| IntroductionLaryngeal carcinoma is one of the most common malignant cancers in the head and neck,about 95% of larynx cancers are laryngeal squamous cell carcinoma(LSCC),which mainly originated in the laryngeal mucosal epithelial tissue.Laryngeal squamous cell carcinoma has a high mortality and poor prognosis,at present,the molecular mechanisms of the occurrence,development and prognosis of laryngeal cancers are not entirely clear.Therefore,it is essential to explore the development of laryngeal cancer and to find biomolecular markers for its diagnosis and prognosis.In recent years,miRNAs(microRNAs)have been reported as a single chain non coding RNA with a length of about 22 nucleotides.It plays a role by binding to target gene mRNA 3'UTR specifically.Studies have shown that miRNA can not only down-regulate the activity of tumor suppressor genes,but also reduce the activity of oncogene as a tumor suppressor gene.miR-200 c is a member of the miR-200 family(miR-200 a,miR-200 b,miR-200 c,miR-429 and miR-141),located on chromosome 12.Studies have shown that miR-200 c plays a decisive role in epithelial-mesenchymal transformation,cell invasion,metastasis,proliferation,apoptosis,autophagy and anti-cancer therapy,and plays different roles in different types of cancer.At present,it is still unclear whether mi R-200 c is involved in the occurrence and development of larynx cancer.In the previous work,we found that the expression of PIN1 increased the centrosome amplification,abnormal cell division and cell migration.Targetscan showed that PIN1 is one of the potential target genes of miR-200 c.In this study,we will explore the influence of miR-200 c on the biological behavior of laryngeal carcinoma Hep-2 cells by molecular biology techniques and whether miR-200 c plays its biological function through PIN1 in laryngeal carcinoma.Materials and Methods Materials1.Laryngeal squamous cell carcinoma cell line Hep-2.2.Tissue samples: Laryngeal cancer tissues and paired normal tissues were obtainedfrom 43 patients at the First Affiliated Hospital of China Medical University,and all patients were not treated with chemotherapy before operation.The samples were stored in the-80°C refrigerator.All samples were confirmed by pathological examination,approved by the Ethics Committee and informed consent of the patients.3.Fundamental molecular biology reagents: RPMI 1640,Serana FBS,RNAiso Plus reagent,SYBR? Premix Ex Taq? II,PrimeScript? RT reagent Kit,SDS-PAGE Gel Kit,Rabbit Anti PIN1 Polyclonal antibody,Mouse Anti beta Actin Monoclonal antibody,Goat Anti-Rabbit IgG(HRP),Goat Anti-Mouse IgG(HRP),Monoclonal Anti-?-Tubulin antibody produced in mouse,DyLight 488 AffiniPure Goat Anti-Mouse IgG,TIANGEN Plasmid Extraction Kit,FITC Annexin V Apoptosis Detection Kit I and so on.Methods1.Luciferase reporter assay2.Cell culture3.Transient gene transfection4.qRT-PCR assay5.Western Blot method6.CCK8 assay7.Transwell chamber method8.Immunofluorescence assay9.Flow cytometry-based apoptosis detcection10.Statistical analysisResults1.Real-time PCR results showed that the expression level of miR-200 c increased in LSCC tissues compared with the controls(P<0.05),suggesting that miR-200 c was involved in the occurrence and development of laryngeal carcinoma.In addition,the expression level of miR-200 c significantly decreased in cancer tissues of the patients with lymphatic metastasis compared with those without lymphatic metastasis(P<0.05),suggesting that mi R-200 c may play a role in the process of tumor metastasis.The expression level of miR-200 c significantly increased and decreased in miR-200 c mimics and inhibitor transfection groups compared with the controls in the Hep-2 cells(P<0.05).These results suggested that transfection was successful.2.CCK8 assay results indicated that there was no significant difference in the growth rate of Hep-2 cells transfected with mi R-200 c mimics and miR-200 c inhibitor(P>0.05).3.Transwell results showed that the number of Hep-2 cells passing through the chamber in miR-200 c mimics transfection group was significantly decreased than control group,while the number of Hep-2 cells passing through the chamber in the miR-200 c inhibitor transfection group was increased than control group(P<0.05).These results suggested that miR-200 c inhibited migration of Hep-2 cells.4.Immunofluorescence results showed that the proportion of abnormal centrosome amplification in miR-200 c mimics transfected group was significantly reduced than comtrol,and the proportion of centrosome amplification in miR-200 c inhibitor transfected group was increased compared with the control group(P<0.05).These results suggested that miR-200 c reduced the abnormal amplification of the centrosome.5.Flow cytometry assay results indicated that there was no significant difference in the early apoptosis rate of Hep-2 cells transfected with miR-200 c mimics and mi R-200 c inhibitor(P>0.05).6.TargetScan results showed that there was a binding site of mi R-200 c in the3'-untranslated region of PIN1,suggesting that PIN1 was one of the potential target genes of miR-200 c.7.Luciferase reporter assay results showed that a significant down-regulation on luciferase activity was found in the presence of miR-200 c mimics co-tranfected with pGL3-PIN1-3'UTR compared with control groups(P<0.05),indicating that PIN1 was a target gene of miR-200 c.8.Real-time PCR and Western Blot showed that PIN1 protein expression level decreased and increased in the Hep-2 cells transfected with mi R-200 c mimics and inhibitor(P<0.05),while the corresponding mRNA expression levels were not significantly different(P>0.05),suggesting that mi R-200 c negatively regulated PIN1 expression at translation level.9.Real-time PCR results showed that the expression level of miR-200 c increased in miR-200 c mimics transfection groups,and the expression level of PIN1 mRNA increased in PIN1 transfection groups,after co-transfection of PIN1 and miR-200 c mimics,PIN1,miR-200 c mimics and mimics NC(P<0.05),suggesting that transfection was successful.10.Western Blot results showed that PIN1 protein expression level in PIN1 and miR-200 c mimics co-transfection group decreased compared with PIN1 group(P<0.05),further indicating that miR-200 c inhibited the expression of PIN1 at translation level.11.CCK8 assay showed that after co-transfection of PIN1 and miR-200 c mimics,there was no significant difference in growth rate of Hep-2 cells compared with other control groups(P>0.05).12.Transwell chamber results showed that the number of Hep-2 cells passing through the chamber in PIN1 and miR-200 c mimics co-transfection group was significantly decreased than PIN1 group(P<0.05),suggesting that miR-200 c regulated PIN1 and inhibited cell migration.13.Immunofluorescence results showed that the proportion of abnormal centrosome amplification in PIN1 and miR-200 c mimics co-transfection group was decreased compared with the PIN1 group(P<0.05),suggesting that miR-200 c regulated PIN1 to reduce abnormal amplification of the centrosome.14.Flow cytometry results showed that after co-transfection of PIN1 and miR-200 c mimics,there was no significant change in the early apoptosis rate compared with other control groups(P>0.05).Conclusions1.miR-200 c is involved in the occurrence and development of laryngeal carcinoma.2.PIN1 is one of the target genes of miR-200 c.3.miR-200 c inhibits Hep-2 cells migration and abnormal centrosome amplification by targeting PIN1. |
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