Molecular Mechanism Of TERC Triggering The Expression Imbalances Of ABCA8-L/-S Splicing Isoforms To Promote Oxaliplatin-resistance In Colorectal Cancer

Objective:Colorectal cancer（CRC）is the leading malignant tumor of the gastrointestinal neoplasm in terms of clinical morbidity and mortality,posing a severe threat to human health.Although the clinical efficacy of conventional chemotherapy regimens for colorectal cancer is widely recognized,the emergence of multiresistance（MDR）in the treatment process has been a bottleneck that has hindered the clinical benefits of treatment for patients.Long non-coding RNAs（Lnc RNAs）were initially considered as a non-functional"dark matter"in the human genome,but in recent years the researches on Lnc RNAs mediating resistance properties through transcriptional and post-transcriptional epistasis modifications have received increasing attention.Telomerase RNA Component（TERC）,with a length of 451nt,playing crucial parts in various tumors,which suggests that elucidating the its roles in cancer has important implications for precise diagnosis and treatment.At present,the gene polymorphisms of TERC have been reported to be associated with telomere prolongation and increased risk of CRC,while biological properties of TERC and molecular mechanisms in CRC development and resistance need to be elucidated.Besides,the frequent alternative splicing（Alternative splicing,AS）events of gene pre-m RNA are important molecular bases for the precise regulation of gene expression and protein diversity.Numbers of evidence indicate that abnormal AS-mediateing dysregulated splicing isoforms,which play different or even contrasting functions in regulating tumor progression and resistance development.ATP Binding Cassette Transporter A8（ABCA8）,characterized initially as a transporter of lipids and cholesterol,was recently reported to play vital roles in regulating tumor progression in various cancers.We recently focused on the distribution pattern of AS and found two types of transcripts,ABCA8-L and ABCA8-S,in CRC cells.However,the regulation of ABCA8-AS in tumor has not been reported,nor have the function mode and specific mechanisms in CRC developing and resistance.Alternative splicing proteins can not only specifically recognize pre-m RNA,but also be recruited or stabilized by upstream regulatory factors,which have become molecules of great interest in the field of epi-modification because of their"on/off"role in precisely regulating AS patterns.Among them,RNA-binding protein 10（RBM10）has been shown to inhibit Exon Inclusion by binding to flanking intron regions adjacent to splice sites,including sites in its own pre-m RNA.However,the mechanisms related to regulating more AS events of target pre-m RNAs by RBM10 have not been studied thoroughly enough.This study aims to elucidate the modes and mechanism of TERC-mediated ABCA8-AS events via RBM10 in CRC drug-resistant cells,to screen potential biomarkers for predicting CRC oxaliplatin chemoresistance,and to provide new therapeutic strategies for reversing CRC resistance.The research purpose is divided into the following three parts.Part I:To clarify the correlation between ABCA8-AS mediated by TERC abnormal upregulation and CRC oxaliplatin resistance.Part II:To elucidate the regulation mode and crucial sites of TERC stabilizing RBM10 triggering skipping splicing of ABCA8Exon 15 in CRC.Part II:To validate the roles of the TERC/RBM10/ABCA8-AS axis in inducing CRC oxaliplatin resistance in vivo and in vitro.Methods:1.Bioinformatics analysis and online platform applications1.1 The significantly dysregulated TERC were identifed based on heatmap-clustering analysis of CRC microarray expression profile data.1.2 The pathways of TERC co-expressed m RNAs were enriched by KEGG analysis.1.3 Venn diagram was plotted to identify ABCA8 based on TERC-mediated key downstreams,factors associated with resistance,and genes where AS occurred.1.4 The tertiary structures of TERC and ABCA8 pre-m RNA（Exon 14-16）were completed using the 3d RNA online platform.1.5 