Study On The Mechanism Of Family With Sequence Similarity 117 Member Ainregulating The Occurrence And Development Of Lung Adenocarcinoma Through DYRK1A

Lung cancer has the highest mortality rate among malignant tumors,Although great advances in targeted therapies for lung cancer have been made in the past few years and survival time of lung cancer patients has been significantly prolonged,however,almost all lung cancer patients eventually die of cancer recurrence and metastasis.The reason is that we do not have enough research on the molecular mechanism of lung cancer.So we conducted in-depth analysis of lung adenocarcinoma patient samples in the TCGA(The Cancer Genome Atlas)database to screen new drug targets and molecular markers.The results were verified by molecular biology,cell biology,nude mouse tumorigenesis experiment and clinical sample analysis.First,after bioinformatics analysis of transcriptome data and prognostic data of lung adenocarcinoma patients,we found that the Family with sequencesimilarity 117 member A(FAM117A)gene with unknown function was significantly correlated with the prognosis of lung adenocarcinoma patients.Further studies found that transcription levels and protein levels of FAM117 A were significantly lower in Patients compared with normal tissues,and the FAM117 A gene expression levels were correlated with the patient’s pathological stage.Patients with lung adenocarcinoma were further divided into groups according to the expression level of FAM117A(high expression group and low expression group).The differentially expressed genes of the two groups were screened and enriched through signal pathway.The results showed that the signaling pathways promoting cell cycle progression and tumor proliferation were significantly enriched in patients with low expression of FAM117 A.On this basis,we established an in vitro cytological model to establish FAM117 A over-expression or silencing lung adenocarcinoma cell line by lentivirus mediated gene transfection.Clonal formation assay and CCK-8 cell proliferation assay showed that FAM117 A silencing significantly promoted the proliferation of lung adenocarcinoma cell line.On the contrary,FAM117 A over-expression significantly inhibited the proliferation of lung adenocarcinoma cell line.In addition,in vivo tumorigenesis experiments confirmed that gene silenced tumor cells grownfasterin nude mice,suggesting that FAM117 A could regulate the proliferation of lung adenocarcinoma cells in vivo.Further studies such as flow cytometry and EDU staining showed that FAM117 A silencing promoted the cell cycle transition from G0/G1 phase to S phase,whereas over-expression of FAM117 A significantly blocked the cell cycle in G0/G1 phase.Western blot assay confirmed that the levels of Cyclin D 、phosphorylated CDK4、p21 and P27,which are related to cell cycle regulation,also showed corresponding changes,suggesting that FAM117 A can change the proliferative activity of tumor by changing the signal pathway of cell cyclerelated proteins.The interaction between FAM117 A and the cell cycle regulator DYRK1 A was confirmed by CO-IP combined with western blot assay.In lung adenocarcinoma cell lines,FAM117 A and DYRK1 A were silenced alone or together,and the cell cycle showed the same phenotype by flow cytometry,which was further verified by EDU staining and clonogenesis assay.Thus,we concluded that FAM117 A regulates the cycle progression of lung adenocarcinoma cells through interaction with DYRK1 A.The silencing of either gene expression leads to the loss of function of controlling cell cycle and promotes the development of lung adenocarcinoma cells.Since FAM117 A silencing causes accelerated cell cycle progression and increases CDK4 phosphorylation and complex activity,we tested the potential role of CDK4/6 inhibitors in FAM117 A silenced cells.Cell cycle detection EDU staining and clonogenesis experiments showed that PD0332991,a small molecule inhibitor of CDK4/6,could completely reverse the accelerated proliferation of lung adenocarcinoma cells induced by FAM117 A.To further study the clinical significance of FAM117 A gene in Chinese patients with lung adenocarcinoma,we collected 57 samples of lung adenocarcinoma tissues treated in the General Hospital of the people’s Liberation Army and underwent radical surgery for lung carcinoma,including cancer tissues and adjacent tissues.The results showed that the expression levels of FAM117 A in the cancer tissues of patients with lung adenocarcinoma were significantly lower by immunohistochemistry and RT-PCR and it was consistent with the expression level of cell cycle inhibitor p21 protein,and is contrary to the expression level of cell cycle promoting protein cyclin D.Statistical analysis of clinical factors and quantitative results of FAM117 A showed that there was a significant correlation between tumor T stage and FAM117 A gene expression,With the increase of T stage,the gene expression showed a decrease trend.This is essentially in agreement with the results of the database.In conclusion,we used bioinformatics technology to analyze TCGA database and found that the expression levels of FAM117 A gene with unknown function were significantly correlated with the prognosis of patients with lung adenocarcinoma.Using cells,animal models and clinical tumor cases,we found that FAM117 A gene silencing can promote the proliferation of lung adenocarcinoma cells and tumor progression by up regulating cell proliferation signals,and the regulatory effect may be completed by interacting with DYRK1 A.CDK4/6 inhibitor can reduce the cell proliferation activity caused by FAM117 A silencing.It is suggested that FAM117 A may be used as a biomarker to predict the sensitivity of lung adenocarcinoma to cell cycle inhibitors,which provides a theoretical basis for studying the molecular mechanisms of tumor occurrence,development,invasion and metastasis.