracarcinoma tissues were collected from37clinical patients in cardiothoracic surgery department of Zhongnan hospital of wuhan university from September2011to June2012. There were25men and12women between43and79years old, and the average age was (61.4±10.2) years. Among them, patients of20cases had a history of smoking, the degree of lung adenocarcinoma differentiation of17cases were moderate and above. The relative level of lncRNA uc001kfo expression in lung adenocarcinoma and the paracarcinoma tissues were detected using real-time fluorescence quantify PCR. Then the relationship between the expression level of long non-coding RNA uc001kfo and lung adenocarcinoma were observed by stastical analysis, the clinical factors of patients, including age, sex, smoking history, lung adenocarcinoma differentiation, lymph node metastasis and neoplasm staging were discussed as well.Results:LncRNA uc001kfo is expressed by lung adenocarcinoma and the paracarcinoma tissues, and the expression level in the former is significantly higher than the latter. In26out of37samples, lncRNA uc001kfo expression level in lung adenocarcinoma tissues were at least twice as high as it in the paracarcinoma tissues. In addition, lncRNA uc001kfo expression level increased depend on the degree of tumor differentiation and lymphatic metastasis, but had nothing related to age, sex, smoking history and neoplasm staging.Conclusion:Expression of long non-coding RNA uc001kfo is high in lung adenocarcinoma, and closely related to the degree of tumor differentiation and lymphatic metastasis. Long non-coding RNA uc001kfo may be involved in the occurrence and development of tumors. Part Ⅱ. Screening of specific short interference RNA (siRNA) for long non-coding RNA uc001kfoObjective:To seek out the specific short interference RNA (siRNA) for lncRNA uc001kfo.Methods:We designed and combined4different pairs of siRNAs specific for silencing lncRNA uc001kfo (numbered1to4), which were seperately transfected into lung adenocarcinoma A549cells using reagent Lipofectamine2000and siRNA-mate of different dosage (0.125ul,0.25ul and0.5μl), then fluorescence microscopy was used to detect the efficiency of transfection, and the interference efficiency of4different pairs of siRNA was analysed by real-time fluorescence quantify PCR.Results:After5pmol siRNA transfected into A549cells using reagent Lipofectamine2000, the cell status was well, transfection efficiency was about70%; Number One of the designed4pairs of siRNAs silenced the expression of lncRNA uc001kfo effectively, the disruption rates were greater than70%at48h and72h, which were obviously higher than the other pairs of siRNAs.Conclusion:Number One of the designed4pairs of siRNA can be used to silence the expression of lncRNA uc001kfo of A549cells. Part Ⅲ. Influence of suppressed long non-coding RNA uc001kfo expression on lung adenocarcinoma A549cells in vitro Objective:To explore the role of lncRNA uc001kfo in the proliferation, migration and invasion of lung adenocarcinoma A549cells.Methods:Transfect the specific siRNA for lncRNA uc001kfo (siRNA-uc001kfo) optimised in the previous experiment into lung adenocarcinoma A549cells, divided into3groups:group Mock(without treatment), negative control group(transfected with5pmol non-specific NC-FAM-siRNA) and experimental group (transfected with5pmol siRNA-uc001kfo).(1) at24h,48h,72h and96h after transfecting, the cell proliferation was observed in vitro by CCK-8cell proliferation bioassay;(2) at24h after lncRNA uc001kfo expression was inhibited, the cell movement was detected by Transwell migration assay;(3) Through Transwell erosion assay at24h after transfecting, invasion ability of cells were determined.Results:(1) Using CCK-8cell proliferation bioassay, at24h,48h,72h and96h after transfecting, the values of OD450nm in group Mock were (0.273±0.018),(0.403±0.027),(0.537±0.011),(0.724±0.017), which in negative control group were (0.250±0.005),(0.368±0.024),(0.512±0.042),(0.708±0.031), and experimental group were (0.374±0.020),(0.506±0.030) and (0.684±0.047), there were no significant difference among the groups.(2) In Transwell migration assay, the cell numbers of group Mock, negative control group and experimental group respectively were (172.3±10.4),(163.8±13.2) and (84.4±11.8) per view, the numbers of A549cells through the basement membrane of experimental group was significantly lower than it of negative control group(p<0.05);(3) In Transwell invasion assay, the cell numbers of group Mock, negative control group and experimental group respectively were (118±11.4),(104.8±15.4) and (59.2±10.7) per view, compared with negative control group, the numbers of A549cells passed through extracellular cell matrix(ECM) of experimental group was significantly lower (p<0.05).Conclusion:lncRNA uc001kfois involved in the regulation of A549cell migration and invasion, while does not affect its proliferation.
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