| Objective:Lung adenocarcinoma(LUAD)is the most common histological type of non-small cell lung cancer and is characterized by a high incidence and poor prognosis.Due to the lack of obvious symptoms in the early stage,most of the diagnosed LUAD patients are in the middle and late stage with multiple metastases,which leads to patients missing the optimal treatment period and the survival rate is still very low.Therefore,exploring the molecular mechanism related to LUAD metastasis is beneficial to the early diagnosis of lung adenocarcinoma and has important guiding significance for finding clinical treatment strategies.Most long non-coding RNAs(Lnc RNAs)play precise regulatory roles in the occurrence and development of cancer.Previously,through data mining and bioinformatics analysis,we obtained the long non-coding RNA LINC00152,which is significantly highly expressed in lung adenocarcinoma tissues and is closely related to survival and prognosis of patients.In this study,we explored the reasons for the high expression of LINC00152 in lung adenocarcinoma and its molecular mechanism of promoting metastasis in lung adenocarcinoma.Methods:Previously,we screened the highly expressed Lnc RNAs in LUAD using bioinformatics analysis and found that LINC00152 was highly expressed in LUAD cells and tissues by RT-q PCR experiments.Then,the effect of LINC00152 on the proliferation and metastasis of lung adenocarcinoma cells in vitro was explored by scratch healing assay,Transwell chamber invasion assay,CCK8 and clone formation assay.The SRAMP website was used to predict the m6 A modification of LINC00152,RNA immunoprecipitation,MS2-Trap,Me RIP,and RNA stability experiments were used to explore the m6 A modification of LINC00152 in LUAD and the effect of m6 A modification on its expression.Star Base predicted Hu R may interact with LINC00152.Using MS2-Trap,RNA immunoprecipitation and Transwell chamber invasion assays,we verified that LINC00152 regualted epithelial-mesenchymal transition(EMT)via Hu R.A subcutaneous tumor model and a lung metastasis tumor model in nude mice were constructed to explore the effect of LINC00152 on the metastasis and proliferation of lung adenocarcinoma cells in vivo.LINC00152 function and downstream target genes were verified in vivo using live animal imaging,H&E staining and immunohistochemistry.Results:1.LINC00152 can promote the proliferation,migration and invasion ability and EMT process of lung adenocarcinoma cells in vitro and in vivo.After successfully overexpressing or knocking down LINC00152 in lung adenocarcinoma cells,we confirmed that LINC00152 significantly promoted the migration and invasion of lung adenocarcinoma cells in vitro by scratch healing assay and Transwell chamber invasion assay.The ability of LINC00152 to promote lung adenocarcinoma cell clone formation and proliferation in vitro was confirmed by CCK8 and clone formation assays.LINC00152 knockout A549 cells and their control A549 cells were constructed by CRISPR/Cas9 technology previously,and a subcutaneous tumor model in nude mice was constructed by subcutaneous injection of A549 stable knockout cell line,Inhibiting the growth of subcutaneous tumors in nude mice.A nude mouse lung metastases model was established by tail vein injection of A549 stable knockout cell line,live animal imaging was performed and the lung tissue was stripped to compare the number and size of lung metastases,The LINC00152 knockout group showed less fluorescence intensity and less lung metastatic nodules.Immunohistochemistry on the dissected subcutaneous tumor tissue showed that the expression of Ki67,ZEB1,Snail and Slug were significantly reduced after LINC00152 knockout,and knockdown of LINC00152 inhibited the proliferation of lung adenocarcinoma cells in vivo.2.ALKBH5 is down-expressed in lung adenocarcinoma cells and leads LINC00152 m6 A modification enhancing and its stability and expression increasing.The SRAMP database predicted that there is some m6 A modification sites on LINC00152,and the Star Base database showed that there is a negative correlation between the canonical demethylase ALKBH5 and LINC00152.ALKBH5 is underexpressed in lung adenocarcinoma cells.MS2-Trap and RNA immunoprecipitation confirmed the direct binding between ALKBH5 and LINC00152.RT-q PCR demonstrated that overexpression of ALKBH5 reduced the expression of LINC00152.It was confirmed by Me RIP experiment that the overexpression of ALKBH5 reduced the m6 A modification on LINC00152,and the RNA stability experiment combined with RT-q PCR confirmed that the overexpression of ALKBH5 reduced the stability of LINC00152.The low expression of ALKBH5 in lung adenocarcinoma cells mediated LINC00152 m6 A modification enhancing and increased its stability to make it highly expressed in lung adenocarcinoma.3.LINC00152 binds Hu R directly and affects the interaction of Hu R with EMT core transcription factors(ZEB1,SNAI1 and SNAI2)to upregulate the expression of these genes,then promoting EMT process and lung adenocarcinoma metastasis.We observed that overexpression of LINC00152 produced a mesenchymal phenotype in lung adenocarcinoma cells,while knockdown of LINC00152 produced an epithelial phenotype.LINC00152 overexpression resulted in upregulation of EMT transcription factors ZEB1,SNAI1,SNAI2 and mesenchymal markers N-cadherin and Vimentin,while downregulation of epithelial markers E-cadherin and Claudin-1.The Star Base website predicted that Hu R could bind to LINC00152,and MS2-Trap and RNA immunoprecipitation verified the direct binding between Hu R and LINC00152,Knockdown of Hu R reversed the EMT transformation caused by LINC00152 overexpression.Importantly,LINC00152 enhanced the interaction of Hu R with EMT core transcription factors(ZEB1,SNAI1 and SNAI2)to upregulate the expression of these genes,thereby promoting the EMT process.Conclusion:1.LINC00152 can enhance the proliferation,migration and invasion of lung adenocarcinoma cells in vitro and in vivo.2.The low expression of ALKBH5 in lung adenocarcinoma cells mediates the enhancing of LINC00152 m6 A modification which upregulated LINC00152 stability and expression in LUAD.3.LINC00152 binds Hu R directly and enhaces the interaction of Hu R with EMT core transcription factors(ZEB1,SNAI1 and SNAI2)which increases the expression of these genes to promot EMT process and LUAD metastasis. |
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