| Objective:To analyze all kinds of genetic variation in esophageal squamous cell carcinoma(ESCC) at the genomic level and depict the landscape of genomic variation and cancer related gene variation. To explore the driver genes and associated signal transduction network in esophageal squamous cell carcinoma.To validate the results of omics sequencing: To study the expression of target gene in ESCC tumor tissue and paired normal esophagus tissues; To clarify the location difference of wild type and mutant type of target gene; To explore the impact of knock-in and knock-down target gene on proliferation, migration and invasion of ESCC cell lines; To preliminarily explore the machanism of target gene in ESCC. Methods:1. Tumor samples and adjacent, histologically normal tissues were obtained from 104 ESCC patients and genomic DNAs were extracted. After the quality control, we conducted whole genome sequencing(WGS) in 14 ESCC cases and whole exome sequencing(WES) in 90 cases. Somatic events identified in the WGS and WES set were selected for target capture-based sequencing validation. We applied various bioinformatics methods to analyze all kinds of genomic variation and cancer related gene and signaling pathway and difference between stage I and stage III.2. PCR sanger sequencing was applied to validate mutation site of ZNF750 in WGS and WES. Quantitative real-time PCR was used to validate copy number deletion of three WGS cases and to study the copy number of cases with ZNF750 point mutation.3. Tissue microarray immunohistochemistry was used to examine the expression and location of ZNF750 in tumor samples and normal esophagus tissues from the cohorts of ESCC patients.4. Immunofluorescence was used to detect the location of wild type and S70 X mutant of ZNF750.5.(1) In vitro, the p LKO.1-puro-sh RNA-ZNF750 vector carrying validated knock-down sequencing were transfected into KYSE150 and KYSE140 cell lines respectively. The knock-down efficacy was confirmed by immunoblotting analysis. Traditonal MTT assay, migration and invasion assays in an x CELLigence RTCA DP system were performed to observe the impact of silencing ZNF750 gene on proliferation, migration and invasion of ESCC cell lines.(2) In vivo, the nude mice tumor xenograft model was establish by injecting ZNF750 knock-down KYSE150 cell line and negative control into the subcutaneous tissue of the nude mice. After 3-4 weeks, the number and volume of tumors were measured.6. Over-expression vector carrying wild type ZNF750 genes was transfected into K2 cells with low ZNF750 expression and immunoblotting was used to validated transfection efficiency. Traditonal MTT assay, colony formation assay and transwell chamber invasion assay were performed to observe the impact of over-expression of ZNF750 gene on proliferation, migration and invasion of ESCC cell lines.7. Western blot was used to observe the expression level of known target gene of ZNF750 in knock-down ESCC cell lines and xenograft tumor tissue. Results:1. We totally identified 10330 somatic point mutations in coding regions: There were 10056 SNVs, of which 67% resulted in missense changes and 6% for nonsense changes; There were 90 small In Dels, of which 84% introduced translational frameshifts and 16% were in-frame; In addition, we identified 184 alterations in splice sites. In total, we identified 7597 non-silent somatic mutation events.2. We next characterized the spectrum of mutations in esophageal squamous cell carcinoma. The high frequency of C>T transitions in coding regions was identified by WGS and WES. The second high frequency is C>G transversion. We identified three kinds of mutational signatures within ESCC genomes by the non-negative matrix factorization method.3. By applying Mut Sig methods eliminating the influence of gene length and background mutation rate, we identified eight significantly mutated genes and 14 significantly altered signal pathway truly associated with ESCC. Among these results, ZN750 is a new ESCC associated significantly mutated gene(P<0.0001,FDR<0.178). 64% of the mutations identified in ZNF750 are inactivating. Additionally, ZNF750 deletions, but no somatic mutations, were observed in 3 out of 14 cases in the WGS set(21%, G score > 0.23).4. Strikingly, a pronounced diversity of NOTCH1 driver mutations was observed in 35% of early stage I tumors but only 8% in late stage III tumors. The difference is statistically significant(P<0.0001). Meanwhile,We identified 7 signal pathways whose frequency difference is statistically significant between stage I and stage III.5. Copy number variation analysis in the whole genome sequencing set yielded universal deletions affecting 4p, 11 p, 16 p, 19 p, 19 q, and frequent gains of 3q, 5p, 7p, 7q, 8p, 8q, 12 p, 14 q, 18 p, 20 q, 21 q, Xp, Xq. By applying GISTIC,we defined 126 significant CNV regions.6. By means of CREST for structure variation analysis, we identified 988 structure variation events, including 257 interchromosomal translocations, 296 intrachromosomal translocations, 27 deletion, 163 insertion and 2 inversion.7. The six mutation sites have been validated successfully by PCR sanger sequencing, the other was validated manually by IGV; Copy number deletions of ZNF750 in three WGS cases have also been validated by q PCR. In addtion,we also found ZNF750 deletion in four of six WES cases with ZNF750 point mutation.8. ZNF750 was aberrantly upregulated in cytoplasm in ESCC tissues compared to that of normal esophagus tissues. Meanwhile, ZNF750 was dramatically down-regulated in nucleus in ESCC tumors compared with normal controls.9. Wild type of ZN750 is located in both nucleus and cytoplasm. S70 X mutant of ZN750 is located in cytoplasm.10.(1) We found that depletion of ZNF750 significantly increased the proliferation of KYSE150 and KYSE140 cells as monitored by MTT assay and colony formation assay. Moreover, ZNF750 knockdown strongly promoted KYSE150 and KYSE140 cell migration and invasion.(2) We injected ZNF750 knock-down KYSE150 cell line and negative control into the subcutaneous tissue of the nude mice establishing the nude mice tumor xenograft model. After 3-4 weeks, Xenograft growth assays showed promotion in tumor incidence and volume with KYSE150 cells stably depleting ZNF750.11. Forced expression of ZNF750 in K2 cells with low ZNF750 expression inhibited cell proliferation, colony formation and invasion,which furtherly indicate that ZNF750 may act as a tumor suppressor gene in ESCC.12. Consistently, knockdown of ZNF750 in KYSE150 and KYSE140 led to a reduced KLF4 level. KLF4 in xenograft tumor tissue of ZNF750 knockdown group is also obviously lower than the control group. Conclusion:1. By means of whole genome sequencing and whole exome sequencing, we identified all kind of genetic variation including single nucleotide varition, copy number variation and structure variations followed by target capture-based sequencing validation; We also identified obvious inactve mutation and copy number deletion in ZNF750 which may be a driver gene in ESCC.2. ZNF750 may be a new tumor suppressor gene closely related to esophageal squamous cell carcinoma.The tumor suppressor role of ZNF750 may be achieved partly through KLF4. |
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