Bioinformatics Analysis Of Alternative Splicing In Esophageal Squamous Cell Carcinoma And Research On The Role And Mechanism Of Splicing Factor HNRNPA1 In Esophageal Squamous Cell Carcinoma

Background As a common malignant tumor of the digestive system,esophageal squamous cell carcinoma(ESCC)occupies the leading position in terms of morbidity and mortality rates.Alternative splicing is the process of splicing pre-RNA to join exons in multiple splicing modes,resulting in the formation of multiple splice isoforms.Alternative splicing is regulated by cis-acting elements and trans-acting factors such as splicing factors.Dysregulation of alternative splicing is one of the important mechanisms of tumor development,and alternative splicing can lead to malignant phenotypes such as tumor proliferation,invasion,immune evasion,and drug resistance.For tumor-specific alternative splicing,m RNA vaccines,Splice-Switching Oligonucleotide(SSO)and other drugs can be developed.At present,the expression characteristics of alternative splicing in ESCC and the regulation of alternative splicing by splicing factors are less studied and the mechanisms are not yet clear.We performed bioinformatics analysis of alternative splicing in ESCC and explored the role and mechanism of splicing factors in ESCC,which is important to identify new targets for diagnosis and treatment of ESCC.Part I.Bioinformatics analysis of alternative splicing in esophageal squamous cell carcinomaObjective To investigate the expression characteristics of alternative splicing events in ESCC and their effects on biological functions.To analyze the association between splicing factors and alternative splicing and to screen for splicing factors that play an important role in ESCC.Methods Eighty-one ESCC tissues and 11 adjacent normal tissues from The Cancer Genome Atlas(TCGA)cohort were studied.Alternative splicing events in the TCGA ESCC cohort were annotated and quantified using the psichomics package in R.To screen for differential alternative splicing events,differences in alternative splicing events between ESCC tissue and adjacent normal tissue were compared using the Wilcoxon rank sum test.Biological function enrichment analysis was performed for genes corresponding to differential alternative splicing in ESCC.Then,one-way Cox regression analysis was used to screen for differential alternative splicing events affecting the prognosis(including overall survival and progression-free interval)of patients with ESCC.Pearson correlation analysis was performed on splicing factor expression levels and alternative splicing events.Subsequently,one-way Cox regression analysis and random forest method were used to screen for splicing factors affecting prognosis of ESCC.Finally,the expression of splicing factors was analyzed in pan-cancer.Results 35144 cases of alternative splicing events were initially detected in 11363 genes of ESCC samples.Among them,15813 cases of skipped exon(SE)accounted for 44.9% of the total alternative splicing events.After screening,using the filtering condition(0.05 <Percent-spliced-in(PSI)< 0.95 and expressed in more than 75% of samples),16856 alternative splicing events were obtained from 6918 genes,and SE remained the most common type of alternative splicing after screening.Comparison of alternative splicing events in ESCC tissue and adjacent normal tissue using Wilcoxon rank sum test the differences in PSI of alternative splicing events between tissues and adjacent normal tissues were screened,and 799 differential alternative splicing events from 584 genes were screened.The alternative splicing events from differential expression of the corresponding genes were excluded and finally,676 differential alternative splicing events from 498 genes were obtained.Among them,291 alternative splicing events were up-regulated and 385 alternative splicing events were down-regulated in ESCC.Gene ontology(GO)enrichment analysis of the genes corresponding to differential alternative splicing events in ESCC showed that the corresponding genes were significantly enriched in cell junction assembly,regulation of cell-substrate adhesion,focal adhesion and cadherin binding.KEGG enrichment analysis showed that the corresponding genes were significantly enriched in focal adhesion,proteoglycans in cancer,and regulation of actin cytoskeleton.One-way Cox