Study On The Function And Mechanism Of MiR-7974 In Proliferation And Invasion Of Lung Adenocarcinoma Cells By Targeting BAP1

Objectives:Lung cancer is the leading cause of tumor-related death worldwide.Lung adenocarcinoma,the most common subtype of lung cancer with occult onset and easy metastasis,is incurable due to the delayed diagnosis in the early stage.For the past few years,targeted therapy followed by the detection of lung cancer driving genes such as EGFR and KRAS has significantly altered the landscape of lung cancer therapy,which is conducive to part of lung adenocarcinoma patients.However,metastasis is still the main cause of treatment failure and death in metastatic lung cancer.The 5-year survival rate of metastatic lung cancer declines sharply to 7% with poor prognosis.Micro RNAs is a kind of non-coding RNAs that regulates gene expression.Abnormal expressions of micro RNA involve almost all tumors.In the previous stage of our research,total transcriptome sequencing was performed on tissues with bone metastasis and without bone metastasis of lung adenocarcinoma patients.We found that miR-7974 was significantly increased in the bone metastasis tissues.Besides,bioinformatics methods couple with double luciferase reporter gene detection identified BAP1 as a direct target gene of miR-7974.So far,there are few studies on miR-7974 in lung adenocarcinoma,and its specific functions and mechanisms are still unclear.This study is designed to explore the function and mechanism of miR-7974 in regulation of proliferation and invasion of lung adenocarcinoma cells,aiming to provide a scientific basis for targeting miR-7974.Methods:1.The UALCAN database was searched to obtain the different expressions of miR-7974 in lung adenocarcinoma and adjacent normal tissues,and we analysed the prognosis with Kaplan-Meierplotter database,then the expression levels of miR-7974 in lung adenocarcinoma tissues and lung adenocarcinoma cells were comfirmed by q RT-PCR.2.To establish the miR-7974 overexpression and knockdown cell models,the miR-7974 mimics and inhibitors were transfected into LAUD cells.Functional assessment including cell clone formation assay,CCK8,scratch wound assay,transwell migration and invasion assay were designed to evaluate the functions of miR-7974 on proliferation,migration and invasion of LAUD cells.3.Followed by overexpressing and knocking down miR-7974,q RT-PCR and WB were performed to detect the expression levels of BAP1 in LAUD cells,then we analysed correlation between miR-7974 expression levels and BAP1 expression levels.4.The UALCAN database was searched to obtain the different expressions of BAP1 in LAUD tissues and adjacent normal tissues,and we analysed the prognosis with Kaplan-Meierplotter database,then the expression levels of BAP1 in LAUD tissues and LAUD cells were comfirmed by q RT-PCR.5.To establish the BAP1 overexpression and knockdown cell models,the overexpression lentivirus vectors and interference lentivirus vectors of BAP1 were transfected into LAUD cells.Functional assessment including cell clone formation assay,CCK8,cell scratching assay,transwell migration and invasion assay were designed to evaluate the functions of BAP1 on the biological behaviors of LAUD cells.6.A series of rescue experiments were designed,concretely,we knocked down miR-7974 in advance,and continued to knock down BAP1,then evaluated whether knocking down BAP1 reversed the inhibitory effects of knocking down miR-7974 on the proliferation,migration and invasion of LAUD cells.Results:1.Bioinformatics analysis showed that miR-7974 was highly expressed in LAUD tissues,and patients with higher expression levels of miR-7974 had shorter survival time;q RT-PCR analysis showed that miR-7974 was highly expressed in LAUD tissues and LAUD cells,consistencing with the results of bioinformatics analysis.2.Overexpressing miR-7974 promoted proliferation,migration and invasion behaviors of LAUD cells;While knocking down miR-7974 inhibited the proliferation,migration and invasion of LAUD cells.3.The m RNA and protein levels of BAP1 in LAUD cells were decreased when miR-7974 was overexpressed;While the m RNA and protein levels of BAP1 were increased when miR-7974 was knocked down.Therefore,miR-7974 downregulated the expression of BAP1.4.Bioinformatics analysis showed that BAP1 was poorly expressed in LAUD tissues,and patients with lower expression levels of BAP1 had shorter survival time.q RT-PCR analysis showed BAP1 was poorly expression in LAUD tissues and cells,consistencing with the results of bioinformatics analysis.5.Overexpressing BAP1 inhibited the proliferation,migration and invasion of LAUD cells,and knocking down BAP1 promoted the proliferation,migration and invasion of LAUD cells.6.Rescue experiments confirmed that knocking down BAP1 reversed the inhibitory effects of knocking down miR-7974 on the proliferation,migration and invasion of LAUD cells.It indicated that miR-7974 promoted the biological behaviors of LAUD cells by negatively regulating BAP1.Conclusions:Mi R-7974 enhanced the ability of proliferation,migration and invasion of LAUD cells.BAP1,as a direct target gene of miR-7974,inhibited the biological behaviors of LAUD cells.The pro-cancer effect of miR-7974 probably related to the targeted regulation of tumor suppressor factor BAP1.