Roles And Mechanisms Of LncRNA MIR100HG In Regulation Of Anti-EGFR Resistance And Metastasis In Colorectal Cancer

【Background】Colorectal cancer（CRC）,with a high morality and mobility worldwide,poses a great threat to human health.Targeted therapy is a novel way of cancer treatment and has significantly improved the survival of patients with advanced CRC.In the clinic,durable responses last no more than one year in most patients treated with cetuximab because of occurrence of acquired drug resistance.Except for the common mechanisms of drug resistance such as mutations in KRAS/NRAS/BRAF and amplification of ERBB2 and MET,about 37%of the cases that are unresponsive to anti-EGFR therapies arise from unknown mechanisms.Effective measures to reverse cetuximab resistance and valid predictive markers of cetuximab resistance remain absent in clinic.Using a three-dimensional（3D）culture system,we previously developed cetuximab sensitive CC cells and cetuximab resistant CC-CR cells and successfully simulated cetuximab resistance phenotype in vitro and in vivo.We found that lnc RNA MIR100HG and its derived mi RNAs mi R-100/125b were significantly upregulated in CC-CR cells compared with CC cells.Mi R-100/125b could collaboratively activate Wnt signaling via repression of five Wnt/β-catenin negative regulators and contribute to cetuximab resistance in CRC.However,whether the MIR100HG transcript itself has an independent role in these events and what’s the underlying mechanism remain unknown.【Objectives】1.To clarify the role of MIR100HG in regulating malignant behaviors of CRC cells.2.To screen and identify the functional binding partner of MIR100HG.3.To elucidate the mechanisms of MIR100HG in regulating malignant behaviors of CRC cells.4.To explore the mechanisms of MIR100HG overexpression in CRC.5.To disclose the expression pattern of MIR100HG and its binding partners in clinic specimens.【Methods】1.The expression of EMT markers in CC and CC-CR cells were detected by IF,Western blot and q PCR.Bioinformatics were conducted to determine the correlation between MIR100HG and EMT genes expression in CRC tissues.MIR100HG-silencing CRC cells were established by ASO transfection,sh RNA infection and CRISPR/Cas9gene editing.MIR100HG overexpressed CRC cells were developed by vector transfection and lentivirus infection.Cetuximab sensitivity of indicated CRC cells was determined by colony formation assay,Ki-67 and cleaved Caspase-3 staining in 3D culture.The in vitro migration and invasion ability of indicated CRC cells were determined by Transwell assay.The in vivo drug resistant and metastatic ability of indicated CRC cells were detected by subcutaneous tumorigenesis experiments and nude mice tail vein injection assay,respectively.2.Potential MIR100HG binding proteins were identified by Ch IRP-MS assay.The direct interaction between MIR100HG and hn RNPA2B1 was confirmed by Ch IRP-Western blot assay and RIP assay.The specific region of MIR100HG that binds to hn RNPA2B1 was determined by MIR100HG truncation pull down assay and hn RNPA2B1truncation RIP assay.HNRNPA2B1-silencing CRC cells were developed by si RNA transfection and sh RNA transduction.HNRNPA2B1 overexpressed CRC cells were established by vector transfection.3D colony formation assay was used to determine the effect of hn RNPA2B1 on cetuximab resistant ability of CRC cells.Transwell assay was used to determine the effect of hn RNPA2B1 on the in vitro migration and invasion ability of CRC cells.Western blot and q PCR assay were used to detect the expression of EMT markers after hn RNPA2B1 silencing in CRC cells.Rescue assays were performed to determine the expression of EMT markers,cetuximab resistance and metastatic ability of MIR100HG overexpressed CRC cell upon hn RNPA2B1 silencing.3.Western blot,q PCR and nuclear and cytoplasmic protein extraction assay were used to determine the effect of MIR100HG