| Part Ⅰ The expression and biological function of LncRNA HOTTIP in nasopharyngeal carcinomaBackgroundNasopharyngeal carcinoma(NPC)is a common malignancy of head and neck,which is derived from epithelial tissue.This disease is prevalent mainly in North Africa,Southeast Asia,and Southern China with high incidence.Since NPCs are highly sensitive to radiation,radiotherapy has become the most promising and effective treatment for early NPCs.However,despite the combination of radiation and chemotherapy,the quality of life and survival of patients with advanced NPC are not satisfactory.Therefore,understanding the molecular mechanism of NPC may offer fresh insights into diagnosis and treatment.The aim of present study was to investigate the expression and biological function of HOXA transcript at the distal tip(HOTTIP)in NPC,and to determine its clinical significance.MethodsThe human NPC cell lines(SUNE-1 and C666-1)and nasopharyngeal epithelial cell line NP69 were incubated under certain conditions.47 NPC samples and matched adjacent non-tumor tissues were collected.Quantifications of HOTTIP expression in cell lines and tissues were carried out by quantitative real time polymerase chain reaction as described by the manufacture.The Chi-square test and Pearson correlation were used to statistically analyze the correlation between clinicopathological parameters and the expression of HOTTIP.Kaplan-Meier curves combined with log-rank test were used to analyze overall survival(OS)rates.Small interfering RNA and plasmid vectors were used to knockdown or overexpress the expression level of HOTTIP in NPC cell lines.Then,the CCK-8 assay,flow cytometry and western blot were carried out to observe the impact of HOTTIP on NPC cells proliferation and apoptosis.In addiation,scratch test and Transwell chamber assay were carried out to observe the impact of HOTTIP on invasion and migration of NPC cells.Western blot was used to detect the effect of HOTTIP on NPC cell apoptosis and the expression of proteins related to epithelial mesenchymal transformation.ResultsThe relative expression levels of HOTTIP in SUNE-1,C666-1 and NP69 cells were detected using qRT-PCR.The results showed that the relative expression levels of HOTTIP in SUNE-1 and C666-1 cells were significantly higher than that in NP69 cells(P<0.05).Similarly,the relative expression values of HOTTIP were significantly higher in NPC tissues than those in the adjacent non-tumor tissues.Moreover,the expression level in phase Ⅲ-Ⅳ NPC tissues with lymph node metastasis and distant metastasis was significantly higher than that in phase Ⅰ-Ⅱ NPC tissues without lymph node metastasis and distant metastasis.The results of Chi-square test showed that the expression value of HOTTIP was related to nasopharyngeal tumor stage,lymph node and distant metastasis.However,the expression of HOTTIP was regardless of gender and age.The kaplan-meier survival curve implicated that the OS of patients with high HOTTIP expression was significantly lower than that of those with low HOTTIP expression.The analysis results of proportional hazard model(COX)implicated that the expression of HOTTIP was an independent factor affecting the prognosis of NPC.The results of CCK-8 assay showed that knockdown of HOTTIP significantly inhibited the proliferation of NPC cells,while overexpression of HOTTIP could significantly promote the proliferation of NPC cells(P<0.05).Moreover,the results of flow cytometry and western blot showed that knockdown of HOTTIP significantly enhanced the apoptosis of NPC cells,while overexpression of HOTTIP significantly decreased the apoptosis of NPC cells(P<0.05).In addiation,the results of scratch test and Transwell chamber assay suggested that the migration and invasion of HOTTIP knockdown cells were significantly inhibited in comparison with the control group.Overexpression of HOTTIP could reverse this phenomenon.Western blot results showed that HOTTIP affected the expression levels of NPC cell apoptosis and epithelial mesenchymal transformation related proteins.ConclusionsLncRNA HOTTIP expression was up-regulated in NPC cell lines and tissues.The relationship between clinical data and HOTTIP expression level indicated that high HOTTIP expression in NPC was negatively correlated with a number of pathological parameters and patient prognosis,suggesting that HOTTIP has a potential to be an important marker for clinicopathological detection of NPC progression and prediction of patient prognosis.Experiments in vitro have shown that HOTTIP could promote the proliferation,migration and invasion,but inhibit apoptosis of NPC cells.Part Ⅱ The mechanism of the action of LncRNA HOTTIP in nasopharyngeal carcinomaBackgroundConsiderable research has confirmed the mechanisms by which lncRNA performs diverse biological functions in human malignant tumors.MicroRNA(miRNA)is endogenous single-stranded RNA between 20 and 25 nucleotides in length,which does not encode proteins but can inhibit gene expression by binding to the sequence of target messenger RNA(mRNA).An increasing number of studies have shown that lncRNAs can act as "sponges" of natural miRNAs,inhibiting the function of miRNAs by competing with one or more miRNA response elements(MREs),thus leading to the occurrence and development of diseases.LncRNA HOTTIP can play an important role in a variety of diseases through miR-455-3p/CCL3,miR-608/DDA1 and Wnt/β-catenin signaling pathways.However,the mechanism of HOTTIP in NPC remains unclear.MethodsThe expression levels of miR-4301 in NPC tissues and cell lines were detected by qRT-PCR,and the effect of HOTTIP on miR-4301 expression was observed.Spearman correlation coefficient was used to analyze the correlation between HOTTIP and miR-4301 expression in NPC tissues.The relationship between HOTTIP and miR-4301 was detected by luciferase reporter experiments.After knockdown of HOTTIP,the expression level of miR-4301 in SUNE-1 cells was detected by qRT-PCR.Moreover,the proliferation,migration and invasion ability of SUNE-1 cells were detected by CCK-8 assay and Transwell assay,and the apoptosis rates of SUNE-1 cells were detected by flow cytometry.ResultsThe expression levels of miR-4301 in NPC tissues and cell lines were detected using qRT-PCR.The results showed that the expression levels of miR-4301 in NPC tissues were significantly lower than that in normal paracancer tissues(P<0.01),and the expression levels in NPC cell lines were significantly lower than that in normal nasopharyngeal epithelial cell(NP69)(P<0.05).Spearman analysis showed a significant negative correlation between HOTTIP and miR-4301 expression.Luciferase reporter result showed that HOTTIP had a binding site for miR-4301 in the gene sequence.The results of qRT-PCR showed that the expression of miR-4301 was up-regulated in HOTTIP-knockdown SUNE-1 cells,and the up-regulated expression of miR-4301 was reversed after transfection of miR-4301 inhibitor(miR-4301 in).The results of CCK-8 assay and Transwell chamber assay showed that silencing miR-4301 in HOTTIP-knockdown SUNE-1 cells could reverse the inhibition of cell proliferation,migration and invasion caused by knockdown of HOTTIP.The results of flow cytometry showed that silencing miR-4301 in HOTTIP-knockdown SUNE-1 cells could reverse the effect of HOTTIP knockdown on apoptosis.ConclusionHOTTIP could play an important role in carcinogenesis of NPC by binding to miR-4301 through MREs and negatively regulating the expression of miR-4301.Therefore,HOTTIP could be a potential target for the treatment of NPC. |
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