| BackgroundAccording to statistics, among the most major cities of our country and developed countries, lung cancer has been in the first site of morbidity and mortality in male malignant tumors, and been in the first site of mortality and second site of morbidity in female malignant tumors already. Nowadays, the mortality and morbidity of lung cancer are still show a rising trend. Among them, non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer. As discovered in studies on the molecular epidemiology of lung cancer, tobacco or industrial emissions and other environmental carcinogens can be used as the initiation factor of bronchial and alveolar epithelial cells to activite proto-oncogenes such as k-ras (called oncogenes) and inactivite tumor suppressor genes such as p53 and RBI. Thus, the proliferation of bronchial and alveolar epithelial become abnormal, which results in disorders of cell proliferation and balance of apoptosis. All these ultimately lead to uncontrolled growth of cell and tumors are formed. Abnormal proliferation of cells is an important factor leading to lung cancer, but its definite mechanisms have not yet been fully elucidated. Therefore, to further discover proliferation-related regulatory factors of lung cancer cells will help us to clarify the molecular mechanisms of lung cancer occurrence and development.EGFL7 (also known as VE-statin, the class of epidermal growth factor domain -7), is an endothelial-specific gene found by V.Mattot in 2006, it expresses in the endothelial cells and endothelial progenitor cells in the process of embryonic development. It is the key elements during the development of blood vessel lumen, reducing its expression can directly lead to stunting of blood vessel lumen. According the studies of Sharon.D et al, expression of EGFL7 in endothelial cells increases very quickly with hypoxia-induced angiogenesis, but this phenomenon do not persist in more mature tumors blood vessels, this suggests that it only plays a role in the early stages of angiogenesis. Moreover, according to studies of Parker LH, supporting the notion that EGFL7 is up-regulated in actively growing/remodeling blood vessels, they found strong expression in the vascular endothelia of many human tumors, including:lung adenocarcinoma, clear cell renal cell carcinoma, prostate and ovarian carcinoma, gastric carcinoma, osteosarcoma, and chondrosarcoma. Furthermore, EGFL7mRNA is barely detectable in the nearby non-tumor vasculature from the same patients. Recent reports have showed that EGFL7 is related to angiogenesis of embryo and tumor, but there are still no clear reports about its relationship with the proliferation of lung cancer cell.miRNAs are a class of small non-coding RNAs of 18-24 nucleotides with a high degree of evolutionary conservation, and make up the RISC (RNA-induced silencing complex) with other proteins together. Once RISC combines with mRNA of target gene, it initiates to incise mRNA or block the translation of mRNA, so as to cause the degradation of mRNA or inhibition of translation. It is widely involved in animal and plant growth and development, cell differentiation, proliferation and apoptosis, hormone secretion, tumor formation and other processes. Bioinformatics prediction results show that each miRNA can control hundreds of target genes, almost each one of these miRNA has a potential impact on each genetic pathway, which regulates different biological processes. Recent research results show that, miRNA mutations and abnormal expression are relevant to a variety of human tumor occurrence and development, miRNA can play a role as oncogenes or tumor suppressor genes. It has been demonstrated that miRNA can influence the expression of a variety of important cancer-related genes (eg. miR-17/20 and IL-8, miR-17-92 and p21, miR-181a and Bcl-2, miR-146a and EGFR, miR-182 and RGS17, miR-199A2/214 and Twistl) in tumors like breast cancer, leukemia, glioma, pancreatic cancer, lung cancer, ovarian cancer respectively. There have been many studies showed many miRNA, among which miR-126 is involved, play important roles in development and progress of lung cancer, level of miR-126 is not the same in different periods of lung development of mice and human, and rises up with maturing of lung tissues.To further analyze the influence of miR-126 on expression of EGFL7, we used bioinformatics software (miRanda and Targetscan software) to predict and found that EGFL7 is one of the target genes of miR-126, miR-126 is located in the intron 6 and 7 of EGFL7 gene 3'-UTR, miR-126 is pairing partly with the 3'-UTR of EGFL7 mRNA and so as to affect its transcription after the translation process. Based on the analysis above, we constructed an over-expressing plasmid of miR-126 targeting EGFL7, and transfected it into the culturing non-small cell lung cancer cell line A549 cells to establish the stable transfected strain by G418 selection; and