The Role And Mechanism Of CDCA3 In Lung Adenocarcinoma

Background and Objective:Lung cancer(LC)is the fastest growing malignant tumor in China in the past 30 years.From the first retrospective survey of the death causes in China in the mid1970 s to the third retrospective survey of the death causes in the 21 st century,the data shows that the death rate of lung cancer has jumped from the fifth to the first among the causes of cancer;According to the China Cancer Registration Center,the incidence of lung cancer in urban and rural areas ranked first among malignant tumors in 2015 and the incidence of lung cancer in men and women showed an upward trend.In terms of regional distribution,the mortality of lung cancer in urban areas is higher than that in rural areas;From the three major economic regions of the east,middle and west,the lung cancer mortality rate in the east is the highest,followed by the middle,and the west is the lowest.The occurrence and development of Lung cancer is closely related to genetic,environmental and other factors.Gene change is the molecular genetic basis of malignant transformation and progress of tumors.Identifying and exploring key genes or targets closely related to the occurrence of Lung tumors can open up a new path for the treatment of malignant tumor.Besides,the study of its pathogenesis,will pr OEide an effective way for early diagnosis and treatment.From the angle of pathology and treatment,LC can be roughly divided into two categories: Non Small Cell Lung Cancer(NSCLC)and Small Cell Lung Cancer(SCLC).Among them,NSCLC accounts for about 80%~85%,including Lung Adenocarcinoma(LUAD),Lung Squamous Cell Carcinoma(LUSC),and Large Cell Lung Cancer(LCC),The rest are Small Cell Lung Cancer(SCLC),accounting for about 15%.LUAD accounts for about 40% of all types of lung cancer and about 55%of NSCLC.The differential expression genes of LUAD were screened based on bioinformatics and the target gene of this study was determined.The expression of CDCA3 in LUAD patients and its correlation with clinicopathological parameters,survival status,prognosis and immune cells infiltration were analyzed by bioinformatics methods and a ce RNA network was constructed based on CDCA3.The possible Wnt/β-Catenin single pathway of CDCA3 was enriched through GSEA.Then,the vivo and vitro experiments were conducted to explore the effects of CDCA3 on proliferation,migration,invasion,apoptosis and other biological functions of LUAD cells.Thirdly,The mechanism of CDCA3 may activated the Wnt/β-Catenin signal pathway to promote the proliferation,migration and invasion of LUAD cells was explored.Finally,the clinical tissues were used to detect the expression of CDCA3.All of the ab OEe hoped to open a new way for the initial screening,early diagnosis and treatment of LUAD.Part Ⅰ The expression and significance of CDCA3 in LUAD based on bioinformatics analysisObjective:To explore the correlation between the expression level of CDCA3 and clinicopathological parameters in LUAD.Methods:1.Download the transcript data of CDCA3 and corresponding clinical data from TCGA database and GSE31210 dataset for the correlation analysis of CDCA3 expression level and clinicopathological parameters;2.The enrichment analysis of differential expresseion genes were carried out through TCGAdatabase、GSE30219 and GSE188423 datasets and the GSEA was conducted at the same time;3.The correlation between the expression level of CDCA3 and immune cells infiltration were analyzed through ESTIMATE,CIBERSORT and TIMER;4.The differential lnc RNAs,mi RNAs and m RNAs were screened by means of TCGA database and the ce RNA network was constructed.Results:1.There existed statistical differences between the expression of CDCA3 and clinical characteristics of LUAD(P<0.05);2.CDCA3 may participate in regulation of Wnt/β-Catenin signal pathway by the analysis of GSEA;3.The high expression level of CDCA3 was correlated with the immune cells infiltration(P<0.05);4.The ce RNA network was constructed based on CDCA3 and 18 lnc RNAs and 7mi RNAs related regulation were screened.Conclusions:CDCA3 was up-regulated in a variety of cancers,including LUAD and may play a certain role in the occurrence and development of LUAD,which pr OEide a theoretical basis for the further study of CDCA3.Part Ⅱ The biological function of CDCA3 in LUAD in vivo and vitro experimentsObjective:To further study the effects of CDCA3 on the proliferation,migration,invasion and apoptosis of NCI-H157 and SK-Lu-1 cells in vitro and in vivo.Hoping to pr OEide a new target and scientific basis for the possible mechanism of LUAD.Methods:1.The expression of CDCA3 was tested in the selected cells,then,CDCA3 knockdown lentivirus interference vector was constructed and transfected into LUAD cells and the mRNA and protein expression level of CDCA3 were detected;2.CCK-8 kit,cell scratch test,Transwell chamber test,flow cytometry and WB were used to detect cell proliferation,cell migration rate,cell invasion,cell apoptosis and the expression level of related proteins during the "EMT";3.Xenograft tumor model was constructed in nude mice to further verify the effect of CDCA3 on NCI-H157 and SK-Lu-1 cells.The character of tumor was detected by HE staining.Results:1.The