| Introduction:Laryngeal carcinoma is a malignant tumor originated from the epithelial cells of laryngeal mucosa and the second most common head and neck tumor in the world.Like most of the cancer,both environmental and genetic factors contribute to laryngeal carcinogenesis.Despite variously advanced treatment interventions used,incidence of laryngeal cancer has been increasing steadily and 5-year survival rate of patients has not been markedly improved.Therefore,studies on the mechanism of laryngeal cancer onset and development at molecular level are still the focus and difficulty of laryngeal cancer research,which will help to discover biological markers for early diagnose and therapy of laryngeal carcinoma and has important theoretical significance and potential clinical application value.It is well known that tumors are caused by excessive proliferation of abnormal cells.The imbalance between cell viability and apoptosis represents an important cause of carcinogenesis,and the degree of the imbalance determines the speed of carcinogenesis.Therefore,the study on cell proliferation and apoptosis mechanism is of importance in tumongenesis.We previously identified and cloned human MYCT1 as a new candidate tumor suppressor gene from laryngeal cancer tissues by electron hybridization and molecular biology methods.Our previous study has also demonstrated that MYCT1 significantly promotes apoptosis and represses viability and invasion in Hep2 cells as a tumor suppressor.However,the molecular mechanism through which MYCT1 promotes laryngeal cancer cell apoptosis remains unknown.Based on laryngeal carcinoma cells stably expressing MYCT1,we found a series of differentially expressed genes related to apoptosis including NPM1 by RNA-seq.NPM1 gene encodes a shuttle protein that travels between nucleus and cytoplasm.In addition to constituting the nucleolus,NPM1 is also involved in regulation of cancer cell apoptosis via both extracellular death receptor pathway and intracellular mitochondrial pathway.In the previous study,we have confirmed that NPM1 is one of miR-181a targets,and have revealed the biological functions of miR-181a in laryngeal cancer,but whether and how NPM1 is involved in the pathogenesis of laryngeal cancer is yet to be investigatedUsing bioinformatics software,we found that MYCT1 has a potential interaction with MAX,and miR-181a is a putative target of MAX.Product encoded by MAX is MYC-associated factor X(MAX),a transcription factor belonging to the basic helix-loop-helix leucine zipper(bHLHZ)family.Studies have shown that MAX regulates variously biological processes including tumor cell apoptosis by manipulating transcription of numerous targets.Whether or not MAX takes part in regulation of laryngeal cancer cell apoptosis is unclear as well.Taken together,we speculate that MYCT1 can mediate apoptosis of laryngeal cancer cells through MAX,miR-181a and NPM1 signal,which is also the major goal and innovation of this researchIn conclusion,we will detect role and function of NPM1 in laryngeal cancer by related molecular biology techniques,and identify relationships among MYCT1,MAX and miR-181a.We will also investigate whether MYCT1/MAX/miR-181a/NPM1 axis promotes laryngeal cancer cell apoptosis through death receptor pathway and mitochondrial pathwayMaterials and methods:1.Materials:human embryo kidney cell line HEK293T,human laryngeal squamous cell cancer cell line Hep2,laryngeal carcinoma and adjacent tissues2.Methods:cell culture,Real-time PCR assay,transient gene transfection,Western blot,luciferase reporter assay,chromatin immunoprecipitation(ChIP),Co-Immunoprecipitation(Co-IP),Immunofluorescence assay,flow cytometry,CCK-8 assay,colony formation assay,caspase activity assay and statistical analysisResults:1.Real-time PCR and Western blot results showed that NPM1 was significantly up-regulated in laryngeal carcinoma tissues compared to the adjacent tissues(P<0.05),and was negatively regulated by MYCT1(P<0.05)2.CCK8 and colony formation detection results showed that overexpression of NPM1 significantly promoted proliferation and colony formation of Hep2 cells compared to