Download the 3D crystal structure of RRM2 and OCRE domains of RBM10 from the PDB website.1.6 Spatial conformations and binding sites of TERC and ABCA8 pre-m RNA（Exon 14-16）binding to RBM10 protein were predicted based on the MOE 3D-docking platform.2.Experiments based on CRC tissue samples2.1 Four pairs of CRC samples were collected and the"lnc RNA-m RNA-AS pattern"microarray expression profile was obtained based on RNA-seq technology.2.2 Large samples of paired CRC tissue samples（n=149）were included to produce tissue microarray（TMA）microarrays,pathology information was collated,and prognosis was regularly followed up.2.3 RNA expression of TERC and ABCA8-L/-S（designed to differentiate probes specifically）were detected by tissue in situ hybridization（ISH）,respectively.2.4 The levels of RBM10 protein in CRC tissues was detected by immunohistochemistry（IHC）.3.Basal phenotypic experiments based on CRC cells3.1 The main cell models involved in this study were CRC oxaliplatin-sensitive cells（HCT116/S and SW480/S）and oxaliplatin-resistant cells（HCT116/R and SW480/R）.3.2 The changes of RNA levels of candidate indicators in different treatment groups were detected and compared using q-PCR and/or RT-PCR assays.3.3 Western blotting（WB）and immunofluorescence（IF）assays were performed to detect and compare the protein expression changes of RBM10 or ABCA8-L/-S isoforms.3.4 Phenotypic changes of CRC cells in different treatment groups were compared upon detecting cell cycle and apoptosis.3.5 MTT assay was performed to detect and analyze the IC₅₀changes of CRC cells in each treatment group after treatment with different concentrations of oxaliplatin.3.6 Comet assay was used to compare the extent of DNA damage in CRC cells of different groups after oxaliplatin treatment.4.Validation experiments on the mechanism of intermolecular interactions4.1 ISH combined with IF assay was used to detect the co-localization of of each candidate factor expression in CRC cells.4.2 Completion of RIP experiments in CRC cells to verify that TERC and RBM10 could form a reciprocal complex.4.3 RNA pull-down assay based on protein lysates from CRC cells and recombinant RBM10 protein to verify the direct binding of TERC and ABCA8 pre-m RNA Exon 15 to RBM10 through specific sequences.4.4 ABCA8 pre-m RNA Exon 15-related wild-type WT and truncated ESE-del overexpression plasmids were designed and co-expressed with RBM10 into CRC cells,respectively.q-PCR assay was performed to detect and analyze the changes in the expression ratio of ABCA8-L/-S in CRC cells.5.In vivo validation experiments on tumor-bearing mice5.1 The axillary tumor-bearing mice（n=6）were tested,and the tumor growth curves were calculated and plotted by measuring the long and short diameters of the tumors at different time points,and the tumor morphology was observed by intravital imaging studies of mouse tumours.5.2 After 40 days of tumor-bearing,the tumors were removed to compare the tumor size,weigh the tumors,and detect the changes of each indicator in different treatment groups by IHC and q-PCR assays.5.3 The axillary tumor-bearing mouse experiment（n=6）was completed,and the survival of nude mice was recorded until day 60,and the K-M survival curve of nude mice was plotted.5.4 Tail vein tumor-bearing mice models were completed（n=6）,and intravital imaging mouse tumours was used to detect and compare the lung metastases of tail vein-injected tumor cells in nude mice from each treatment group.5.5 The metastatic tumors were removed after 60 days of tumor-bearing,and the IHC and ISH methods were used to detect each indicator in different groups.6.Statistics and Analysis6.1 For results based on ISH and IHC,after the expression scores were tallied under the microscope,a table was created using Excel in the WPS office 2019 suite to enter the tallied expressions,which were statistically analyzed using the statistical software SPSS26.0 or Graph Pad Prism 8.0.1 Specifically,linear regression was used to analyze the association between the expression of TERC,RBM10 protein,and