Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

Figures & data

Figure 1 Schematic features of miR cassettes and knockdown efficacy in N2a cells. (A) Each miR cassette in pcDNA6.2-GW/EmGFP vector. (B) Western blot of N2a cells transfected with each miR expression construct. (C) Quantitation of PrP^C expression. When miRdual was introduced into the cells, PrP^C expression was the most efficiently decreased as compared with wild-type cells (*p < 0.05, **P <0.01).

Figure 2 Transfection of miR cassettes and Prnp knockdown efficacy at mRNA and protein level. (A) PCR of genomic DNA shows miR cassette-specific amplicons. Lane L, 100 bp DNA ladder; lane 1, expression vector with miRdual cassette; lane 2, expression vector with miRscr; lane 3, wild-type cells; lane 4, N2amiRdual; lane 5, N2amiRscr; lane 6, no template. (B) qPCR analysis of PrP^C transcript abundance showing knockdown efficiency of miRdual in mRNA levels. (C) Western blot analysis of PrP^C protein abundance. Top part is the western blot and bottom panel is densitometric evaluation. dual1 and dual2 are N2amiRdual cell clones (*p < 0.05, **p < 0.01).

Figure 3 Quantitative PCR analysis of PrP^C interacting and/or associated molecules. 6720460F02Rik, Plk3, Ppp2r2b, Csnk2a1 transcripts were upregulated in prion knockdown N2amiRdual cells compared to wild-type N2a cells. The increased Mpg transcripts in both N2amiRdual and N2amiRscr seemed to have no correlation with decreased PrP^C (*p < 0.05, **p < 0.01).

Figure 4 Cell proliferation, viability and GFP expression test. (A) Viability and proliferation of N2a cells stably transformed with miRdual or miRscr (*p < 0.05). (B) Confocal microscopy images of clonal expanded cells. N2amiRdual and N2amiRscr showed green fluorescent signals. Cell nuclei were stained with DAPI (blue). Photos were taken at 20 doubling passages. GFP signals stably expressed over 135 doubling passages. Scale bar, 50 um.

Figure 5 Apoptotic resistance to serum deprivation. Cells were cultured under conditions of serum deprivation and collected at indicated time points. (A) Detection of Drp1 expression levels analyzed by densitometric analysis of western blot. (B) Cytochrome C expression in cytosolic fractions detected by immunoassay. (C) Analysis of caspase 3 activity calculated by comparison with the free pNA. dual, N2amiRdual; scr, N2amiRscr; wt, miR non-treated wild-type N2a (*p < 0.05, **p < 0.01).

Figure 6 Detection of apoptotic cells following serum deprivation using Annexin V-Cy3 assay. Cells were collected at the indicated time points and 5 × 10⁵ cell suspensions were treated with Annexin V. The proportion of cells showing apoptotic change was increased in N2amiRdual whereas most of cells showed the resistance against apoptosis stimulation in N2amiRscr and wild-type N2a cells (*p < 0.05).

Figure 7 Downregulation pattern of PrP^C in RK13-moPrnp cells harvested at different confluences. RK13-moPrnp cells were lentiviral-transduced with miR expression cassettes and the relative abundances of PrP^C and GFP processed were determined by western blot (*p < 0.05, **p < 0.01).

Table 1 Target genes of qPCR for characterizing the established N2amiRdual cell line

Gene Symbol	Gene Name	GenBank Accession No.	General Putative Function	Ref.
Prnp	prion protein	NM_011170	The function of PrP^C is not known. PrP^C is encoded in the host genome and is expressed both in normal and infected cells.
6720460 F02Rik	RIKEN cDNA 6720460F02 gene	AK049710	Unknown	30
Cdr34	Cerebellar degeneration-related antigen 1	NM_001166658	In fission yeast, acts as a mitotic inducer. In G2 it negatively regulates wee1, a mitotic inhibitor. Also has a role in cytokinesis where it required for proper septum formation.	31
Csnk2a1	casein kinase 2, alpha1 polypeptide	AK011501	Casein kinases are operationally defined by their preferential utilization of acidic proteins such as caseins as substrates. The alpha and alpha′ chains contain the catalytic site.	32
Gfap	Glial fibrillary acidic protein	AF332061	GFAP, a class-III intermediate filament, is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells.	33
Mpg	N-methylpurine-DNA glycosylase	NM_010822	Hydrolysis of the deoxyribose N-glycosidic bond to excise 3-methyladenine, and 7-methylguanine from the damaged DNA polymer formed by alkylation lesions.	30
Plk3	Polo-like kinase 3 (Drosophila)	NM_013807	Serine/threonine protein kinase involved in regulating M phase functions during the cell cycle. May also be part of the signaling network controlling cellular adhesion.	30
Ppp2r2b	protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform	NM_028392	The B regulatory subunit might modulate substrate selectivity and catalytic activity, and also might direct the localization of the catalytic enzyme to a particular subcellular compartment.	31

