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Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
MiR-195 reverses 5-FU resistance through targeting HMGA1 in gastric cancer cells
1Department of Pharmacy, Luoyang Orthopedic Hospital of Henan Province, Orthopedic Hospital of Henan Province, Luoyang, China. wangchunqiuluoyang@163.com.
Objective:
To investigate the role of micro ribonucleic acid (miR)-195 in acquired resistance to 5-fluorouracil (5-FU) in gastric cancer and its potential mechanism.
Materials And Methods:
The drug resistance of AGS/5-FU and SGC-7901/5-FU cells compared with their parental cells was verified via methyl thiazolyl tetrazolium (MTT) assay, and the expression levels of miR-195 and high-mobility group protein A1 (HMGA1) in AGS/5-FU and SGC-7901/5-FU cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. MiR-195 mimic and miR-195 inhibitor were transfected into AGS/5-FU and AGS cells, respectively, the changes in HMGA1 expression were detected via qRT-PCR and Western blotting, and the sensitivity of cells to 5-FU after transfection was detected via MTT assay. After the wild-type and mutant-type luciferase reporter plasmids of HMGA1 were co-transfected with miR-195 mimic or miR-195 NC into cells, the luciferase activity was analyzed using the dual-luciferase reporter system. Finally, the rescue experiment was performed to confirm whether the changes in HMGA1 expression promote the formation of drug resistance in gastric cancer.
Results:
Both AGS/5-FU and SGC-7901/5-FU cells were significantly resistant to 5-FU compared with their parental cells, and miR-195 was down-regulated in AGS/5-FU and SGC-7901/5-FU cells, while HMGA1 was up-regulated in AGS and SGC-7901 cells. The transfection with miR-195 mimic could suppress the expression level of HMGA1 in AGS/5-FU cells, while the transfection with miR-195 inhibitor could up-regulate the expression level of HMGA1 in AGS cells. Moreover, miR-195 could bind to HMGA1 3'-untranslated region (3'UTR) in a targeted way, thereby inhibiting its expression. It was confirmed via a rescue experiment that the changes in HMGA1 expression promoted the formation of drug resistance in gastric cancer.
Conclusions:
The down-regulation of miR-195 induces the resistance to 5-FU in gastric cancer through promoting the expression of HMGA1.
Insights
Down-regulation of microRNA-195 (miR-195) promotes acquired resistance to 5-fluorouracil (5-FU) in gastric cancer. This occurs by increasing the expression of high-mobility group A1 (HMGA1), a key factor in developing drug resistance.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Gastric cancer exhibits acquired resistance to 5-fluorouracil (5-FU), a common chemotherapeutic agent.
- The precise molecular mechanisms underlying this drug resistance remain incompletely understood.
Purpose of the Study:
- To investigate the role of microRNA-195 (miR-195) in acquired 5-FU resistance in gastric cancer.
- To elucidate the potential mechanism involving high-mobility group A1 (HMGA1) expression.
Main Methods:
- Cell lines (AGS/5-FU, SGC-7901/5-FU) were used to model 5-FU resistance.
- Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting assessed miR-195 and HMGA1 expression.
- Transfection with miR-195 mimic/inhibitor and luciferase reporter assays were performed.
Main Results:
- miR-195 was significantly down-regulated, while HMGA1 was up-regulated in 5-FU resistant gastric cancer cells.
- miR-195 directly targeted and suppressed HMGA1 expression.
- Restoring miR-195 inhibited HMGA1 expression and resensitized cells to 5-FU.
Conclusions:
- Down-regulation of miR-195 is a key driver of acquired 5-FU resistance in gastric cancer.
- The mechanism involves the promotion of HMGA1 expression by reduced miR-195 levels.
- Targeting the miR-195/HMGA1 axis may offer therapeutic strategies for overcoming 5-FU resistance.
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