Jian Liu1, Yanqin Huang2, Hongqian Wang1, Denghai Wu1. 1. Department of General Surgery, The Second Affiliated Hospital of Zhejiang Chinese Medical University (Xinhua Hospital of Zhejiang Province) Hangzhou, China. 2. Cancer Institute, Second Affiliated Hospital, College of Medicine, Zhejiang University Hangzhou, China.
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. IJCEP
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. IJCEP
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