Investigating the mechanisms underlying stemness regulation by miR-203 in MCF-7 human breast cancer cells

• Main Authors: Wen-Yu Lin, 林玟妤
• Other Authors: Yeu Su
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/55htjj

碩士 === 國立陽明大學 === 生物藥學研究所 === 105 === Breast cancer is one of the main causes of female mortality worldwide. Although surgery coupled with hormone therapy, chemotherapy or target therapy based on tumor subtypes markedly improves survival rates, many patients are still refractory to the present therapies. In this regard, the identification of breast cancer stem cells (BCSCs) opens a new door for developing better treatments. Cancer stem cells, also called tumor initiating cells (TICs), are a small group of cancer cells responsible for tumor initiation, metastasis, relapse after treatment, and drug resistance. Previous studies in our laboratory found that miR-203 inhibits the stemness of HT-15 and HCT-116 human colorectal cancer (CRC) cells. By contrast, the role of this miR in the malignant progression of breast cancer is still controversial. Hence, I tried to dissect the role of miR-203 in the stemness of human breast cancer cells as well as the underlying mechanism. As the first step to address this question, both miR-203-overexpressing and -sequestered stable clones were established from the ER+, PR+ , HER-2- MCF-7 human breast cancer cells. After RT-qPCR analysis, two clones were chosen from each group and designated as miR-203-OE1 & 8 and miR-203-SQ1 & 6, respectively. Intriguingly, the protein levels of Bmi-1, a known miR-203 target, were only increased in the miR-203-SQ1 clone. Subsequently, the sphere-forming abilities in miR-203-OE and miR-203-SQ clones were found to be lower and higher than the vector control clone, respectively. While the inhibiting effects of miR-203 on the soft agar colony forming abilities of MCF-7 cells were clearly detected, its sequestration didn’t significantly increase the anchorage–independent growth of these cells. In the meantime, flow cytometric analysis demonstrated that side populations were decreased and increased, respectively, in miR-203-OE and miR-203-SQ clones. Regarding the action mechanism of miR-203, I found that the protein levels of ΔNp63, a potential miR-203 target, were decreased and increased, respectively, in miR-203-OE1 and miR-203-SQ1 clones. Moreover, the mRNA levels of DKK1 and BRCA1, two known mediators of ΔNp63, in miR-203-OE and miR-203-SQ clones were increased and reduced, respectively. Since DKK1 is an inhibitors of Wnt signaling, I subsequently analyzed the nuclear level of β-catenin as well as it two targets, cyclin D1 and c-Myc in these clones. Surprisingly, no changes in the levels of three proteins were found. Taken together, miR-203 appears to be a stemness inhibitor for MCF-7 cells. However, while the involvement of the Wnt signaling in the stemness inhibitory effect of miR-203 in MCF-7 human breast cancer cells was ruled out, the role of ΔNp63 in the process needs further investigate.