The Mechanism Of DONSON-CCNE2 In The Occurrence And Progression Of Lung Adenocarcinoma

Background and purpose:Lung cancer is by far the second most common cancer and the leading cause of death globally,while NSCLC(Non–small cell lung cancer)accounts for 85% of all lung cancer cases.The main subtypes of NSCLC are adenocarcinoma,squamous cell carcinoma,and large cell carcinoma,LUAD(Lung adenocarcinoma)comprises about 40% of all lung cancer cases.Because of the lack of early diagnosis technology,only a small percentage of lung cancer patients are diagnosed at early stage,while with around 60% of lung cancer patients have been detected with advanced stages-stage III or IV,and therefore lose chance of curative surgical resection.Treatments for advanced lung cancer include chemotherapy,targeted therapy,immunotherapy,local radiation therapy and surgery based on systemic treatments.However,the efficacy of platinum-based chemotherapy in patients with advanced NSCLC is only about 30%-40%.Though the rapid development of targeted therapy and immunotherapy has dramatically improved progression-free survival(PFS)and quality of life(Qo L)of NSCLC patients in recent years,they are still facing high risk of tumor recurrence and metastasis.Therefore,discovery of new driving genes which can provide an improved understanding of the molecular mechanism of lung cancer and be used as therapeutic targets are currently still the most important contents in the lung cancer research.DONSON(Downstream of neighbor SON)is a newly discovered gene whose main function is work as part of the replication fork to maintain genome stability by protecting and stabilizing the established replication fork,further promoting and activating the cell cycle checkpoint,thereby ensuring the correct DNA repair process,avoiding gene mutation and chromosomal breakage.Mutations and deletions in the DONSON gene are associated with congenital developmental disorders such as MPD(Microcephalic primordial dwarfism).Recent studies have shown that DONSON may play an important role in the development of cc RCC(clear cell renal cell carcinoma)and prostate cancer,which is closely related to pathological grade,tumor stage,lymph node metastasis and clinical prognosis,although the molecular mechanism remains to be further studied.Our research found that DONSON gene is abnormally highly expressed in LUAD tissues,there has been no relevant report on the function and mechanism of DONSON in LUAD until now.In this study,we chose focused on DONSON and its downstream target gene to explore the roles and mechanisms of DONSON in the tumorigenesis and progression of LUAD.We conducted in-depth research from gene chip,bioinformatics analysis,molecular biology,cell function and experimental zoology,it will help to clarify the pathogenesis of lung cancer and provide theoretical basis for the discovery of new therapeutic targets.Methods:1.LUAD tissues analysis and TCGA(The Cancer Genome Atlas)database analysis(1)Detection of differentially expression genes in LUAD and para-carcinoma tissues by gene chip;(2)The TCGA database was used to search the gene expression profile changes between LUAD and normal samples,analyzed the difference of DONSON expression,investigated the relationship between DONSON expression and overall survival of LUAD patients by Kaplan survival curve;(3)qRT-PCR was used to detect the different levels of DONSON expression between LUAD and para-carcinoma tissues;(4)The expression of DONSON protein in LUAD and para-carcinoma tissues was examined by tissue microarray,inspected the correlation between the expression of DONSON protein and tumor pathological characteristics,the connection of DONSON protein with overall survival in patients was also studied.2.Effects of DONSON on biological behavior of lung cancer cell lines(1)DONSON knockdown lung cancer cell lines were generated by using the lentivirus infection system with short hairpin RNA;(2)The effects of DONSON on proliferation,apoptosis,cell cycle,cell migration and invasion of lung cancer cell lines were examined by Celigo cell proliferation assay,cell apoptosis assay,cell cycle analysis,cell scratch assay and transwell invasion assay;(3)The effect of DONSON on the proliferation of lung cancer cell lines was deeply verified by the nude mice tumor model in vivo,the expression of Ki-67 was detected