To Explore The Roles Of Klf4in Proliferation、EMT、invasion And Metastasis Of Nasopharyngeal Carcinoma

Background and objectiveNasopharyngeal carcinoma (NPC) which has a remarkably unusual geographic distribution in Southern China and Southeast Asia, is a malignant tumor originating from nasopharyngeal mucosa. The major etiological factors of NPC include environmental factors, diet, genetic and Epstein-Barr virus (EBV) infection. Epidemiological studies have found that the incidence of NPC has geographical, ethnic and familial aggregation phenomenon. The treatment of NPC has radiotherapy and chemotherapy, and has a high cure rate in the early stage; however, because of its high degree of malignancy and early lymph node metastasis, it’s cure rate is quite low in that time. At present, the pathogenesis of NPC is not very clear. Therefore, it is worth for us to study the pathogenesis of NPC.Kriippel-like factor4(Klf4) belongs to the Klf family with a zinc finger-type transcription factor and is widely expressed in eukaryotes. Klf4was first described by Shields, JM in1996, which was isolated from cDNA library of mouse NIH3T3cells. Klf4widely expresses in the digestive tract. Thus, Klf4is also called the gut-enriched Kriippel-like factor. Klf4protein contain many of functional areas, including the N-terminal acidic amino which able to participate in the interaction between the protein-protein; and C-terminal zinc finger region which can bind to the DNA binding region; there is also a transcriptional repressor area.Klf4mainly expresses in the skin, mouth, gastrointestinal epithelium, vascular endothelium, etc. Klf4plays important roles in many aspects of the normal tissues, such as cell proliferation, differentiation, apoptosis, and other biological behaviors.Klf4can either activate or repress transcription by different mechanisms, depending on the target gene(s). Klf4can maintain homeostasis of the colonic epithelium by inhibiing cell proliferation and promoting their differentiation. In addition, Klf4is an important molecule in skin epithelial differentiation; some studies found that Klf4expression could rapidly upregulate in smooth muscle cells. In the tumor tissue, Klf4can function as an oncogene or a tumor suppressor by combing with different target genes. In human colorectal carcinoma, Klf4is down-regulated, with evidence of both hypermethylation and loss of heterozygosity; what’s more, Klf4can inhibit the Wnt pathway by combing with the APC gene, which can suppress the development of colon cancer; strong evidence also implicates Klf4as a tumor suppressor in the gastric cancer. As Klf4was downregulated and played as a tumor suppressor in Esophageal cancer, bladder cancer, and lung cancer; but it was amplified and promote breast cancer. Additionally, Klf4is overexpressed in laryngeal squamous cell carcinoma as an early event in its progression.However, there is little known about the functions of Klf4in pathogenesis of NPC, which encourages us to explore the roles of Klf4in proliferation、EMT、 invasion and metastasis of nasopharyngeal carcinoma.Methods:Part I Expression profile of Klf4in NPC cell lines and tissues detected by qRT-PCR.1. qRT-PCR was used to detect the expression of Klf4in NPC-derived cell lines (i.e., CNE1, CNE2, HONE1, HNE1,5-8F,6-1OB, SUNE1and C666-1) and in immortalized nasopharyngeal epithelial cell strain NP69.2. Klf4expression in21human NPC tissues and6human chronic nasopharyngitis tissues was detected by qRT-PCR.Part Ⅱ The effects of Klf4on NPC cell proliferation1. Generation of NPC cell lines overexpressing Klf4transgeneLentiviruses carrying Klf4and enhancand green fluorescent protein (EGFP) genes were produced, followed by infecting5-8F, HONE1and SUNE1using lentivirus supernatant. Finally, NPC cell lines overexpressing Klf4transgene were confirmed to be generated by EGFP assay under inverted fluorescence microscope and qRT-PCR, Werstern blot used to detect the expression of Klf4transgene in5-8F, HONE1and SUNE1.2. The effects of Klf4overexpression on NPC cell proliferation in vitroCCK8assay, cell cycle and colony formation assay were used to detect the ability of NPC cell proliferation3. The effects of siRNA-mediated Klf4down-regulation on NPC cell proliferation in vitroChemical synthesis of two Klf4siRNA, by transient transfecting NPC cell (5-8F),to detect the inhibition efficiency of Klf4siRNA.Then, detect the ability of cell proliferation by cck8assay4. The effects of Klf4overexpression on NPC cell proliferation in vivo5-8F-Klf4and5-8F-con were cultured in vitro. The cells were inoculated subcutaneously in nude mice to establish the NPC ectopic transplantation tumor model.When the tumor lenth is about0.5cm, we need to measure the lenth of tumor after day and calculate tumor volume. In addition, using immunohistochemical method to detect the expression level of Ki-67, BrdU and p21gene in tumor tissues.Part Ⅲ The effects of Klf4on MET and migration of NPC cellsTranswell chamber assay was used to detect the migration of Klf4-expressing NPC cells in vitro. qRT-PCR and Western blot were used to detect the expression of EMT-related genes, such as E-cadherin, α-catenin, fibronectin, N-cadherin and vimentin. The influences of Klf4downregulation on EMT and migration were evaluated by Western blot and transwell migration assay, respectively.Results:Part I Expression profile of Klf4in NPC cell lines and tissuesqRT-PCR was used to detect the expression of Klf4in NPC cell lines and human NPC tissues. The results indicated that the levels of Klf4expression was significantly lower in NPC cell lines (such as CNE1, CNE2,5-8F,6-1OB, C666-1, HNE1, SUNE1and HONE1) than in NP69cells. qRT-PCR results showed that the levels of the Klf4expression in NPC tissues was also lower than in chronic nasopharyngitis tissues (t=-2.599,p=0.015)Part Ⅱ The effects of Klf4on NPC cell proliferation1. Generation of NPC cell lines overexpressing KIf4transgeneNPC cell lines (i.e.,5-8F, HONE1and SUNE1) stably overexpressing Klf4transgene were confirmed to be successfully generated by EGFP assay under inverted fluorescence microscope, qRT-PCR and Western blot were used to detect the expression of Klf4transgene in5-8F, HONE1and SUNE1.2. The overexpression of Klf4in NPC cells can suppress cell proliferation in vitroCompared with the control group, CCk8assay, cell cycle and colony formation assay strongly supported that Klf4overpression could suppress cell proliferation.3. The down-regulation of Klf4in NPC cells can suppress cell proliferation The results from CCk8assay indicated that down-regulated Klf4expression in5-8F and HONE1cells induced cell proliferation (p<0.01)4. The overexpression of Klf4can inhibit NPC cell proliferation in vivoOur results showed that only five mice obtained tumor which injected5-8F-Klf4cells;while,six moice were obtained tumor in control group.Additionally,both the staining intensity and the number of hyperproliferative Ki-67positive and BrdU positive tumor cells were significantly decreased compared with control (p<0.01), on the contrary, p21positive cells were increased (p<0.01). Part III Klf4can lead to MET and suppress NPC cells migration1. Compared with control group, Klf4overpression can induce the expression of E-cadherin in NPC cells (HONE1and5-8F)(p<0.05), while the expression of N-cadherin was downregulated in HONE1and5-8F cells (p<0.05)On the contrary, the downregulation of Klf4can induce an EMT, as shown by upregulation of epithelial protein E-cadherin, and downregulation of mesenchymal proteins vimentin and N-cadherin.2. Klf4overexpression can suppress cell motility (p<0.01), as indicated byTranswell migration assay, while down-regulated expression of Klf4caused opposite results.Conclusion:Taken together, these findings demonstrate that Klf4can suppress cell proliferation, trigger MET, and inhibit cell motility in NPC cells.