Study On MYCT1 In Laryngeal Carcinoma Cell Migration Via MiR-92b/TOB1

Introduction: Laryngeal carcinoma is a common cancer of the head and neck.Due to unobvious early symptoms,patients are often accompanied by invasion and metastasis when diagnosed.With present treatment,patients suffer from low survival quality and poor prognosis.Therefore,searching for invasion and metastasis related molecular markers of laryngeal cancer and study on the molecular mechanisms of laryngeal cancer cell invasion and metastasis is quite necessary.MYCT1 is a laryngeal cancer-related gene cloned in our group.We also found that MYCT1 inhibits laryngeal cancer cell proliferation,migration and promotes apoptosis,indicating that it acts as a tumor suppressor gene in laryngeal cancer.However,its molecular mechanism in laryngeal cancer is still unclear.miRNAs are a family of non-coding small RNA molecules of 18-25 nucleotides,which inhibit m RNA translation or degrades it by binding to the 3′-untranslated region(3′ UTR)of the target m RNA.Studies have shown that miRNAs are dysregulated in rheumatoid arthritis,chronic pancreatitis,neuralgia and tumor,regulating the occurrence and progression of diseases through complex regulatory networks.In laryngeal cancer cells,we found that MYCT1 over-expression results in differential expression of a series of miRNAs.miR-92b is one of the down-regulated miRNAs,indicating that it may serve as a downstream regulator of MYCT1 signaling pathway.Studies have shown that miR-92b promotes the proliferation of Ep CAM+ embryonic liver cell proliferation and inhibits its differentiation.miR-92b also inhibits the hypertrophy of the myocardium.In the meantime,miR-92b is differentially expressed in non-small cell lung cancer,osteosarcoma,glioma,hepatocellular carcinoma,gastric cancer,pancreatic cancer and nasopharyngeal carcinoma,acting as either an oncogene or a tumor suppressor gene.At present,whether miR-92b parcitipates in the occurance and development of laryngeal cancer has not yet been reported.Based on the prediction of bioinformatics software such as Target Scan and miRDB,we found that there is a miR-92b binding site at the 3′ UTR of TOB1,suggesting that TOB1 is one of the potential target genes of miR-92b.TOB1 is a member of the BTG/TOB anti-proliferative protein family members,and can bind to deadenylation enzyme to form a protein complex,regulating a variety of normal biological processes.TOB1 is down-regulated in lots of cancer,such as gastric cancer,breast cancer,lung cancer,and so on.TOB1 inhibits cancer cell proliferation,migration and invasion,enchances apoptosis,increases sensitivity to radiotherapy of tumor cells,indicating that it acts as a tumor suppressor gene.However,study on the mechanism of TOB1 in the migration and invasion of laryngeal carcinoma has not been reported.Therefore,we speculate that the decreased MYCT1 expression in laryngeal cancer leads to miR-92b up-regulation,latter of which inhibits TOB1 expression,leading to laryngeal cancer cell migration via some TOB1 related signal pathways.In the present study,we will detect miR-92b and TOB1 expression in laryngeal cancer,confirm the binding of miR-92b to 3′ UTR of TOB1 and explore whether MYCT1 regulates laryngeal carcinoma cell migration through miR-92b/TOB1 –associated pathway,which provides novel clue in finding potentially molecular biomarkers related to the invasion and metastasis of laryngeal cancer.Materials and methods: Forty-six pairs of laryngeal carcinoma and adjacent tissue samples provided by Department of ENT,463 Hospital of PLA were collected.Total RNA was extracted and Real-time quantative PCR was used to detect miR-92b and TOB1 transcription level in laryngeal carcinoma tissues and adjacent normal tissues.Human laryngeal squamous cell carcinoma Hep2 cell line was cultured in RPMI1640 medium containing 10% fetal bovine serum,cultured in 5%CO2 cell incubator at 37℃.Plasmid overpressing MYCT1 was transfected into Hep2 cells.Real-time q PCR was used to detect MYCT1,miR-92b and TOB1 transcription level in MYCT1-treated and untreated Hep2 cells.Western blot was applied to monitor MYCT1 and TOB1 protein in MYCT1-treated and untreated Hep2 cells.Bioinformatics softwares were used to predict the potential downstream target gene of miR-92b,TOB1.We then constructed wild and mutant type TOB1 3 ’ UTR luciferase reporter gene plasmids,which were co-transfected with miR-92b mimic into Hep2 cells,respectively.Dual-luciferase reporter gene assay was performed and relative fluorescence values were calculated.Transfection of miR-92b mimic and inhibitor was then performed in Hep2 cells.Real-time q PCR was used to detect the RNA level of both miR-92b and TOB1 and western blot was used to detect TOB1 protein expression in Hep2 cells.Real-time q PCR and western blot were used to detect TOB1 m RNA and protein level after MYCT1 plasmid and miR-92b mimic were transfected,respcetively,or co-transfected into Hep2 cells.miR-92b inhibitor and si TOB1 were transfected,respectively,or co-transfected into Hep2 cells.Migration ability of laryngeal cancer cell was then detected by Transwell chamber method.Finally,results above were analyzed by different statistical methods.Results: 1.Real-time q PCR result displayed that miR-92b and TOB1 transcription levels in laryngeal cancer tissue was significantly higher and lower than that in normal adjacent tissue,respectively(P <0.05),which showed negative correlation(r=-0.347,P <0.05).2.Real-time q PCR and western blot results showed that both MYCT1 m RNA and protein levels in Hep2 cells transfected with MYCT1 overexpressing plasmid significantly increased,and miR-92b expression was significantly decreased(P <0.05).3.Bioinformatics prediction results of Target Scan and miRDB showed that there is a potential miR-92b binding site within TOB1 3′ UTR and the sequence is highly conservative among species.Dual luciferase reporter assay results showed that compared with control group,the luciferase activity of miR-92b mimic and TOB1 3′ UTR wild type luciferase plasmid cotransfection group was significantly decreased(P <0.05).4.Real-time q PCR and Western blot results showed that in Hep2 cells transfected with miR-92b mimic,inhibitor and the corresponding control fragment,compared with the control group,the level of miR-92b increased and decreased respectively(P <0.05),TOB1 m RNA and protein levels were decreased and increased(P <0.05).5.Real-time q PCR and Western blot results indicated that in Hep2 cells transfected with MYCT1 plasmid and miR-92b mimic,TOB1 m RNA and protein levels were both significantly increased and decreased(P <0.05).However,there was no significant difference at both TOB1 m RNA and protein levels in Hep2 cells co-transfected with MYCT1 plasmid and miR-92b mimic compared to the controls(P >0.05).6.Transwell results revealed that the number of migrated Hep2 cells significantly decreased and increased in Hep2 cells transfected with MYCT1 expression plasmid and miR-92b mimic compared to the controls,respectively(P <0.05).However,there was no significant difference in Hep2 cells co-transfected with MYCT1 plasmid and miR-92b mimic compared to the controls(P >0.05).7.Transwell results showed that the number of migrated cells significantly decreased and increased in Hep2 cells transfected with miR-92b inhibitor and si TOB1 compared to the controls,respectively(P <0.05).However,there was no significant difference in Hep2 cells co-transfected with miR-92b inhibitor and si TOB1 compared to the controls(P >0.05).Conclusions: 1.miR-92b and TOB1 are up-regulated and down-regulated in laryngeal cancer,respectively,whose expression levels show a negative correlation,implying that they play oncogenic and suppressive roles in layngeal tumorigenesis.2.TOB1 is a direct target of miR-92b.3.MYCT1 inhibits laryngeal cancer cell migration via miR-92b/TOB1.