RNAi for contactin 2 inhibits proliferation of U87-glioma stem cells by downregulating AICD, EGFR, and HES1

Abstract

Glioblastoma is the most common form of malignant brain tumors and has a poor prognosis. Glioma stem cells (GSCs) are thought to be responsible for the aberrant proliferation and invasion. Targeting the signaling pathways that promote proliferation in GSCs is one of the strategies for glioma treatment. In this study, we found increased expression of contactin 2 (CNTN2) and amyloid β precursor protein (APP) in U87-derived GSCs (U87-GSCs). RNA interference (RNAi) for CNTN2 downregulated the expression of APP intracellular domain (AICD), which is the proteolytic product of APP. Treatment with CNTN2 RNAi inhibited the proliferation of U87-GSCs. CNTN2 RNAi decreased the expression of epidermal growth factor receptor and HES1, which are potential targets of AICD. In summary, inhibition of the CNTN2/APP signaling pathway may repress the proliferation in U87-GSCs via downregulating the expression of HES1 and epidermal growth factor receptor. CNTN2/APP/AICD signaling pathway plays an important role in U87 glial tumorigenesis. Further studies are warranted to elucidate the role of these signaling pathways in other sources of GSCs. Depending on their role in proliferation in other sources of GSCs, members of the CNTN2/APP/AICD signaling pathway may provide novel targets for the development of therapy for glioblastomas.

Keywords: CNTN2; TAG1; contactin 2; glioma stem cells; transient axonal glycoprotein-1.

Figure 1

Characterization of U87-GSCs. Notes: (A) U87-GSCs are cultured in serum-free medium for 1 month, and the P5 spheres are shown with phase-contrast, HE staining (violet), and immunostaining with GSC markers CD133 and Nestin (brown). (B) Flow cytometry showed that the percentage of CD133+/Nestin+ cells were 0.540%±0.308% and 91.993%±0.238% in U87 cells (upper) and U87-GSCs (lower), respectively. (C) U87-GSCs are cultured in differentiation medium containing serum for 7 days, and the differentiated neurons, astrocytes, and oligodendrocytes are demonstrated by immunostaining with β-tubulin III, GFAP, and O1, respectively (brown). Scale bar =50 μm for (A) and 100 μm for (C). Abbreviations: U87-GSCs, U87-derived glioma stem cells; HE, hematoxylin and eosin; GFAP, glial fibrillary acidic protein; PE, phycoerythrin.

Figure 2

Expression of CNTN2 and APP in U87-GSCs and U87 cells. Notes: (A) The melt curves of qPCR showed more CNTN2 (left) and APP (right) mRNA expression in U87-GSCs than those in U87 cells with the quantification graphs below. The different colored lines represent the different samples. (B) The Western blot demonstrated a higher CNTN2 and APP protein expression in U87-GSCs than those in U87 cells with the quantification graphs below. *P≤0.05. Abbreviations: CNTN2, contactin 2; APP, amyloid β precursor protein; U87-GSCs, U87-derived glioma stem cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

CNTN2 RNAi inhibited AICD expression and U87-GSCs proliferation. Notes: (A) The Western blot demonstrates that lentiviral infection with CNTN2 RNAi-2 does not change the APP expression but significantly downregulates the AICD expression in U87-GSCs, with quantification graphs below. (B) The U87-GSCs were cultured for 72 h after the lentiviral infection with CNTN2 RNAi-2. The CNTN2 RNAi-2-infected U87-GSCs showed lower proliferation by the phase-contrast micro-imaging (upper lane) with the quantifications of number and diameter of spheres (middle lane) and the time-dependent growth curves (lower lane). Scale bar =100 μm for (B). *P≤0.05. Abbreviations: CNTN2, contactin 2; RNAi, RNA interference; AICD, APP intracellular domain; U87-GSCs, U87-derived glioma stem cells; APP, amyloid β precursor protein; OD, optical density; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

CNTN2 RNAi inhibited EGFR and HES1 expressions in U87-GSCs. Notes: (A) The quantification graphs of qPCR demonstrate that lentiviral infection with CNTN2 RNAi-2 significantly decreased EGFR and HES1 mRNA levels in U87-GSCs. (B) The Western blot demonstrates that lentiviral infection with CNTN2 RNAi-2 significantly downregulated the EGFR and HES1 protein expression in U87-GSCs, with quantification graphs below. *P≤0.05. Abbreviations: CNTN2, contactin 2; RNAi, RNA interference; EGFR, epidermal growth factor receptor; U87-GSCs, U87-derived glioma stem cells; qPCR, quantitative polymerase chain reaction; mRNA, messenger RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.