| Objective:Laryngeal cancer,as one of the common malignant tumors of head and neck carcinoma,has ranked the third in the incidence of head and neck malignant tumors in China.It is widely believed in the medical community that it is closely related to smoking,drinking,viral infection and other factors,and may also be related to genetic changes and cell signaling pathway disorders.Human papillomavirus(HPV)has a strong affinity for mucosa and skin,especially the carcinogenic effect of high-risk HPV with the potential to cause malignant transformation on oropharyngeal tumors has attracted more and more attention from scholars.So far,a growing number of studies have confirmed that the high-risk types of HPV,such as HPV-16 and HPV-18 closely related with the occurrence of laryngeal cancer and prognosis,these high-risk type of virus itself E6/E7protein can make excessive proliferation and differentiation of normal cells and activate cell cancer related because,at the same time,the virus itself DNA and can play a role of cancer with tumor cell gene integration.Although surgery,chemoradiotherapy and targeted drug therapy can improve the cure rate of patients with laryngeal cancer,the overall clinical treatment effect is also very difficult to be satisfactory.Therefore,improving the diagnosis rate of early HPV and timely and effective intervention are of vital importance for the prevention and treatment of laryngeal cancer.Method to detect HPV at present,such as HC2 method,fluorescence quantitative PCR method and immunohistochemical method for experimental conditions or operating technology such as the demand is higher,the long cycle of testing costs more expensive or diagnosis,are not able to achieve in the grass-roots hospital popularity or ability to undertake the task to deal with the incident detection,research and development applicable to clinical HPV diagnosis method is simple,rapid,efficient is imminent.In addition,is generally believed that the poor prognosis of laryngeal cancer is usually clinically is closely connected with late diagnosis,tumor recurrence and metastasis,along with the advance of image technology and the popularity of hd endoscopic applications,although to a certain extent,improved the clinical doctor to the diagnosis of laryngeal cancer,but traditional way of pathological diagnosis is still not science reflects the biological characteristics of tumor lesions,further research at the molecular level to molecular expression in patients with laryngeal cancer spectrum of clinical looking for specific therapeutic targets,is very important to improve the survival rates of patients with laryngeal cancer.Micro RNAs(mi RNAs),as single-stranded non-coding regulatory RNA molecules that play a role in post-transcription regulation,perform a variety of biological functions and participate in epigenetic regulation.In many diseases,mi RNA expression profiles have certain characteristic changes,especially in the paracancerous tissues of tumors and tumor tissues have significant differences in mi RNA expression profiles.Therefore,in-depth study of mi RNAs is of great theoretical and practical significance to explore the invasion and metastasis mechanism of laryngeal cancer,search for new diagnostic markers and therapeutic targets,and realize effective individualized treatment of tumors.In this study,we first established an isothermal nucleic acid amplification method based on color determination with independent intellectual property rights--multiple cross-linked spiral amplification method(MCLSA),which can quickly and efficiently detect HPV-16 gene from laryngeal cancer clinical samples.At the same time,mi RNAs carrying HPV-infected laryngeal cancer samples from Northeast China were sequencing and screened to provide a theoretical basis for further research on mi RNAs and the occurrence and development of laryngeal cancer.Methods:According to the characteristics of BST polymerase and the principle of the currently reported isothermal amplification method,a novel multiple cross-linking spiral amplification method was designed,and specific primers were designed for the conserved E6/E7 gene sequence of HPV-16,and the visual verification of the method was performed.The established MCLSA method needs to go through the evaluation procedures of specific primer conditions optimization,specificity,sensitivity and detection limit determination,and then use the clinically collected DNA of laryngeal cancer samples to verify and evaluate the application effect.Then,using the commercial nano colloidal gold reagent and FITC and biotin biotin labeling was prepared based on MCLSA high specificity and sensitivity of the new type of biosensor based on nano colloidal gold method in fast detection techniques of good application effect,the above characteristics of the preparation of biosensor can be used to quickly and efficiently achieve the HPV-16 in laryngeal cancer samples testing purpose.At the same time,the gene profiles of mi RNAs in laryngeal cancer samples associated with HPV infection and paracancillary tissues were sequenced,all mi RNAs were analyzed by bioinformatics method,and the mi