Mechanism Of E2F7-HNRNPC-BOLA3 Alternative Splicing Axis On Promoting Esophageal Squamous Cell Carcinogenesis

Esophageal carcinoma(ESCA)is one of the major malignant cancers.The incidence and death of esophageal squamous cell carcinoma(ESCC)cases in China accounts for more than half of the world.ESCC is insidious in origin and progresses rapidly.ESCC patients are mostly in the middle and late stages when diagnosed,requiring multiple interventions such as surgery,radiotherapy and immunotherapy.Due to the limitations such as surgical complications,treatment resistance and poor sensitivity of immunotherapy,the treatment effect of progressive ESCC is poor,and the 5-year survival rate is only about 20%-30%.Therefore,it is important to investigate the molecular mechanism of ESCC development and find precise and efficient targets for ESCC diagnosis and treatment,further improving the prognosis of patients.Alternative splicing(AS)is a form of RNA processing in which multiple exons or other sequences of m RNA precursors are induced to generate multiple mature transcripts in specific combinations.AS plays an important role in promoting normal organ development and maintaining tissue homeostasis.The AS process is precisely regulated by the spliceosome complex.When abnormal regulations such as mutations of cis-elements and disruptions of splicing factor expression occur,it often leads to the development of various pathological states including carcinogenesis.The formation of pro-oncogenic transcripts has an important role in promoting the malignant progression of ESCC.Some of the aberrantly expressed pro-oncogenic transcripts can predict the poor prognosis of ESCC patients.The AS-related molecules and splicing factors can be used as specific targets for small molecule targeted inhibitors and antisense oligonucleotide therapy.Current researches on the regulatory mechanisms of abnormal AS events in ESCC are still limited.It is urgent to explore more cancer-promoting splicing molecules and splicing regulatory axes to provide new directions for the exploration of key therapeutic targets in ESCC.In this study,we collected 11 pairs of ESCC and paraneoplastic tissues(CH-Cohort)for transcriptome sequencing.The differential AS events showed that the third exon(exon3)of Bol A family member 3(BOLA3)was over-inclusion in ESCC and the long transcript BOLA3-L was elevated in ESCC.No mechanisms have been reported for BOLA3 promoting ESCC progression,and the biological functions played by different transcripts of BOLA3 in ESCC progression remain to be clarified.We hypothesized that BOLA3exon3 over-inclusion was a key abnormal AS event in ESCC progression,BOLA3-L might be a key transcript driving ESCC development.BOLA3 alternative splicing axis might be a key regulatory axis promoting ESCC progression.Based on the above hypotheses,the main contents and results of this study were as follows:Part Ⅰ Screening and functional exploration of BOLA3 exon3 abberant skipping events in ESCCObjective: To explore the key aberrant AS events and the pro-oncogenic function of transcripts that are critical in the development of ESCC.Methods:1.Combine CH-Cohort sequencing and TCGA data to clarify key abnormal alternative splicing events in ESCC tissues.2.Validate the expression of different transcripts of BOLA3-L/S in tissues by Q-PCR and gel electrophoresis.Expressions of BOLA3-L/S and relationships with prognosis of BOLA3-L/S at esophageal cancer and pan-cancer level were explored.3.Construct stable knockdown line of BOLA3-L and stable overexpression lines of BOLA3-L and BOLA3-S.Perform malignant phenotype assays on different treated cell lines,and take CCK-8,colony assay,Edu assay to detect proliferation ability.Perform scratch assay and transwell assay to detect the migration ability.Subcutaneous transplantation tumor model was constructed in nude mice to evaluate the subcutaneous tumorigenic ability.Results:1.There was a large number of ES-based differential AS events in ESCC,mainly involving in various cellular pathways and physiological functions such as intercellular adhesion,energy metabolism,ubiquitination,mitochondrial autophagy,etc.2.BOLA3 exon3 skipping splicing generated long transcript BOLA3-L and short transcript BOLA3-S.The over-inclusion of BOLA3 exon3 in ESCC led to an elevated proportion of BOLA3-L and was a key aberrant ES event that promoted the development of ESCC.3.The BOLA3-L transcript promoted the progression of the malignant phenotype of ESCC,whereas BOLA3-S had no significant pro-oncogenic