Identification Of LTF Gene On The Region Of NPC Susceptibility Gene-Chromosome 3p21 And The Function Study Of LTF Gene That Inhibited Proliferation In The Nasopharyngeal Carcinoma Cells

【Identification of LTF Gene on the Region of NPC Susceptibility Gene—Chromosome 3p21】In our previous study, linkage analysis of 18 pedigrees from Hunan Province of China suggests that potential susceptibility loci linked to NPC are located on chromosome 3p21. The putative NPC susceptibility locus is located a region from D3S1289 to D3S1298 on chromosome 3p21 through haplotype analysis. These results show that the candidate NPC susceptibility genes may be on chromosome 3p21.To scan and identify the candidate NPC susceptibility genes on chromosome 3p21 further, two different gene chips were manufactured. One was cDNA microarray of subtracted cDNA libraries constructed by suppression subtractive hybridization, the other was a custom-made chromosome 3p21-specific cDNA microarray. Two different cDNA microarray were used to scran the same samples for improving their reliability.First, the colonies used to made cDNA microarray were prepared by PCR from 4 NPC's subtracted cDNA libraries (NNTDH12, TRP19 DHNE1, TNPC DNN119, and TNN119 DNPC). There were more than 1,200 colonies including 300 colonies from every library to produce cDNA microarray. The purified colonies were used to made cDNA microarray of NPC's subtracted cDNA libraries. After 19 NPCs, 3 NPC derived cell lines, and 10 chronic inflammation of nasopharyngeal mucosa tissue samples hybridized the cDNA microarray, primary data collection and analysis were carrded out via Genepix Pro 3.0 (Axon Instruments). The area of the array with obvious blemish was manually flagged and excluded from subsequent analysis. To minimize artifacts arising from low expression values, only genes with raw intensity values for both Cy3 and Cy5 of＞200 counts were chosen for differential analysis. Then overall intensifies were normalized with a correction coefficient obtained using the ratios of these genes. One thousand and ninety-seven valid colonies were obtained.For the above preprocessed hierarchical clustering, colonies for which＞85% of measurements among all the samples were selected for further analysis. Altogether 898 of the 1,097 colonies passed this variation filter for the initial, then mean-centered and normalized with respect to other colonies in the sample and corresponding colonies in other samples. First, this list of colonies was used in Genesis software (http://genome.tugraz.at) to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mucosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, and they were clustered tightly.Though chronic inflammation of nasopharyngeal mucosa tissue and nasopharyngeal carcinoma tissue samples could be distinguished by 898 colonies, we hoped to find the smallest colony number to reach the aim. Genesis software was used for the following in ANOVA analysis to obtain the differentially expressed colonies among chronic inflammation of nasopharyngeal mueosa, NPCs and NPC-dedved cell lines. We obtained 105 differentially expression colonies. At last, these differentially expressed colonies were used in Genesis software to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mueosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, which was clustered tightly. It showed that 105 colonies had the same test efficiency.To identify the 105 colonies differentially expressed in NPC, all the cDNA colonies were amplified by using of the SSH secondary primers. The PCR products were purified from the gel anddirectly sequenced. The cDNA sequences were compared to GenBank by Blast search (http://www.nebi.nlm.nih.gov/BLAST/). There were 14 known genes, including STAT5A, RAB25, SPARC, and SPRR3, et al. to be identified in all the 37 colonies expressed highly in NPC and NPC_derived cell lines. Different frequency exists in every gene. STAT5A gene had the highest frequency of 18.92%, the second was RAB25(13.51%), followed by SPARC, SPRR3 and so on. These 14 genes were distributed on 9 chromosomes, including 1q, 2q, 4p, 5q, 7p, 7q, 12p, 12q, 13q, 15q and 17q. Eighteen known genes, including LTF, PLUNC, CDC37L1, HNRPDL genes, et al. were identified in 68 colonies which expressed poorly in N-PC and NPC_derived cell lines. The frequency to every gene was different. PLUNC gene had the highest frequency of 30.88%, the second was CDC37L1 (13.24%), followed by HNRPDL and so on. These 