RNA m6A Methyltransferase METTL3 Promotes The Growth Of Prostate Cancer By Regulating Hedgehog Pathway

Abstract

Purpose: N⁶-methyladenosine (m⁶A) is the most abundant internal modification on eukaryotic mRNA and gained increasing attention recently. More and more evidence suggest that m⁶A methylation plays crucial role in tumor genesis and development. However, its role in prostate cancer remains largely unknown.

Methods: METTL3 expression status in prostate cancer was analyzed by using TCGA database and Western blotting. m⁶A content was analyzed by using RNA Methylation Quantification Kit. The role of METTL3 in prostate cancer cells was determined by proliferation, survival, colony formation, and invasion assays. The m⁶A level of GLI1 RNA was detected by methylated RNA immunoprecipitation (MeRIP) assay. In vivo role of METTL3 was studied on xenograft models.

Results: We found that m⁶A methyltransferase METTL3 was overexpressed in prostate cancer cell lines, together with increased m⁶A content. Functionally, silencing of METTL3 by shRNA in prostate cancer cell lines resulted in decreased m⁶A content, cell proliferation, survival, colony formation, and invasion. Interestingly, overexpression of wild-type METTL3 abrogated the repression effect of METTL3 depletion on m⁶A content, cell proliferation, survival, colony formation, and invasion, while the overexpression of m⁶A catalytic site mutant METTL3 was unable to rescue the inhibitory effect caused by METTL3 depletion. Further mechanism analysis demonstrated that METTL3 silence decreased the m⁶A modification and expression of GLI1, an important component of hedgehog pathway, which led to cell apoptosis. Moreover, depletion of METTL3 inhibited tumor growth in vivo.

Conclusion: Our results suggested that the m⁶A methyltransferase METTL3 promotes the growth and motility of prostate cancer cells by regulating hedgehog pathway.

Keywords: GLI1; METTL3; RNA methylation; hedgehog; prostate cancer.

Figure 1

METTL3 is deregulated in prostate cancer. (A, B) As shown in TCGA database, expression of METTL3 is increased in several types of cancers including prostate cancer (A) and different studies showed different gene alteration frequencies of METTL3 (B). (C) Expression level of METTL3 in tumor is higher than in normal tissue, as seen on GEPIA database. (D) The protein level of METTL3 in human normal prostate epithelial cell and prostate cancer cell lines was detected by Western blotting. (E) The methylated RNA (m⁶A) level in human normal prostate epithelial cell and prostate cancer cell lines. **P ˂ 0.01.

Figure 2

Effects of METTL3 depletion on cells proliferation, survival, colony formation, and invasion. (A) The knockdown efficiency of METTL3 in LNCaP and PC3 cells was detected by Western blotting. (B) The methylated RNA (m⁶A) level in LNCaP and PC3 cells after METTL3 depletion. *P ˂ 0.05, **P ˂ 0.01. (C) The proliferation of LNCaP and PC3 cells was analyzed by CCK-8 assay after METTL3 depletion. **P ˂ 0.01. (D) The survival of LNCaP and PC3 cells was analyzed by trypan blue staining after METTL3 depletion. *P ˂ 0.05. (E) LNCaP and PC3 cells were performed colony formation assay after transduced with control (Scr) or METTL3 shRNA. *P˂ 0.05. (F) The invasion ability of LNCaP and PC3 cells was analyzed by invasion assay after METTL3 depletion. *P ˂ 0.05.

Figure 3

Effects of METTL3 depletion on cells proliferation, survival, colony formation, and invasion were m⁶A activity-dependent. (A) Protein structure of METTL3 showed location of catalytic domain and the mutation site for disruption of catalytic site. (B-G) LNCaP and PC3 cells were transduced with Scr or METTL3 shRNA for 48 hrs followed by overexpression with wild type or mutant METTL3 plasmid for 24 hrs. METTL3 protein level was detected by Western blotting (B); the m⁶A content was analyzed by kit (C); cell proliferation was analyzed by CCK-8 assay (D); cell survival was analyzed by trypan blue staining (E); colony formation ability was analyzed by colony formation assay (F); invasion ability was analyzed by invasion assay (G). *P ˂ 0.05.

Figure 4

Effects of METTL3 depletion on SHH-GLI pathway and apoptosis. (A)The protein level of SHH-GLI pathway was analyzed by Western blotting after METTL3 depletion. (B, C) The mRNA level of c-Myc and cycling D1 was analyzed by qRT-PCR. *P ˂ 0.05, **P ˂ 0.01. (D, E) The protein level of Bak, Bax, Bcl-2, Bcl-xL, and cleaved PARP was analyzed by Western blotting after METTL3 depletion. (F) The relative caspase-3/7 activity was measured using Apo-One™ homogenous caspase-3/7 assay. **P ˂ 0.01. (G, H) LNCaP and PC3 cells were transduced with Scr or METTL3 shRNA for 48 hrs followed by overexpression with wild type or mutant METTL3 plasmid for 24 hrs. GLI1 protein level was detected by Western blotting (G) and the methylated GLI1 mRNA level was analyzed by Me-RIP assay (H). *P ˂ 0.05.

Figure 5

Effects of METTL3 depletion on tumor growth in vivo. (A) Typical photos of tumors on day 28 from Scr and METTL3 shRNA groups. (B, C) METTL3 depletion decreased tumor size and weight. *P ˂ 0.05, **P ˂ 0.01. (D) Mice body weight of Scr and METTL3 shRNA groups was measured at indicated time.