regression analysis showed that 27 alternative splicing events were effective factors for overall survival and 31 alternative splicing events were effective factors for progression-free interval in patients with ESCC.The alternative splicing events of REPS1 and NIPAL3 were strongly associated with both overall survival and progression-free interval in patients with ESCC.Using Pearson correlation coefficient analysis,653 differential alternative splicing events in ESCC were regulated by 58 splicing factors.In addition,one alternative splicing event was regulated by up to 9 splicing factors and one splicing factor regulated up to 36 alternative splicing events.17 and 16 splicing factors were screened using the random forest method and were strongly associated with overall survival and progression-free interval of patients with ESCC,respectively.Ranked by relative importance,RBM17 and ZC3H14 played an important role in the overall survival prognosis,whereas HNRNPH3,PRRC1 and SRSF2 played an important role in the progression-free interval prognosis of patients with ESCC.HNRNPA1 was an important factor in the overall survival and progression-free interval prognosis of patients with ESCC.Pan-cancer Analysis showed that expression levels of HNRNPA1 were significantly higher in most tumors than in the corresponding normal or adjacent normal tissues.Conclusion Alternative splicing events are specifically expressed in ESCC and associated with tumor characteristics such as metastasis and prognosis of ESCC.HNRNPA1 and other splicing factors play an important role in the prognosis of patients with ESCC.Part II Exploration of the effect of splicing factor HNRNPA1 on the malignant phenotype of esophageal squamous carcinoma cellsObjective To investigate the expression of HNRNPA1 in ESCC and its effect on the malignant phenotype of ESCC cells such as proliferation,migration and invasion.Methods ESCC tissues were collected and the expression of HNRNPA1 in ESCC tissues was analyzed by RT-q PCR.ESCC cells with stable knockdown of HNRNPA1 were constructed using sh RNA lentiviral vector.The effect of stable knockdown of HNRNPA1 on the proliferation of ESCC cells was examined by CCK8 cell proliferation assay and cloneformation assay.The effect of stable knockdown of HNRNPA1 on the migration and invasion ability of ESCC cells was examined using Transwell assay.The effect of stable knockdown of HNRNPA1 on the proliferation of ESCC cells in vivo was examined using a tumorigenesis assay in nude mice.Results In 35 ESCC tissues and the corresponding precancerous tissues,RT-q PCR showed that the expression level of HNRNPA1 was significantly higher in the ESCC tissues than in the precancerous tissues.The lentiviral sh RNA vectors were used to construct stable knockdown of HNRNPA1 in ESCC cell lines KYSE-410 and KYSE-30.CCK8 cell proliferation assay and clone formation assay showed that the proliferation ability of KYSE-410 and KYSE-30 was significantly reduced after stable knockdown of HNRNPA1.Knockdown of HNRNPA1 inhibited the migration and invasion ability of KYSE-410 and KYSE-30.Nude mice subcutaneous tumorigenesis assay showed that knockdown of HNRNPA1 inhibited the proliferation ability of KYSE-30 in ESCC cells in vivo.Conclusion The expression of splicing factor HNRNPA1 was upregulated in ESCC tissues.Knockdown of HNRNPA1 inhibited the malignant phenotype of ESCC cells in terms of proliferation,migration and invasion.Part III Study on the role of splicing factor HNRNPA1 in regulating KIF23 alternative splicing in the malignant phenotype of esophageal squamous carcinoma cellsObjective To investigate the molecular mechanism by which the splicing factor HNRNPA1 promotes the malignant phenotype of ESCC cells by regulating alternative splicing.Methods The alternative splicing events associated with HNRNPA1 were screened by RNA sequencing of HNRNPA1 knockdown ESCC cells.Signaling pathways which were significantly altered by HNRNPA1 knockdown were investigated using GSVA enrichment analysis.The trend of alternative splicing events in ESCC cells after HNRNPA1 knockdown was examined using RT-PCR assay,while the expression of alternative splicing events was verified in ESCC tissues using RT-PCR assay.The association of HNRNPA1 gene expressionwith alternative splicing was analyzed in the TCGA ESCC cohort.Binding of HNRNPA1 to genes