R-205-3p with significantly different expression was verified and analyzed by q RT-PCR.The expression of ZEB1 protein was verified by western blot.At the cell level,MTT,clone formation assay,cell cycle detection and other related functional experiments were performed.Meanwhile,the role of mi R-205-3p in the proliferation and invasion of TU212 laryngeal cancer cells was verified by CCK-8 and transwell tests.Results:Primer Premier 5.0 software was used to design the specific primers for the MCLSA method.The design principle was similar to that of the LAMP method.During the amplification of MCLSA,the positive amplification products showed bright Green fluorescence after the system was mixed with SYBR Green I(SGI)dye under optimized conditions and incubated at 62℃for 45min,while the negative amplification products showed no significant change.The specificity test showed that the established MCLSA technique had high specificity,and could effectively distinguish the 5 strains of HPV-16from other pathogenic microorganisms,without cross-reaction.In terms of sensitivity analysis,the limit of detection(Lo D)of MCLSA was about 5.4×10~1copies/tube,which was 10 times higher than that of LAMP and fluorescence quantitative PCR.The positive detection rate of MCLSA and HC2 kit was 32.61%(15/46)in 46 cases of laryngeal cancer suspected of HPV infection in Liaoning area.The positive rate of MCLSA was higher than that of fluorescence quantitative PCR(100%vs 93.33%)and LAMP(100%vs 86.67%).Therefore,the MCLSA technique developed in this study is a potentially useful tool for HR-HPV screening of Po Cs.Subsequently,MCLSA-LFB method was developed by combining the lateral flow biosensor(LFB)based on nanoparticles with the established MCLSA method.FITC labeled DNA probe and nanoparticle based LFB were used to detect the E6/E7 gene of HPV-16.After the optimization of the amplification conditions,the amplification product of MCLSA was based on the biotin/streptavidin interaction to generate two red bands,and the negative amplification was a reaction band.The specificity test showed that the established MCLSA-LFB assay had good specificity for HPV-16,and could distinguish all HPV-16 from other pathogenic microorganisms.In terms of sensitivity,the detection limit of MCLSA-LFB method was 6 copies/tube,which was 10 times that of fluorescence quantitative PCR and LAMP.In addition,the positive rate of MCLSA-LFB was higher than that of fluorescent quantitative PCR and LAMP.The procedure includes sample DNA processing(30 min),isothermal reaction(20 min)and result visualization(5 min),and can be completed in 60 minutes.Therefore,the established MCLSA-LFB method has good specificity and sensitivity for the diagnosis of HPV-16 in clinical samples,and is suitable for the promotion and application of HPVs detection in primary medical units.In addition,9298 differentially expressed mi RNAs were obtained by mi RNA microarray screening.According to the expression levels of known and newly predicted mi RNAs,the differentially expressed mi RNAs were screened by the bioinformatics R software package DESEQ2.After bioinformatics comparison,6 mi RNA expressions were significantly up-regulated and 8 mi RNA expressions were significantly down-regulated in laryngeal cancer tissues.Fluorescence quantitative PCR was used to analyze 14mi RNAs differentially expressed in laryngeal cancer tissues and paracancerous tissues.The results showed that mirna-205-3p could significantly up regulate the expression of ZEB1 gene,induce EMT and accelerate the process of tumor migration and invasion.mi R-205-3p may be a potential biomarker for the diagnosis and treatment of laryngeal cancer.Conclusion:This study established a isothermal amplification technologies with independent intellectual property rights MCLSA method,this new visualization based on Bst polymerase isothermal amplification techniques to quickly and effectively identify HPV-16 E6/E7 gene,with published LAMP and fluorescent quantitative polymerase chain reaction(PCR),specificity and sensitivity analysis MCLSA not only has the characteristics of high specificity and sensitivity,and the ability to accurately identify HPV-16 genes in clinical specimens of laryngeal cancer.In addition,the commercial nano colloidal gold,made of biotin labeling in MCLSA method combining with new type of biosensor,also has the advantages of high sensitivity and specificity,can not only shorten the testing time,improve the detection limit,and easy to store more make MCLSA-LFB method has the advantages of the clinical screening for HPV provide new test kit.For laryngeal cancer associated with HPV infected samples and tissue adjacent to carcinoma into the mi RNA expression means of sequencing and bioinformatics analysis of significant difference expression of micrornas,tip can be mi R-205-3p in the development process of laryngeal cancer-b expression may play the role of tumor suppressor genes,and through the increase of epithelial-between qualitative(EMT)affect the effect of laryngeal cancer proliferation,but the specific mechanism remains to be further research. |
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