Expression Of LncRNA-UCA1 In Hypopharyngeal Carcinoma And Its Influence On Cell Function

BackgroundHypopharyngeal squamous cell carcinoma(HSCC)is a malignant tumor arising from the mucosa of the upper aerodigestive tract and accounts for 5%of all the squamous cell carcinomas in the head and neck area.The HSCC is seldom diagnosed in early stage,due to its atypia symptom,although the 5-year survival rate of patients in early stage(stage ?-?)is as high as 70%.Approximately 70-85%of the patients are diagnosed at stage ?-?,whereas the 5-year survival rate is only about 15-45%.The treatment of the HSCC includes primary surgery with pre-or postoperative radiotherapy and primary radiotherapy with chemotherapy.Although the treatment of the HSCC has been improved in recent years,the prognosis is still far from satisfactory.Therefore,it is imperative to identify high sensitive and specific carcinogenesis-associated biomarkers for early-stage diagnosis and potential targets for therapy.Long noncoding RNAs(LncRNAs)are defined as non-protein-coding RNAs with more than 200 nucleotides in length.Previous studies show that LncRNAs have low sequence conservation and regulate gene expression at a transcriptional and posttranscriptional level through interaction with nucleic acids and proteins.And LncRNAs are widely implicated in diverse biological processes including cell proliferation,differentiation and chromosome inactivation.Increasing evidence demonstrates that the aberrant expression of LncRNAs have been reported in various types of cancers including breast cancer,lung cancer,pancreatic cancer,osteosarcoma,hepatocellular carcinoma and leukaemia.These results suggest that LncRNAs can be used as novel biomarkers and targets specifically for cancers and offer new avenues for therapeutic intervention against cancer progression.Urothelial carcinoma-associated 1(UCA1)is a well-characterized LncRNA initially identified in urinary bladder cancer(UBC)tissues and found to significantly enhance tumorigenicity and invasive potential in vitro and in vivo.In addition,LncRNA-UCA1 also functions as tumor-promoters in breast cancer,colorectal cancer,gastric cancer,and esophageal squamous cell carcinoma and has close relationship with prognosis of a variety of cancers.For example,Han et al.found high LncRNA-UCAl expression indicated a worse prognosis in colorectal cancer.In our previous microarray analysis,LncRNA-UCA1 was found to be overly expressed in the HSCC tissue.However,the role of LncRNA-UCA1 in HSCC has not fullyclarified yet.In this study,we first confirmed LncRNA-UCA1 was upregulated in HSCC tissues in comparison to adjacent non-tumor tissues and found its expression wasclosely linked to clinicopathological characteristics and prognosis of HSCC patients.Further,in vitro experiments established its role in cell apoptosis,proliferation and migration in HSCC,suggesting that LncRNA-UCA1 might be an important prognostic biomarker and treatment target.PART I The expression and the clinical significance of the LncRNA-UCA1 in hypopharyngeal carcinomaObjectiveTo study the expression of LncRNA-UCA1 in hypopharyngeal carcinoma and its relation with the clinicopathologic characteristics and prognosis.MethodQuantitative real-time PCR was performed to investigate the relative expression in the HSCC tissues in comparison to adjacent non-tumor tissues in 53 patients.Standard statistical analysis was used to analyse the relation between the expression level of the LncRNA-UCA1 and clinicopathologic characteristics and prognosis of the HSCC.ResultsThe relative expression level of LncRNA-UCA1 in HSCC tissues was 0.0088 ±0.0050,while it was 0.0051 ± 0.0040 in paired adjacent normal tissues(p<0.05).The higher expression levels of LncRNA-UCA1 were significantly correlated with advanced T category(p = 0.034),late clinical stage(p = 0.021)and worse lymphoid node metastasis(p = 0.02).However,no significant correlation was observed between LncRNA-UCA1 expression and other clinicopathological features,such as age and differentiation.The overall survival rate of patients with higher expression of LncRNA-UCA1 was significantly lower than those with lower expression(p<0.0001).ConclusionLncRNA-UCA1 was highly expressed in the HSCC and the level of expression of LncRNA-UCA1 was associated with the T classification,clinical staging and lymph node metastasis and the overall survival rate of the patients.The differentiation of the tumor and the age of the patients were not significantly associated with the expression of the LncRNA-UCA1.The LncRNA-UCA1 may have promoted the development of HSCC and could be a potential biaomarker of prognosis for HSCC patients.Part ? The influence of LncRNA-UCA1 expression on the biological behaviours of hypopharyngeal carcinoma cells.ObjectiveTo investigate the influence of LncRNA-UCA1 expression on the proliferation,migration and apoptosis of HSCC cells.MethordsDNA oligonucleotides to produce plasmid-based small hairpin RNA(shRNA)were cloned into the pLeno-GFP vector,while the scrambled sequence was used as a negative control.The pLeno-GFP vector together with packing vectors(pRsv-REV,pMDIg-pRRE and PMD2G)was cotransfected into 293T cells.FaDu cells were infected with constructed lentiviruses and the efficiency of silencing LncRNA-UCA1 was subsequently quantified by qRT-PCR.Apoptosis analysis by Annexin V staining,Cell cycle analysis by PI staining,Colony formation assay and Transwell assays were performed to analysed the influence of LncRNA-UCA1 expression on cell proliferation,migration and invasion in HSCC.ResultsThe expression levels of LncRNA-UCA1 were significantly decreased in Fadu cells transfected with interferenced UAC1(Sh-1 and Sh-2)in comparison with the scrambled sequence.The colony formation assay demonstrated that the colony numbers of UCA1-depletion Fadu cells were significantly less than those transfected with scramble sequence.Silencing of LncRNA-UCA1(Sh-1 and Sh-2)obviously induces apoptosis in Fadu cells measured by a flow cytometry.In the cell cycle analysis,UCA1 interference(Sh-1 and Sh-2)significantly caused accumulation of Fadu cells in G0/G1 phase and a reduction in S phase in contrast with control.In the transwell assays,knockdown of LncRNA-UCA1 in Fadu cells significantly attenuated the invasive and migratory ability of cells.ConclusionUpregulation of LncRNA-UCA1 contribute to proliferation,invasion and migration of hypopharyngeal carcinoma cells,which demonstrated that LncRNA-UCA1 may acts as as an oncogene role in hypopharyngeal carcinoma cells.