| Objective:Nasopharyngeal carcinoma is one of common head and neck cancers, which is insidious onset. Most patients are found late, and the prognosis is not good. Treatment of nasopharyngeal cancer is mainly by radiotherapy. Although there is some progress in treatment, for 5-year survival rate of the patients, it is not significantly improved. So far, the pathogenesis of nasopharyngeal carcinoma is not clear enough. Long non-coding RNA has an important adjustment function to life activity. We focus on the study of the relationship between long non-coding HNF1A-AS and the nasopharyngeal carcinoma. To study the expression changes of HNF1A-AS in nasopharyngeal carcinoma, and to prove HNF1A-AS can promote the abilities of nasopharyngeal carcinoma cell proliferation, migration and invasion, and study its possible mechanism of promoting the cell proliferation, migration and invasion. Methods:First of all, the content of the HNF1A-AS was detected through the quantitative reverse transcription polymerase chain reaction(q RT-PCR) in tissue of the experimental group and the control group, which collected from 20 patients with NPC and its adjacent normal tissues respectively. After the research in human tissue, further study at a cellular level was then carried out. Cultivating nasopharyngeal carcinoma cell line 5-8F, 6-10 B, HONE-1, CNE-2, SUNE-1 and C666-2 as the experimental group, human nasopharyngeal epithelial cell NP69 as the non cancerous cells control group, through the q RT-PCR experiments, to prove expression changes of the HNF1A-AS at a cellular level in the two groups. Two cell lines, CNE-2, SUNE-1, which changed more obvious were selected for the subsequent experiments.A sequence targeted HNF1A-AS was designed and applied as the experimental group, and the commonly used negative control sequence was selected in the RNA interference experiment as the control group(scrambled si RNA). Followed by Packaging, purification, and determination of HNF1A-AS interference viral titer, We made the slow virus vector which had already carried the HNF1A-AS-si RNA(AS well AS the scrambled si RNA) transfect CNE-2, SUNE-1 cells. q RT-PCR experiment was used to detect the expression changes of the HNF1A-AS in cell lines after transfection.Secondly, the function of the HNF1A-AS in two groups was tested. Cell proliferation migration and invasion abilities were estimated in vitro by Cell proliferation capacity(determined by MTT), colony-formation, wound-healing, and transwell assays. Cell cycle analysis was used to further examine the role of HNF1A-AS in cell proliferation. The tumor size of 24 male mice with or without HNF1A-AS interference was monitored once a week. Lastly, the underlying mechanism of HNF1A-AS mediated cell migration was studied by western blot analysis. Results:Relative HNF1A-AS m RNA levels in tumor tissues and cultured NPC cells were significantly higher than those in the adjacent tissues. Compared with the control group, HNF1A-AS in clinical NPC tissues showed 3-fold higher expression than noncancerous tissues on average, p<0.001. HNF1A-AS was observed to be upregulated in these NPC cell lines, of which CNE-2 and SUNE-1 exhibited highest HNF1A-AS expression. The HNF1A-AS level in SUNE-1 cells was increased by 8-fold and even 9.2-fold higher in CNE-2 cells, p < 0.05(5-8F,HONE-1); P < 0.01(6-10 B,CNE-2,SUNE-1,C666-1).We infected the NPC cells, CNE-2 and SUNE-1 with lentivirus carrier, stably expressing scrambled and specific HNF1A-AS si RNA. Fluorescence microscopy indicated high infection efficiency(> 70%) for both cell lines. q RT-PCR analysis showed that m RNA levels of HNF1A-AS were remarkably decreased in CNE-2 and SUNE-1 cells. In CNE-2 cells, compared with the control group, the HNF1A-AS level of HNF1A-AS-si RNA group was decreased by 76%. In SUNE-1 cells, total HNF1A-AS m RNA level fell