Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

Abstract

Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.

Keywords: Alternative splicing; Colorectal cancer; RAC1; RBFOX2; RNA-binding proteins; lncH19.

Fig. 1

LncH19 interactors network. (A) Summary of sequences obtained by RNASeq from lncH19 Antisense Precipitation. (B) Western blot for the indicated proteins from lncH19 Antisense Precipitation in CRC cell lines (SW620 and HCT116). One representative experiment of three is shown. (C) RIP assay with anti-Fox2 (left) and anti-hnRNPM (right) antibodies to assess the binding of the RNABPs to lncH19 RNA in CRC cell lines (SW620 and HCT116); IgG was used as control. LncH19 levels were determined by qRT–PCR, normalized with input and presented as fold enrichment in RBFOX2 or hnRNPM relative to IgG. Statistical analyses were performed using normality test and t-test, p-value is shown in the graphs. (D) Binding affinity prediction between lncH19 and the indicated Splicing Factors by the use of catRAPID algorithm (E) Venn Diagram of lists of genes whose mRNA interacts with lncH19 (interactions are determined by the analysis of our RAP/RNA sequencing data and in-silico predictions), RBFOX2, and hnRNPM (interactions are determined by the analysis of eCLIP data from ENCODE portal

Fig. 2

RBFOX2, hnRNPM and lncH19 bind RAC1 mRNA. (A-B) RIP assay with anti-Fox2 and anti-hnRNPM antibodies to assess the binding of the RNABPs to RAC1 RNA in HCT-116 and SW620 cells, IgG was used as control. RAC1 levels were determined by qRT–PCR normalized with input and presented as fold enrichment in RBFOX2 or hnRNPM relative to IgG. (Normality test and subsequent t-test (A) or Wilcox test (B). (C) RNA pull-down with biotin-labeled lncH19 oligonucleotides (lncH19 RAP) in CRC cell lines (SW620 and HCT116) to analyze the interaction between lncH19 and RAC1 mRNA. RAC1 levels were determined by qRT–PCR and presented as fold enrichment in lncH19 samples respect to RNA pull-down obtained with scrambled oligonucleotides (Normality test and t-test). (D) Agarose electrophoresis of splice-sensitive PCR RAC1-RAC1B from lncH19 RAP in CRC cell lines (SW620 and HCT116). One representative experiment of three is shown. (E) Quantitative analysis of RAC1B levels determined by qRT–PCR and presented as fold enrichment in lncH19 samples relative to input. Statistical analyses were performed using one sample t-test, the p-value is shown in the graphs

Fig. 3

RBFOX2 is involved in RAC1 alternative splicing in CRC cells. (A-B-D, E-F-H) QRT-PCR for the indicated mRNA in CRC cells, silenced for RBFOX2 with two different siRNA. Graphs show 2-ΔΔct calculated in silenced cells respect to relative controls (Normality test and t-test). (C, F) Western Blot and densitometric analyses for RAC1B in SW620 and HCT116 silenced for RBFOX2 and relative controls. For densitometric analysis data are represented as normalized OD. (I-L) qRT-PCR of the indicated genes in SW620 and HCT116 silenced for H19, the graphs represent the 2^- ΔΔ ct of the indicated calculated respect the expression in control cells. (M) Western Blot of RAC1b protein levels in CRC cells (SW620 and HCT116) in H19 silenced cells and relative control cells. Statistical analyses were performed using normality test and t-test, p-value is shown in th e graphs

Fig. 4

Overexpression of lncH19 upregulates RAC1B expression. (A) qRT-PCR to analyze H19 mRNA levels in CRC cells (SW620 and HCT116) transfected with lncH19 or empty vector. (B) qRT-PCR to analyze RAC1B mRNA levels in CRC cells (SW620 and HCT116) transfected with lncH19 or empty vector. (C) Western Blot for RAC1B in CRC cells (SW620 and HCT116), transfected with lncH19 or empty vector. lncH19 overexpression promotes RAC1B nuclear localization. (D) Representative confocal microscopy images of anti-RAC1b immunofluorescence showed nuclear localization of RAC1b in lncH19-overexpressing CRC cells, SW620 (upper panel) and HCT116 (lower panel). The analysis of the RAC1B nuclear signal is reported on the histogram. (E) Western blot analysis and densitometric analyses for RAC1B in nuclear protein fractions of CRC cells (SW620 and HCT116) overexpressing H19 cells and relative control cells. E-F) Overexpression of lncH19 upregulate CiclinD and c-Myc expression through RAC1B activity qRT-PCR for the indicated genes in SW620 (F) and HCT116 (G) transfected with pH19 or empty vector and treated or not with RAC1B inhibitor. Statistical analyses were performed using: normality test, one sample t-test to compare different conditions to pEmpty untreated cells, and Two-tails unpaired t-test to compare two groups *; p-value is shown in the graphs

Fig. 5

CRC tissues present higher levels of lncH19 compared to respective marginal non-tumor. (A-C) Gene expression levels for the indicated genes were examined by qRT-PCR in tumor and paired marginal non-tumor samples (n = 20). (D) Pearson correlation between lncH19, RAC1, and RAC1B expression analyzed in 14 colorectal cancer samples with lncH19 overexpressed compared to marginal non tumor tissue. (E-F) qRT-PCR for the indicated genes in colorectal cancer samples with lncH19 levels. All statistical analyses were performed using two-tail paired t-test, p-value is shown in the graphs

Fig. 6

LncH19 is required to drive splicing factors on RAC1 mRNA. (A-D) QRT-PCR for RAC and RAC1b from RNA-immunoprecipitation (RIP) with anti-Fox2 (A, C) or anti-hnRNPM (B, D) antibodies in SW620 and HCT116 cells, RAC1 and RAC1B levels presented as fold enrichment in RBFOX2 or hnRNPM IP relative to IgG IP. Statistical analyses were performed using two-tail unpaired t-test to compare the binding between wt and shH19 silenced cells, p-value is shown in the graphs. (E) Schematic representation of the proposed model. Representation of binding sites position of the complex lncH19-hnRNPM-RBFOX2 with RAC1 unspliced mRNA