then detected the change of expression of its target genes EGFL7 in transfected strains led by over-expression of miR-126; finally, we detected changes of cell proliferation before and after transfection with in vitro and vivo experiments.Objects1. Detecting the targeting of miR-126 on expression of EGFL72. Detecting the proliferation influences of miR-126 overexpression on A549 cell by experiments in vitro.3. Detecting the tumorgenesis influences of miR-126 overexpression on A549 cell by experiments in vivo.Methods1. Firstly constructing a miR-126 overexpression plasmid and transfect them together with mock plamids into A549 cells to establish two transfectants, after selected by G418, we got two transfectants.2. Detecting the efficiency of the stable transfection by flow cytometry.3. Analyzing the morphology difference between A549 cells and A549/miR-126 cells with light microscope.4. By flow cytometry, real-time fluorescence quantitative PCR and Western blot et al, we detected the expression of EGFL7 in three groups.5. Experiments by cell counting, cell proliferation experiments (including experiments and soft agar colony cloning experiments) were used to observe the impact of over-expressing plasmid on the proliferation of A549 cells.6. Using flow cytometry to test the cell cycle of the three groups before and after transfection, analyzing the difference of the cell cycle between the three groups.7. By subcutaneous injection of transfected cells of three groups before and after transfection to establish lung cancer in vivo transplantation model in nude mice, we observed and analyzed the impact of overexpression plasmid on tumor forming, including tumor growth curves, tumor volume and weight comparison.8. Frozen sections of tumor tissue from three groups were taken and then carried out immunohistochemistry to compare the expression of EGFL7 between three groups before and after transfection.9. Statistical analysis:The results were treated by SPSS13.0 software statistics. The experiment were repeated independently for at least three times, and got a good reproducibility. Three selected chart were a result of repeated experiments. Data was show as mean±standard deviation, analysis of variance between groups used one-way ANOVA method. P<0.05 indicates statistically significant.Results1. Establishing two stable transfectants, including A549/mock cell and A549/miR-126 cell.2. By light microscope, we observed the changes of shape of cells before and after transfection, unlike the polygon of A549 cell and A549/mock cell, the shape of A549/miR-126 cell appears spindle, which suggests that shape of cells transfected with overexpression plasmid are more narrow than other groups.3. With flow cytometry, we observed the efficiency of transfection is up to 90%, compared with A549 cell.4. By real-time fluorescence quantitative PCR, detection of miR-126 and EGFL7 mRNA of the three groups shows that miR-126 and EGFL7 mRNA in A549/miR-126 cells are higher than any of A549 cells and A549/mock cells.5. Using Western blot and flow cytometry, we detected EGFL7 protein in three groups before and after transfection, and found that EGFL7 were high in A549/miR-126 cells, compared with any of the cells from A549 group and A549/mock group,.6. Using cell counting method to map the adherent cell growth curves of cultured cells from three groups, results showed that, compared with any of A549 cells and A549/mock cells, growth rate of A549/miR-126 cells is much slower.7. The result of plate cloning experiments of three groups showed that proliferation clones of A549/miR-126 cells were significantly less than any of the other two groups.8. By soft agar cloning experiments, three groups of cells, we found that proliferation clones of A549/miR-126 cell were significantly less than any of A549 cells and A549/mock cells.9. By flow cytometry on cell cycle analysis, results showed that proliferation index of A549/miR-126 cells in S phase was significantly lower than any of A549 cells and A549/mock cells, suggesting that DNA synthesis of cell in S phase decreased significantly after miR-126 expression(F=60.150, P<0.001).10. Tumor transplantation model in nude mice showed that, comparing with any of the A549 group and A549/mock group, tumor size of A549/miR-126 group were significantly smaller, tumor weight of A549/miR-126 group were lighter, and growth rate of A549/miR-126 group was slower.11. Immunohistochemistry results of tumor tissue from three groups showed that, comparing with any of the A549 group and A549/mock group, EGFL7 level of tumor tissue from A549/miR-126 group was higher.Conclusions1. miR-126 regulate positively the expression of EGFL7 at both mRNA and protein level.2. Overexpression of miR-126 may inhibit the proliferation of A549 cell in vitro.3. Overexpression of miR-126 may inhibit the tumorgenesis of A549 cell in vivo. |
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