to 14% of the control group, p < 0.01(CNE-2), p < 0.001(SUNE-1).HNF1A-AS interference inhibits cell proliferation and colonies formation in vitro. MTT assay revealed the proliferation rate of CNE-2 cells was suppressed by 32.5% at the 4th day, while droped to 33.3% at the 3rd day for SUNE-1 cells. Colony-formation assay showed HNF1A-AS si RNA other than scrambled infection blocked the formation of clones in CNE-2 and SUNE-1 cell lines. Quantification result showed that about 70 colonies were formed in case of scrambled si RNA-treated(the control groups)CNE-2 cells, while only 21 colonies were observed in case of specific si RNA-treated cells. Meanwhile, in SUNE-1 cells, about 60 colonies were formed in case of scrambled si RNA-treated CNE-2 cells, while only 19 colonies were observed in case of specific si RNA-treated cells, p < 0.01(CEN-2, SUNE-1).HNF1A-AS promoted the abilities of cell migration and invasion in vitro. Scratched wound healing assay revealed that in both CNE-2 and SUNE-1 cells, cell migration ability was suppressed when HNF1A-AS-si RNA was knocked down. In CNE-2 cells, the scrambled group filled 67% of the scratched wound as compared to only 44% when HNF1A-AS-si RNA was knocked down. Simultaneously, in SUNE-1 cells, the scrambled group filled 51% of the scratched wound as compared to only 25% when HNF1A-AS-si RNA was knocked down, p < 0.01(CEN-2,SUNE-1). Representative images of migration and Matrigel invasion assay for both cell lines. Quantification of the transwell assay results revealed thatcells numbers migrating to the lower chamber decreased by 66.7% for CNE-2 cells and by 62.5% for SUNE-1 cells. In the invasion assays, the differences were 69.6% and 62.5%, p < 0.01(CEN-2,SUNE-1).Cell cycle analysis experiments demostrated HNF1A-AS increased the cell number of CNE-2 in G0/G1 phase by approximate 1.5-fold, but decreased the percentage of cells in S and G2/M phases by 65% and 66.7%, respectively. Likewise, more than 25.7% cells in S and G2/M phases shifted to G0/G1 phase when SUNE-1 cells were treated with lentivirus expressing specific si RNA against HNF1A-AS, p < 0.01(CEN-2,SUNE-1).HNF1A-AS interference suppressed tumor growth in NPC mouse model:Weekly measurement of tumor volumes revealed a large extent of difference in tumor sizes between HNF1A-AS-si RNA and control groups from the 3rd week in both, cell line-injected mice,specifically, the tumor sizes in CNE-2-injected mice were reduced by 58.8% and 64% at the 3rd and 4th week, respectively. Similar results were obtained for the SUNE-1 injected mice, where difference between HNF1A-ASsi RNA and control groups was 54% at the 3rd week and 69.7% at the 4th week, p < 0.05(CEN-2,SUNE-1).Western blot:HNF1A-AS interference reversed the EMT process. Expression of EMT markers, including Cd C25 C, cyclin B1, Snail, N-cad- herin and vimentin were significantly suppressed when HNF1A-AS was knocked down in both CNE-2 and SUNE-1 cell lines, while the expression of E-cadherin was increased. Conclusion:HNF1A-AS is overexpressed in both clinical NPC tissues and cultured NPC cells. HNF1A-AS promotes cell proliferation, colonies formation, the abilities of cell migration and invasion in vitro. HNF1A-AS interference caused cell cycle arrest in G0/G1 phase in NPC cell lines. HNF1A-AS interference suppressed tumor growth in NPC mouse model. HNF1A-AS interference reversed the EMT process. To sum up, long chain non-coding RNA HNF1A-AS factors were closely related to the cell proliferation, migration and invasion in nasopharyngeal carcinoma. The mechanism caused cell migration and invasion might be involved in the EMT process. This noticed us that the further research on the long non-coding RNA HNF1A-AS could provide a new direction for the diagnosis and treatment of nasopharyngeal carcinoma. |
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