MiR-106a-5p promotes 5-FU resistance and the metastasis of colorectal cancer by targeting TGFβR2

Abstract

Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. 5-Fluorouracil (5-FU)-based chemotherapy has always been the first-line treatment. However, development of 5-FU resistance seriously affects its curative effect. The aim of this study was to elucidate the molecular mechanisms of 5-FU resistance through miR-106a-5p in CRC.

Methods: Colorectal cancer tissues were collected to analyze miR-106a-5p and TGFβR2 expressions by qPCR. Functional experiments for evaluating cell survival and metastasis were conducted to observe the biological effects of miR-106a-5p and TGFβR2. The cell survival rate was calculated using an MTT assay; the metastasis was confirmed with a Transwell invasion assay and Western blotting, which we used to measure the expression levels of the epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin. The combination of miR-106a to TGFβR2 was predicted using Targetscan, and confirmed through the construction of the luciferase reporter plasmid pGL3-basic. The interplay between miR-106a-5p and TGFβR2 was tested with qPCR and Western blotting. A Spearman rank analysis was employed to verify the correlation of miR-106a-5p and TGFβR2 expressions.

Results: MiR-106a-5p was up-regulated and TGFβR2 was down-regulated in 5-FU resistant CRC tissues and HT-29 cells. MiR-106a-5p promoted cell survival and suppressed the apoptosis rate and caspase 3 activity. Additionally, cell invasion was promoted by miR-106a-5p overexpression in the HT-29 cells and was inhibited by miR-106a-5p knockdown in the 5-FU resistant HT-29 cells; miR-106a-5p overexpression contributed to migration by increasing vimentin expression and by decreasing E-cadherin expression in the HT-29 cells; miR-106a-5p functioned by directly binding to TGFβR2. The TGFβR2 knockdown conferred chemoresistance of 5-FU and metastasis in 5-FU resistant HT-29 cells, and TGFβR2 overexpression reduced cell survival, invasion numbers, vimentin expression, and increased the cell apoptosis rate and caspase 3 activity in 5-FU resistant HT-29 cells. Also, miR-106a-5p negatively regulated TGFβR2 in a linear correlation way in the CRC tissues.

Conclusion: The up-regulation of miR-106a-5p contributes to the pathomechanism of colorectal cancer by promoting 5-FU resistance and metastasis via inhibiting target TGFβR2. Our findings provide new promising ways for the clinical application of the TGFβR2-miR-106a axis in clinical chemotherapy for 5-FU resistant colorectal cancer.

Keywords: 5-FU resistance; MiR-106a-5p; TGFβR2; colorectal cancer; metastasis.

Figure 1

The 5-FU-resistance role of miR-106a-5p in CRC tissues and cells. A. The expressions of miR-106a-5p were compared among 5-FU-resistant CRC patient specimens (n = 24) with 5-FU-sensitive specimens (n = 32). MiR-106a-5p was up-regulated in 5-FU-resistant tissues. B. The survival of HT-29 cells and HT-29-5-FU resistant cells treated with 0, 10, 20, 40, 60, 80, 100 μm of 5-FU for 48 h. The IC 50 values of HT-29 and HT-29-FU were 14.09 and 66.26 μm respectively. The MTT assay showed the higher cell survival of the HT-29-5-FU cells. C. The mRNA expressions of miR-106a-5p in the HT-29 cells and the HT-29-5-FU resistant cells were examined by qPCR. The statistical analysis results were expressed as the mean ± SEM. *P < 0.05 as compared with control group.

Figure 2

MiR-106a-5p regulated 5-FU-resistance in the HT-29 cells. (A) Knockdown of miR-106-5p in HT-29-5-FU cells. (B) Overexpression of miR-106-5p in the HT-29 cells. After transfection of anti-miR-106-5p and miR-106-5p into the cells, the relative expressions of miR-106-5p were examined by qPCR respectively. The cell survival of HT-29-5-FU cells (C) transfected of the anti-miR-106-5p and HT-29 cells (D) transfected of miR-106-5p was measured by MTT assay. The apoptosis of HT-29-5-FU cells (E) overexpressed of anti-miR-106-5p and HT-29 cells (F) overexpressed of miR-106-5p was detected by flow cytometry, and the rates of the apoptotic cells were counted and analyzed. 15 μm 5-FU was for the HT-29 cells, and 60 μm 5-FU was for the HT-29-5-FU cells. The caspase 3 activity of the anti-miR-106-5p overexpression HT-29-5-FU cells (G) and the miR-106-5p overexpression of the HT-29 cells (H) were monitored by a Caspase 3 Activity Assay Kit. Statistical analyses results are shown as mean ± SEM. *P < 0.05, compared with control group.

