A Preliminary Study On Genetic And Epigenetic Alterations Of LF Gene And Their Functional Correction In The Nasopharyngeal Carcinoma Cell Line

Nasopharyngeal carcinoma(NPC) is a malignancy derived from epithelium with high incidence in Southeast Asia and Southern China. The epidemiological and etiological studies indicate that genetic alterations, Epstein-Barr virus (EBV) infection, dietary and environmental factors are closely associated with its occurrence and development. The ethnic susceptibility and familial aggregations of NPC manifest that genetic alterations may play very important roles in NPC, but relatively little is known about the exact molecular genetic changes associated with NPC. Studies to date have shown that no point mutations or low frequency of point mutations can be found in many all the well-known tumor suppressor genes (TSGs), such as p53, Rb, p21, MTS/p16, VHL, FHIT. The overexpression of p53 and loss of p16 may participate in the occurrence and development of NPC, but it cannot be concluded that these alterations play key roles in tumorigenesis of nasopharyngeal epithelia, according to our current knowledge.Based on the microarray experiments conducted on NPC and other human malignancies, LF gene located at 3p21.3-22(which is a common eliminated region in NPC), evoked our interest. Previous results in our laboratory indicated LF was down-regulated or absent in 76ï¼…of NPC tissues due to LOH, gene mutation and/or promoter methylation.In this study, we further analyzed the expression, genetic and epigenetic alterations of LF in NPC cell lines. Subsequently, the expression of LF was restored in 5-8F cells via gene transfection and the changes of biological characteristics of transfected 5-8F cells were examined for studying the possible role of LF in NPC. The exprimental methods and results were as follows:â… . The expression, genetic and epigenetic alterations of LF in NPC cell linesRT-PCR and Real-time RT-PCR were performed to analyze the expression of LF at the transcription level in 8 chronic nasopharyngitis biopsy specimens tissues and 7 NPC cell lines (including HNE1, HNE2, HNE3, CNE1, CNE2, 5-8F, 6-10B). The results showed that mRNA expression level of LF was stable in chronic nasopharyngitis tissues, while aberrant expression (loss or down-regulation) of LF was detected in 100ï¼…(7 of 7) of NPC cell lines (P=0.001). To elucidate the molecular mechanisms underlying the aberrant expression of LF in NPC cell lines, we detected the genetic and epigenetic alterations of LF in NPC cell lines, including LOH, mutation and promoter methylation status of LF.By use of PCR-SSCP and subsequent sequencing analysis, we examined whether there exsisted mutations in the promoter region, exon 1 and exon 2 of LF in 7 NPC cell lines. In consequence, a mutation (1076delC) in the promoter region was only found in HNE1 cell line and no mutation was found in other NPC cell lines or at other sites, resulting in a total mutation frequency of 14ï¼…in N-PC cell lines (1/7). We speculated that 1076delC mutation in the promoter region might influence the binding ability of the trans-acting factors to the LF promoter, leading to the transcription inactivation of LF. In addition, our results verified that polymorphism could be found in exon 2 of LF.We detected the LOH status using a PCR approach with primers for amplification of polymorphic microsatellites flanking the LF gene (D3S4169, GBD:181215, D3S1478, G59627 and RH119558) in 7 NPC cell lines. The results showed only one allele loss was found at the D3S1478 site in CNE1 and no allele loss was found at other sites or other cell lines, i.e. the total allele loss frequency of LF gene in NPC cell lines was 14ï¼…(1/7), indicating that LOH may play a certain role for aberrant expression of LF in NPC cell lines.To evaluate the relationship between promoter methylation of LF and its expression in NPC cell lines, methylation specific-PCR (MSP) was applied to examine the status of CpG islands methylation in LF promoter in 7 NPC cell lines. The results showed that promoter methylation could be found in all the NPC cell lines examined, and unmethylation of CpG islands also could be found in CNE1 and 5-8F cell lines, thus the promoter methylation rate in NPC cell lines was calculated as 100ï¼…(7/7), suggesting that the promoter CpG islands methylation may be considered as a key mechanism causing inactivation of LF in NPC cell lines.â…¡. Preliminary investigation on the functions of LF in nasopharyngeal carcinoma cell linesIn order to explore the function of LF in NPC, gene transfection experiment was performed. First, the open reading frame (ORF) sequence of LF was amplified from the chronic nasopharyngitis tissues by RT-PCR. The amplified ORF sequence was showed to have two single nucleotide polymorphism (SNP) sites and one mutation (9523A→T). Subsequently, the eukaryotic expression vector of LF (designated pcLF) was constructed by inserting the LF ORF into the pcDNA3.1(-) vector. Then the pcLF vector was transferred into 5-8F cells by Lipofectamine 2000. After G418 selection, we obtained several G418-resistant cell clones. It was demonstrated that LF expressed stably both at mRNA and protein level in the G418-resistant cell clones detected by RT-PCR and western blotting, respectively, showing that the 5-8F cell line stably expressing LF gene (named 5-8F-LF) was successfully established. The recombinant LF protein was detected mainly in the cytoplasm by immunocytochemistry.To illustrate the possible changes in biological characteristics of 5-8F-LF cells, flow cytometry (FCM) analysis was carried out. The results showed that LF expression could block the cell cycle progression of 5-8F-LF cells in G₁ phase, as the pencentage of 5-8F-LF cells in G₁ phase increased by～10ï¼…(62.28ï¼…vs 51.81ï¼…) while that in S phase and G₂-M phase decreased by～3ï¼…(24.54ï¼…vs 27.80ï¼…) and～7ï¼…(13.19ï¼…vs 20.38ï¼…), respectively.The proliferation and clonality abilites of 5-8F-LF cells were measured by MTT analysis and colony formation assay, respectively. Compared with 5-8F cells transfected with blank vector, 5-8F-LF cells proliferated much more slowly (P＜0.05) and had a much lower cloning efficiency (28ï¼…vs 54.7ï¼…), providing evidence that LF had an antiproliferative activity on 5-8F-LF cells. In summary, based on previous work of our laboratory, we further demonstrated that LF mRNA level was markedly down-regulated in NPC cell lines compared to the chronic nasopharyngitis tissues, and then we analyzed the molecular mechanisms of LF aberrant expression in NPC cell lines. According to our results, the aberrant expression of LF in NPC cell lines was partially ascribed to LOH and gene mutation but mainly due to LF promoter methylation. Moreover, stable expression of LF could block the cell cycle progression of 5-8F-LF cells in G₁ phase, and decrease the proliferation and clonal abilites of 5-8F-LF cells, indicating LF might be a promising NPC-associated candidate TSG, but more work is required to be done to confirm it.