MicroRNA-365 Promotes Lung Adenocarcinoma By Downregulating The USP33/SLIT2/ROBO1 Signaling Pathway

According to WHO,cancer is the leading cause of death worldwide,and lung cancer is the main cause of cancer-related deaths.1 In 2017,there were an estimated 222,500 new diagnoses of lung cancer in the United States,and 155,870 people died of lung cancer.With advances in medical technology,lung cancer treatment has entered the era of precision medicine.Therefore,identifying genes that drive cancer progression and providing effective treatments may prolong the survival of patients with NSCLC.Abnormal microRNA expression is closely related to cancer occurrence and de velopment.miR-365 plays an oncogenic role in skin cancer,but its role in lun g cancer remains unclear.In this study,we aimed to investigate its role and u nderlying molecular mechanisms in lung cancer.miR-365 was upregulated in 1 ung adenocarcinoma and lung cancer cell lines.Overexpression of miR-365 pro moted and inhibition of miR-365 suppressed the proliferation,migration,and in vasion of lung cancer cells.A nude mouse model of lung cancer further verifi ed these findings.We identified USP33 as the downstream target of miR-365 and observed a negative correlation between miR-365 and USP33 expression in lung adenocarcinoma patients.Further experiments indicated that miR-365 may downregulate USP33 expression in a targeted manner.The USP33/SLIT2/ROB 01 signaling pathway has been previously reported to inhibit the invasion and metastasis of lung cancer.Following the overexpression of miR-365,SLIT2 a nd ROBO1 were downregulated,whereas the simultaneous overexpression of U SP33 and miR-365 mitigated this effect.In summary,miR-365 acts as an onco gene by promoting lung carcinogenesis via the downregulation of the USP33/S LIT2/ROBO1 signaling pathway,making the miR-365/USP33/SLIT2/ROBO1 axi s a new mechanism of lung cancer promotion and a novel therapeutic target f or predicting prognosis and response to gene therapy.Materials and Methods:1.PCR and Western blot assay were used to detect the expression of miR-365 in lung cell lines and lung cancer tissues.2.To investigate the effect of high expression of miR-365 on the proliferation of lung cancer cells,we used plate clone formation and Edu assays.Transwell migration and invasion assay were consistent with those of the wound healing assay,indicating that miR-365 promotes the migration and invasion activities of lung cancer cells.3.A western blot assay showed that the expression of USP33 was significantly downregulated following the over expression of miR-365.Next,qRT-PCR was used to detect the expression of miR-365 and USP33 in tissues derived from 20 cases of lung adenocarcinoma.Relative luciferase activity was significantly decreased in cells co-transfected with WT USP33 3' UTR and agomiR-365.4.Functional rescue assays were performed to further verify that the miR-365-mediated downregulation of USP33 promotes the proliferation,migration,and invasion of lung cancer cells in plate clone formation and Edu assays,transwell migration and invasion assay and wound healing assay.5.A western blot assay was performed to observe changes in the expression of SLIT2 and ROBO1 in A549 and SPC-A-1 cells overexpressing USP33 or miR-365 alone or in combination.6.The subcutaneous implantation tumor model further demonstrated that miR-365 promotes tumor formation in vivo.Results:1.MiR-365 is upregulated in NSCLC tissues and cell lines.2.MiR-365 promotes proliferation,migration,and invasion in lung cancer cells.3.USP33 is a downstream target gene of miR-365 and is downregulated by miR-365.4.MiR-365 promotes lung cancer cell proliferation,migration,and invasion by downregulating USP33.5.MiR-365 regulates the USP33/SLIT2/ROBO1 signaling pathway.6.MiR-365 promotes subcutaneous tumor formation in mice.Conclusions:1.We aimed to determine the role and mechanism of miR-365 in lung cancer.2.miR-365 was upregulated in lung adenocarcinoma and lung cancer cell lines.3.miR-365 overexpression promoted lung cancer proliferation,migration,and invasion.4.USP33 was identified as the downstream target of miR-365.5.The miR-365/USP33/SLIT2/ROBO1 axis is a new mechanism of lung cancer.