on hn RNPA2B1 expression and subcellular distribution.RNA-seq was used to screen the downstream targets regulated by both MIR100HG and hn RNPA2B1.m RNA stability assay was used to determine the effect of MIR100HG and hn RNPA2B1 on the half-life of TCF7L2 m RNA.q PCR was used to detect the expression of Wnt targeted genes in CRC cells upon MIR100HG and hn RNPA2B1 overexpressing or silencing.The activity of Wnt pathway upon MIR100HG or hn RNPA2B1 silencing was determined by Top/Fop flash assay.me RIP-q PCR was used to detect the m⁶A modification level in CRC cells transfected with METTL3 si RNA.RIP assay was performed to examine the interaction between hn RNPA2B1 and TCF7L2m RNA in METTL3-silencing or MIR100HG knockout CRC cells.The effect of m⁶A modification on the interaction between hn RNPA2B1 and TCF7L2 m RNA was determined by RNA pull down assay.Luciferase reporter assay was used to testify the effect of m⁶A modification and MIR100HG on the interaction between hn RNPA2B1 and TCF7L2.4.Potential transcription factor binding sites in the MIR100HG promoter region were predicted by JASPAR database.q PCR assay was used to detect the expression of MIR100HG upon TCF7L2 overexpressing or silencing.Luciferase reporter assay were performed to determine the transcriptional regulation of TCF7L2 on MIR100HG.Serial deletion analysis and site-directed mutagenesis of the MIR100HG promoter were conducted to determine the specific binding sites of TCF7L2 on MIR100HG promoter region.The direct binding between TCF7L2 and MIR100HG promoter was confirmed by Ch IP assay.Bioinformatics analysis was performed to determine the correlation between TCF7L2 and MIR100HG expression in TCGA CRC dataset.5.MIR100HG and TCF7L2 m RNA were detected by RNAscope assay in paired tumor specimens from 12 individuals before the start of cetuximab treatment and at the time of tumor progression and in 14 paired specimens of primary CRC tissues,adjacent nontumor tissues and their matched lymph node or distant metastasis samples.hn RNPA2B1 expression was detected by IHC in the same samples.【Results】1.Cetuximab resistant CC-CR cells showed significantly increased expression of mesenchymal markers（N-cadherin,Vimentin,Slug,Zeb1）and decreased expression of epithelial markers（E-cadherin,ZO-1）compared to cetuximab sensitive CC cells as demonstrated by IF,Western blot and q PCR assay.MIR100HG and EMT score were highly correlated in a large human CRC dataset（n=2,375）.GSEA demonstrated that EMT gene expression patterns were significantly enriched in CRC samples with a high level of MIR100HG.Analysis of TCGA CRC data repository（n=433）revealed that MIR100HG was tightly correlated with the expression of mesenchymal genes.Western blot and q PCR revealed that upregulation of MIR100HG reduced the expression of epithelial markers and increased the expression of mesenchymal markers,while downregulation of MIR100HG produced the opposite effect.In 3D culture,in the presence of cetuximab,MIR100HG^KOE4cells showed significant reduction in colony numbers compared to WT cells.Decreased Ki-67 and increased cleaved Caspase-3 staining were observed in MIR100HG^KOE4 CC-CR cells following cetuximab treatment.In vivo drug resistance assay using subcutaneous xenografts with MIR100HG^KOE4 CC-CR cells in nude mice showed that MIR100HG knockout significant impaired the sensitivity of CC-CR cells as demonstrated by decreased tumor volume and weights and fewer Ki-67-positive cells and more cleaved Caspase-3-positive cells after cetuximab treatment.Transwell assay and nude mice tail vein injection assay revealed that downregulation of MIR100HG significantly reduced the migration,invasion and metastasis of CRC cells.2.Five potential differentially expressed MIR100HG binding proteins were identified by Ch IRP-MS assay.Ch IRP Western blot assay confirmed the existence of hn RNPA2B1.RIP assay demonstrated that MIR100HG can