| BackgroundLung cancer has the highest rates of Morbidity and mortality of all cancers, and theincidence of pulmonary malignant cancer increased significantly in our country. The majortype of pulmonary malignant cancers is non-small cell lung cancer (NSCLC) and mostpatients are diagnosed at late stage. Although great progress has been made in Surgery,radiotherapy, chemotherapy and molecular targeted drugs, comprehensive treatment andprognosis are still unsatisfactory.The occurrence and development of lung cancer involves in mutations of a variety ofgenes. Individualized targeted therapy is imperative in diagnosis and treatment of lungcancer. Recently, effects of MicroRNA (miRNA) on the occurrence, development,diagnosis and treatment of lung cancer, especially NSCLC draw much attention. MiRNAsare a class of small non-protein-coding RNAs, approximately18to25nt long and playroles in many biological process. Expression levels of microRNAs show great changes inmany cancers hinting that they are associated with occurrence, development, metastasis ofthe cancer. MiRNA-145shows effects of tumor suppression and regulates manytumor-related genes, which correlate with genesis and progress of tumor, as well asaggressive biological behaviors.Kruppel-like factor4(Klf4) and Oct4(also known as Oct3/4) are members oftranscription factors family, and their expression levels varies in different tumors. Klf4is atwo-way regulated gene and recent studies show that it acts as a tumor suppressor. Oct4overexpression in lung cancer inhibits cell apoptosis and thus promotes cell proliferation.Previous research has suggested that miRNA-145is closely related with Klf4and Oct4.MiRNA-145may be one of up-stream regulators of Klf4and Oct4, which promotes orinhibits cell proliferation, differentiation and apoptosis via regulating expressions of Klf4and Oct4. Due to low awareness of the mechanism of miR-145regulation of Klf4and Oct4,further work is need.ObjectivesLentiviral vector, A549human lung cancer cell line and nude mice were used to studythe possible mechanism of miR-145regulation of Klf4and Oct4. Effects of miRNA-145on cell cycle, proliferation, apoptosis, invasion and metastasis of lung cancer cell line were detected. Then the A549transfected were transplanted into subcutaneous of nude mice.Methods1. Lentiviral expression vector of miRNA-145gene were constructed, and A549cellswere transfected. qRT-PCR were performed to verify its expression efficacy.2. Expression of Klf4and Oct4protein were detected by western blots.3. Cell cycle and apoptosis were detected by flow cytometry. CCK-8assay, cellmigration assay, cell invasion assay and plate clone formation assay were performed todetect proliferation, invasion, migration ability of A549cells. Then the A549transfectedwere transplanted into subcutaneous of nude mice for further verification.Results1. Lentiviral vector carrying miRNA-145gene has been successfully constructed andA549maintains high expression of hsa-miR-145-5p.2. After transfection, expressions of Oct4and Klf4in A549cells were inhibited(P<0.0001) and the miRNA-145induced inhibition was down-regulated as the timeprolongs.3. Cell cycle analysis showed S and G2phase cycle arrest after transfection（P<0.0001）.4. Increased apoptosis, especially early apoptosis of A549cells was detected aftertransfection（P<0.0001）.5. Proliferation ability of A549cells decreased significantly after transfection shownby CCK-8analysis（P<0.0001）.6. Migration ability of A549cells decreased significantly after transfection shown bycell migration analysis (P<0.0001).7. Invasion ability of A549cells decreased significantly after transfection shown bycell invasion analysis (P<0.0001).8. Colony forming ability of A549cells decreased significantly after transfectionshown by plate clone formation assay.9. Nude mice experiment showed that weight of the nude mice in BLANKã€NCã€MIR-145groups had no statistical difference (P>0.05). As the time prolongs, weight of thenude mice in MIR-145group was heavier than the other two groups (P<0.05). Tumorvolume of the nude mice in MIR-145group was significantly smaller than the other two groups (P<0.0001). Weight of tumor in MIR-145group was heavier than the other twogroups (P<0.05)ConclusionKlf4and Oct4are target genes of miRNA-145. miRNA-145inhibits proliferation,invasion and migration ability of A549cells by regulating expression of Klf4and Oct4.The tumorigenicity of A549cells was attenuated after transfection with miRNA-145.Thiswork offers an important theoretical basis for treatment targets miRNA-145. |