controls(P<0.05);whereas,inhibition of NPM1 expression significantly inhibited proliferation and clonal formation of Hep2 cells(P<0.05)3.Flow cytometry assay result indicated that inhibition of NPM1 expression significantly promoted the early apoptosis rate of Hep2 cells compared to controls(P<0.05),but overexpression of NPM1 has no significant effect on early apoptosis of Hep2 cells(P>0.05)4.Bioinformatics software prediction result showed that MYCT1 has potential interaction with MAX.Co-IP result showed that MYCT1 interacted with MAX in Hep2 cells5.Immunofluorescence result revealed that co-localization of MYCT1 and MAX proteins was enhanced in nuclei of Hep2 cells overexpressing MYCT1 compared to controls,suggestting that MYCT1 promoted nuclear translocation of MAX6.Bioinformatics prediction result showed several potential MAX binding sites in miR-181a promoter region.Result of Real-time PCR showed that the expression level of miR-181a was markedly reduced in Hep2 cells after MAX knockdown(P<0.05)7.ChIP result confirmed that MAX could bind to miR-181a promoter,and MAX siRNA could significantly reduce the ability of MAX binding to the promoter in Hep2 cells(P<0.05)8.Result of luciferase reporter assay showed that MAX or MYCT1 knockdwon significantly decreased transcription activity of miR-181a promoter(P<0.05),suggesting that MAX could directly promote miR-181a transcription9.Real-time PCR result showed that miR-181a was significantly down-regulated in laryngeal carcinoma tissues compared to the adjacent tissues(P<0.05),and was up-regulated by MYCT1(P<0.05)10.CCK8 and colony formation detection results showed that miR-181a inhibition significantly promoted proliferation and colony formation of Hep2 cells compared to controls(P<0.05),whereas its overexpression significantly inhibited proliferation and clonal formation of Hep2 cells(P<0.05).However,NPM1 overexpression significantly recovered the influence of miR-181a on the above functions(P<0.05)11.Flow cytometry assay result indicated that miR-181a inhibition significantly inhibited early apoptosis of Hep2 cells(P<0.05),whereas its overexpression significantly promoted early apoptosis of Hep2 cells compared to controls(P<0.05).However,NPM1 overexpression significantly recovered the influence of miR-181a on early apoptosis of Hep2 cells(P<0.05)12.CCK8 and colony formation detection results showed that MYCT1 overexpression significantly inhibited proliferation and colony formation of Hep2 cells compared to controls(P<0.05).However,NPM1 overexpression or miR-181a inhibition significantly rescued the influence of MYCT1 on the above functions(P<0.05).13.Flow cytometry assay result indicated that compared to controls,MYCT1 overexpression significantly promoted early apoptosis of Hep2 cells(P<0.05).However,NPM1 overexpression or miR-181a inhibition significantly rescued the influence of MYCT1 on early apoptosis of Hep2 cells(P<0.05).14.Caspase activity assay and Western blot results showed that overexpression of MYCT1 significantly increased the activities and expression of Caspase-related proteins compared to controls(P<0.05).However,NPM1 overexpression or miR-181a inhibition could significantly recover the effect of MYCT1 on the activities and expression of Caspase-related proteins(P<0.05).Conclusions:1.NPM1 is up-regulated in laryngeal carcinoma,which is negatively correlated with MYCT1 expression,promotes proliferation and inhibits apoptosis of laryngeal carcinoma cells,suggesting that it plays an oncogenic role in laryngeal cancer.2.MYCT1 interacts with MAX in Hep2 cell nuclei.3.MAX acts as a transcription factor that binds to the miR-181a promoter region and promotes its transcription.4.miR-181a is down-regulated in laryngeal cancer,positively correlated with MYCT1 expression,inhibits Hep2 cell proliferation and promotes apoptosis through NPM1,suggesting that miR-181a plays a suppressive role in laryngeal cancer.5.MYCT1 binding to MAX promotes miR-181a expression whereas in turn inhibits NPM1 expression leading to laryngeal cancer cell apoptosis via the death receptor and mitochondrial signal pathways. |
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