| Introduction: Laryngeal squamous cell carcinoma is the biggest problem that plagues patients with head and neck squamous cell carcinoma,and the incidence is about 1/3 of head and neck squamous cell carcinoma.In China,there are about 20,000 new cases of laryngeal cancer each year,and the incidence rate remains high.The major risk factor for laryngeal cancer was smoking,followed by multiple risk factors including HPV infection,environmental and work exposure.The easy recurrence and metastasis of laryngeal cancer has always been a huge problem for patients with laryngeal cancer.In recent years,laryngeal surgery,radiotherapy,chemotherapy,gene therapy and other comprehensive treatment measures have achieved a high 5-year survival rate for patients with laryngeal cancer,but 30-40% of patients with laryngeal cancer still die of tumor recurrence or metastasis.The development of new treatment modalities,combined with biomarkers,and the selective application of this new treatment approach in these patients may be a new modality to help determine improved survival.Therefore,finding the important molecular biological mechanism in the occurrence and development of laryngeal cancer,exploring the genes related to laryngeal cancer invasion and metastasis,and introducing new biomarkers as the predictors of laryngeal cancer,so as to carry out the diagnosis and treatment of laryngeal cancer in its early stage are the key issues that people need to pay attention to.MYCT1(MYC Target 1)is a potential tumor suppressor gene that has been the focus of our research group.Studies have shown that MYCT1 gene is under-expressed in gastric cancer,liver cancer,laryngeal cancer and cervical cancer,and overexpression of MYCT1 can significantly promote the apoptosis of tumor cells,suggesting that MYCT1 has a potential antitumor effect.Our previous research results showed that MYCT1 overexpression inhibited the migration and invasion ability of laryngeal cancer Hep-2cells,and promoted cell apoptosis.However,there have been few reports on the mechanism of MYCT1 regulating the biological function of laryngeal cancer cells. Therefore,the study on the molecular mechanism of MYCT1 regulating downstream genes will lay a theoretical foundation as a target for the early diagnosis and treatment of laryngeal cancer.In our research group,we previously constructed laryngeal cancer Hep-2 cell lines that expressed MYCT1 stably,and screened the downstream differentially expressed genes of MYCT1 through protein microarray.It was found that the expression level of ADAM20(a disintegrin and metalloproteinase 20)was significantly decreased in the overexpression cell lines of MYCT1,suggesting that MYCT1 may have a negative regulation effect on ADAM20.ADAM20 is one of the Cancer/Testis Antigen(CTA)genes,which are normally only expressed in male testicular tissues,but are reactivated in some malignant tumors.Due to the immunogenicity of these reactivated testicular proteins,they are named as tumor/testicular Antigen(CTA).Studies have shown that CTA are reactivated in a significant proportion in lung,ovary,bladder,melanoma and breast tumors,and to a lesser extent in leukemia and lymphoma,as well as in kidney,colon and pancreas tumors.The reexpression of CTA gene is one of the important markers of tumor growth,but the specific role of CTA gene in tumor remains unclear.ADAM20 is a member of the protein family containing the deintegrin domain and the metalloproteinase depolymerization domain(ADAM).It has a transmembrane structure and secretory function.The cell is anchored to the cell membrane and is involved in cell adhesion and proteolysis of various cell surface receptors and signaling molecules.ADAM20 is up-regulated in thyroid cancer,colorectal cancer and other malignant tumors,and may act as a target gene of MYCT1 to play the role of oncogene downstream and promote tumor cell proliferation,invasion and metastasis at the initial stage of tumor.Therefore,the study of ADAM20 gene is helpful to discover the mechanism of laryngeal cancer development and potential targets for cancer treatment,which is of great significance for the clinical diagnosis and treatment of laryngeal cancer.Real-time PCR results showed that the expression level of ADAM20 significantly decreased after the overexpression of MYCT1 in Hep-2 cells,which proved that MYCT1 could negatively regulate the expression of ADAM20 gene,but the specific molecular mechanism remains unknown.Combined with the previous studies of our group,we noted that the binding site of the downstream gene miR-181 a of MYCT1 and the 5’-UTR region of ADAM20 exists.It may be involved in the occurrence,development and biological function