The Screening And Preliminarily Functional Study Of Candidate Tumor Suppressor Genes Located At 3p21.3 In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) remains to be the major malignant tumor in head and neck tumor. Although the traditional surgery, radiotherapy, chemotherapy and combined therapy have a great therapeutic efficacy to early oral cancer, a satisfied effect can not be achieved to those patients who were in advanced stage or recurrence stage. Therefore, studying the molecular mechanism of carcinogenesis of oral mucous membrane and finding the molecular marker of early diagnosis and treatment can attribute to acquire new therapy and raise survival rate and quality.Increasing study suggested that malignant tumor is actually a gene disease and the occurrence and development involved the change of several genes. Tumor suppressor gene (TSG) always plays an important role in physiological function and its inactivation and deletion are closely related to the occurrence and development of tumor. Many tumor suppressor genes are found by studying the loss of heterozygosity or homozygosity of allele. Study showed high frequency deletion of chromosome 3p21.3 was existed in several tumors such as lung cancer, nasopharyngeal carcinoma (NPC), and so on. Several groups find that the region of chromosome 3p21.3 also exist different degree of high frequency deletion in OSCC. Currently, little study has done to the role of TSGs at this region in the occurrence and development of OSCC and amount of information remain to further be mined.Based on the reasons above, in this study, we prepare to choose the candidate TSGs little reported as the research object. Screening the candidate genes down-expression in OSCC and further analyze the possible reason of expression inactivation and the biological function in OSCC.[The screening of candidate TSGs located at 3p21.3 in OSCC]We preliminarily determined 11 genes as the research object, such as AUXD1,BAP1,FUS1,GNAT1,LARS2,LTF,NPRL2,RASSF1A,SEMA3B,SEMA3F and ZNF1, by literature review and analyzing the possible function of genes with bioinformatics. RT-PCR was performed to analyze the expression of 11 genes at the transcription level in primary OSCC biopsies and matched tumor-adjacent normal tissues. The results showed that LTF, GNAT1, SEMA3B, AUXD1 were down-regulation or deletion in 50%(6/12),58.3%(7/12),41.7%(5/12),41.7%(5/12) respectively in OSCC and there were significant difference compared to matched tumor-adjacent normal tissues (P<0.05). SEMA3F, RASSF1A were down-regulated or deletion in 25%(3/12), while ZNF35, LARS2 were up-regulated in 33.3%(4/12) and 25%(3/12) in OSCC. No significant difference in transcriptional levels of other 3 genes (NPRL2,BAP1,FUS1) was found between OSCC and matched tumor-adjacent normal tissues (P>0.05). Results of preliminary screening suggested LTF, GNAT1, SEMA3B, AUXD1 were highly down-regulated in OSCC. Therefore, we choose these 4 gens as the object for further research.[The role of aberrant promoter methylation in gene expression]To understand exactly the expression statues of 4 genes (LTF, GNAT1, SEMA3B, AUXD1) in OSCC, we analyzed the expression levels of 4 genes in tongue cancer cell line-TCA8113 and 36 OSCC and matched tumor-adjacent normal tissues by RT-PCR. Results revealed LTF, GNAT1, SEMA3B were all down-regulated or deleted except AUXD1 expressed in TCA8113. Compared to matched tumor-adjacent normal tissues, LTF, GNAT1, SEMA3B, AUXD1 were down-regulated or deleted in 61.1%(22/36),44.4%(16/36),50%(18/36),47.2%(17/36) in OSCC biopsies. Statistical analysis showed that the 4 genes have statistical significance between the two groups (P<0.05).These data were further analyzed to determine the relationship between the aberrant expression of 4 genes and clinical related factor withχ2 test.Statistical analysis indicated that no any significant correlation was found between gender and expression of LTF,AXUD1,GNAT1 and SEMA3B (P>0.05); Only the under-expression of LTF but not other three genes was significantly correlated with metastasis. The expression level of LTF in patients with metastasis was significantly lower than those with no metastasis (P<0.05).To further explore the role of promoter methylation in abnormal expression of genes in OSCC biopsies or cell lines, we analyzed the promoter methylation statues of LTF, GNAT and SEMA3B in OSCC patients with down-regulated expression of each 3 genes. MSPCR analysis revealed LTF, GNAT, SEMA3B promoter hypermethylation in 72.7%(16/22),75%(12/16),77.8%(14/18) primary OSCCs respectively, and in 22.7%(5/22),56.25%(9/16) and 38.9%(8/18) matched tumor-adjacent normal tissues respectively.