Figure 3

Cell invasion and EMT was promoted by miR-106a-5p in the HT-29 cells. The cell invasion number (A) and the expressions of the EMT markers, E-cadherin and vimentin, (C) in the HT-29-5-FU cells were tested using a Transwell invasion assay and Western blotting. The effects of miR-106a-5p knockdown/overexpression on cell invasion (B) and EMT markers expression (D) in HT-29-5-FU/HT-29 cells were determined. All data above were represented as the mean ± SEM. *P < 0.05, compared with control groups.

Figure 4

MiR-106a-5p down-regulated TGFβR2 by potentially binding in HT-29 cells. (A) Prediction of the potential target binding sites between miR-106a-5p and TGFβR2 3’ UTR using Targetscan. The cells were collected and analyzed with a dual-luciferase reporter assay. (B) The co-transfection of anti-miR-106a-5p with plasmids overexpressed of TGFβR2 and TGFβR2-Mut in HT-29-5-FU cells, respectively. The luciferase activity of co-expressed TGFβR2-Wt and anti-miR-106a-5p was higher. (C) The transfection of miR-106a-5p into TGFβR2/TGFβR2-Mut overexpression HT-29 cells. The luciferase activity of co-expressed TGFβR2-Wt and miR-106a-5p was lower. Detection of the expression of TGFβR2 in HT-29-5-FU cells expressed of anti-miR-106a-5p and HT-29 cells expressed of miR-106a-5p, both in mRNA (D) and protein (E) level. The relative TGFβR2 expression level was lower in the HT-29-5-FU cells, compared with the level in HT-29 cells. The expression of TGFβR2 was raised by anti-miR-106a-5p in the HT-29-5-FU cells and reduced by miR-106a-5p in the HT-29 cells. (F) The quantification of TGFβR2 protein expression was normalized by Image J. All data shown represented the mean ± SEM. *P < 0.05, compared with control groups.

Figure 5

TGFβR2 regulated 5-FU resistance and invasion in colorectal cancer. TGFβR2 was overexpressed in the HT-29-5-FU cells (A) and knocked down in the HT-29 cells (B) by transfection of the reconstruction plasmid. A cell survival assay was performed to monitor the effects of TGFβR2 (C) and shTGFβR2 (D) on cell activity. The survival was decreased/increased at different concentrations of 5-FU in HT-29-5-FU cells and HT-29 cells. The apoptosis rate was recorded by flow cytometry in the HT-29-5-FU cells (E) treated with 60 μm 5-FU, and in HT-29 cells (F) treated with 15 μm 5-FU. TGFβR2 promoted the 5-FU-induced apoptosis of HT-29-5-FU cells, and shTGFβR2 rescued the apoptosis of the HT-29 cells. The caspase 3 activity induced by 5-FU was measured in HT-29-5-FU cells (G) treated with 60 μm 5-FU, and in HT-29 cells (H) treated with 15 μm 5-FU. Similarly, TGFβR2 facilitated 5-FU-stimulated caspase 3 activity in HT-29-5-FU cells, and shTGFβR2 inhibited caspase 3 activity in HT-29 cells. A Transwell invasion assay was carried out to clarify the role of TGFβR2 (I) and shTGFβR2 (J) on cell invasion. Conversely, TGFβR2 attenuated cell invasion in HT-29-5-FU cells, and shTGFβR2 contributed to cell invasion in HT-29 cells by the knockdown of TGFβR2. The data above are the mean ± SEM. *P < 0.05, compared with control groups.

Figure 6

TGFβR2 expression was negatively associated with the miR-106a-5p expression level in colorectal cancer tissues. (A) Western blotting displayed TGFβR2 expressions in 2 specimens of 5-FU-sensitive and 2 samples of 5-FU-resistance, which were randomly selected. TGFβR2 was down-regulated in 5-FU-resistant CRC tissues, both on the mRNA expression level (B) and the protein level. (C) TGFβR2 expression, to a certain extent, is in a manner of negative correlation with miR-106a-5p expression in CRC tissues. Spearman rank analysis was chosen to explain the correlation. The results of the statistical analyses were expressed as the mean ± SEM. *P < 0.05 as compared with control group.