| Introduction: Laryngeal carcinoma is a common malignant tumor,of which squamous cell carcinoma is the most common.Causes of laryngeal cancer are complex and studies have shown that the occurrence and development of laryngeal cancer are related to smoking,drinking,exposure to harmful dust and human papilloma virus infection.Despite advances in treatment techniques including surgery,chemotherapy,radiotherapy and gene therapy,five years of total survival in laryngeal cancer patients have not been improved significantly in the past decade.Therefore,search for new biomarkers in early diagnosis and individualized treatments for laryngeal cancer is a hotspot at present.Micro RNAs(mi RNAs)are a kind of non-coding small RNAs with 18 ~ 24 nucleotides in length and have important biology function.mi RNA are involved in regulation of many life processes such as cell survival,apoptosis,proliferation,differentiation and migration.mi RNA,present in lots of liquids in human body and abnormally expressed many human tumors,are useful biomarkers in human diseases including cancers.mi R-181 a is a member of mi R-181 family and can influence cancer cell proliferation,apoptosis,adhesion,invasion,metastasis and modulating multiple target genes.mi R-181 a plays roles as a tumor suppressor gene in glioma,cervical,lung and breast cancer,and as an oncogene in liver and stomach cancer.At present,related study on mi R-181 a in laryngeal cancer is not reported.We predicted that NPM1 is a potential target gene of mi R-181 a by several softwares such as Mi Randa.In recent years,studies of NPM1 mainly focused on its relationship with blood system tumor.In addition,NPM1 has been shown to be over-expressed in some solid cancers including liver,prostate and lung cancer,indicating its role as an oncogene in cancer.However,the role of NPM1 in the oncogenesis and development of laryngeal cancer has not been studied either.In the study,we examined mi R-181 a and NPM1 expression,identified the target NPM1 of mi R-181 a and examined biological functions of mi R-181 a and NPM1 in laryngeal cancer via molecular biology technology,which might provide a basis in further investigation of functional mechanism and probability of mi R-181 a as a potential biomarker in laryngeal cancer.Materials and methods: 1.Cell lines and tissue samples: human laryngeal carcinoma cell line Hep2,human embryonic kidney cell line HEK293 T and laryngeal cancer tissues.2.Experimental methods: cell culture,gene transient transfection experiment,q RT-PCR detection,flow cytometry,CCK8 detection and cloning formation assay,luciferase reporter gene activity test and statistical analysis.Results: 1.q RT-PCR result showed that mi R-181 a levels in laryngeal cancer tissues and Hep2 cells were significantly reduced compared to the controls(P<0.05).NPM1 m RNA levels in laryngeal cancer tissues and Hep2 cells were increased significantly compared to the controls(P<0.05).mi R-181 a and NPM1 transcription levels in laryngeal cancer were negatively correlated(r =-0.4185,P<0.01).Western blot result indicated that NPM1 protein levels in laryngeal cancer tissues were significantly higher than controls(P<0.05).These results suggested that mi R-181 a and NPM1 act as a tumor suppressor and an oncogene in laryngeal cancer,respectively.2.Luciferase reporter assay result showed that luciferase activity of Hep2 cells co-transfected with Lut-NPM1-Wt and mi R-181 a mimic significantly decreased compared to the controls(P<0.05).Real-time PCR and Western blot results revealed that both NPM1 m RNA and protein levels were significantly decreased or increased,respectively,in Hep2 cells transfected with mi R-181 a mimic and inhibitor at 48 h compared to the controls(P<0.05).The results above indicated that NPM1 is a direct target of mi R-181 a.3.CCK8 and clone formation detection results displayed that proliferation and clone formation abilities of Hep2 cells in mi R-181 a mimic transfection group decreased significantly compared to the controls(P<0.05).On the contrary,proliferation and clone formation abilities of Hep2 cells in mi R181 a inhibitor group were significantly higher than those in the controls(P<0.05),suggesting mi R-181 a inhibits proliferation of Hep2 cells.4.Flow cytometry assay result indicated that Hep2 cell early apoptosis at 48 h after transfection of mi R-181 a mimic or mi R-181 a inhibitor was significantly higher or lower compared to the control groups(P<0.05),implying that mi R-181 a promotes Hep2 cell apoptosis.5.Flow cytometry result in cell cycle detection showed that Hep2 cells in S phase increased significantly at 48 h after transfection of mi R-181 a mimic compared to the controls(P<0.05),while cells in G0/G1 phase and the G2/M phase decreased significantly(P<0.05),speculating that mi R-181 a inhibits laryngeal cancer cell proliferation by inducing S phase arrest.6.CCK8 and clone formation detection results showed that proliferation and clony formation abilities of Hep2 cells transfected with NPM1 significantly increased compared to the controls(P<0.05).While in Hep2 cells transfected with si-NPM1,the proliferation and clone formation abilities decreased significantly compared to the controls(P<0.05),suggesting NPM1 promotes Hep2 cell proliferation.7.Flow cytometry results in detecting apoptosis displayed that early apoptosis of Hep2 cells transfected with si-NPM1 significantly reduced compared to the controls(P<0.05).However,Hep2 cells transfected with NPM1 plasmid showed no significant change in early apoptosis compared to the controls(P>0.05),suggesting NPM1 knockdown increases Hep2 cell early apoptosis.8.Flow cytometry result in cell cycle detection demonstrated that the ratios of Hep2 cells in G0/G1 phase at 48 h after transfected with si-NPM1 and NPM1 plasmid significantly increased and decreased compared to the controls,respectively(P<0.05),those of cells in S phase and G2/M phase significantly decreased and increased,respectively(P<0.05),suggesting NPM1 promotes the proliferation of laryngeal carcinoma cells by inducing the cell to evolve from G0/ G1 phase to S phase.Conclusion: 1.mi R-181 a and NPM1 are down-regulated and up-regulated in laryngeal cancer,respectively,and they are negatively correlated at transcription level.These suggest that mi R-181 a and NPM1 act as a tumor suppressor and an oncogene in laryngeal cancer,respectively.2.NPM1 is a direct target of mi R-181 a.3.mi R-181 a and NPM1 respectively inhibit and promote the proliferation of laryngeal carcinoma cells,of which the former inhibits the proliferation of laryngeal carcinoma cells through S phase arrest,while the latter promotes the proliferation of laryngeal carcinoma cells by inducing the G0 / G1 phase to S phase.4.mi R-181 a and NPM1 promote and inhibit laryngeal cancer cell early apoptosis,respectively,moreover,NPM1 represses the promoting effect of mi R-181 a on the apoptosis of laryngeal carcinoma cells,suggesting that mi R-181 a could influence laryngeal cancer cell early apoptosis via targeting NPM1. |
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