m RNA and protein expression level of CDCA3 in LUAD cells NCI-H157 and SK-Lu-1 were higher than that in LUAD cells Calu-3,NCI-H1975 and control cells BEAS-2B(P<0.05);2.The knockdown of CDCA3 will significantly inhibited the proliferation,migration and invasion of LUAD cells,while increased the apoptosis rate of cells markedly;The knockdown of CDCA3 also decreased the expression of N-cadherin and Vimentin but up-regulated the expression of E-cadherin protein on the contrary during "EMT" process(P<0.05).In vivo experiment,the knockdown of CDCA3 in LUAD cells in nude mice led to a significant decrease of tumor formation rate(P<0.05);3.Further validation of the effect of CDCA3 on the tumor formation rate of LUAD cells transplanted into nude mice in vivo and detection of tumor properties through HE staining.Conclusions:The knockdown of CDCA3 may inhibit the proliferation,migration and invasion of LUAD cells,while accelerate the apoptosis rate of cells and lead to a significant decrease in tumor formation rate in nude mice,suggesting that CDCA3 may play an important role in the occurrence and development of LUAD.Part Ⅲ The mechanism of CDCA3 activates Wnt/β-catenin signal pathway to promote the proliferation and migration in LUAD cellsObjective:Aiming to furtherly discuss the possible molecular mechanism of the CDCA3 may activate the regulation of Wnt/β-catenin signal pathway to promote the proliferation,migration and invasion of LUAD cells.Methods:1.The OEer-expressed of CDCA3 lentivirus interference vector was constructed and transfected into LUAD cells and the m RNA and protein expression level of CDCA3 were tested;2.The key proteins in Wnt/β-catenin signal pathway and the process of "EMT" were detected by WB;3.CCK-8 kit,cell scratch test and Transwell chamber test were applied to detect the cell proliferation,cell migration rate,cell invasion respectively.To deeply explore the possible mechanism on how the CDCA3 activates the Wnt/β-catenin signal pathway in LUAD.Results:1.The koocking down of CDCA3 significantly inhibits the expression level of c-Myc and β-Catenin,which were the major proteins in the Wnt/β-catenin signal pathway;The OEer-expressed of CDCA3 will up-regulate this two important proteins expression level conversely(P<0.05);2.The OEer-expressed of CDCA3 may up-regulated the level of N-cadherin and Vimentin and down-regulated the E-cadherin protein during "EMT" in NCI-H157 and SK-Lu-1 cells(P<0.05);After adding in the Wnt/β-catenin signal pathway inhibitor FH535,the opposite results occured(P<0.05);3.The OEer-expressed of CDCA3 could significantly enhanceed the proliferation,migration and invasion of NCI-H157 and SK-Lu-1 cells,while the adding of Wnt/ β-Catenin signaling pathway inhibitor FH535 could significantly inhibit the promotion effect caused by CDCA3 OEer expressed(P<0.05).Conclusions:The OEer-expressed of CDCA3 may accelerate the proliferation,migration and invasion of LUAD cells and regulate the major proteins in the Wnt/β-catenin signal pathway.CDCA3 may play an important role in the occurrence and development of LUAD by activating Wnt/β-catenin signal pathway.Part Ⅳ The correlation between the expression of CDCA3 and the clinicopathologic characteristics and prognosis in LUADObjective:In order to detect the expression level of CDCA3 in clinical tissues and analyze its relationship with prognosis in LUAD.Methods:1.Download the transcript data and corresponding survival data of CDCA3 in TCGA database and GSE30219,GSE18842 and GSE31210 datasets for survival and independent prognosis analysis;2.33 LUAD patients tissues and the paired normal lung tissues were collected.All the patients underwent surgical treatment in the Department of Thoracic Surgery of the Second Affiliated Hospital of Kunming Medical University and comply with the inclusion and exclusion criteria strictly.The m RNA expression level of CDCA3 was detected by quantitative real-time PCR(q RT-PCR).Immunohistochemistry(IHC)and Western blotting(WB)were used to detect the protein expression of CDCA3 in LUAD and normal lung tissue;3.Clinical prognosis analysis:the archived wax blocks were kept by the Pathology Department of the Second Affiliated Hospital of Kunming Medical University.421 cases of LUAD tissues wax blocks preserved from 2016.9 to 2018.3 were selected for follow-up of clinical related studies by immunohistochemical score.Results:1.The K-M analysis showed that the high expression level of CDCA3 was negatively correlated with the OEerall survival(OS)and disease-free survival(DFS)and it maybe a new prognostic marker according to the results of Cox regression analysis;2.The mRNA and protein expression level of CDCA3 in LUAD tissues were higher than that in normal lung tissues,there existed statistically differences(P<0.05);3.Patients with low expression of CDCA3 had longer OS and DFS than patients with high expression of CDCA3,there existed statistically differences(P<0.05).Conclusions:The m RNA and protein expression level of CDCA3 in clinical tissues was higher than that in normal lung tissues.Patients with low expression of CDCA3 in clinical patients had longer OS and DFS,which further indicated that CDCA3 may play an important role in the development of LUAD.