be significantly enriched by hn RNPA2B1antibody.RNA pull down assay using biotin-labeled MIR100HG fragments showed that hn RNPA2B1 mainly binds to a fragment of MIR100HG from nucleotides 2,170 to 3,100.RIP assay using antibodies against Flag-tagged full length or truncated hn RNPA2B1demonstrated that the RNA recognition motif 2（RRM2）domain of hn RNPA2B1 was primarily responsible for interaction with MIR100HG.Western blot assay revealed that silencing hn RNPA2B1 repressed expression of mesenchymal marker and enhanced expression of epithelial marker.In 3D culture,colony formation assay showed that downregulation of hn RNPA2B1 remarkably impaired the drug resistant ability of CC-CR cells to cetuximab treatment.Transwell assay demonstrated that migration and invasion ability of CRC cells was decreased upon hn RNPA2B1 silencing.Rescues assay indicated that MIR100HG regulated EMT,cetuximab resistance and metastasis through hn RNPA2B1.3.Western blot,q PCR and nuclear and cytoplasmic protein extraction assay demonstrated that MIR100HG had no effect on hn RNPA2B1 expression and subcellular distribution.TCF7L2 was the only transcript downregulated in all the MIR100HG-and hn RNPA2B1-silencing groups as revealed by RNA-seq.m RNA stability assay demonstrated that MIR100HG and hn RNPA2B1 promoted the stability of TCF7L2 m RNA.q PCR showed that MIR100HG and hn RNPA2B1 increased the expression of Wnt target genes.Top/Fop flash assay confirmed that MIR100HG and hn RNPA2B1 activated the Wnt signaling.The m⁶A modification level in CRC cells transfected with METTL3 si RNA was markedly reduced.RIP assays demonstrated that TCF7L2 m RNA enriched by the hn RNPA2B1 antibody was significantly decreased when METTL3 was knocked down or MIR100HG was knocked out.RNA pull down assay showed that m⁶A modification site mutant TCF7L2 could not bind hn RNPA2B1.Luciferase reporter assay revealed that the interaction between hn RNPA2B1 and TCF7L2 was dependent on m⁶A modification and MIR100HG.4.Four potential TCF7L2 binding sites in the MIR100HG promoter region were predicted by JASPAR database.q PCR assay demonstrated that overexpression of TCF7L2enhanced MIR100HG expression while silencing of TCF7L2 in CRC cells resulted in reduced MIR100HG expression.Serial deletion analysis and site-directed mutagenesis of the MIR100HG promoter showed that TCF7L2-binding sites 1 and 2 are the predominant sites for TCF7L2-mediated transcriptional activation.Chromatin immunoprecipitation（Ch IP）assays showed that TCF7L2 protein bound directly to the MIR100HG promoter at TCF7L2-binding sites 1 and 2.A positive correlation was observed between TCF7L2 and MIR100HG expression in a panel of CRC cell lines,as well as in the TCGA data repository.5.RNAscope assay and IHC assay indicated that MIR100HG and TCF7L2 were significantly overexpressed in tumors that progressed on cetuximab treatment compared with the pre-treatment counterparts,and positive correlations between MIR100HG and TCF7L2 expression were observed.Compared to primary lesions,lymph nodes and distant metastases exhibited significantly higher expression of MIR100HG,hn RNPA2B1and TCF7L2.Positive associations were also identified between MIR100HG or hn RNPA2B1 with TCF7L2 expression.【Conclusions】1.MIR100HG promotes EMT,cetuximab resistance and metastasis in CRC in a mi RNA-independent manner.2.MIR100HG promotes CRC EMT,cetuximab resistance and metastasis via its interaction with hn RNPA2B1.3.MIR100HG and hn RNPA2B1 collaboratively regulate TCF7L2 m RNA stability and activate Wnt signaling through TCF7L2.m⁶A modification of TCF7L2 m RNA is required for its binding to hn RNPA2B1,with MIR100HG an indispensable partner for the interaction.4.TCF7L2 activates MIR100HG transcription and forms a reciprocal positive feedback loop.5.Upregulation of MIR100HG,hn RNPA2B1 and TCF7L2 occurs in the setting of cetuximab resistance and metastatic CRC patients.