ABCA8-L/-S;ROC curves were plotted to determine the optimal threshold values of high/low expression of each of the above indicators;Pearson chi-square test and unconditional logistic regression were further applied to analyze the correlation between high and low expression of candidate factors and clinicopathological parameters;Log-rank one-way K-M method and adjusted multi-factor COX regression were applied to analyze the relationshipof high or low expression of TERC,RBM10 protein and ABCA8-L/-S with prognosis of CRC patients,respectively.6.2 All other data in this experiment were counted and plotted in Graph Pad Prism 8.0.1or Excel in WPS office 2019 suite with specific statistical graphs such as line graphs and bar graphs.The differences between the comparison groups were compared using Student’s t-test and expressed as mean±standard deviation（±SD）,with P<0.05 was considered a statistically significant difference.Results:1.Abnormal TERC upregulation mediating ABCA8-AS associated with oxaliplatin resistance in CRC.1.1 TERC high expression was associated with oxaliplatin resistance in CRC tissues and cells:First,TERC was found to be significantly highly expressed in CRC tissues based on 4 pairs of CRC microarray expression profiles（FC=3.17）.Subsequently,detection of paired large samples of CRC tissue samples（n=149）revealed that abnormally high expression of TERC in CRC tissue was strongly associated with tumors occupying>1/2 of the intestinal perimeter,tumor diameter>6 cm,and the occurrence of metastases.In addition,patients with high TERC expression had a significantly shorter survival time after oxaliplatin chemotherapy.In contrast to oxaliplatin-sensitive ones,TERC was indeed highly expressed in CRC oxaliplatin-resistant cells.1.2 TERC mediated ABCA8-L/-S imbalance to promote CRC oxaliplatin resistance:TERC co-expressed m RNAs were mainly enriched on the"variable spliceosome"and resistance-associated"ABC transporter protein"pathways.The key downstream ABCA8was obtained by intersecting multi-resistance,the occurrence of AS,and significantly down-regulated genes in the Wayne diagram.Among them,the highest ABCA8pre-m RNA Exon 15 skipping splice score generated a new ABCA8-S variant.Detection of paired large CRC tissue samples（n=149）based on a custom probe revealed that low expression of ABCA8-L in CRC tissues was negatively associated with TERC,whereas high expression of ABCA8-S was positively associated with TERC.High expression of ABCA8-S was associated with CRC progression and poor prognosis in patients receiving oxaliplatin chemotherapy,whereas the opposite was true for ABCA8-L.In addition,ABCA8-L levels were significantly reduced in CRC oxaliplatin-resistant cells,while ABCA8-S was increased considerably,and the proportion of ABCA8-S expression gradually increased with the decrease of TERC.1.3 TERC regulated ABCA8-L/-S variant expression and induced CRC resistance:Overexpression of TERC resulted in a significant increase in ABCA8-S expression and a significant decrease in ABCA8-L expression in CRC cells and vice versa.Cycle arrest and apoptosis induced by oxaliplatin treatment were significantly attenuated after upregulated TERC expression and vice versa.In addition,TERC overexpression significantly attenuated the cytotoxic effect of oxaliplatin on CRC cells and the extent of DNA damage.2.Mechanism and crucial sites of TERC stabilization of RBM10 triggering skipping splicing of ABCA8 pre-m RNA Exon 152.1 AS protein RBM10 was a key factor in TERC-mediated ABCA8-AS events.Based on CRC cell lysates,RBM10 protein was obtained after sending the protein dragged by the TERC probe to mass spectrometry.Abnormally high expression of RBM10 in CRC tissues（n=149）was significantly positively correlated with TERC and ABCA8-S and negatively correlated with ABCA8-L.Furthermore,high RBM10 expression was strongly associated with CRC progression and poor prognosis in patients（n=86）receiving oxaliplatin chemotherapy.Finally,the WB method clarified that RBM10 protein was indeed highly expressed in CRC-resistant