χ2 analysis indicated that there was a significant difference in methylation frequency of LTF (P=0.000<0.01) between OSCCs and tumor-adjacent normal tissues, while no difference in that of GNAT1 between this two groups (P=0.264>0.01). In the other hand, restoration of LTF or SEMA3B expression along with decrease in methylated allele of LTF or SEMA3B could be achieved in TCA8113 cell line after the treatment with 5-aza-2'-deoxycytidine (Aza), a DNA methyltransferase inhibitor. It suggested that promoter hypermethylation should be the important mechanism responsible for inactivation of LTF or SEMA3B in OSCC.[Investigation of LTF functions in tongue cancer cells]Real-time PCR can detect more exactly than RT-PCR in mRNA expression levels. Based on previous analysis, we further determined the expression levels of LTF in OSCC biopsies and matched tumor-adjacent normal tissues using real-time PCR method. Results showed the average of Ct value (9.490±1.675) denoting the expression level of LTF in OSCC biopsies was higher than that in matched tumor-adjacent normal tissues (6.212±4.00). It is similar to previous results of RT-PCR that LTF expression was obviously down-regulation (P<0.01) and the ratio of down-regulation was 63.3%(19/30).To better understand the role of LTF protein in OSCC, immunohistochemical method was also used to detect the protein levels and location of LTF in 30 OSCC specimens. The results showed LTF protein was mainly located in the cytoplasm and stratified squamous epithelial cell. Additionally, positive strong signal (++)or(+++) can be detected in 26 tumor-adjacent normal tissues besides 4 expressed weak signal, while 60%(18/30) of the OSCC tissues showed negative (-) or very low expression level (+) of LTF protein.In order to investigate the functions of LTF in tongue cancer cells, using enzyme digestion methods, we obtained-2.8kb cDNA containing the open reading frame (ORF) sequence of LTF gene from bought pCMV6-XL5-LTF. Sequencing results showed that LTF cDNA sequence contained three missense SNP site (23R, T29A, R47K), but these had no influence on LTF function according to bioinformatic analysis. Subsequently, the eukaryotic expression vector of LTF (named pcLTF) was constructed by inserting the LTF ORF into the pcDNA3.1(-) vector. Then the pcLTF vector was transferred into TCA8113 cells by Lipofectamine transfection technology. After G418 selection, several G418-resistant cell clones were obtained. It was demonstrated that LTF 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 TCA8113 cell line stably expressing LTF gene (named TCA-LTF) was successfully established. The recombinant LTF protein was detected mainly in the cytoplasm of TCA-LTF by immunocytochemistry.Subsequently, we examined the changes in biological characteristics TCA-LTF cells. The proliferative activity and clonality ability of TCA-LTF were measured by MTT analysis and colony formation assay, respectively. Compared with TCA8113 cells transfected with blank vector (named TCA-pc3.1) or untransfected TCA8113 cells, TCA-LTF cells showed especial proliferation inhibition (P<0.05) and reduction in colony formation efficiency (27.7% vs 51.1%/47.7%). Flow cytometry (FCM) analysis showed that TCA-LTF could block the cell cycle progression of TCA8113 cells in G0-G1 phase, as the pencentage of G0-G1 phase cells were increased (66.37% vs 53.7%) while S phase (24.6% vs 32.83%) and G2-M phase cells (9.05%vs 13.47%) were reduced. These results indicated that LTF overexpression blocked TCA8113 cells at G0-G1 phase resulting in attenuated proliferation and clony forming ability in vitro.In summary, we elected the deletion region of chromosome 3p21.3 as a breakthrough point to find other TSG candidates closely related OSCC, expression of 11 genes from which in OSCC biopsies and matched tumor-adjacent normal tissues have been done. Our results suggest that 4 genes, such as LTF, GNAT1, SEMA3B, AUXD1, expresses is significantly down-regulated or absent in OSCC tissues. Promoter hypermethylation plays a key role in inactivating LTF and SEMA3B in OSCC, but not in down-regulation expression of GNAT1. Re-expression of LTF in TCA8113 cells can confer partial reversion of the malignant phenotype of tongue cance cells. These results will help us comprehensively understand molecular mechanism underlying OSCC carcinogenesis, and provide the theoretical and experimental evidence for finding new therapy method and biomarker used in early diagnosis or therapy.