Emerging roles of RNA-binding protein hnRNPM in alternative splicing regulation and UTMD mediated sh-hnRNPM/CMBs suppress the proliferation of colorectal cancer.

Abstract

Heterogeneous nuclear ribonucleoprotein M (hnRNPM) is an RNA-binding protein that is aberrantly expressed in tumorigenesis. However, the alternative splicing regulation by hnRNPM in human colorectal cancer (CRC) remain unclear. Herein, we observed that hnRNPM was highly expressed in CRC tissues. The knockdown of hnRNPM inhibited the proliferation of colon cancer cells both in vivo and in vitro. Using RNA-seq, we screened and identified several alternative splicing events regulated by hnRNPM. Knockdown of PLEKHB2-S splice isoform could reduce the growth of colon cancer cells in vitro and in vivo, predicting its role in malignant proliferation of colon cancer cells. Mechanically, the minigene reporter assay indicated the predominant regulatory roles of hnRNPM in PLEKHB2 splicing. The in vivo crosslinking immunoprecipitation (CLIP) assay demonstrated the direct binding of the RNA recognition motif RRM2 of hnRNPM protein to exon 9 of PLEKHB2 pre-mRNA. HnRNPM facilitated the skipping of alternative exon 8 in PLEKHB2 by binding to the constitutive exon 9. Furthermore, we developed cationic microbubbles shRNA/CMBs and transferred to colon cancer cells via ultrasound-targeted microbubble disruption (UTMD). The size and zeta potentials of CMBs and shRNA-CMBs were measured and the optimal concentration range of shRNA/CMBs were screened with low cytotoxicity. The introduction of sh-hnRNPM/CMBs or sh-PLEKHB2-S/CMBs suppressed the proliferation of colon cancer cells in vitro and in vivo. Collectively, our findings identify that hnRNPM dysregulates colorectal carcinoma proliferation at the molecular level of splicing regulation and predicate sh-hnRNPM/CMBs and its splicing target sh-PLEKHB2-S/CMBs as promising therapeutic drugs and innovative strategies for treating colorectal cancer. Schematic diagram of the functions of UTMD mediated sh-hnRNPM/CMBs in colorectal cancer and emerging mechanism by which hnRNPM promotes the malignant proliferation of CRC by regulating the alternative splicing of PLEKHB2 pre-mRNA. The picture was created with BioRender.com.