18 genes were distributed on 12 chromosomes, including lq, 3p, 4q, 7q, 8q, 9p, 11q, 12p, 12q, 14q, 16p, 20q and 22q. We scanned 32 genes differentially expressed in NPC using the cDNA mieroarray of subtracted cDNA libraries constructed by SSH, including LTF gene on the region of NPC susceptibility gene—ehromosome 3p21.In addition, the results showed that lactotransferrin (LTF), dystroglyean 1, FLJ34969, and TU3A genes were down-regulated in NPC samples, glutamate receptor, metabotropie 2(GRM2) and interleukin 17 receptor B (IL17RB) were up-regulated in NPC samples by a custom-made chromosome 3p21-specifie cDNA mieroarray including 288 genes in this region in the same samples.Through eompositive analysis of these results, we found that LTF was down-regulated in NPC samples in the two different gene chips. There was the parallel change in the same samples by different cDNA mieroarray. So we could believe that LTF gene might play an important role in pathogenesis of NPC.To confirm the cDNA mieroarray results, we chose the same samples of cDNA mieroarray to perform real-time quantitative RT-PCR of LTF, PLUNC, CDC37L1, and HNRPDL genes, which were down-regulated in NPC samples, and the same was done to STAT5A, RAB25, and SPARC genes, which were up-regulated in NPC samples. The expression of LTF, PLUNC, CDC37L1, and HNRPDL genes were low in NPC and the expression of STAT5A, RAB25 and SPARC genes were high in NPC. It corresponded with the results of cDNA mieroarray and proved its reliability.To validate the genes differentially expressed in NPC by cDNA mieroarray, we used in situ hybridization technique to confirm once more the expression of PLUNC, CDC37L1 and STAT5A genes with two NPC-tissue arrays. The positive expression rate of LTF and PLUNC genes in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly higher than that of NPCs (p＜0.01). The positive expression rate of STAT5A gene in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly lower than that of NPCs (p＜0.01). It corresponded with the results of cDNA microaray and proved its reliability.【Function Study of LTF Gene that Inhibited Proliferation in the Nasopharyngeal Carcinoma Cells】LTF was the parallel change in the different cDNA mieroarray. With compositive analysis of it and the previous results by linkage analysis of pedigrees, we had reasons to believe that LTF gene might play an important role in pathogenesis of NPC. At the same time, the expression of lactotransferrin protein was studied using immunoehemisty in NPC-tissue microarray by Dr. Zhao-yang ZENG. The results showed that laetotransferrin protein was down-regulated in NPC and up-regulated in NPC adjacent epitheliums and chronic inflammation of nasopharyngeal mueosas. Laetotransferrin protein could be detected in almost all of gland in nasopharynx, confirming that lactotransferrin protein was a secretory protein up-regulated in nasopharynx. In addition, the expression of laetotransferfin protein was negative correlated to the neck metastasis states (p＜0.05) and was significantly correlated to the clinical stages of NPC (p＜0.01). The expression of lactotransferrin protein in clinical stageⅠandⅡis obviously higher than that of the clinical stageⅢandⅣ. These findings suggest that LIF may be correlated with invading, metastasis and clinical evlovement and it is a good candidate tumor suppressor gene in NPC, and it may be a molecular marker for prediction of NPC invading, metastasis and clinical evlovement.The results of cDNA mieroarray and TMA showed that LTF gene was down-regulated in NPC samples. To investigate the mechanism of LTF to NPC, we investigated the effects of LTF in the Nasopharyngeal Carcinoma Cells in vitro.First, we used recombinant human laetoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, we chose the reduction of the yellow tetrazolium salt3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay to assessment of proliferation- suppressing effect of LIF in NPC cell lines. The results of MTT assay showed that LTF significantly inhibited 5-8F, CNE1, and HNE1 cell proliferation after treated with lactoferrin. The growth of 5-8F, CNE1, and HNE1 cell treated with laetoferrin was suppressive compared with the control group. It was significantly different after treated with laetoferrin for 24 hours and 48 hours. It validated the results of cDNA microarray and TMA in part I from NPC cell lines in vitro. These findings suggested that LTF could reduce the growth of NPC cell lines in