Study On The Effect Of Genetic And Epigenetic On Alternative Pre-mRNA Splicing Of Tumor Suppressor Genes CDH1 And HMLH1 In Gastric Cancer Cells

Gastric cancer(GC)is a leading cause of cancer-related death worldwide,which arises from accumulated genetic alterations and various factors.Previous studies show that CDH1 germline mutations is the genetic basis of hereditary GC,while perturbation of E-cadherin expression is the most frequent genetic alteration in sporadic GC.Intestinal gastric cancers have been recognized as common extracolonic tumors in the hereditary nonpolyposis colorectal cancer syndrome,which are often caused by germline mutations of mismatch repair genes,predominantly hMLH1.However,several groups have indicated the association between hMLH1 mutations and hereditary GC in East Asians.In our previous work,we detected the germline mutations and methylation status of CDH1 and hMLH1 Nonetheless,in some GC patients,reduced expression of E-cadherin or hMLH1 protein was detected without pathological germline and somatic mutationsor hypermethylation.The regulatory mechanism needs to be studied.Alternative splicing of pre-mRNA plays a crutial role in differentiation,development,and disease and is a major source for protein diversity in eukaryotes.The balance of alternative splicing keeps normal function,and disordering of this balance might be harmul and leading to cancer.Recent studies revealed that,besides traditionally,RNA regulatory elements and spliceosome,DNA methylation and histone modification had been also implicated in alternative splicing regulation.Based on our genetic mutations in patients with gastric cancer,in this study we probed the role of genetic and epigenetic alteration on alternative pre-mRNA splicing of tumor suppressor genes CDH1 and ZhMLH1 in order to provide a new traint in the initiation and etiology of gastric cancer.Part one:Study on the effect of genetic and epigenetic on alternative pre-mRNA splicing of CDH1 geneWe screened CDH1 gene germline mutations in 282 cases with gastric cancer and 425 controls from peripheral blood DNA samples,and analyzed its alternative pre-mRNA splicing in tumor and paired normal tissues,then further study the effect of genetic and epigenetic alteration on alternative pre-mRNA splicing of CDH1 gene.1.Mutation screening of CDH1 exon 9 in GC patients revealed two novel germline missense mutations including c.1296C>G(N432K)and c.1297G>A(D433N).RT-PCR for E-cadherin in patient 278(harboring the c.1296 C>G mutation)revealed the coexistence of the normal,delEx9 transcripts.However,RT-PCR for E-cadherin in patient 201(harboring the c.1297 G>A mutation)just yielded normal transcript.The results of bio informatics analysis and RT-PCR indicated that c.1296 C>G mutation was pathological germline mutation,but c,1297 G>A mutation was neutral mutaiton.2.RT-PCR for E-cadherin in patients revealed the coexistence of the normal and 1054del83 transcripts as well.We further detected the CDH1 alternative splicing products in tumor and paired normal tissues from 50 gastric patients by qRT-PCR.The results indicated the coexistence of the CDH1 normal and 1054de183 transcript in majority of gastric patients.These results suggested CDHI 1054del83 transcript was widely existed in GC,and verifying in gastric cell lines.3.We did not detect mutations in CDH1 exon 8 and flanking sequence,thus searching for another factors engaged our interest.Various evidence confirm that transcription and splicing is simultaneously and close coupling.Alternative pre-mRNA splicing is apparently regulated via epigenetic changs.First,we detected higher DNA methylation pattern of CDH1 exon 8 and flanking sequence in GES-1 and GC cell lines.AZA treatment did not affect CDH1 1054del83 transcript products,thus illustrating no direct relation between DNA methylation and CDH1 alternative splicing.4.ChIP results demonstrated a lower H3ac and H4K16Ac in internal region of CDH1 gene surrounding the exon 8 both in SGC-7901 and BGC-823 compared to GES-1.TSA treatment decreased ratios of CDH1 1054del83 vs normal transcript in BGC-823 and SGC-7901.So it suggesting histone acetylation was involved in the splicing regulation through transcriptional activation and the rate of RNA polymerase II elongation.5.Furthermore,higher level of H3K36me3 around the CDH1 exon 8 regions was discovered in SGC-7901 and BGC-823 compared to GES-1.siSETD2 