RUNX3-regulated circRNA METTL3 inhibits colorectal cancer proliferation and metastasis via miR-107/PER3 axis

Abstract

Colorectal cancer (CRC) is one of the most prevalent and lethal malignancies. Exploring the underlying molecular mechanisms is very helpful for the development of new therapy. Here, we investigated the function of circMETTL3/miR-107/PER3 in CRC. Human CRC tissues from diagnosed CRC patients and six CRC cell lines, one normal human colon cell line were used. qRT-PCR and western blotting were performed to determine expression levels of RUNX3, circMETTL3, miR-107, PER3, and proliferation-, and migration-related proteins. CCK-8, colony formation assay, transwell assay, and scratch wound assay were utilized to assess CRC cell proliferation and invasion. ChIP, EMSA, biotin-pull down, RIP assay, and dual luciferase reporter assay were performed to validate interactions of RUNX3/METTL3 promoter, circMETTL3/miR-107, and miR-107/PER3. FISH was used to characterize circMETTL3. MSP was employed to measure methylation level. Nude mouse xenograft model was used to determine the effects on tumor growth and metastasis in vivo. RUNX3, circMETTL3, and PER3 were diminished while miR-107 was elevated in CRC tissues and cells. Low levels of RUNX3 and circMETTL3 correlated with poor prognosis of CRC. Overexpression of RUNX3, circMETTL3, or PER3 suppressed while miR-107 mimics promoted, CRC cell proliferation and invasion, as well as tumor growth and metastasis in vivo. Mechanistically, RUNX3 bound to METTL3 promoter and activated circMETTL3 transcription. circMETTL3 directly bound with miR-107 which targeted PER3. circMETTL3/miR-107 regulated CRC cell proliferation and invasion via PER3. CircMETTL3, transcriptionally activated by RUNX3, restrains CRC development and metastasis via acting as a miR-107 sponge to regulate PER3 signaling.

Fig. 1. RUNX3 and circMETTL3 were reduced in CRC tissues and cells.

A Relative circMETTL3 levels in CRC tissues. Left: mean level; Right: paired comparison. B Relative circMETTL3 levels in CRC cell lines. C Detection of circMETTL3 in CRC cells. D Sanger sequencing of circMETTL3. E RT-PCR to characterize circMETTL3 circular features. F Characterize the stability of circMETTL3 by RNase digestion. G FISH to analyze the localization of circMETTL3. H Correlation between circMETTL3 level and overall survival rate and disease free survival rate of CRC patients. I Relative RUNX3 mRNA levels in CRC tissues from TCGA database. J Relative RUNX3 mRNA levels in CRC tissues. Left: mean level; Right: paired comparison. K Relative RUNX3 mRNA and protein levels in CRC cell lines. L Positive correlation between RUNX3 level and circMETTL3 level in CRC cells.

Fig. 2. Overexpression of RUNX3 suppressed proliferation and migration of CRC cells.

A Relative RUNX3 levels in transfected Caco2 and HCT15 cells. B CCK-8 assay to measure cell viability. C Colony formation assay to assess cell proliferation. D Scratch wound assay to analyze cell migration ability. E Transwell assay to evaluate cell invasion ability. F Representative tumor images in mice bearing transfected Caco2 and HCT15 cells. G Tumor volume in each group of mice. H Tumor weight in each group of mice. I Representative lung images in each group of mice, and H&E staining to measure the number of tumor nodules in the lung from each group of mice. J Quantification of the number of tumor nodules in the lung. K IHC staining and TUNEL staining to analyze RUNX3/Ki67 expression and apoptosis of cancer cells in each group.

Fig. 3. Overexpression of circMETTL3 inhibited proliferation and migration of CRC cells.

A Relative circMETTL3 levels in transfected Caco2 and HCT15 cells. B CCK-8 assay to measure cell viability. C Colony formation assay to assess cell proliferation. D Scratch wound assay to analyze cell migration ability. E Transwell assay to evaluate cell invasion ability. F Representative tumor images in mice bearing transfected Caco2 and HCT15 cells. G Tumor volume in each group of mice. H Tumor weight in each group of mice. I Representative lung images in each group of mice, and H&E staining to measure the number of tumor nodules in the lung from each group of mice. J Quantification of the number of tumor nodules in the lung. K qRT-PCR to measure circMETTL3 level in cancer tissues at the end. L IHC staining and TUNEL staining to analyze Ki67 expression and apoptosis of cancer cells in each group.

Fig. 4. RUNX3 regulated CRC cell prolfeiration and migration via circ-METTL3.

A Relative circMETTL3 levels in transfected HCT116 and SW620 cells. B CCK-8 assay to measure cell viability. C Colony formation assay to assess cell proliferation. D Scratch wound assay to analyze cell migration ability. E Transwell assay to evaluate cell invasion ability.

Fig. 5. circMETTL3 regulated CRC cell proliferation and migration via miR-107 in vitro.

A Relative miR-107 levels in transfected Caco2 and HCT15 cells. B Relative miR-107 levels in circMETTL3 overexpression Caco2 and HCT15 cells. C CCK-8 assay to measure cell viability. D Colony formation assay to assess cell proliferation. E Scratch wound assay to analyze cell migration ability. F Transwell assay to evaluate cell invasion ability.

Fig. 6. miR-107 regulated CRC growth and metastasis via PER3 in vitro.

A Relative PER3 mRNA levels in transfected Caco2 and HCT15 cells. B Relative PER3 protein levels in transfected Caco2 and HCT15 cells. C CCK-8 assay to measure cell viability. D Colony formation assay to assess cell proliferation. E Scratch wound assay to analyze cell migration ability. F Transwell assay to evaluate cell invasion ability.