| BackgroundLung cancer is one of the malignant tumors with the highest morbidity and mortality in the world.Because of atypical early symptoms,most patients with lung cancer have been diagnosed in the middle and late stages,and the 5-year survival rate is very low.It is extremely important to seek new and effective therapeutic targets and targeted drugs.Chemotherapy is a classical method to inhibit the growth of cancer cells,but it brings a series of side effects,such as gastrointestinal reaction,liver and kidney damage,hair loss and so on,which seriously affects the living quality of patients.Although some targeted drugs,such as EGFR-TKIs,have achieved good therapeutic effects,drug resistance will arise after a period of time,which suggests that we must continue to promote cancer research.Moreover,as one of the common mutation sites of non-small cell lung cancer,Kras-mutation gene has no effective therapeutic drugs nowadays,which provides a clear direction for our experimental.As an important member of the small G protein family,cell division cycle 42(Cdc42)plays an important role in regulating cell movement,cytoskeleton,cell proliferation,cell transformation and metastasis of malignant tumors.In recent years,Cdc42 has been found to be highly expressed in a variety of malignant tumors,which is involved in regulating the proliferation,transformation and metastasis of cancer cells.However,the role and molecular mechanism of Cdc42 in lung cancer remain unclear.Methods1.Animal experiment1.1 Tet-On system and Cre/loxp technology were used to construct lung cancer mice model and lung cancer based-type ? alveolar epithelium-specific knockout Cdc42 mice model,and subsequent experiments were carried out.1.2 The left lungs were fixed,paraffin embedded and sliced as HE staining.The right lungs were performed for Western blot to detected the expression of Cdc42 protein.1.3 The mice were inhaled with ML 14,and the growth of the tumors was observed by HE staining.2.Cell experiments2.1 A549 lung cancer cell lines were cultured and knocked down Cdc42 by small RNA interference technology.CCK8 test,Ki67 fluorescence staining,flow cytometry and colony formation tests were used to detect cell proliferation,scratch test was used to detect cell migration ability,and Transwell assays were used to detect cell migration and invasion ability.2.2 The cells were harvested for Western blot to detect Cdc42 level.2.3 Cells were treated with diff-erent concentrations of'ML141 and the proliferation ability was detected by CCK8 assay.2.4 Cell RNA was extracted and transcriptome sequencing was used to analyze the changes of downstream genes and signaling pathways of Cdc42.3.Statistical analysis was conducted using SPSS.Results1.The Kras mutant lung cancer mouse model was successfully constructed.The lung cancer model can be obtained by crossing the target gene mice and fed with Dox for 3 months2.A mouse model of Cdc42 gene knockout in type ? alveolar epithelium based on lung cancer was successfully constructed.After the establishment of animal models,the number and size of tumors in Cdc42 knockout group was significantly less than those of non-knockout group,and the difference was statistically significant(P<0.05)3.The A549 cell line knocking down Cdc42 protein was constructed stably.The expression of Cdc42 protein in siCdc42 group was significantly less than that in control group,and the difference was statistically significant(P<0.05).4.Effect of knockdown Cdc42 protein in on cell proliferation,migration and invasion in A549 cell line.Compared with the control group,the cell proliferation,migration and invasion ability of siCdc42 group decreased significantly in A549,and the difference was statistically significant(P<0.05).5.The effect of ML 141 on cell and lung cancer modelAfter treatment with ML141,the proliferation of A549 cells was significantly inhibited by high concentration of ML141(P<0.05).Compared with lung cancer group,the number of lung cancer was significantly reduced after treatment with ML141.6.Cdc42 knockdown in A549 cells affects many genes and signaling pathwaysTranscriptome sequencing analysis showed that Cdc42 knockdown regulated many downstream genes and signaling pathways,including ECM-receptor interaction,microRNAs in cancer,p53 signaling pathway,focal adhesions and PI3K-Akt signaling pathway.Conclusion1.Conditional gene knockout technique was used to overexpress Kras gene on alveolar epithelium in mice,which could successfully construct in situ lung cancer model in mice.The model also confirmed that the oncogene KRAS has a strong ability to induce lung cancer formation.2.The relationship between Cdc42 and lung cancer:In vivo experiments,knocking out Cdc42 protein reduced the proliferation ability and migration ability of lung cance.Therefore,Cdc42 may become one of the targets for the treatment of lung cancer in the future.3.The mechanism of Cdc42 and lung cancer:Downregulation of Cdc42 expression in A549 lung cancer cells affected many downstream genes and signaling pathways.Thus,Cdc42 may play a role through various targets,in which the changes of extracellular matrix-related genes play the most important role. |
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