Study On The Expression And Function Of Long Non-coding RNA KAT7 In Nasopharyngeal Carcinoma

Backgroud and purposeLong non-coding RNA,as a new participant in the regulation of physiological processes,now was attracted wide attention.LncRNAs have been reported to be involved in tumor proliferation,invasion and metastasis and are considered to be potential tumor biomarkers and therapeutic targets for human cancers.Nasopharyngeal carcinoma is one of the most common head-neck neoplasms in southern China.Radiotherapy is an effective method,especially in patients with early nasopharyngeal carcinoma.However,most of patients were diagnosed as suffering from terminal cancer.It is of great significance to study the expression of IncRNA and its biological effects in nasopharyngeal carcinoma for the diagnosis and treatment of nasopharyngeal carcinoma.Our study was aimed to investigate the expression of LncRNA KAT7 in nasopharyngeal carcinoma tissues and cells and its biological behaviours innasopharyngeal carcinoma cells.The main ideas of the research are:investigate the expression of LncRNA KAT7 in nasopharyngeal carcinoma tissues and CNE-2 and 5-8F cells;two nasopharyngeal carcinoma cell lines overexpressed LncRNA KAT7 were constructed to study the biological effect of LncRNA KAT7,to detect EMT markers and to explore the target of LncRNA KAT7.Method1.Collecting the remaining samples from the endoscopic biopsy of the otolaryngologist,11 cases of benign nasopharyngeal tissues and 27 cases of nasopharyngeal carcinoma tissues were recruited.The patients’information was obtained from the case information management system.2.qRT-PCR was used to detect the expression of LncRNA KAT7 in chronic inflammatory nasopharyngeal tissues and nasopharyngeal carcinoma tissues.Total RNA was extracted with trizol and reversed to cDNA using a reverse transcription kit,and the expression of LncRNA KAT7 was detected by real-time PCR.3.qRT-PCR was used to detect the expression of LncRNA KAT7 in human immortalized nasopharyngeal epithelial cell line NP69 and nasopharyngeal carcinoma cell lines CNE-2,5-8F.4.Two nasopharyngeal carcinoma cell lines stably expressing LncRNA KAT7 were established using lipofectamineTM2000 transfection reagent:negative control plasmid and LncRNA KAT7 expression plasmid were stably transfected into CNE-2 and 5-8F cells with lipofectaminerTM2000 transfection reagent.Monoclonal cell lines were selected after G-418 screening and detection of LncRNA KAT7 expression in CNE-2 and 5-8F cells by qRT-PCR.5.CCK8 proliferation assay and colony formation assay were used to assess the capability of proliferation in control group and experiment group.Migration assay and wound healing assay were used to estimate the role of LncRNA KAT7 in cell migration.Transwell assay with matrigel was used to further examine the role of LncRNA KAT7 in cancer cells’invasion.6.The underlying mechanism of LncRNA KAT7 mediated cell migration and invasion was preliminarily studied by Western Blot.Western Blot was performed to detect the expression level of ZEB1,E-cadherin,MMP-2,NF-κB P65 and p-NF-κB P65 in two groups.Results1.The results of qRT-PCR showed that the expression of LncRNA KAT7 in nasopharyngeal carcinoma tissues"was significantly lower than chronic inflammatory nasopharyngeal tissues,and there was statistically significant difference(P<0.01).The expression of LncRNA KAT7 in CNE-2 and 5-8F cells was significantly lower than that in NP69 cells(P<0.01)2.The stable transfection results showed that the expression levels of LncRNA KAT7 in CNE-2/pcDNA-KAT7 and 5-8F/pcDNA-KAT7 cells were significantly increased(P<0.01).3.CCK8 proliferation assays showed that LncRNA KAT7 overexpression decreased the proliferation rates of CNE-2 and 5-8F cells compared with the control group.Meanwhile,the results of colony formation assay showed that the number of cloned cells were decreased in LncRNA KAT7 overexpression cells compared with the control group,and there was statistically significant difference(P<0.01).Compared with the control group,the results of migration assay and wound healing assay showed that the number of migrated cells was decreased in LncRNA KAT7 overexpression cells,and there was statistically significant difference(P<0.01).The results of Transwell assays with matrigel showed that the number of migrated cells in LncRNA KAT7 overexpression cells was decreased compared with the control group.and there was statistically significant difference(P<0.05).4.Western Blot was performed to detect the expression levels of NF-κB P65,p-NF-κB P65.ZEB1,E-cadherin and MMP-2.The results showed that LncRNA KAT7 overexpression inhibited EMT process in CNE-2 cells comparedwith the control group.There was no significant difference in the expression of NF-κB in CNE-2/pcDNA-KAT7 compared with the control group.The expression of p-NF-KB P65,MMP-2,and ZEB1 were suppressed in CNE-2/pcDNA-KAT7 cells,while the expression of E-cadherin CNE-2/pcDNA-KAT7 cells was increased.Conclusion1.The expression levels of LncRNA KAT7 RNA were significantly down-regulated in nasopharyngeal carcinoma tissues and cells.2.Overexpression of LncRNA KAT7 can inhibit the abilities of proliferation,migration and invasion in CNE-2 and 5-8F cells.LncRNA KAT7 reversely regulated EMT process in CNE-2 cells.It is suggested that LncRNA KAT7 may inhibit the progression of nasopharyngeal carcinoma.