Fig 2: MMSET upregulation promotes sphere formation and invasion in EC cells(A) qPCR analysis of MMSET mRNA expression in the immortalized human endometrial epithelial EM cell line and EC cell lines. (B and C) qPCR analysis and western blotting analysis of MMSET expression in Ishikawa cells after transient overexpression of MMSET, and in HEC-1 cells after transient knockdown of MMSET. (D and E) Sphere formation (D) and invasion (E) in Ishikawa cells after transient overexpression of MMSET. (F and G) Sphere formation (F) and invasion (G) in HEC-1 cells after transient knockdown of MMSET. (H) Western blotting analysis of indicated proteins in Ishikawa and HEC-1 cells after overexpression or knockdown of MMSET. (I) Ishikawa cells were transfected with MMSET expression vector or the control vector in culture plates. 48 hours later, MMSET-overexpressing Ishikawa cells were injected into nude mice. Tumor volume and weight were measured. (J) HEC-1 cells were transfected with MMSET siRNA or the control siRNA in culture plates. 48 hours later, MMSET-knockdown HEC-1 cells were injected into nude mice. Tumor volume and weight were shown. **P < 0.01.

Fig 3: miR-34a, miR-424 and miR-513 directly target MMSET 3'-UTR to downregulate MMSET expression in EC cells(A) Alignment of miR-34a, miR-424 and miR-513 and their corresponding complementary binding sequences in MMSET 3'-UTR by bioinformatics algorithms. (B) Relative miR-34a/miR-424/miR-513 levels in EC cell lines and EM cells. (C) HEC-1 cells were transfected with reporter constructs containing either wild-type (WT) MMSET, or MMSET 3'-UTR with mutation (MUT), along with miR-34a/miR-424/miR-513 mimic or negative control mimic, respectively. Relative luciferase activity was measured. (D) Western blotting analysis of MMSET expression in EC cells after overexpression or knockdown of miR-34a/miR-424/miR-513. (E and F) Cell invasion (E) and sphere formation (F) of EC cells after overexpression or knockdown of miR-34a/miR-424/miR-513. **P < 0.01.

Fig 4: Elevated MMSET expression predicts poor survival in EC(A) Relative mRNA expression of MMSET, Twist1, Vimentin and E-cadherin in 50 matched human normal endometrial tissues and EC tissues. (B and C) Relative MMSET mRNA levels in EC samples were categorized based on tumor grade (B) and stage (C). (D) Meta-analysis of MMSET and Twist1 mRNA in EC samples and adjacent normal tissues from the BioExpress database. (E) Kaplan–Meier overall survival curves for high and low MMSET mRNA expression in EC patients. **P < 0.01.

Fig 5: miR-34a, miR-424 and miR-513 inhibit EMT, invasion and the sphere-forming ability of EC cells through targeting MMSET(A) miR-34a/miR-424/miR-513 mimic or control mimic was co-transfected into HEC-1 cells, together with (or without) MMSET cDNA vector lacking the 3'-UTR region. (B) miR-34a/miR-424/miR-513 inhibitor or control inhibitor was co-transfected into Ishikawa cells, together with (or without) MMSET siRNA. Cell invasion and sphere formation assays were performed. (C and D) qPCR analysis of indicated genes in HEC-1 (C) and Ishikawa (D) cells treated as described above. *P < 0.05, **P < 0.01.