MicroRNA-22 suppresses the growth, migration and invasion of colorectal cancer cells through a Sp1 negative feedback loop

Abstract

MicroRNAs have recently emerged as regulators of many biological processes including cell proliferation, development and differentiation. This study identified that miR-22 was statistically decreased in colorectal cancer clinical specimens and highly metastatic cell lines. Moreover, low miR-22 expression was associated with tumor metastasis, advanced clinical stage and relapse. Consistent with clinical observations, miR-22 significantly suppressed the ability of colorectal cancer cells to growth and metastasize in vitro and in vivo. Sp1 was validated as a target of miR-22, and ectopic expression of Sp1 compromised the inhibitory effects of miR-22. In addition, Sp1 repressed miR-22 transcription by binding to the miR-22 promoter, hence forming a negative feedback loop. Further study has shown that miR-22 suppresses the activity of PTEN/AKT pathway by Sp1. Our present results implicate the newly indentified miR-22/Sp1/PTEN/AKT axis might represent a potential therapeutic target for colorectal cancer.

Keywords: AKT; PTEN; Sp1; colorectal cancer; miR-22.

Figure 1. MiR-22 level is associated with CRC progression, metastasis and relapse

(A) Comparison of miR-22 expression level between primary CRC samples and paired adjacent normal tissues. A log₂ fold change (CRC/normal) less than −2 was considered a significant down-regulation (dotted lines). (B) miR-22 expression levels in different clinical stages of CRC patients. (C) miR-22 is differentially expressed in the lymph node metastasis negative group (LN-Negative) compared with the positive group (LN-Positive). (D) The relative expression of miR-22 in matched primary CRC tissues and liver metastatic tissues. (E) The relative expression of miR-22 in five CRC cell lines (HT29, Caco-2, SW480, SW620 and LoVo). (F) The correlation of miR-22 expression and CRC recurrence was analyzed. (G) Kaplan-Meier analysis of relapses-free survival of Stage I–III CRC patients by miR-22 expression. *P < 0.05, **P < 0.01.

Figure 2. MiR-22 modulates CRC cell proliferation, colony formation, migration and invasion

(A, B) MTT and colony formation assays were performed in SW620 and LoVo cells expressing miR-22 or the control. (C, D) MTT and colony formation assays were performed in SW480 and HT29 cells transiently transfected with miR-22 inhibitor or with control inhibitor. (E, F) SW620 and LoVo cells transfected with miR-22 mimics or with control mimics were subjected to transwell migration and invasion assays. (G, H) SW480 and HT29 cells transfected with miR-22 inhibitor or with control inhibitor were subjected to transwell migration and invasion assays. *P < 0.05, **P < 0.01.

Figure 3. MiR-22 suppresses the growth and metastatic ability of CRC cells in vivo

(A) Effects of miR-22 on subcutaneous tumor generation. (B) Tumor sizes were measured on the indicated days (d). (C) Representative H&E staining images using a dissection microscope showed metastatic lesions in the lungs of mice injected with miR-22 overexpressing SW620 cells or control cells (×100 magnification). (D) The number of lung metastatic nodules per mouse was counted under the microscope.

Figure 4. Sp1 is a direct target of miR-22

(A) The wild-type and mutant of putative miR-22 target sequences of Sp1 3′UTR. (B, C) Analysis of the luciferase activity of psicheck-2-Sp1 3′UTR WT and MUT vector in HEK293T cells by miR-22 or anti-miR-22. (D) The Sp1 mRNA levels in the indicated cells was analyzed by qRT-PCR. (E) The Sp1 protein levels in the indicated cells were examined by western blot. *P < 0.05, **P < 0.01.

Figure 5. The levels of Sp1 influence the effects of miR-22 in CRC cells

(A) Sp1 protein level was detected by Western blot analysis after transfection with Sp1 in SW620 and LoVo cells. (B, C) MTT and colony formation assays were performed in SW620 and LoVo cells transfected with miR-22, Sp1 or miR-22/Sp1. (D, E) Transwell migration and invasion assays were performed in SW620 and LoVo cells transfected as above. *P < 0.05, **P < 0.01.

Figure 6. Sp1 binds to the promoter of miR-22 and depends on the miR-22 to promote CRC aggressiveness

(A) miR-22 levels were analyzed by qRT-PCR in SW480 cells transfected with Sp1 or siSp1. (B) schematic diagram indicates the location and sequences of 3 putative Sp1-binding sites on miR-22 promoter region. (C) ChIP assays were performed in SW620 and SW480 cells with anti-Sp1 antibody. (D) ChIP assay was performed in SW480 and HT29 cells transfected with a control or siSp1. (E) Analysis of the luciferase activity of pGL3-miR-22 promoter report plasmid cotransfected with an empty vector or Sp1 plasmid in SW480 and HT29 cells. (F, G) MTT and colony formation assays were performed in SW480 and HT29 cells transfected with Sp1 alone or in combination with miR-22. (H, I) Transwell migration and invasion assays were performed in SW480 and HT29 cells transfected as above. *P < 0.05, **P < 0.01.

Figure 7. MiR-22 affects PTEN/AKT pathway through Sp1

(A) The PTEN, p-AKT and T-AKT protein levels were analyzed by Western blot in SW480 and SW620 cells transfected with miR-22 and Sp1 expression plasmid. (B) The Sp1 binding site on the PTEN promoter. The wild-type and mutant Sp1 binding site are shown. (C, D) Relative luciferase activity in Sp1 or control expressing HEK293T and SW480 cells cotransfected with a wild-type or mutant PTEN promoter. *P < 0.05.

Figure 8. Schematic of pathways involved in the tumor-suppressor role of miR-22 in CRC cells