The Study On Molecular Mechanism Of LINC00665 Regulating MiR-361-5p/HOXB7 In Lung Adenocarcinoma

Background:Long non-coding RNA(lncRNA),a member of the non-coding RNA family,is more than 200 nucleotides long and usually acts as a proto-oncogene or tumor suppressor during tumor development,such as cancer cell proliferation,invasion and apoptosis.Recent studies have shown that lncRNA LINC00665 is involved in cell cycle regulation in lung cancer.Due to the complexity of the tumorigenesis network,the potential role of LINC00665 in the progression of lung adenocarcinoma(LUAD)needs further investigation..Objective:In this study,we investigated the expression and biological activity of LINC00665 in LUAD tissues and cell lines,as well as its internal regulatory mechanism.Methods:The LUAD tissues and adjacent non-tumor tissues in clinical were coletecd,and quantitative real-time PCR(qRT-PCR)was applied to detect the expression of LINC00665 in LUAD tissues and cell lines.LINC00665 overexpressed recombinant vector and siRNA were constructed and synthesized,and transfected into LUAD cells to up-regulate and knock down LINC00665.The CCK-8 assay,colony formation assay,flow cytometry assay,transwell and wound healing assay were used to find out the effects of LINC00665 on the cell viability,proliferation,apoptosis,migration and invasion in LUAD cells.microrna-361-5p(miR-361-5p)that may interact with LINC00665 was analyzed by bioinformatics methods and verified by luciferase reporter assay.QRT-PCR was applied to detect the expression of miR-361-5p in LUAD tissues and cell lines.The biological function of LINC00665 regulating LUAD cells as mir-361-5p molecular sponge was verified by a series of molecular biological methods.The possible target gene HOXB7 of miR-361-5p was analyzed by bioinformatics methods,and the interaction between miR-361-5p and the target gene HOXB7 was verified by luciferase reporter gene system.The immunohistochemistry(IHC)staining,qRT-PCR and Western blot was used to test the expression of HOXB7 in LUAD tissues and cell lines.The recombined overexpression vector of HOXB7 was constructed and the siRNA for knockdown of HOXB7 was synthesized.A series of molecular biological assay methods were applied to detect the biological roles of HOXB7.The rescue experiments were applied to verify the biological roles of miR-361-5p through regulating target gene HOXB7.The Western blot was used to test the signal pathway of HOXB7 in LUAD cells.Results:The results of qRT-PCR showed that the expressions of LINC00665 were dramatically increased in LUAD tissues and cells,compared with adjacent non-tumor tissues and human normal lung epithelial cells(BEAS-2B).The expressions of LINC00665 were significantly upregulated or downregulated in the LUAD cells transfected with recombined overexpression vector of LINC00665 or siRNA,respectively.The results of a series of molecular biological assay showed that the overexpression of LINC00665 promoted the cell viability,colony formation,migration and invasion,but inhibited apoptosis.The results of luciferase reporter assay showed that there was a negative interaction between LINC00665 and miR-361-5p.The results of qRT-PCR revealed that the expression of miR-361-5p was dramatically decreased in LUAD tissues and cells,compared with adjacent non-tumor tissues and BEAS-2B.And there was a negative correlation between LINC00665 and miR-361-5p expression in LUAD cells.The expressions of miR-361-5p were significantly upregulated or downregulated after transfections with miR-361-5p mimic or inhibitor in LUAD cells.The results of a series of molecular biological assay showed that LINC00665 functioned as a molecular sponge of miR-361-5p to regulate cell viability,colony formation,migration,invasion and apoptosis in LUAD cells.The bioinformatics analysis and luciferase reporter gene system verified that HOXB7 was the target gene regulated by miR-361-5p.The results of IHC staining,qRT-PCR and Western blot showed that the expressions of HOXB7 were obviously increased in LUAD tissues and cells.And there was a positive correlation between LINC00665 and HOXB7 expression in LUAD cells.Overexpression of HOXB7 promoted cell viability,colony formation,migration and invasion,but inhibited apotosis in LUAD cells.The results of rescue experiments showed that miR-361-5p inhibited tumor progression by regulating HOXB7 in LUAD cells.The results of Western blot suggested that overexpression or knockdown of HOXB7 leaded to the upregulated or downregulated of AKT and Cyclin D1 in LUAD cells.Conclusion:LncRNA LINC00665 was upregulated in LUAD tissues and cells,and promoted cell viability,colony formation,migration and invasion,but inhibited apoptosis in LUAD cells.LINC00665 promoted tumor progression by targeting the miR-361-5p/HOXB7 axis in LUAD.HOXB7 may regulate the expression of cellular Cyclin D1 by binding to AKT protein.LINC00665 may become a potential target for drug treatment of LUAD in the future.