Table 2 Sequences of artificial miRs targeting Mus musculus Prnp ORF

			Sequence
		L	Mature miRNA Sequence	Loop Sequence	Sense Sequence	L
miR1	T	TGC TG	ACA TCA GTC CAC ATA GTC ACA	GTT TTG GCC ACT GAC TGA C	TGT GAC TAT GGA CTG ATG T
	B	C	TGT AGT CAG GTG TAT CAG TGT	CAA AAC CGG TGA CTG ACT G	ACA CTG ATA CCT GAC TAC A	GTC C
miR2	T	TGC TG	ATC TTC TCC CGT CGT AAT AGG	GTT TTG GCC ACT GAC TGA C	CCT ATT ACC GGG AGA AGA T
	B	C	TAG AAG AGG GCA GCA TTA TCC	CAA AAC CGG TGA CTG ACT G	GGA TAA TGG CCC TCT TCT A	GTC C
miRscr	T	TGC TG	AAA TGT ACT GCG CGT GGA GAC	GTT TTG GCC ACT GAC TGA C	GTC TCC ACG CAG TAC ATT T
	B	C	TTT ACA TGA CGC GCA CCT CTG	CAA AAC CGG TGA CTG ACT G	CAG AGG TGC GTC ATG TAA A	GTC C

L, linker; T, top strand; B, bottom strand.

Table 3 Primer sequences used in this study

Target Gene	Purpose	Label		Sequence (5′ to 3′)	Size	Product Size
miR cassette	PCR	-	F	TGC TGC TGC CCG ACA ACC ACT ACC TGA GCA	30	412
		-	R	ATC AGC GAA CCG CGG GCC CTC TAG ATC AAC	30
6720460 F02Rik	PCR	-	F	GAT CAC TCC TAA AGG AGG AAC AGC TAG AG	29	643
		-	R	ATA AGG GAG ACG GTC ATG TCA CCA C	25
	qPCR	FAM	F	CGT TAC TCA GCC TGA ACC TCG GTA A	25	68
		-	R	CTG CTC TGG CTC TGG ACT TTC C	22
Cdr34	PCR	-	F	ATG CTG GCA GAT AAC CTA GTG GAG G	25	1,240
		-	R	AAC TGA TGG ACT CTG GGC TTA CAG G	25
	qPCR	FAM	F	CGT GGA GAG CTA CTG AAG AAG TGC CA	26	67
		-	R	AGG TCT GCA CAG CCT TGT GTG	21
Csnk2a1	PCR	-	F	AAG CAG GGC CAG AGT TTA CAC AGA T	25
		-	R	GCT GGA ACA GGT ATC CCA AGT GAG T	25
	qPCR	FAM	F	CGA AGT TTC TGG ACA AGC TGC TT	23
		-	R	AGC CTG GTC CTT CAC AAC AGT G	22
Gfap	PCR	-	F	ACG CTT CTC CTT GTC TCG AAT GAC T	25
		-	R	GCT CCT GCT TCG AGT CCT TAA TGA C	25
	qPCR	FAM	F	CGG ATA CTT TCT CCA ACC TCC AGA TC	26
		-	R	CCT CTT GAG GTG GCC TTC TGA C	22
Mpg	PCR	-	F	GGG CAG AGG ATC CCT AAA ACC GGT G	25
		-	R	CAG GCT GTT TGC TGA GGC TGA TCC A	25
	qPCR	FAM	F	CGA CAG CCC TAA AGA GAG ACT CCT GT	26
		-	R	GGT CCT CTG GGC TGG AGA AGT	21
Plk3	PCR	-	F	CTA TGA AGC CAC TGA CAC CGA GTC T	25
		-	R	GAA GCG AGG TAA GTA CAA GCA CTG C	25
	qPCR	-	F	CTT GGT GAG TGG CCT CAT GC	20
		FAM	R	CGG AGT GAA ACT ACA GGA GCC TC	23
Prnp	PCR	-	F	CTG CTG GCC CTC TTT GTG ACT ATG T	25
		-	R	CGA TCA GGA AGA TGA GGA AGG AGA T	25
	qPCR	FAM	F	CGA AGC AGC AAC CAG AAC AAC TT	23
		-	R	ACC GTG TGC TGC TTG ATG GT	20
Ppp2r2b	PCR	-	F	AGG AGG ACA TTG ATA CCC GCA AAA T	25
		-	R	ATG TTT TCT GAA GGA TGC CAA GCT G	25
	qPCR	FAM	F	CGG TCT TCT TCA GGA TGT TCG AC	23
		-	R	AGG ATG CCA AGC TGT ATG CAA G	22

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