activity.Part Ⅱ Prediction and functional exploration of HNRNPC,the upstream splicing regulator of BOLA3Objective: To explore the upstream splicing factors that regulate aberrant splicing of BOLA3 and clarify their expression and biological functions.Methods:1.Combine CH-Cohort sequencing,TCGA data,and POSTAR3 database to screen key RNA-binding proteins that regulate BOLA3 splicing.2.To characterize the expression of BOLA3 splicing factor HNRNPC at m RNA and protein levels.To explore the association of HNRNPC expression with ESCC prognosis.3.Construct HNRNPC stable knockdown cell line and perform transcriptome sequence of HNRNPC after knockdown.Clarify the differential AS events and BOLA3-L/S m RNA expression changes after HNRNPC knockdown.4.To detect changes in proliferation,migration phenotype and subcutaneous tumorigenic ability of ESCC cells after HNRNPC knockdown.To investigate the effect of overexpression of BOLA3-L/S on the reversion of malignant phenotype after HNRNPC knockdown.Results:1.In ESCC,HNRNPC was a key molecule regulating abnormal splicing of BOLA3,and its expression showed a positive correlation with the degree of exon3 inclusion.2.HNRNPC expression was elevated at both m RNA and protein levels in ESCC.High expression of HNRNPC correlated with poor prognosis of ESCC.3.The malignant phenotypes such as cell proliferation and migration ability were reduced after HNRNPC knockdown.Overexpression of BOLA3-L transcript could partially revert the diminished malignant phenotypes after HNRNPC knockdown,while BOLA3-S had no obvious ability to revert.Part Ⅲ Exploration of the mechanism of HNRNPC-regulated BOLA3 splicing interactions and establishment of the E2F7-HNRNPC-BOLA3 pro-oncogenic splicing axisObjective: To explore the specific binding sites of BOLA3 and HNRNPC.To clarify the basic mechanism of the BOLA3 pro-oncogenic splicing axis.Methods:1.RIP assay was performed to clarify the binding evidence of HNRNPC and BOLA3.The RRM binding domain deletion plasmid of HNRNPC was constructed to clarify the binding domain of HNRNPC and BOLA3 m RNA.2.Multiple mini-gene mutant plasmids of the BOLA3 exon2-exon4 region were constructed,and the mutant plasmids contained sequence deletion mutations at each potential binding site.The mini-gene plasmids were cotransfected with HNRNPC wild-type and RRM mutant plasmids to clarify the key binding motifs of HNRNPC regulating BOLA3 exon3 skipping.3.Construct BOLA3-L stable knockdown cell line and perform transcriptome sequencing to explore the downstream pathways and biological functions of BOLA3-L.4.To explore the transcription factors regulating the upregulation of splicing factor HNRNPC.Perform CHIP assay and dual luciferase assay to detect the binding sites and interactions between E2F7 and HNRNPC promoter.Results:1.RIP experiments and electrophoresis results after transfection of different mutant plasmids suggested that HNRNPC could bind to BOLA3 L/S m RNA through the RRM structural domain.2.HNRNPC promoted BOLA3 exon3 inclusion by binding to the UGCAU motif of BOLA3 exon4.3.BOLA3-L protein isoform was mainly localized in mitochondria,which was closely related to mitochondrial structure,function and membrane potential stability.BOLA3-L might play a pro-oncogenic role through WNT/β-catenin pathway.4.E2F7 was a key transcription factor regulating the upregulation of HNRNPC,and showed a positive correlation with HNRNPC and BOLA3-L expression.E2F7 bound to the TTTCCCCGCCCCCGCGCA region of HNRNPC promoter to play a role of pro-transcription.Conclusions:1.A large number of aberrant ES events existed in ESCC,involving in intercellular adhesion,mitochondrial autophagy and other pathways.The BOLA3 exon3 skipping produced two transcripts,BOLA3-L and BOLA3-S.BOLA3 exon3 over-inclusion occured in ESCC,resulting in an elevated percentage of BOLA3-L expression in ESCC,further driving the malignant phenotype of ESCC.2.HNRNPC was a key splicing factor that regulated BOLA3 exon3 splicing.HNRNPC promoted BOLA3 exon3 inclusion by interacting with the UGCAU sequence of exon4 through the RRM domain.3.BOLA3-L protein localized in mitochondria and had a critical role in maintaining mitochondrial structure,function,and membrane electrical stability.BOLA3-L might exert pro-oncogenic effects through the WNT/β-catenin pathway.4.E2F7 acted as a key transcription factor to regulate the transcriptional upregulation of HNRNPC and promoted the inclusion of BOLA3 exon3.The E2F7-HNRNPC-BOLA3 splicing axis was an important regulatory axis driving the progression of ESCC.