MicroRNA-34a inhibits cells proliferation and invasion by downregulating Notch1 in endometrial cancer

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs composed of 18-25 nucleotides that regulate the expression of approximately 30% of human protein coding genes. Dysregulation of miRNAs plays a pivotal role in the initiation and progression of malignancies. Our study has shown that microRNA-34a (miR-34a) was upregulated in human endometrial cancer stem cells (ECSCs). However, it is unknown how miR-34a regulates endometrial cancer itself. Here, we report that miR-34a directly and functionally targeted Notch1. MiR-34a inhibited the proliferation, migration, invasion, EMT-associated phenotypes by downregulating Notch1 in endometrial cancer cells. Overexpression of miR-34a also suppressed tumor growth in nude mice. Importantly, further results suggested miR-34a was significantly downregulated in endometrial cancer tissues and negatively correlated with Notch1 expression. There was a significant association between decreased miR-34a expression and worse patient prognosis. Taken together, our results suggest that miR-34a plays tumor-suppressive roles in endometrial cancer through downregulating Notch1. Thus miR-34a could be a potential therapeutic target for prevention and treatment of endometrial cancer.

Keywords: Notch1; endometrial cancer; invasion; microRNA-34a; proliferation.

Figure 1. Tumorsphere formation and differentiation

(A) HEC-1-B cells grew as an adherent monolayer. (B) Primary tumorspheres formed in SFM medium. (C) Typical tumorspheres formed in SFM at 10 days. (D) Tumorsphere differentiated at 3 days in DMEM/F12 medium supplemented with 10% FBS. (E) High-level expression of stem cell marker CD133 in tumorspheres. (F) Oct4 and Sox2 were significantly elevated in tumorspheres compared to 5-day and 10-day differentiated cells. (Scale bar=100 μm).

Figure 2. Microarray analysis of miRNAs expression level in ECSCs and its differentiated cells

(A) Hierachical clustering analysis of miRNAs expression profiles in ECSCs and 10-day differentiated cells. Green represents low expression, red represents high expression, and black represents intermediate expression. (B) The expression of miR-34a was detected in ECSCs and its differentiated cells by RT-qPCR. Three independent experiments were performed. ^** P < 0.01.

Figure 3. MiR-34a directly targets Notch1

(A, B) Notch1 mRNA and protein expression were determined in ECSCs and 10-day- differentiated cells. (C) The wild type (wt) and mutant (mut) binding sites for miR-34a in the 3’-UTR of the Notch1 gene. (D) Luciferase reporter assays. NC: non-specific negative control. Three independent experiments were performed. ^**P < 0.01.

Figure 4. The transfection of miR3-4a mimics or Notch1 siRNA

(A) HEC-1-B cells were transfected with miR-34a mimics, Notch1 siRNA and their corresponding controls for 48 h. The mRNA levels of Notch1 was determined by RT-qPCR. (B) The cells were treated as indicated above, and the protein levels of Notch1 were determined by Western blotting. NC: non-specific negative control. Three independent experiments were performed. ^** P < 0.01.

Figure 5. Overexpression of miR-34a suppressed HEC-1-B cells proliferation, migration, invasion and EMT by targeting Notch1

(A) HEC-1-B cells were transfected with miR-34a mimics, Notch1 siRNA and corresponding controls for 48 h. The cell proliferation ability was determined by CCK-8 assay. (B, C) Colony formation assays. (D-F) Cell migration and invasion were detected using Transwell assay. (G, H) Wound healing assay. Scale bar=100 μm. (I) Protein expressions of E-cadheirn and Vimentin were measured by Western blotting. NC: non-specific negative control. Three independent experiments were performed. ^*P < 0.05, ^**P < 0.01.

Figure 6. Overexpression of miR-34a inhibited tumor growth and EMT in BALB/c nude mice

(A) Average of tumor volumes were measured after cells were injected. (B, C) Photograph of typical tumors grown in mice. (D) The protein expression of Notch1, E-cadherin and Vimentin was detected by Western blotting. (E) The expression of Notch1 was detected by IHC. Scale bar=190 μm. ^*P < 0.05, ^**P < 0.01.

Figure 7. Down-regulation of miR-34a in endometrial cancer tissues is negatively correlated with expression of Notch1

(A) Low expression of miR-34a in endometrial cancer tissues (EC tissues) compared with normal endometrial tissues (NETs). (B) The expression of miR-34a in different clinical stages of endometrial cancer patients. (C) Low expression of miR-34a in patients with lymph node metastases. (D) The expression of Notch1 was detected in normal endometrial tissues and endometrial cancer tissue by IHC. a. Normal endometrial tissues samples; b. Endometrial cancer tissues samples from patients with stage I; c. Endometrial cancer tissues samples from patients with stage II; d. Endometrial cancer tissues samples from patients with stage III ; e Endometrial cancer tissues samples from patients with stage IV. (Scale bar=70μm). (E) The pathologic score of Notch1 expression in endometrial cancer tissues. (F) The expression of miR-34a was negatively correlated with Notch1 scores in endometrial cancer. ^*P < 0.05, ^**P < 0.01.