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The Expression of CD147 in Squamous Cell Carcinoma、Adenocarcinoma of Lung and Human Lung Adenocarcinoma Cell Line A2 and the Association with Biological Behaviors of Cell Line A2

Author: LiBo
Tutor: LiYu
School: China Medical University
Course: Surgery
Keywords: CD147 MMP-2 squamous cell carcinoma of lung adenocarcinoma of lung RNAi
CLC: R734.2
Type: PhD thesis
Year: 2007
Downloads: 127
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Abstract


BackgroundLung cancer is a malignant tumor with high incidence rate and death rate and its death rate increases by 118.0%of city malignant tumors in recent 20 years.Now general treatments involving operation、radiotherapy、chemotherapy and immunotherapy have unsatisfactory therapeutic efficacy.Invasion and metastasis of lung lung cancer is the main block of clinical treatment and one of the main reasons of death of lung cancer pateints.During the invasion and metastasis of tumors,basement and extracellular matrix was degraded by proteinases of which matrix metalloproteinases（MMPs）were most important because of wide substrates.MMPs in most malignant tumors were secreted from matrix cell,especial fibroblast.Fibroblst general secreted very little MMPs but can secrete much MMPs when there were tumor cells and degraded extrcellular matrix to provide a convinent condition fortumor’s invasion and metastasis.CD 147 is a stimulatory factor which can make fibroblasts secrete much MMPs and becomes one of research foucus.CD147 was separated from human lung cancer cell LX-1 by Biswas and is a transemembrane glycoprotein along,to immunoglobulin super family which locates on the surface of tumor cell.CD147 is also named extracellular matrix metalloproteinase inducer（EMMPRIN）、basic immunoglobulin（basigin）and M6;and is named HT7、neutothelin、5A11 in chick;and is named OX-47、CE9 in big mouse and Basigin、gp42 in small mouse.CD147 is unexpressed or very little expressed in normal cell while is high expressed in tumors especially in malignant tumors and CD147 plays an important role in tumor’s invasion and metastasis and angiogenesis.Now CD147 has been cloned in lung cancer cell、hepacarcinoma cells.RNA interference（RNAi）is a progress of a double-stranded RNA molecule close a certain gene expression in mRNA level and is an especial post-transcriptional gene silencing（PTGS）.A small interfering RNA or shorting interfering RNA（siRNA）is an important effective molecule by which RNA interference plays an important role.We study the expression of CD147 in squamous cell carcinoma and adenocarcinoma of lung and the association with MMP-2.We also study the association of the expression of CD147 with biological behaviors of cell line A2Materials and Methods1.Materials55 NSCLC patients for operation from 3 to 12,2005 were seleted,34 of which were men and 21 were women.31 cases were squamous and 24 were adenocarcinoma. 28 patients were in stageⅠ;9,in stageⅡ,and 18,in stageⅢ.Tumor tissues and normal tissues next to them were resected,which were put into liquid nitrogen,and then were put into refrigerator of-70℃.2.Methods（1）ImmunohistochemistrySamples were put into 3%H2O2 and antigens were repaired in microwave oven by 10 minutes in 95-96℃.All samples were blocked in 10%goat serum for 20 minutes. First and secondary antibodies were put onto the slices consecutively,the and DAB were put on them for color display.（2）RT-PCRA little cancer and normal tissues were cut.Total RNA were distilled and PCR was conducted.The reversal transcription system was as follows:primer:CD147（229bp）:5’-GATACTCCTCACCTGCTCCTTG-3’ 5’-CGACTTCACAGCCTTCACTCT-3’β-actin（498bp）:5’-GTGGGGCGCCCCAGGCACCA-3’ 5’-CTCCTTAATGTCACGCACGATTTC-3’The product was for agar gel electrophoresis.The relative quantity of each mRNA was determined by the ratio ofβ-actin mRNA by Bandleader software.（3）Cell cultureUsing the 10%RPMI 1640 medium of 56℃inactivated foetus cattle’s serum, cultured in the 37℃of saturation degree of wetness,5%of the CO2 incubator,the cell grew as single storey sticks to the wall,fetches growth period of logarithm cells for the experiment.（4）TransfectionThe cells（1×105）were cultured in 12 plastic flasks,with RPMI 1640 medium, without 10%fetal calf serum,at 37℃in a humidified atmosphere（5%CO2 and 95% air）for 24 hours.Then,0.8μg siRNA,98μL RPMI1640 and 2μL Lipofectarnine were added in test group,respectively.After transfection,followed by the condition:5%CO2 and 95%air for 4 hours,,new RPMI1640 with 10%fetal calf serum was added in and cultured continuely.The four groups were A2 group、A2-Lipofectamine group、negative group and test group.（5）MTTAfter transfection,the liquid containing single cell of each test groups was regulated by PBS into 2×105/mL cell/ml.The cells（2×105）were cultured in 96 plastic flasks respectively,with RPMI 1640 medium containing 10%fetal calf serum,2g/L NaHCO2 at 37℃in a humidified atmosphere（5%CO2 and 95%air）for 24 hours.At the time of 24h,48h,72h,new RPMI1640（80μL）and MTT（20μL,5mg/mL）were added in,respectively.Cultured 4h and then added in DMSO（100μL）,the each groups were tested（OD,490nm）.（6）Statistical analysisThe data were analyzed by the SPSS 11.5 software.Chi-squared test and Fisher test were used in datas of quantity.One-way ANOVA test was used in data of quality.Cox regression was used to analyse prognosis.Results1.The expression of CD 147 in squamous cell carcinoma、adenocarcinoma of lung CD147 is unexpressed in normal lung tissue（0/3）and the positive expression rate is 66.7%（16/24）in adenocarcinoma of lung and 58.1%（18/31）in squamous cell carcinoma. 2.CD147 is unexpressed in normal lung tissue and high expressed in lung carcinoma.The amount of CD147 is 1.0907±0.2159.3.Compared with control group,both the expression of mRNA of CD147 in A2 cells were obviously decreased in siRNA transfected cells,respectively.4.The cell growth and viability of CD147siRNA transfected group were significantly inhibited（P＜0.05）.Conclusion1.CD147 is unexpressed in normal lung tissue and high expressed in squamous cell carcinoma、adenocarcinoma of lung.2.The expression of CD147 is association with lymph nodes metastasis and TNM stage and CD147 maybe play an important role in invasion and metastasis of lung carcinoma.3.Interference with siRNA against the specific gene can effectively inhibit its expression in cells.4.Inhibiting expression of CD 147 maybe inhibit the proliferation of cell line A2.

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