of laryngeal carcinoma by inhibiting ADAM20 transcription.Micro RNAs(miRNAs)are a class of conserved endogenous non-coding RNAs,consisting of 21 to 23 nucleotides in length,which are generally believed to be involved in the genesis and development of many malignant tumors,including the proliferation,invasion and metastasis of cancer cells.The role played by micro RNAs in tumors can be used as targets for tumor diagnosis and treatment.In laryngeal cancer,miR-181 a also plays a tumor suppressive role and promotes apoptosis of laryngeal cancer cells by inhibiting NPM1 expression at the transcriptional level.However,the mechanism of this action remains to be further studied.Previous research results of our group showed that MYCT1 directly activated miR-181 a by binding with MAX,which has anti-tumor effect in laryngeal cancer cells,inhibits the biological functions of laryngeal cancer Hep-2 cell proliferation,and promotes the apoptosis of laryngeal cancer cells.Bioinformatics software predicted that there were binding sites between miR-181 a and ADAM20 in the upstream 5’-UTR,which might inhibit the transcriptional activity of ADAM20.In this study,we intended to identify the downstream gene of MYCT1,ADAM20,through relevant molecular biological techniques,to explore the expression and biological function of ADAM20 in laryngeal cancer,and to clarify the influence of MYCT1 on the biological function of laryngeal cancer cells through the MYCT1/miR-181a/ADAM20 regulatory axis,so as to help to discover new molecular targets for diagnosis and treatment of laryngeal cancer,which has potential clinical significance.Materials and methods:1.Materials: human laryngeal squamous cell carcinoma Hep-2 cell line,human laryngeal cancer TU212 cell line.2.Methods: cell culture,plasmid extraction,cell transfection,real-time PCR,Western blot,Transwell,cell scratch assay,CCK8 cell proliferation detection,clone formation assay,immunofluorescence,double luciferase reporter gene activity detection and statistical analysis.Results:1.Protein microarray analysis and verification of the downstream gene of MYCT1 showed that ADAM20 expression was decreased downstream of MYCT1(P<0.05).2.Real-time PCR and Western Blot results showed that the expression level of ADAM20 was decreased after the overexpression of MYCT1.The expression level of ADAM20 increased after MYCT1 knockdown(P<0.05).3.Immunofluorescence results showed that ADAM20 was mainly expressed in the cell membrane,partly in the cytoplasm;MYCT1 was mainly expressed in the cytoplasm,and partly in the cell membrane.Both of them had intracellular co-localization,and the expression level of ADAM20 was decreased when MYCT1 was overexpressed.When MYCT1 was knocked down,the expression of ADAM20 on the cell membrane was increased(P<0.05).4.CCK8 and clone formation experiments showed that the proliferation activity of laryngeal cancer cells in si ADAM20 group was significantly decreased(P<0.05).5.Transwell and cell scratch healing experiment results show that knocking down ADAM20 significantly reduces the migration ability of laryngeal cancer cells(P<0.05).6.UCSC and Jaspar predicted that the downstream target gene miR-181 a of MYCT1 might have a potential binding site with the upstream 5 ’-UTR of ADAM20.Real-time PCR results showed that transfection of miR-181 a mimics significantly reduced the expression level of ADAM20 m RNA in laryngeal carcinoma Hep-2 cells(P<0.05).7.The luciferase reporter gene results showed that miR-181 A significantly reduced the transcriptional activity of ADAM20 gene(P<0.05).8.CCK8 and clone formation experiments showed that inhibition of MYCT1 or miR-181 a expression promoted the proliferation of laryngeal cancer cells compared with the control group,and ADAM20 knockdown rescued the phenotypic effect of MYCT1 and miR-181 a on the proliferation of laryngeal cancer cells(P<0.05).9.Transwell and cell scar healing experiments showed that inhibition of MYCT1 or miR-181 a expression promoted laryngeal cancer cell migration compared with control group,and ADAM20 knockdown could rescued the effect of MYCT1 and miR-181 a on laryngeal cancer cell migration phenotype(P<0.05).Conclusions:1.ADAM20 is abnormally activated in laryngeal cancer cells,and its expression is increased,which plays an oncogene role and is negatively regulated by MYCT1.2.As a transcription factor,miR-181 a directly inhibited the transcription activity of ADAM20.3.In laryngeal cancer cells,MYCT1 transcriptional inhibition of ADAM20 expression through target gene miR-181 a,thus affecting the biological functions of laryngeal cancer cell proliferation and migration. |
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