Downregulation of N6-methyladenosine binding YTHDF2 protein mediated by miR-493-3p suppresses prostate cancer by elevating N6-methyladenosine levels

Abstract

Recent evidence suggests that m6A modifications regulate the progressions of several types of tumors. YTHDF2, an m6A reader, has been implicated in the regulation of hepatocellular carcinoma (HCC). miR-493-3p has been defined as tumor suppressor that inhibits the progressions of several types of cancers. However, the functions and mechanisms of YTHDF2 and the indirect m6A regulated role of miR-493-3p in prostate cancer (PCa) remains to be elusive. In this study, immuno-histochemical (IHC) staining and chromogenic in situ hybridization (CISH) were performed to find YTHDF2 was frequently upregulated but miR-493-3p was downregulated in both PCa tissues and cell lines (DU-145 and PC3) which was negatively correlated with each other. Knock down of YTHDF2 significantly elevated m6A levels, and inhibited the cell proliferation and migration of DU-145 and PC3 cell lines. The dual-luciferase reporter assay confirmed YTHDF2 as the direct target of miR-493-3p. In addition, forced expression of miR-493-3p consistently elevated the m6A levels and inhibited proliferation and migration with the knock down of YTHDF2. In contrast, overexpression of YTHDF2 and inhibition of miR-493-3p conversely reduced m6A levels. Additionally, the rescue experiments revealed that inhibition of miR-493-3p abrogated the suppression of proliferation and migration induced by si-YTHDF2. To conclude, YTHDF2 and miR-493-3p, as two crucial m6A regulators, are involved in the progression of PCa by indirectly modulating m6A levels. In view of these promising results, YTHDF2 and miR-493-3p may provide new insights into the carcinogenesis and new potential therapeutic targets for PCa.

Keywords: PCa; YTHDF2; epigenetics; m6A; miR-493-3p.

Figure 1. YTHDF2 is upregulated in PCa tissues and cell lines and negatively correlated with miR-493-3p

(A) IHC of YTHDF2. The results of IHC demonstrated that YTHDF2 was upregulated in PCa tissues than adjacent normal tissues. (B) The representative images of IHC. YTHDF2 protein was located in the cytoplasm and nucleus in PCa. (C) CISH of miR-493-3p. The results of CISH revealed miR-493-3p was downregulated in PCa. (D) The representative images of CISH of miR-493-3p. (E) q-RT-PCR. miR-493-3p was downregulated in PCa cell lines (DU-145, PC3) compared with normal prostate cell line (RWPE-1). (F) Statistical analysis indicated a significant negative correlation between YTHDF2 and miR-493-3p. Error bars represent the S.E. obtained from three independent experiments; ^*; P < 0.05. Scale bar = 100 μm.

Figure 2. Knock-down of YTHDF2 significantly elevates the global mRNA m6A levels and inhibits proliferation and migration of PCa cell lines

(A) m6A dot-blot assay. A significant elevation of m6A levels in DU-145 and PC3 cell lines transfected with si-YTHDF2 was detected. And the band intensity was measured and the result was shown behind. (B) m6A dot-blot assay. A significant reduction of m6A levels in DU-145 and PC3 cell lines transfected with pYTHDF2. And the band intensity was measured and the result was shown behind. (C) CCK-8 test. A significant suppression of proliferation at 48h and 72h was observed in DU-145 and PC3 cell lines transfected with si-YTHDF2. (D) Colony formation assay. Knock-down of YTHDF2 inhibited colony formation ability in DU-145 and PC3 cell lines. And the migration rate was calculated and shown behind. (E) Trans-well assay. Knock -down of YTHDF2 inhibited the migration in DU-145 and PC3 cell lines. And the migration rate was calculated and shown behind. (F) Western blot assay. A significant upregulation of E-cadherin and downregulation of N-cadherin was observed at protein level. And the band intensity of proteins was measured and the result was shown behind. Error bars represent the S.E. obtained from three independent experiments; ^*; P < 0.05. Photographs of trans-well assay were obtained under 10x objective, and the scale bar = 100 μm.

Figure 3. YTHDF2 is the direct target gene of miR-493-3p

(A) Dual-luciferase reporter assay. miR-493-3p significantly reduced the luciferase activity of wild-type group but no significant reduction was observed in mutated-type group in both DU-145 and PC3 cell lines. (B) q-RT-PCR assay. The expression of YTHDF2 was significantly inhibited at mRNA level by overexpressed miR-493-3p. (C) Western blot assay. The expression of YTHDF2 was suppressed at protein level by miR-493-3p. And the band intensity was measured and the result was shown behind. (D) The schematic diagram showed the sequences of 3′-UTR of YTHDF2 (wild type and mutated type). Error bars represent the S.E. obtained from three independent experiments; ^*; P < 0.05.

Figure 4. Forced expression of miR-493-3p significantly elevated the global m6A levels and inhibited cell proliferation and migration of PCa

(A) m6A dot-blot assay. Forced expression of miR-493-3p significantly elevated m6A level in DU-145 and PC3 cell lines. And the band intensity was measured and the result was shown behind. (B) m6A dot-blot assay. Inhibition of miR-493-3p conversely reduced the m6A level in DU-145 and PC3 cell lines. And the band intensity was measured and the result was shown behind. (C) CCK-8 test. Overexpression of miR-493-3p significantly suppressed the proliferation at 48h and 72h in DU-145 and PC3 cell lines. (D) Colony formation assay. Overexpression of miR-493-3p significantly inhibited the colony formation ability in DU-145 and PC3 cell lines. And the migration rate was calculated and shown behind. (E) Trans-well assay. Overexpression of miR-493-3p inhibited the migration of DU-145 and PC3 cell lines. And the migration rate was calculated and shown behind. (F) Western blot assay. E-cadherin was upregulated, N-cadherin and Vimentin were downregulated in DU-145 and PC3 cell lines transfected with miR-493-3p mimics. And the band intensity of proteins was measured and the result was shown behind. Error bars represent the S.E. obtained from three independent experiments; ^*; P < 0.05. Photographs of trans-well assay were obtained under 10x objective, and the scale bar = 100 μm.

Figure 5. Inhibition of miR-493-3p partially abrogated the cell proliferation and migration suppressed by si-YTHDF2

(A) Colony formation assay. Inhibition of miR-493-3p reversed the colony formation ability repressed by si-YTHDF2. (B) The colony formation rate was calculated. (C) Trans-well assay. Inhibition of miR-493-3p reversed the migration repressed by si-YTHDF2. And the migration rate was calculated and shown behind. (D) and (E) q-RT-PCR and western blot. The expression of YTDHF2 at mRNA and protein levels were all detected a significant upregulation by inhibition of miR-493-3p. And the band intensity of proteins was measured and the result was shown behind. Error bars represent the S.E. obtained from three independent experiments; ^*; P < 0.05. Photographs of trans-well assay were obtained under 10x objective, and the scale bar = 100 μm

Figure 6. The schematic diagram of YTHDF2 and miR-493-3p in regulating the progression of PCa

In PCa, total RNAs are transcribed and catalyzed by m6A writer (WTAP, METTL3 and METTL14) to produce the m6A modifications. YTHDF2, an m6A reader, recognizes and binds to the m6A modified sites to degrade the mRNAs, subsequently resulting in the reduction of the m6A levels, and consequently induced the progression of PCa. However, miR-493-3p transcribed from DLK1-DIO3 genomic region targets the 3’-UTR of YTHDF2 to suppress the translation of YTHDF2, thereby elevating the global RNAs m6A levels and inhibiting the progression of PCa.