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

Figures & data

Table 1 CNTN2 RNAi sequences

CNTN2-RNAi-1F	TGCTGTTCATCTTCCACCGATAGGTGGTTTTGGCCACTGACTGACCACCTATCTGGAAGATGAA
CNTN2-RNAi-1R	CCTGTTCATCTTCCAGATAGGTGGTCAGTCAGTGGCCAAAACCACCTATCGGTGGAAGATGAAC
CNTN2-RNAi-2F	TGCTGTCCACTTTGCGCCACTTGATCGTTTTGGCCACTGACTGACGATCAAGTCGCAAAGTGGA
CNTN2-RNAi-2R	CCTGTCCACTTTGCGACTTGATCGTCAGTCAGTGGCCAAAACGATCAAGTGGCGCAAAGTGGAC
CNTN2-RNAi-3F	TGCTGTTTGGTTGCATCTCGCACAGAGTTTTGGCCACTGACTGACTCTGTGCGATGCAACCAAA
CNTN2-RNAi-3R	CCTGTTTGGTTGCATCGCACAGAGTCAGTCAGTGGCCAAAACTCTGTGCGAGATGCAACCAAAC
CNTN2-RNAi-4F	TGCTGTCTTATAGCCGGTGACTGCAGGTTTTGGCCACTGACTGACCTGCAGTCCGGCTATAAGA
CNTN2-RNAi-4R	CCTGTCTTATAGCCGGACTGCAGGTCAGTCAGTGGCCAAAACCTGCAGTCACCGGCTATAAGAC

Abbreviations: CNTN2, contactin 2; RNAi, RNA interference; F, forward; R, reverse.

Table 2 Primer sequences

APP-F	GTGGCTGAGGAGATTCAAG
APP-R	AAAGAAGGCATGAGAGCATC
CNTN2-F	AGTCACAGCCTGTCCTCTAG
CNTN2-R	ATCTGCCTATGCCTTGGTTG
EGFR-F	AACACAGTGGAGCGAATTCCTTT
EGFR-R	GGAAGTCCATCGACATGTTGCT
HES1-F	TTCTGGAAATGACAGTGAAGCA
HES1-R	CCTCGGTATTAACGCCCTC
GAPDH-F	TCATGGGTGTGAACCATGAGAA
GAPDH-R	GGCAGGACTGTGGTCATGAG

Abbreviations: APP, amyloid β precursor protein; CNTN2, contactin 2; EGFR, epidermal growth factor receptor; F, forward; R, reverse.

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.

Figure S1 CNTN2 RNAi selection.

Notes: (A) The Western blot shows that CNTN2 RNAi-2 was the most effective among the four RNAi vectors to inhibit CNTN2 expression in U87-GSCs. (B) Gene sequencing results of CNTN2 RNAi-2 after lentiviral infection of U87-GSCs.

Abbreviations: CNTN2, contactin 2; RNAi, RNA interference; U87-GSCs, U87-derived glioma stem cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.