LncRNA MSTO2P promotes colorectal cancer progression through epigenetically silencing CDKN1A mediated by EZH2

Abstract

Background: Pseudogene-derived long non-coding RNAs (lncRNAs) have been reported to act as key regulatory factors of cancers. However, the study focused on pseudogene misato family member 2 (MSTO2P) in the occurrence and development of colorectal cancer (CRC) remains unclear.

Methods: CCK-8, colony formation, and transwell assays clarified HT-29 and SW480 cell proliferation and invasion. Furthermore, flow cytometry was carried out to detect cell cycle and cell apoptosis. Subcellular localization assay indicated the location of MSTO2P in HT-29 cells. RIP and CHIP assays clarified the relationship of MSTO2P with target protein and gene in HT-29 cells.

Results: MSTO2P expression was upregulated in CRC tissues and cells. Functional experiments revealed that inhibition of MSTO2P suppressed HT-29 and SW480 cell proliferation and invasion, and promoted cell cycle arrest and cell apoptosis. Besides, MSTO2P epigenetically down-regulated cyclin-dependent kinase inhibitor 1A (CDKN1A) via binding to the enhancer of zeste homolog 2 (EZH2) in the nucleus. At last, rescue experiments proved the anti-tumor effect of inhibition of MSTO2P was partially recovered due to the knockdown of CDKN1A in HT-29 cells.

Conclusion: LncRNA MSTO2P promoted colorectal cancer progression through epigenetically silencing CDKN1A mediated by EZH2.

Keywords: CDKN1A; CRC; MSTO2P; Pseudogene.

Fig. 1

LncRNA MSTO2P is aberrantly overexpressed in CRC tissues and cells. A TCGA database showed that MSTO2P level was increased in primary CRC tissues. B RT-qPCR assay revealed MSTO2P level was markedly upregulated in CRC tissues. C RT-qPCR assay indicated high level of MSTO2P in CRC cell lines. *p<0.05, **p<0.01, and ***p<0.001

Fig. 2

Inhibition of MSTO2P suppresses proliferation and invasion of CRC cells. MSTO2P was knocked down in HT-29 and SW480 cells by transfecting cells with si-MSTO2P for 24 h. A The transfection efficiency of si-MSTO2P was confirmed by RT-qPCR assay. B CCK-8 assay demonstrated that knockdown of MSTO2P dramatically suppressed cell viability. C, D Colony formation assay clarified that suppression of MSTO2P reduced cell colonies. E, F Transwell assay displayed that the downregulation of MSTO2P significantly suppressed cell invasion. Bar values= 100 μm. *p<0.05, **p<0.01, and ***p<0.001

Fig. 3

Inhibition of MSTO2P promotes cell cycle arrest and cell apoptosis. A, B Flow cytometry assay showed that the proportion of HT-29 and SW480 cells at the G0/G1 phase was elevated by si-MSTO2P. C, D Flow cytometry assay demonstrated that si-MSTO2P enhanced cell apoptosis. *p<0.05, **p<0.01, and ***p<0.001

Fig. 4

MSTO2P interacts with EZH2 to inhibit CDKN1A transcription. A The distribution of MSTO2P in HT-29 cells was evidenced by RT-qPCR assay. B RT-qPCR assay showed that si-MSTO2P upregulated CDKN2B, CDKN1A, Bax, EMP1, and PTEN expression level and decreased Bcl-2 level in HT-29 cells. C Western blot assay showed that si-MSTO2P upregulated CDKN1A protein level. D Bioinformatics were used to predict the interaction probabilities of lncRNA MSTO2P and EZH2 via RNA-protein interaction prediction (http://pridb.gdcb.iastate.edu/rpiseq/). Predictions with probabilities >0.5 were considered positive. E RIP assay confirmed that MSTO2P was directly bound to EZH2 in HT-29 cells. F RT-qPCR assay showed si-EZH2 significantly enhanced CDKN1A expression level. G Western blot assay showed si-EZH2 significantly enhanced CDKN1A expression level. H CHIP assay showed that si-MSTO2P suppressed binding of EZH2 and H3K27me3. *p<0.05, **p<0.01, and ***p<0.001

Fig. 5

Inhibition of MSTO2P suppresses CRC progression through upregulating CDKN1A expression. si-MSTO2P and si-CDKN1A were co-transfected into HT-29 cells. A CCK-8 assay showed that cell viability was inhibited by si-MSTO2P, which was reversed by si-CDKN1A. B, C Colony formation assay clarified that si-MSTO2P decreased the number of colonies in HT-29, while si-CDKN1A partially recovered clone formation. D, E Transwell assay showed cell invasion in HT-29 cells. Bar values= 100 μm. F, G The cell cycle and (H–I) cell apoptosis were detected by flow cytometry assay