MicroRNA-509 Acts As A Tumor Suppressor In Tongue Squamous Cell Carcinoma By Targeting Epidermal Growth Factor Receptor

Tongue squamous cell carcinoma(TSCC)is the most frequent type of oral carcinoma,and is characterized by high metastatic and growth capabilities.Previous studies have demonstrated that miRNAs act as essential regulators of numerous biological processes,including cell growth,development,differentiation,apoptosis and endocrine homeostasis.In the past research aberrantly expressed cancer-associated microRNAs(miRs)may be associated with tumorigenesis and tumor development in various types of cancer,including TSCC.MiRNAs suppress the expression of their target genes through the action of the RNA-induced silencing complex,following binding of the miRNA molecule to the 3' untranslated region(UTR)of its target mRNA;this results in the degradation of the target mRNA or the repression of mRNA translation.Previous studies have demonstrated that oncogene activation and tumor suppressor gene inactivation may be implicated in the pathogenesis of TSCC.However,the detailed molecular mechanisms underlying TSCC development and progression have yet to be elucidated.Therefore,it is essential to investigate the molecular mechanisms underlying the pathogenesis of TSCC and to develop novel therapeutic approaches,in order to improve the diagnosis,treatment and prognosis of patients with TSCC.MiR-509 has been identified as a critical regulator in tumorigenesis and tumor development,via its tumor-suppressing actions in several types of human cancer.In the present study,miR-509 expression in TSCC tissues and cell lines was determined by qRT-PCR.The effects of miR-509 on TSCC cell proliferation and invasion were evaluated via MTT and invasion assays,respectively.In addition,the direct target of miR-509 in TSCC was investigated by qRT-PCR,western blot and dual luciferase reporter assays.The present study demonstrated that miR-509 expression was downregulated in TSCC tissue samples and cell lines(Tca8113,SCC-15,CAL-27),whereas its ectopic expression suppressed TSCC cell proliferation and invasion in vitro.In addition,epidermal growth factor receptor(EGFR)was identified as a direct target gene of miR-509 in TSCC cells.EGFR downregulation was demonstrated to suppress the proliferation and invasion of TSCC cells,similar to miR-509 overexpression.Furthermore,EGFR was significantly upregulated in TSCC tissues,and the levels of miR-509 were revealed to be negatively correlated with EGFR expression in TSCC tissues.Following transfec-tion with miR-509 mimics,signaling pathways downstream of EGFR appeared to be suppressed,as phosphorylated(p)-extracellular signal-regulated kinase and p-Akt were downregulated in TSCC cells.In conclusion,the results of the present study suggested that miR-509 may inhibit the proliferation and invasion of TSCC cells via directly targeting EGFR,thus suggesting that the miR-509/EGFR axis may have potential as a novel therapeutic target for the development of a treatment for patients with TSCC.Part 1.miR-509 inhibits TSCC cells proliferation and migration aswell as invasion in vitro[Objectives]MiR-509 has been identified as a critical regulator in tumorigenesis and tumor development,via its tumor-suppressing actions in several types of human cancer.In this part we aimed to detect whether miR-509 could inhibit TSCC cell proliferation,migration and invasion through in vitro experiments,and further explored underlying mechanisms.[Methods]1)qRT-PCR was used to detect the expression of miR-509 in TSCC tumor tissues and cell lines.2)MTT method was used to detect the effect of overexpression of miR-509 on the proliferation in TSCC cell lines(Tca8113,CAL-27).3)Transwell cell migration and invasion assays were used to detect the impact of overexpression of miR-509 on the migration and invasion in TSCC cell lines(Tca8113,CAL-27).[Results]1)miR-509 was down-regulated expressed in 28 paired TSCC tissues and TSCC cell lines(Tca8113,SCC-15,CAL-27).2)In TSCC cell lines(Tca8113,Cal-27),overexpression of miR-509 could inhibit cell proliferation.After the transfection of 2 days and 3 days,the proliferation of cells decreased significantly,and the differences were statistically significant.3)Transwell cell migration and invasion assays showed that overexpression of miR-509 significantly inhibited the migration and invasion of Tca8113,Cal-27 cells.[Conclusions]MiR-509 was down-regulated expressed in TSCC tissues and TSCC cell lines(Tca8113,SCC-15,CAL-27).MiR-509 inhibited the proliferation,migration and invasion of Tca8113,Cal-27 cells and played the role of tumor suppressor in TSCC.Part 2.Identification of target genes directly targeting miR-509[Objectives]Through target gene prediction software,we searched for the target genes which were directly targeted to miR-509 but not been identified to regulate miR-509 in TSCC.We verified whether the expression of selected target genes EGFR mRNA and protein were regulated by miR-509 in TSCC tissues and cell lines,and further explored underlying mechanisms.[Methods]1)Using target gene prediction software microma.org(http://www.microrna.org)and TargetScan(www.targetscan.org),we screened target genes that might directly target miR-509,and further screened based on mirSVR values.2)The expression of EGFR protein and downstream signal pathway of EGFR was detected by Westemblot in TSCC cell line(Tca8113,CAL-27)after transfection with miR-509 mimics and miR-NC,and the expression of EGFR mRNA was detected by qRT-PCR after transfection with miR-509mimics.3)Dual luciferase reporter assays were performed to test whether the 3 'UTR side of EGFR mRNA was the direct target of miR-509.4)RNAi was performed to test that whether or not silenced EGFR could mimic the declined capacity of proliferation,migration and invasion of TSCC cell line(Tca8113,CAL-27)caused by overexpression of miR-509.5)qRT-PCR was performed to detect the expression of EGFR mRNA in TSCC tissue.Spearman's correlation analysis was used to evaluate the correlation between miR-509 and EGFR mRNA expression in TSCC tissues.[Results]1)The candidate target gene(EGFR)for miR-509 were selected through Computational algorithms.2)After transfection with miR-509 mimics and miR-NC 72 hours,westernblot experiments showed that transfection with miR-509mimics group significantly inhibited the expression of EGFR,P-AKT and P-ERK1/2 protein in Tca8113 and CAL-27 cells.QRT-PCR confirmed that transfection with miR-509 mimics group downregulated the expression of EGFRmRNA in Tca8113 and CAL-27 cells.3)Dual luciferase reporter assays suggested that miR-509 regulated EGFR expression by targeting 3'UTR of EGFR mRNA directly.4)RNAi experiments confirmed that knocking down of EGFR could significantly inhibit Tca8113,CAL-27 cell proliferation and migration as well as invasion,similar to the biological changes of the tumor cells caused by the overexpression of miR-509.5)EGFRmRNA were highly expressed in 28 paired TSCC tissues.Spearman's correlation analysis showed that an inverse correlation was detected between EGFR mRNA and miR-509 expression in TSCC tissues.[Conclusions]1)Overexpression of miR-509 could downregulate EGFR and downstream signal pathway of EGFR(P-AKT,P-ERK1/2)expression in TSCC cell line(Tca8113,CAL-27).2)The 3'UTR side of EGFRmRNA is the direct target of miR-509.3)MiR-509 is involved in the development and progression of TSCC tumors by negatively regulating the expression of EGFR.