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Screening for alternative splicing of lncRNA Dleu2 in the mouse liver cell line AML-12.

ABSTRACT: The long non coding RNA deleted in leukemia 2 gene (Dleu2) has recently been demonstrated to be an active player in the progression of several types of cancer, including hepatocellular carcinoma. Dleu2 may serve a role in modulating the downstream effects-mediated by alternative splicing of its multiple exons. However, the proportional expression of these alternative splicing populations of the Dleu2 exons is currently unknown. To determine how Dleu2 could be affected by alternative splicing, a series of alternative splicing primer sets were designed to investigate which transcripts were preferentially activated when Dleu2 was targeted for downregulation or upregulation. A specific Dleu2 small interfering RNA that targeted an exon upstream of the tumor suppressor microRNA site significantly knocked down Dleu2 expression across all the primer sets used, which targeted 13 different alternative splicing transcripts over 5 different promoter sites in the mouse liver cell line, AML-12. Similarly, 50 µM Resveratrol led to significant upregulation of Dleu2 in 11 alternative splicing transcripts. These results show that Dleu2 is capable of successful modulation across alternative splicing transcripts that can be screened, and also that Resveratrol can be a potential nutraceutical, which may potentially lead to novel approaches in the use of lncRNA Dleu2 for diagnostics and regulation.

SUBMITTER: Kay MK

PROVIDER: S-EPMC8042668 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe therapeutic value of targeted therapies in patients with lung cancer is reduced when tumours acquire secondary resistance after an initial period of successful treatment. However, the molecular events behind the resistance to targeted therapies in lung cancer remain largely unknown.AimsTo discover the important role and mechanism of lncRNA BC in promoting tumor metastasis and influencing clinical prognosis of LUAD.Materials & methodsMicroarrays were used to screen a comprehensive set of lncRNAs with differential expression profiles in lung cancer cells. The functional role and mechanism of lncRNA were further investigated by gain- and loss-of-function assays. RNA pull-down, protein assays, and mass spectrometry were used to identify proteins that interacted with lncRNA. TaqMan PCR was used to measure lncRNA in lung adenocarcinoma and adjacent nontumor tissues from 428 patients. The clinical significance of lncRNA identified was statistically confirmed in this cohort of patients.ResultsIn this study, we show that the long non-coding RNA BC009639 (BC) is involved in acquired resistance to EGFR-targeted therapies. Among the 235 long non-coding RNAs that were differentially expressed in lung cancer cell lines, with different metastatic potentials, BC promoted growth, invasion, metastasis, and resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), both in vitro and in vivo. BC was highly expressed in 428 patients with lung adenocarcinoma (LUAD) and high BC expression correlated with reduced efficacy of EGFR-TKI therapy. To uncover the molecular mechanism of BC-mediated EGFR-TKI resistance in lung cancer, we screened and identified nucleolin and hnRNPK that interact with BC. BC formed the splicing complex with nucleolin and hnRNPK to facilitate the production of a non-protein-coding inositol monophosphatase domain containing 1 (IMPAD1) splice variant, instead of the protein-coding variant. The BC-mediated alternative splicing (AS) of IMPAD1 resulted in the induction of the epithelial-mesenchymal transition and resistance to EGFR-TKI in lung cancer. High BC expression correlated with clinical progress and poor survival among 402 patients with LUAD.DisscussionThrough alternative splicing, BC boosted the non-coding IMPAD1-203 transcript variant while suppressing the IMPAD1-201 variant. In order to control the processing of pre-mRNA, BC not only attracted RNA binding proteins (NCL, IGF2BP1) or splicing factors (hnRNPK), but also controlled the formation of the splicing-regulator complex by creating RNA-RNA-duplexes.ConclusionOur results reveal an important role for BC in mediating resistance to EGFR-targeted therapy in LUAD through IMPAD1 AS and in implication for the targeted therapy resistance.

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Screening for alternative splicing of lncRNA Dleu2 in the mouse liver cell line AML-12.

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