vitro and suppress the development of tumor, in addition, LTF was down-regulated in NPC samples.To follow, we chose 10μmol/L recombinant human lactoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, the experiment of flow cytometry was applied for cell cycle analysis of NPC cell lines to evaluate the effect of LTF. The findings showed that the percentage of G0-G1 phase of the cell cycle in 5-8F, CNE1, and HNE1 cells was significantly increased after treated with lactoferrin (10μmol/L) for 24 hours or 48 hours, but the percent of G2-M phase and S phase was reduced respectively. But it had no significantly effect to 6-10B cell after treated with lactoferrin. Among 5-8F, CNE1, HNE1, and 6-10B cells, 6-10B cell was the only one that had no characteristic of metastasis. It corresponded with the results of immunochemisty in NPC-tissue microarray- LTF might be correlated with invading, metastasis and clinical evolvement. The results hinted again that LTF mainly functioned in the mid-terminal stages of NPC and played an important role in the stages. These data were consistent with results of MTT army. Moreover, it proved that LTF could suppress the growth and proliferation of NPC cells in vitro.To reveal underlying mechanism that LTF delayed G0-G1 phase progress to inhibit growth of NPC cells in vitro, we chose 5-8F cell line with high characteristic of metastasis as investigation object. After 5-8F cells were treated with lactoferrin, we examined the expression levels of Cyclin D1, p-Rb, p21, p27, p53, total star3, p-stat3(Tyr705) and signal molecules of mitogen-activated protein kinase (MAPK) signal pathway including JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. At last, we hoped to reveal some underlying mechanism that LTF inhibited the growth and proliferation of NPC cells in vitro.After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of cyclin D1 and phosphorylated Rb by western blot. After treated with lactoferrin, the expression levels of cyclin D1 and phosphorylated Rb were reduced distinctly compared with the corresponding levels in the untreated. The results suggested that LTF affected the expression of cyclin D1 and p-Rb. Subsequently, we studied the expression of p21 and p27 proteins that were cyclin-dependent kinase inhibitor. After treated with lactoferrin for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, the expression levels of p21 and p27 proteins were increased significantly compared with the corresponding levels in the untreated. In conclusion, these findings suggested that LTF could suppress the proliferation of NPC derived cell lines 5-8F through regulating the moeulars of cyclin D1, p-Rb, cyclin-dependent kinase, and cyclin-dependent kinase inhibitor.To additionally realize the molecular changes associated with cyclin D1 and p-Rb, we examined the signal molecules of mitogen-activated protein kinase (MAPK) signal pathway by western blot according to our previous results and accumulated document evidences. After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. The expression level of p-ERK1/2 was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. But we found that LTF gene had no effect on the expression level of total ERK. The expression level of JNK2 was reduced obviously after treated with lactoferrin (10μmol/L) for 12h. LTF gene affected the expression level of JNK2 protein. LTF gene cut down the expression levels of not only JNK2 protein but also c-Jun protein. The expression level of c-Jun was decreased obviously after treated with lactoferrin (10μmol/L) for 30min, and then was kept at an invariablenes level. In addition, LTF gene had an important effect on the expression of c-fos. The expression level of c-fos was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. It corresponded with the changes ofp-ERK1/2.In summary, LTF negatively modulated sub-families of JNK2, and ERK of MAPK signal pathway to suppress the expression of cyelin D1, p-Rb, and Cyelin-Dependent Kinase and to delay the G0-G1 progression of nasopharyngeal carcinoma cells. LTF inhibited growth and proliferation of 5-8F cells in vitro by modulating the G1/S phase regulation systems with regulating eyelins/CDKs/CKI/pRb as the key point. Subsequently, regulation systems of gene expression are very complicated and organic contacted. There are some cross-talks among different pathways anddifferent moleeulars. To reveal the reality ftmetions of LTF, we must do more experiments to verify it farther.