MIR-145 Inhibits Invasion By Directly Targeting Smad3 In Nasopharyngeal Cancer

Background and Objective: Nasopharyngeal carcinoma is a highly malignant tumor with the strong ability of invasive and metastatic in head and neck carcinomas, which is prevalent in Asian, particularly in southern China.With the development of epigenetics, more and more attention was paied to the role of mi RNAs in many kinds of cancers in recent years. It has been reported that mi R-145 act as a tumor suppressor in many cancersand down-regulated in nasopharyngeal carcinoma tissues, however, there is presently not verymuch known about mi R-145 involvement in nasopharyngeal carcinoma. In this study, we verify the expression of mi R- 145 in nasopharyngeal carcinoma tissuesamples andnasopharyngeal cell lines,furtherexplore the biological functions of mi R-145 in nasopharyngeal carcinoma and its possible regulatory mechanism,providean experimental basisfor diagnosis and treatment of nasopharyngeal carcinoma.Methods:(1) To research the relative level of mi R-145 innasopharyngeal carcinoma, quantitative real-time PCR and Western Blot were initially performed in 3nasopharyngeal carcinoma cells and NP69 and 11 pairednasopharyngeal carcinoma tissue specimens.(2). mi R-145 mimics(or mi R-NC) and mi R-145 inhibitor(or mi R-NC inhibitor).were transfected high metastatic nasopharyngeal cell lines(CNE-2, CNE-2Z). Then, we evaluated the cell proliferation, migration, and invasion using Cell Counting Kit-8 assay and transwell assay in nasopharyngeal carcinoma cells.(3) Firstly, we predicted the potential gene targets of mi RNA-145 by Targetscan, Pic Tarand mi Randabioinformatics software, then performed dual-luciferase assay to verify whether Smad3 is a direct target gene of mi R-145 in nasopharyngeal carcinoma.Finally, we detected the expression of Smad3 in transfected with mi R-145 mimics(or mi R-NC) and mi R-145 inhibitor(or mi R-NC inhibitor)in CNE-2 and CNE-2Z cells.(4)Transfected si-Smad3(or si-NC)to CNE-2 and CNE-2Z cell lines, we detect the expression of Smad3 to ensure the success of the transfection, and then evaluated the cell proliferation, migration, and invasion.(5)Detected the relative expression of the Smad3 in NP69 and three nasopharyngeal carcinoma cell lines and 11 pairednasopharyngeal carcinoma tissue samples by using quantitative real-time PCR.Meanwhile,performed regression analysis of the target gene Smad3 and mi R-145 expression to explore their correlation.Results:(1) The level of mi R-145 wasobviously decreased in CNE-1, CNE-2 and CNE-2Z cells when compared with NP69.The mi R-145 wassignificantly lower in 11 pairednasopharyngeal carcinoma tissues when compared with the non-tumor nasopharyngeal tissues.In a word,the level of mi R-145 is decreased in nasopharyngeal carcinoma cells and tissues.(2)After transfected with mi R-145 mimics, the proliferation,migration and invasion of CNE-2 and CNE-2Z cells was reduced; whereasknockdown of mi R-145 with mi R-145 inhibitor, show theopposite results.(3) Bioinformatics software analysis showed that multiple genes are able to be potentially target of mi R-145.It was validated that mi R-145 can decreased Smad3 expression bydirectly targeting the seed sequence of Smad33’UTR in CNE-2 and CNE-2Z cells through mi R-145(or mi R-NC) and mi R-145 inhibitor(or mi R-NC inhibitor) transient transfection and Dual Luciferase Reporter Assay experiment. At last, Smad3 was chosen as the target gene of mi R-145 in this study.(4)The expression of Smad3 protein was obviously decreased after transfected with si-Smad3( or si-NC), suggests that the transfection iseffective. The proliferation ability show no difference between si-Smad3 group and si-NC group cells. However, the migration and invasion ability of si-Smad3 transfected cells was decreased, indicated that Smad3 may promote metastasis of nasopharyngeal carcinoma.Apparently higher expression of Smad3 wasdetected in CNE-1,CNE-2 and CNE-2Z cell lines when compairedto that in NP69. Meanwhile,the expression level of Smad3 was found to be increased in 11 pairednasopharyngeal carcinoma tissue compared with thenon-tumor nasopharyngeal tissues.In conclusion,the level of Smad3 is elevated in nasopharyngeal carcinoma cells and tissues. Furthermore, regression analysis revealed that the ratio of mi R-145 and Smad3 m RNA level was negative correlated in 11 paired tissues.In this study, we have characterized that mi R-145 is down-regulated in nasopharyngeal carcinoma, and is able to inhibit the proliferation and invasion ability of nasopharyngeal carcinoma cells.By bioinformatics and biologicalfunctiontest we verified that Smad3 isthedirecttarget genesofmi R-145.This provides a theoretical basisforfurtherin-depth studyofnasopharyngeal carcinoma pathogenesis and treatment.