treatment decreased ratios of CDH1 1054del83 vs normal transcript in GES-1,SGC-7901 and BGC-823.Thus the high lever of H3K36me3 correlates with increasing CDH11054del83 transcript.But siSRSF2 treatment did not influence ratios of CDH11054del83 vs normal transcript in GES-1 and SGC-7901,BGC-823.Additional studies were needed to disclose the mechanism how the level of H3K36me3 regulated alternative splicing of CDH1 exon 8.Part two:The genetic and epigenetic on alternative pre-mRNA splicing of hMLHl gene in gastric cellsWe screened hMLH1 gene germline mutations in 236 cases with gastric cancer and 240 controls from peripheral blood DNA samples,and analyzed its alternative pre-mRNA splicing in tumor and paired normal tissues,then further study the effect of genetic and epigenetic on alternative pre-mRNA splicing of hMLH1 gene.1.Case-control analysis showed that hMLH1 c.2101C>A(Q701K)variation might increase the risk of gastric cancer in Chinese males.RT-PCR in patients with mutaion and without mutation detected c.2101C>A mutation affecting alternative pre-mRNA splicing of hMLHl gene,and leading to hMLH1 Exon 18 skipping.2.RT-PCR in patients with and without mutation detected coexistence of the hMLH1 normal,delEx10,delEx11,delEx10-11 delExl6 and delExl7 transcripts.We confirmed these aberrant transcripts were widespread in gastric cell lines.3.We did not detected the mutations in hMLHl exon10,exon11,exon16,exon17 and flanking sequence,thus genetic changes could be ruled out.Therefore,we investigated the relation between epigenetics and alternative pre-mRNA splicing in hMLHl gene.Firstly,the aberrant splicing regions of hMLHl were hypermethylated in human gastric mucosa cell line and gastric cancer cell lines.There is no significant change in the transcription level of alternative splicing transcripts after the use of DNA methyltransferase inhibitor AZA.These data showed no direct relation between DNA methylation and hMLHl alternative splicing.4.Secondly,ChIP results displayed H4K16ac and H3ac were lower in hMLH1 exon10-11 region in SGC-7901 and BGC-823 when compared with GES-1.Consequently,chromatin structure was compacted,and the rate of RNA polymerase Ⅱelongation was slow in this region,then leading to weak splicing sites identified,thus conducive to exons jumping.While after the adding of histone deacetylase inhibitors TSA,the amount of alternative splicing transcript including hMLHl delEx10-11 in gastric cancer cell line MGC80-3 decreased significantly,further manifesting correlation of lower histone acetylation with exon skipping.Nevertheless,the enrichment of acetylation in hMLHl exon16-17 region in SGC-7901 was similar to GES-1.And there was no significant change in the transcription lever of alternative splicing alteration.5.Then,the enrichment of H3K36me3 was lower in hMLHl exon 10-11 and exon16-17 regions in gastric cell lines when compared with GES-1.Treatment with siRNA of SETD2(interference of specific methylation transferase of H3K36me3)increased the ratio of aberrent transcripts such as hMLHI delEx 10,delEx 11 and delEx10-11.Overall,lower enrichment of H3K36me3 was benificial to aberrant transcript.In comparision with GES-1,the enrichment of H3K4me2 in hMLHl exon 10-11 and exon 16-17 regions was also lower in gastric cell lines.Thereby,the lever of H3K4me2 possibly modulated hMLH1 alternative splicing as well.Treatment with siSRSF2,the ratio of hMLHl delEx10 and delfx10-11 transcripts were increased.When SRSF2 expression reducing,normal splicing site could not be recgnized,and increasing aberrant transcripts.However,the expression lever of hMLH1 delEx16 and delEx17 transcripts did not change after siSRSF2,thus SRSF2 did not affect alternative splicing site in hMLHl exon 16-17 region.In conclusion,our study revealed CDH1 germline mutation c.1296C>G created CDH1 exon 9 skipping and hMLH1 germline mutation c.2101C>A caused hMLH0 exon 18 jumping.CDH1 1054del83,MLH1 delEx10,MLH1 delEx11,MLH1 delEx10-11,MLH1 delEx16 and MLH1 delEx17 transcripts were ubiquitous in gastric cells.Histone acetylation and specific histone methylation regulated the selection of these aberrant transcriptss through the rate of RNA polymerase II elongation or effect of splicing factors.Therefore,it was essential to study the effect of both genetic and epigenetic alteration on alternative pre-mRNA splicing of CDH1 and hMLHl in gastric carcinogenesis.