Expression Of MiR-362-3p In Laryngeal Cancer And Preliminary Study Of Its Effect On Laryngeal Cancer Cell Migration

Introduction: Laryngeal cancer is one of the most common malignant neoplasms of the head and neck,accounting for 1% of cancer-related deaths worldwide.Over 90% of laryngeal carcinoma is squamous cell carcinoma.Local invasion and distant lymph node metastasis are main causes of death in laryngeal cancer patients.Although treatment for laryngeal cancer is improving,the five-year survival rate is still not optimistic.Therefore,it is significant to further study molecular mechanism of laryngeal cancer development and find novel therapeutic target for diagnosis,treatment and prognostic assessment of laryngeal cancer.miR-362 is associated with progression of gestational diabetes mellitus,epidermal cell senescence,atherosclerosis,cancer and various other diseases.miR-362-3p is reported to play an important role in migration of renal cancer cells and gastric cancer cells.However,less is understood about function of miR-362-3p in laryngeal cancer.Previously,we cloned MYCT1 gene by electronic hybridization technique.Report has shown that MYCT1 suppresses poliferation,clone formation,migration of cancer cells and promotes apoptosis of cancer cells,which plays an inportant role in development of laryngeal cancer and other cancers.In addition,by Next Generation Sequencing,we found that miR-362-3p expression was significantly difference in overexpressed MYCT1 laryngeal cancer cells compared to control group,which suggests that miR-362-3p is a downstream gene of MYCT1.Based on Targetscan and Starbase programs,we found that ALDOA is a potential target of miR-362-3p.Studies have shown that ALDOA is abnormally expressed and related to cancer cell migration in many cancers such as pancreatic cancer,lung cancer,colorectal cancer,gastric cancer and renal cell carcinoma.At present,role of ALDOA in laryngeal cancer is not clear.Here,we will detect miR-362-3p expression in laryngeal cancer,explore its clinical significance in laryngeal cancer and investigate its effects on laryngeal cancer migration.We will also explore whether miR-362-3p regulates laryngeal cancer cell migration via ALDOA,which will provide a clue for finding biomarker for early diagnosis and prognosis of laryngeal cancer.Materials and methods Materials:Cell lines: Human laryngeal squamous cell carcinoma cell line Hep2,human embryo kidney cell line HEK293,human pharynx squamous cell carcinoma cell line FADU,human laryngeal carcinoma cell line TU212. Tissue samples: Laryngeal cancer and paired normal tissues were obtained from 50 patients at the otolaryngology department of People's Liberation Army No.463 Hospital from January 2015 to November 2017.All tissue samples were immediately frozen at-80? after removed from patients.Verification of the specimens was performed by a pathologist.Detailed clinical data was also collected with informed consents of patients.The research was approved by the Ethics Committee of China Medical University.Methods: 1.Cell culture 2.Transient gene transfection 3.qRT-PCR assay 4.Western blot 5.Cell wound-healing assay 6.Transwell chamber method 7.Analysis of clinical data 8.Statistical analysis Result: 1.qRT-PCR results showed that miR-362-3p was significantly overexpressed in laryngeal cancer tissues and cells compared to controls(P<0.05).The results suggests that miR-362-3p plays an oncogenic role in laryngeal cancer.2.One-Way ANOVA result showed that miR-362-3p was significantly upregulated in cancer tissues of laryngeal cancer patients with lymph node metastasis and higher clinical stage compared to controls(P<0.05),but there was no significant difference in miR-362-3p expression in cancer tissues related to different age,sex,differentiation degree and invasion depth(P>0.05).These results indicates that miR-362-3p is related to malignant degree in laryngeal cancer.3.qRT-PCR result revealed that miR-362-3p was significantly upregulated or downregulated in Hep2 cells transfected with miR-362-3p mimic or inhibitor compared to control group(nc),respectively(P<0.05),indicating the success of transfection.Migration assay and cell wound-healing assay results displayed that both cell migration number and distance were significantly increased or decreased in Hep2 cells transfected with miR-362-3p mimic or inhibitor compared to control group(nc),respectively(P<0.05),suggesting that miR-362-3p promotes laryngeal cancer migration.4.Based on Targetscan and Starbase programs,miR-362-3p binding site was found in the 3'-untranslated region of ALDOA mRNA,implying that ALDOA is a potential target of miR-362-3p.qRT-PCR and Western blot results revealed that ALDOA was downregulated or upregulated both at mRNA and protein levels in Hep2 cells transfected miR-362-3p mimic or inhibitor compared to control,respectively(P<0.05).These results indicate that ALDOA is a possible target of miR-362-3p.5.Western blot result showed that ALDOA was downregulated in Hep2 cells transfected with siALDOA,suggesting that transfection is successful.Migration and cell wound-healing assay results revealed that both migration number and distance of Hep2 cells transfected with siALDOA were significantly increased,whereas the effects of miR-362-3p inhibitor on laryngeal cancer cell migration were rescued in Hep2 cells when co-transfected with siALDOA and miR-362-3p inhibitor,suggesting that miR-362-3p influences laryngeal cancer cell migration via ALDOA.Conclusion: 1.miR-362-3p plays an oncogenic roles in laryngeal cancer tissues and its expression is associated with malignant degree of laryngeal cancer.2.miR-362-3p promotes laryngeal cancer cell migration.3.ALDOA is a possible target of miR-362-3p.4.miR-362-3p promotes laryngeal cancer cell migration partly via ALDOA.