Expression,Downregulation Mechanism And Function Of Kank1Gene In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a kind of epithelial malignancy with high incidence in Southeast Asia and Southern China, while seldom in Northern China, Europe and America, characterised as a distinct ethnic aggregation and geographic distribution. The research of epidemiology and etiology revealed that varied factors involved in the occurrence and development of NPC nowadays, including Epstein-Barr virus(EBV) infection, environmental factors(mainly chemical carcinogens,etc.) and genetic factors (genetic susceptibility). The activation of oncology and inactivation of tumor suppressor genes are the two resons of genetic susceptibility which play an important role in NPC development. So it is of great significance to look for and identify the genes closely related to the development of NPC and study their functions which is the key way to reveal the molecular mechanism of NPC.Kankl gene is an important member of kank family mapped to chromosome9at p24.3. The research shows that kankl gene exits point mutation, LOH(loss of heterozygous) or promoter hypermethylation in the tumor tissues of brain, lung and kidney, which leads to reduce or lost of expression and function of the gene. The results indicated that kankl may act as a tumor suppressor gene associated with tumorigenesis. Recently high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in NPC samples. High incidences of LOH were identified on chromosomes9p and following research narrowed the region of high frequency of LOH in9p21.3-p24.3. All the results suggested that there may be associated with the NPC tumor suppressor gene in this region. Several tumor suppressor genes have been reported in this region including p16, NGX6and kankl. While till now the relationship between the kankl gene and NPC has not been reported.Therefore, we performed to examine the expression, the possible mechanisms leading to its aberrant expression, biological function and pathological significance of kankl in NPC[The expression status of kankl in NPC]The expression of kankl at the transcription level were investigated by RT-PCR and real-time RT-PCR in a normal nasopharyngeal cell line NP69,4NPC cell lines (CNE1,CNE2,5-8F,6-10B),11normal nasopharyngitis biopsies and77NPC biopsies. The results showed that kankl was expressed stably in NP69cells and almost all of the normal nasopharyngitis tissues, while down-expression of kankl was detected in100%(4/4) of NPC cell lines and50%above of NPC biopsies. Western Blotting results indicate that kankl was downregulated in NPC tissues contrast to normal nasopharyngitis tissues, the downregulation ratio was60%(12/20). The results indicate that kankl was high frequency of down-regulation or loss in NPC.[Molecular mechanisms causing kankl aberrant expression in NPC]Kankl located at chromosomes9p24which is high incidences of LOH region in NPC. Microsatellite markers located at gene locus or flanking the gene (upstream and downstream) were usually used to indirectly reflect deletion. Using PCR amplification, followed by denaturing poly-acrylamide gel electrophoresis (PAGE) and silver staining, two microsatellite markers, D9S1858and WI12779were used for LOH analysis in35Primary NPC biopsies as well as their matched control peripheral blood samples. Aberrant promoter methylation is one of the most common reasons for epigenetic alteration of TSGs. CpG islands were found in the promoter region through CpG Island Searcher software. MSPCR analysis was performed to assess the kankl methylation status. The results showed that D9S1858and WI12779loss frequeney of kankl were respectively detected in at least31.4%(11/35) and34.3%(12/35) of NPC tissues. And the total loss frequeney of kankl was detected in at least51.4%(18/35) of NPC tissues. MSPCR analysis revealed kankl promoter hypermethylation in4NPC cells, all the NPC tissues(35/35), and60%(6/10) normal tissues. It suggested that LOH and promoter methylation were both due to inactivation of kankl in NPC.[Investigation of kankl functions in NPC cells]In order to investigate the functions of kankl in NPC, kankl plasmids and empty vectors were transferred into5-8F cells by lipofectamine2000to establish the5-8F cells stably expressing kankl (5-8Fkank1) and control vector cells(5-8Fvector). Then, we analyzed the affection of biological characteristics by overexpression of kankl in5-8F cells. The MTT analysis indicated that5-8Fkank1cells showed significant growth inhibition compared with5-8Fvector or untransfected5-8F cells (P<0.05).And colony formation assay invealed5-8Fkank1cells showed significant reduction in the cloning efficiency compared with the control groups(P<0.05). The results of flow cytometry (FCM) analysis showed that5-8F kank1cells could be arrested in G0/G1phase, as the G0/G1phase cells increased while S phase and G2/M phase cells reduced compared with the control groups(P<0.05). The flow cytometry (FCM) and hoechst33258staining analysis showed that apoptosis cells in5-8Fkank1cells increased significantly than5-8Fvector or5-8F cells(P<0.05).The cell migration assay and invasion assay revealed that the mobility and invasion ability of5-8Fkank1cells were inhibited significantly compared with5-8Fvector or5-8F cells(P<0.05). All the results suggested that kankl could significantly decrease the cells growth, promote the cells apoptosis, inhibit the cells migration and invasion in vitro in NPC.[Clinical significance of kankl in NPC]In order to investigate the clinical significance of kankl in NPC Immuno-histochemistry(IHC) were performed to analyze the expression and location of kankl at the protein levels in14cases of normal nasopharynx and69cases of NPC. The positive signal was mainly located in the cytoplasm.The results showed that72.5%(50/69)of the NPC specimens showed negative or very low expression level of kankl protein while only1case among14normal nasopharynx showed downregulation of kankl. Additionally, Kankl expression levels were negatively associated with regional lymph node and clinical stages (p<0.05), tumors with kankl expression tend to have a more advanced clinical stage and lymph node metastasis. However it was unrelated to the histological type/grade, primary tumor (T) stage, the patients'age and gender.In summary, our results suggest that kankl expresses stably in normal nasopharyngeal epithelia, but is significantly down-regulated or absent in NPC cell lines and tissues. LOH and promoter high hypermethylation play a certain role in aberrant expression of kankl in NPC. Overexpression of kankl in5-8F cells can inhibit the ability of proliferation, migration, invasion and promote the apoptosis of NPC cells. Down-regulation of kankl gene may play an important role in the development of nasopharyngeal carcinoma.The results suggest that kankl may be a candidated tumor suppressor gene of nasopharyngeal carcinoma.