corresponding to alternative splicing events was verified using RIP experiments.ESCC cells were transfected using Splice-Switching Oligonucleotide(SSO)to regulate alternative splicing.The efficiency of SSO with alternative splicing was examined using RT-PCR assay.The effects of SSO on the malignant phenotype of ESCC cells were verified using CCK8 cell proliferation assay,clone formation assay,Transwell migration and invasion assay and tumorigenesis assay in nude mice.SSO transfection was performed in stable knockdown HNRNPA1 ESCC cells,and the reversion effect on the malignant phenotype of ESCC cells was examined using CCK8 cell proliferation assay,clone formation assay,Transwell migration and invasion assay.Results RNA sequencing results showed that knockdown of HNRNPA1 in KYSE-30 cells resulted in upregulation of PSI values in 1206 alternative splicing events and downregulation of PSI values in 1076 alternative splicing events.GSVA enrichment analysis showed that tumor-associated MYC signaling pathway was significantly inhibited after knockdown of HNRNPA1 in KYSE-30.The RNA sequencing results intersected with the differential alternative splicing events in TCGA ESCC samples.RT-PCR experiments showed that stable knockdown of HNRNPA1 in KYSE-410 and KYSE-30 resulted in increased expression of the KIF23 long isoform and a significant increase in PSI values,verifying that HNRNPA1 regulates KIF23 alternative splicing.The PSI values of KIF23 alternative splicing in TCGA ESCC samples were negatively correlated with the expression levels of HNRNPA1.RT-PCR showed that the PSI values of KIF23 alternative splicing in ESCC tissues were significantly lower than those in adjacent normal tissues.Transfection with SSO-KIF23 promoted KIF23 exon 19 skipping and increased expression of KIF23 short isoform in ESCC cells.The proliferation,migration and invasion ability of KYSE-410 and KYSE-30 cells were enhanced after transfection with SSO-KIF23.Intratumoral injection of SSO-KIF23 in nude mice promoted ESCC proliferation.After transfection of SSO-KIF23 in HNRNPA1 knockdown ESCC cells,the malignant phenotypes of cell proliferation,migration and invasion could be restored.Conclusion HNRNPA1 promotes the malignant phenotype of ESCC cells by regulating the alternative splicing of KIF23.Part IV The splicing factor HNRNPA1 regulates KIF23 alternative splicing to play a tumor promoting role in esophageal squamous carcinoma cells by mediating MYC activationObjective To investigate the molecular mechanism by which HNRNPA1 regulates KIF23 alternative splicing and exerts pro-cancer effects in ESCC cells.Methods The expression of MYC and its downstream genes after knocking down HNRNPA1 or interfering with KIF23 alternative splicing in ESCC cells was examined by RT-q PCR.Western Blot was used to verify the expression trends of KIF23,MYC and their downstream target genes at the protein level after knocking down HNRNPA1 or interfering with KIF23 alternative splicing in ESCC cells.MYC was knocked down in ESCC cells with disrupted KIF23 alternative splicing.The role of MYC in KIF23 mediated proliferation and other malignant phenotypes of ESCC cells were examined using CCK8 cell proliferation assay,clone formation assay,Transwell migration and invasion assay.Results RT-qPCR experiments suggest that knockdown of HNRNPA1 in KYSE-410 and KYSE-30 reduces the expression of MYC and its downstream genes AIMP2,CDC25 A,EIF4E and SRM;interference with KIF23 alternative splicing restores the expression of MYC and its downstream genes;Western Blot show that the expression of KIF23,MYC and the downstream regulatory genes CDC25 A and SRM are downregulated at the protein level after knockdown of HNRNPA1 in KYSE-410 and KYSE-30.Interfering with KIF23 alternative splicing restored the protein expression of KIF23,MYC and downstream genes.Knockdown of MYC in ESCC cells restored the enhanced malignant phenotype of cell proliferation,migration and invasion induced by interference with KIF23.Conclusion Knockdown of HNRNPA1 in ESCC cells can cause repression of MYC and its downstream genes expression and interference with KIF23 can restore the expression levels of MYC and its downstream genes.MYC plays an important role in HNRNPA1 regulation of KIF23 alternative splicing mediated malignant phenotype of ESCC cells such as proliferation,migration and invasion.