cell lines and that ABCA8-L/-S heterodimer expression was restored upon silencing of RBM10 after overexpression of TERC.2.2 Validation of the mechanism by which TERC stabilizes AS factor RBM10 by specific sequences:TERC overexpression significantly elevated RBM10 levels in CRC cells and vice versa.RBM10 protein stability was reduced considerably after TERC silencing,and there was high co-localization of TERC with RBM10 expression in the nucleus.RIP experiments showed that the RBM10 antibody could significantly drag to TERC,and when TERC was overexpressed,TERC abundance was significantly increased（P<0.05）.After predicting TERC interactions with RBM10-OCRE via GGGCCC（-249～-243）sequences,the results based on RNA pull-down assay revealed that TERC-WT could significantly enrich RBM10 protein in CRC cells compared to TERC-Mut,and the RBM10 abundance was reduced considerably after silencing TERC.2.3 Validation of the mechanism by which RBM10 recognized ESE elements that promote skipping splicing of ABCA8 pre-m RNA Exon 15:m RNA and protein expression of ABCA8-S was down-regulated after silencing RBM10 expression,and the opposite was true for ABCA8-L,and there was a high degree of co-localization between RBM10and ABCA8 pre-m RNA expression in the nucleus.After predicting the discovery of UUCA as a critical sequence for RBM10-RRM2 to recognize ABCA8 pre-m RNA Exon15,RNA pull-down experiments revealed that pre-A8-WT significantly enriched RBM10protein in CRC cells compared to pre-A8-Mut,and its abundance was significantly reduced after silencing TERC.The ABCA8 pre-m RNA wild-type and ESE element knockdown plasmids were constructed,and co-expression expression with RBM10significantly increased the percentage of ABCA8-S isoform expression obtained by splicing in the pre-A8-WT sequence,which was not altered in the pre-A8-Mut sequence.3.To verify the role of the TERC/RBM10/ABCA8-AS axis in developing CRC oxaliplatin resistance in vivo and in vitro.3.1 RBM10 promoted oxaliplatin resistance in CRC cells through key sites involved in the TERC/ABCA8-AS axis.Design of RBM10-OCRE with TERC binding site（Thr571and Gln583）mutated plasmid R10-Mut1 and RBM10-RRM2 with ABCA8 pre-m RNA binding site（Lys400 and Lys402）mutated plasmid R10-Mut2.Overexpression of R10-WT after silencing TERC in CRC resistant cells significantly restored ABCA8-L/-S m RNA and protein expression,while R10-Mut1 and R10-Mut2 had no such effect.In addition,overexpression of R10-WT after silencing TERC in CRC-resistant cells significantly restored cell proliferation,apoptosis,IC₅₀,and DNA damage levels,while R10-Mut1 and R10-Mut2 had no such effect.3.2 In vivo validation of the TERC/RBM10/ABCA8-AS axis in CRC oxaliplatin resistance:Silencing of TERC significantly enhanced the inhibitory effect of oxaliplatin on the proliferation rate of CRC tumors.In addition,silencing TERC significantly shortened the survival time of CRC transplanted nude tumor mice after oxaliplatin treatment.Tumor dissection after 40 days of loading revealed a significant reduction in tumor size and tumor weight.Besides,ABCA8-S restored the various drug-sensitizing effects caused by silencing TERC,while ABCA8-L did not have this effect.Further examination of each index in situ tumors and lung metastases revealed that TERC silencing suppressed RBM10 protein expression in CRC tumors and downregulated the proportion of ABCA8-S expression.Conclusions:1.Upregulation of TERC expression in CRC tissues and cells mediates imbalance in ABCA8-L/-S isoforms expression and promotes oxaliplatin resistance formation.2.RBM10 acts as an intermediate link,directly bound and stabilized by TERC through the crucial motif GGGCCC（-249～-243）and promotes exon skipping by recognizing the ABCA8 pre-m RNA Exon 15-ESE element UUCA（+38～+41）.3.TERC induces oxaliplatin resistance in CRC cells by increasing the expression ratio of ABCA8-S isoform depending on key amino acids on RBM10（OCRE:Thr571 and Gln583）and（RRM2:Lys400 and Lys402）,and finally elucidates the"TERC/ABCA8-AS/CRC resistance"regulation axis.