by immunohistochemistry to understand the upshot of DONSON knockd-own on cell proliferation furtherly.3.Molecular mechanism of DONSON-CCNE2 on biological behavior of lung cancer cell lines(1)Gene chip was used to detect the changes of gene expression profile of lung cancer cell lines with DONSON knockdown;(2)The expression of CCNE2 protein in LUAD and normal tissues was detected by tissue microarray,analyzed the correlation of CCNE2 protein expression and tumor pathological features,examined its association with overall survival in patients carefully;(3)Construct CCNE2 knockdown(sh CCNE2)and DONSON overexpress(DONSON OE)lentivirus,then transfect sh CCNE2,DONSON OE,DONSON OE plus sh CCNE2 lentivirus into lung cancer cell lines individually;(4)The effects of CCNE2 knockdown,DONSON overexpress,DONSON overexpress plus CCNE2 knockdown on the cell proliferation and clonogenicity,apoptosis,migration and invasion of lung cancer cell lines were detected by Celigo cell proliferation assay,cell clonogenic assay,cell apoptosis assay,cell scratch assay and transwell invasion assay respectively.Results:1.The expression of DONSON was significantly higher expressed in LUAD tissues and negatively correlated with the clinical prognosis of patients(1)Gene chip detection found that,there were 2,551 differentially expressed genes between LUAD and matched para-carcinoma tissues,of which 925 were up-regulated and 1626 were down-regulated genes,the expression of DONSON in LUAD tissues was significantly higher than that in para-carcinoma tissues;(2)From the TCGA data analysis,we found the expression of DONSON in LUAD tissues was significantly higher than that in normal tissues,and negatively related to the overall survival of patients;(3)The qRT-PCR results showed that,compared to para-carcinoma tissues,DONSON gene was highly expressed in LUAD tissues;(4)The results of tissue microarray showed that,the expression of DONSON protein was significantly higher in LUAD in comparison with para-carcinoma tissues,similarly,it was negatively correlated with the tumor pathological and clinical prognosis of LUAD;2.DONSON promotes the malignant proliferation,migration and invasion of lung cancer cells,inhibits the apoptosis of cells significantly(1)The cell proliferation experiment showed that,knockdown of DONSON in lung cancer cells can significantly inhibited cell proliferation in vitro;(2)The cell apoptosis rate was significantly increased in DONSON knock down cells;the cell cycle could not transfer from G0/G1 phase to S phase fluently and was abnormally arrested at G2/M phase;(3)Results of cell migration assay and transwell invasion assay showed that,cell migration and invasion decreased significantly after DONSON expression was interfered;(4)The nude mice xenograft model was successfully established,the results verified that the tumor formation rate and the tumor volume decreased significantly in the mice those implanted with DONSON knock down lung cancer cells,the expression of Ki-67,a marker of cell proliferation,was also notably decreased in the DONSON knockdown group.3.Effects of DONSON-CCNE2 on the biological behavior of lung cancer cells(1)Gene chip analysis revealed that the expression of CCNE2 was remarkably down-regulated in lung cancer cells which transfected with sh DONSON lentivirus;(2)The expression of CCNE2 was significantly higher in patients with lung adenocarcinoma,and negatively correlated with tumor pathological stage,lymph node metastasis and clinical prognosis;(3)CCNE2 could inhibit cell proliferation and cell clone formation,promote cell apoptosis,inhibit cell migration and invasion;(4)Overexpression of DONSON could rise the proliferation and clonogenicity of lung cancer cells,inhibit cell apoptosis,promote cell migration and invasion.The knockdown of CCNE2 on the basis of DONSON overexpression could notably blocked the biological behavior of lung cancer cells.Conclusions:1.DONSON and CCNE2 were abnormally high expressed in LUAD tissues,the expression of CCNE2 was positively correlated with that of DONSON,both DONSON and CCNE2 were negatively correlated with pathological characteristics and overall survival of LUAD patients;2.CCNE2 was the potential downstream target gene of DONSON;3.DONSON promotes proliferation of lung cancer cells in vitro and in vivo by regulating the expression of CCNE2.