MiR-590-3p promotes proliferation and metastasis of colorectal cancer via Hippo pathway

Abstract

Studies reported that miR-590-3p was involved in human cancer progression. However, its roles of oncogene or anti-oncogene in malignancies still remain elusive. This study was aimed to investigate the effect of miR-590-3p on the cell proliferation and metastasis via Hippo pathway in colorectal cancer (CRC). In our study, miR-590-3p was demonstrated highly expressed in CRC tissues, compared with adjacent normal tissues (P<0.05). In addition, miR-590-3p was positively associated with TNM stage and distant metastasis. Survival analysis showed that high miR-590-3p was related with poor overall survival rate. Then, over-expressed miR-590-3p was demonstrated to promote proliferation, invasion and migration of colon caner cells. What's more, MST1, LATS1 and SAV1 mRNA were showed lowly expressed and YAP1 expression in mRNA and protein levels were highly expressed in CRC tissues, compared with adjacent normal tissues (all P<0.05). miR-590-3p expression was negatively associated with LATS1 and SAV1 mRNA respectively and positively related with YAP1 mRNA in CRC tissues, meanwhile, there was no relationship between miR-590-3p and MST1 mRNA. Furthermore, over-expressing miR-590-3p inhibited expressions of LATS1 and SAV1, promoted YAP1 expression and didn't effect MST1 expression in colon cancer cells. And luciferase assay showed that miR-590-3p over-expression inhibited the luciferase activity of LATS1 and SAV1 3'UTR, meanwhile it had no effect on the mutated form of these two plasmids. Taken together, these data suggest that highly-expressed miR-590-3p promotes biological effect of proliferation and metastasis via targeting Hippo pathway, and predicts worse clinical outcomes of CRC patients.

Keywords: Hippo pathway; colorectal cancer; metastasis; miR-590-3p; proliferation.

Figure 1. Statistical comparisons of the miR-590-3p expressions in tumor tissues and normal colorectal mucosae

miR-590-3p expression level was detected usingqRT-PCR assay in a total of 46 patients with CRC (normalized to GAPDH). (A) miR-590-3p was highly expressed in CRC tissues, compared with adjacent normal ones. (B) miR-590-3p expression was displayed difference in different TNM stages of 46 CRC tissues. (C) Survival analysis of Log-rank assay and Kaplan-Meier curve were carried out and showed that higher miR-590-3p expression was associated with lower 5-year survival rate of patients with colorectal cancer. All graphs summarize the data from three independent experiments. ^ns referred to no significance, *P<0.05, ^*P<0.01, compared with control, using Student's t-test.

Figure 2. miR-590-3p mimics facilitate the proliferation of CRC cells

HCT116 cells were transfected with the indicated miRNA mimics or plasmids for 24 h. (A) Clone formation experiments were employed between miR-590-3p mimics group and negative control group. (B) The transfected HCT116 cells were cultured for 4 days to perform MTT test. (C),(D) EdU staining and DAPI staining were detected in HCT116 and SW480 after transfection of miR-590-3p mimics or negative mimic control for 48h. Representative images (left) and quantification of migrating cells (right) are shown. All graphs summarize the data from three independent experiments. *P<0.05, ^*P<0.01, compared with control, using Student's t-test.

Figure 3. Over-expressed miR-590-3p promotes cell invasion and migration in colon cancer cells

miR-590-3p mimics or negative control were transfected into HCT116 cells and SW480 cells, respectively. These transfected cells were cultured for subsequent assays. (A),(B) The wound healing assay was performed to evaluate migration ability of both HCT116 cells and SW480 cells. (C) Transwell assays were carried out to assess effect of miR-590-3p on invasion and migration capacity. The cell number of invasion and migration were presented in the right panel. All graphs summarize the data from three independent experiments. **P<0.01, compared with control, using Student's t-test.

Figure 4. Expression measurement of the core factors of Hippo pathway in CRC

A total of 46 cases of CRC tissues and adjacent normal tissues were fresh quick frozen tissues. They were used to examine expression of core factors of Hippo pathway. (A),(B),(C) The core factors of Hippo pathway of MST1, SAV1 and LATS1 mRNAs were measured by qRT-PCR assay and showed that they were all lowly expressed in CRC tissues, compared with adjacent normal tissues. (D),(E) (×200) YAP1 mRNA and protein level were detected by qRT-PCR test and immunohistochemistry assay, and indicated that YAP1 was highly expressed in CRC tissues, compared with adjacent normal tissues. All graphs summarize the data from three independent experiments. *P<0.05, ^*P<0.01, compared with control, using Student's t-test.

Figure 5. Scatter plots showing the statistical association between miR-590-3p and expression levels of key molecule mRNAs of Hippo pathway

A total of 46 cases of CRC tissues and adjacent normal tissues were fresh quick frozen tissues. They were used to analyze the correlation between miR-590-3p expression and core factors of Hippo pathway by qRT-PCR assay. (A) miR-590-3p expression level was not related with MST1 mRNA expression in CRC tissues. (B),(C) The negative association between miR-590-3p and SAV1 and LATS1 mRNA expression were demonstrated respectively. (D) The positive association between miR-590-3p and YAP1 mRNA expression was demonstrated.

Figure 6. miR-590-3p inhibits Hippo pathway by targeting SAV1 and LATS1 in CRC

(A) Bioinformatics analysis was performed and showed that 3’UTR of SAV1 and LATS1 mRNA had binding sequence of miR-590-3p. (B),(C) HCT116 cells or SW480 cells were transfected with miR-590-3p mimics or negative control and cultured for 48 hours. QRT-PCR and western blotting assay were carried out and the results indicated that over-expressed miR-590-3p inhibited expressions of LATS1 and SAV1, and promoted YAP1 expression (Normalized to GAPDH) in HCT116 cells or SW480 cells, respectively. (D) Immunofluorescent test was employed and the representative image demonstrated that over-expressed miR-590-3p improved the YAP1 expression in cell nuclear. (E),(F) Luciferase assay showed that miR-590-3p mimics significantly inhibited the luciferase activity of SAV1 or LATS1, meanwhile, it had less effect on the mutated form of SAV1 or LATS1 (Normalized to Rellina). All graphs summarize the data from three independent experiments. ^ns referred to no significance, *P<0.05, ^*P<0.01, compared with control, using Student's t-test.

Figure 7. Schematic representation of a model for the major molecular mechanism of biological effect of miR-590-3p via Hippo pathway in CRC