| [Background] Long non-coding RNA(lnc RNA)is a class of RNA with a length of more than 200 nt,which itself does not encode a protein,but in the form of RNA at multiple levels(epigenetic regulation,transcriptional regulation,and transcription Post-regulation,etc.)regulation of gene expression [1].Recent studies have shown that lnc RNA is widely involved in a variety of biological processes.Moreover,the abnormal expression of lnc RNA is closely related to a variety of diseases,including tumors.There are evidence that lnc RNA involved in liver cancer,pancreatic cancer,glioma and other malignant tumor development [2-5].However,the global expression profile and functional significance of lnc RNAs in locally advanced NPC remain unclear.Nasopharyngeal carcinoma(NPC)is a unique epithelial malignancy arising from the superior aspect of the pharyngeal mucosal space,and is much more common in southern China(with an incidence of 25 to 50 in 100,000)and Southeast Asia,having remarkably distinct ethnic and geographic distributions.The prognosis of NPC is closely related to the stage of disease.Because of the concealment of NPC,there are no symptoms and signs in the early stage.70% of the patients with are locally advanced NPC.In recent years,although the level of diagnosis and treatment of NPC has been greatly improved,the molecular mechanism of its pathogenesis is vague.Moreover,because the majority of NPC cases are diagnosed at an advanced stage and they are often found with distal metastasis,the prognosis and survival of patients with NPC remain unsatisfactory,with a 5-year overall survival rate of approximately 70% [7],for most patients develop with local treatment failure and distant metastasis.Thus,treatment of patients with advanced HNSCC remains challenging.In recent years,lnc RNA more and more people to receive more and more concern,but the role of lnc RNA in NPC,especially locally advanced NPC,remains to be uncovered.In light of these factors,the unraveling and elucidation of the gene expression networks and the molecular regulatory mechanism for locally advanced NPC should be fully completed.Lnc RNA gene chip is an ideal and effective method for extensive application and study of 1nc RNA expression profile.Through the lnc RNA gene chip,rapid and high-throughput access to specific biological processes or disease-related Lnc RNA expression changes,so as to follow-up Lnc RNA function research or biomarker screening to provide great convenience.[Objective ] The aim is to study the lnc RNA and its related biological functions related to local advanced NPC,and to explore its mechanism,and to provide theoretical and scientific basis for the study of NPC.[methods] 1.The Human lnc RNA Microarray version 4.0,integrated NCBI Refseq,UCSC,RNAdb and Lnc RNAs and other database resources,was used to detect the differential expression profile of lnc RNA and m RNA between NPC tissue and its adjacent tissues,the two tissues between the,Then,cluster analysis,GO analysis,pathway analysis and other preliminary bioinformatics analysis were performed.2.CNE1 and HONE1 of NPC cell lines and NP69 of nasopharyngeal normal epithelial cells were cultured in vitro.Real-time PCR was used to verify the expression level of target lnc RNA in CNE1,HONE1 and NP69 cell lines.3.si RNA-interfering technique was used to down-regulated expression of ENST00000501541,to investigage its effect on the biological characteristics of NPC cells.Furthermore,real-time PCR was used to detect the transfection efficiency of si RNA.4.The proliferation,invasion,and apoptosis of NPC cell lines were detected by CCK8 method,boyden and transwell experiment,and apoptosis test,respectively.[Results] Part I: 1.324 differentially expressed lnc RNAs and 188 differentially expressed m RNAs were found by the gene chip technology,with the screening criteria: P value ≤ 0.05,Foldchange ≥ 1.5,screened2.2.Pathway results showed that: lnc RNA potential target gene m RNA may participate in a total of 22 signaling pathways,which up the expression of the signal pathway 6,down 13.GO analysis showed that the highest level of m RNA expression,cell composition and molecular function enrichment were cell chemotaxis,collagen trimer and chemokine activity.The down-regulated expression was protein localization to cytoskeleton,cell cortex and protein homodimerization activity.Part II: 1.The results of real-time quantitative PCR showed that the relative expression of AW450413,ENST00000501541,ENST00000457325 and uc003 owl.1 increased,and the relative expression of ENST00000448834,ENST00000554458 and ENST00000433377 were consistent with that of gene chip.More importantly,the expression level of ENST00000501541 on CNE1 and HONE1 was significantly higher than that of NP69,and there was significant difference(p <0.05).2.The proliferation of CNE1 and HONE1 in the experimental group(si RNA-1)and the experimental group(si RNA-2)were significantly decreased(P <0.05)compared with the control group(si RNA-NC).3.Ranswell migration experiments showed that the number of cells per high magnification in the experimental group(si RNA-1)was significantly lower than that in the control group(P <0.05)compared with the control group(si RNA-NC).4.Boyden invasion experiments showed that compared with the control group (si RNA-NC),the number of cells passing through the Matrigel membrane was significantly lower in the experimental group(si RNA-1)than in the control group(P <0.05).5.The results of scratches showed that the sclerotic healing ability of the experimental group(si RNA-1)was significantly lower than that of the control group(P <0.05)at 48 hours and 72 hours compared with the blank group and the negative control group.6.The expression of CNE1 and HONE1 in nasopharyngeal carcinoma cells was significantly increased(p <0.05)compared with the negative control group(sh RNA-NC)in the experimental group(si RNA-1).[Conclusions] 1.We used lnc RNA gene chip found that 324 differentially expressed lnc RNAs,and in nasopharyngeal carcinoma cell lines to verify the reliability of gene chip results.2.The expression of ENST0000050154 in nasopharyngeal carcinoma and nasopharyngeal carcinoma cell line was confirmed.The expression level of ENST0000050154 could inhibit the proliferation,migration and invasion of nasopharyngeal carcinoma cell line and promote apoptosis.The results suggest that ENST0000050154 may participate.The development of nasopharyngeal carcinoma.In summary,our experiments have initially studied the effect of ENST0000050154 on nasopharyngeal carcinoma cell lines in vitro and laid a foundation for the study of the functional mechanism of nasopharyngeal carcinoma.It also provides a new perspective for the study of locally advanced nasopharyngeal carcinoma。... |
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