Assessment of the influences of CD44 overexpression on the stemness of HCT-15 and HT-29 human colon cancer cells

• Main Authors: Yu-Yin Chang, 張毓尹
• Other Authors: Yeu Su
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/23049626286897529072

碩士 === 國立陽明大學 === 生物藥學研究所 === 101 === Cancer stem cells (CSCs) are a small group of cancer cells which have the ability of self-renewal and differentiation. CSCs are considered to be responsible for the initiation and the subsequent malignant progression of tumor. CD44 is a cell surface glycoprotein involved primarily in cell-cell and cell-matrix interactions and cell adhesion. CD44 also positively regulates the migration, proliferation, invasion and metastasis of cancer cells. In a previous study, we found that CD44+ HCT-15 human colon cancer cells have the characteristics of cancer stem cells. These indicate that CD44 not only can be used as a marker for cancer stem cells but also plays an important role in cancer progression. This study aimed to assess whether CD44s overexpression in HCT-15 and HT-29 human colon cancer cells could enhance their stemness. My results showed that, mRNA levels of several stemness-related genes such as Oct4, Nanog and Klf4 were increased in CD44s-overexpressing HCT-15 S1 clone, compared to the parental cells. Protein levels of Nanog, Snail and Twist were increased compared to the vector control (V) clone. In addition, the nuclear levels of Oct4, Nanog and Bmi1 proteins were increased compared to the vector control clone. Both tumor sphere formation and soft agar colony formation abilities were far more superior to the vector control clone, indicating better abilities in self-renewal and anchorage-independent growth of HCT-15 S1 cells. Moreover, the mRNA levels of Oct4 and Nanog were slightly increased in CD44s-overexpressing HT-29 clones S1 and S2 compared to the vector control clone. On the other hand, the mRNA level of Twist was significantly increased in HT-29 S1 and S2 cells. Interestingly, protein levels of the stemness-related proteins such as Snail, Bmi1 and Twist were also increased, while E-cadherin was decreased in CD44s-overexpressing HT-29 clones. In addition, the nuclear levels of Oct4 and Nanog proteins were increased. The abilities of self-renewal and anchorage-independent growth in HT-29 S1 and S2 cells were also much higher than the vector control clone. By contrast, no significant changes in the aforementioned abilities were found in CD44s-knockdowned clones, sh2 and sh4. Through DNA microarray analysis, upregulated expression of Wnt11, Wnt16 and IL-8 gene was detected in HT-29 S2 cells, implying that Wnt signaling may be more active in CD44s-overexpressing HT-29 cells. Additionally, an increased migration ability of parental HT-29 cells was observed after being cultured in the conditioned media (CM) collected from HT-29 S1 or S2 cells, suggesting the presence of some chemoattractants in them. Therefore, I will examine the IL-8 protein levels in these conditioned media. Moreover, knockdown of Twist1 or Twist2 in HT-29 S1 cells, T1sh and T2sh clones, their mRNA levels of Twsit1, Bmi1 and IL-8 were significantly reduced than HT-29 S1 clone. The abilities of self-renewal in HT-29 T1sh and T2sh were lower than the HT-29 S1 clone. By contrast, no significant changes in the anchorage-independent growth ability were found in HT-29 T1sh and T2sh comparing to HT-29 S1 clone. In addition, knockdown of Bmi1 in HT-29 S1 or HT-29 S2 cells, S1 B1sh and S2 B1sh clones, their mRNA levels of Nanog, Twsit1, Bmi1 and IL-8 were significantly reduced than HT-29 S1 clone. The abilities of self-renewal in HT-29 S1 B1sh and S2 B1sh were lower than the HT-29 S1 and HT-29 S2 clones. By contrast, no significant changes in the anchorage-independent growth ability were found in HT-29 S1 B1sh and S2 B1sh comparing to HT-29 S1 and HT-29 S2 clones.