Expression profiling of wild type and β-catenin gene disrupted human BxPC-3 pancreatic adenocarcinoma cells

Abstract

To study the role of WNT/β-catenin signaling in pancreatic adenocarcinoma, human BxPC-3 cell lines deficient of the central canonical WNT signaling protein β-catenin were established by using zinc-finger nuclease mediated targeted genomic disruption of the β-catenin gene (CTNNB1). Comparison of the global transcription levels in wild type cells with two β-catenin gene disrupted clones identified 85 transcripts that were the most differentially regulated. Gene ontology (GO) term enrichment analysis of these transcripts identified “cell adhesion” as the most significantly enriched GO term. Here we describe the data from the transcription profiling analysis published in the article “Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells” [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE63072.

Specifications
Organism/cell line/tissue	Human BxPC-3 pancreatic adenocarcinoma cell line
Sex	Female
Sequencer or array type	Illumina HumanHT-12 V4.0 expression beadchip
Data format	Raw and processed
Experimental factors	Wild type versus β-catenin deficient BxPC-3 cells
Experimental features	BxPC-3 cells deficient of β-catenin was generated by zinc-finger nuclease mediated targeted gene disruption.
Consent	N/A
Sample source location	BxPC-3 wild type cell line was obtained from ATCC (CRL-1687)

Direct link to deposited data

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63072.

Experimental design, materials and methods

Cell culture

The human pancreatic adenocarcinoma BxPC-3 (ATCC CRL-1687) epithelial cell line was grown in RPMI-1640 (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 0.002 × insulin–transferrin–selenium (Life Technologies, Carlsbad, CA, USA) cells were propagated at 37 °C in a humidified atmosphere containing 5% CO₂.

Generation of β-catenin deficient cells

BxPC-3 cells with targeted disruption of the β-catenin gene (CTNNB1) were established using CompoZr custom Zinc Finger Nucleases (ZFNs) (Sigma-Aldrich). Briefly, following transfection of the cells with ZFN mRNA targeting exon 3 of the CTNNB1 gene, monoclonal cell populations were obtained by limiting dilution cloning and analyzed for β-catenin expression. From 150 initial clones five β-catenin gene disrupted clones negative for β-catenin expression were identified and selected for further analysis (clone #4, #31, #79, #93 and #111).

RNA isolation microarray analysis

Total RNA from exponentially growing wild type BxPC-3 cells and β-catenin gene disrupted clones #4 and #111 was isolated using the GenElute Mammalian Total RNA Purification Kit (Sigma-Aldrich). The RNA was subjected to microarray analysis using Illumina HumanHT-12 v4 Expression BeadChips (Illumina, CA, USA) at the Norwegian Genomics Consortium core facility (Oslo University Hospital, Norway). For each sample 6 biological replicates were analyzed. Data extraction and quality control was performed in GenomeStudio (Illumina) and the data analysis was performed using J-Express [2].

Differential expression quantification and classification

To identify the most differentially expressed genes between wild type BxPC-3 cells and the β-catenin gene disrupted clones #4 and #111 (average) Significance Analysis of Microarrays (SAM) analysis was carried out [3]. From the SAM analysis a threshold of fold change > 2 and q-value = 0 was selected to identify the most regulated probes. In Table 1 the resulting list of the 85 most differentially regulated probes is shown. To identify relevant shared biological functions associated with the identified 85 most differentially regulated transcripts, Gene Ontology (GO) term enrichment analysis was done using DAVID [4] with the GOTERM_BP_2 annotation (Table 2).

Table 1.

List of the most differentially regulated probes from significance analysis of microarrays (SAM) comparing wild type BxPC3 cells and the gene disrupted clones #4 and #111 (average).

The list was generated by selecting the probes that displayed a fold change > 2 and q-value = 0 in the SAM analysis of all probes. The list is sorted by the d-score and genes up- and down-regulated in the gene disrupted clones have a positive and a negative fold change value, respectively.

Probe_Id	Symbol	ILMN_GENE	d-Score	Fold change	q-Value
ILMN_1686664	MT2A	MT2A	22.155	2.45	0
ILMN_1659688	LGALS3BP	LGALS3BP	15.656	2.358	0
ILMN_2042771	PTTG1	PTTG1	14.42	2.109	0
ILMN_1672503	DPYSL2	DPYSL2	12.37	2.368	0
ILMN_2183409	SCARB1	SCARB1	− 12.283	− 2.43	0
ILMN_1673356	FAM83C	FAM83C	− 11.125	− 2.327	0
ILMN_3247895	LOC728188	LOC728188	10.777	2.446	0
ILMN_1713147	MCRS1	MCRS1	10.703	2.178	0
ILMN_1655347	SCGB1A1	SCGB1A1	− 10.414	− 2.055	0
ILMN_2320250	NOL6	NOL6	− 10.404	− 2.087	0
ILMN_1799098	LOC652846	LOC652846	10.398	2.289	0
ILMN_1750324	IGFBP5	IGFBP5	− 9.748	− 6.458	0
ILMN_1733756	COL12A1	COL12A1	− 9.342	− 2.076	0
ILMN_2145116	TMEM173	TMEM173	9.246	2.264	0
ILMN_1811972	MYCBP2	MYCBP2	− 9.226	− 2.024	0
ILMN_1678707	TAF15	TAF15	− 9.141	− 2.081	0
ILMN_1765641	SEMA3A	SEMA3A	− 9.018	− 2.024	0
ILMN_1753196	PTTG1	PTTG1	8.995	2.523	0
ILMN_1673023	EP400	EP400	− 8.897	− 2.068	0
ILMN_1765701	LOC399942	LOC399942	8.74	2.077	0
ILMN_2400759	CPVL	CPVL	8.644	2.023	0
ILMN_1661366	PGAM1	PGAM1	8.606	2.736	0
ILMN_1740233	UGT1A10	UGT1A10	8.476	2.096	0
ILMN_1676358	RALB	RALB	8.265	2.493	0
ILMN_2321153	MUC4	MUC4	− 8.15	− 2.477	0
ILMN_3247578	FAT1	FAT1	− 8.034	− 2.08	0
ILMN_2411915	ATG4B	ATG4B	7.981	2.191	0
ILMN_1754795	FAT1	FAT1	− 7.879	− 3.211	0
ILMN_1678757	BCYRN1	BCYRN1	− 7.775	− 3.991	0
ILMN_1695917	C5orf15	C5ORF15	7.679	2.148	0
ILMN_2395389	PSMC4	PSMC4	7.197	2.627	0
ILMN_2132982	IGFBP5	IGFBP5	− 7.174	− 4.242	0
ILMN_1676763	PIPSL	PIPSL	7.021	2.131	0
ILMN_2109708	ECGF1	ECGF1	6.793	2.086	0
ILMN_1795778	P4HA2	P4HA2	6.646	2.211	0
ILMN_2095610	ANXA8	ANXA8	6.489	2.124	0
ILMN_1691563	GAGE12I	GAGE12I	− 6.241	− 2.119	0
ILMN_1704342	UBE3C	UBE3C	− 6.183	− 2.136	0
ILMN_1779353	PUS7	PUS7	− 6.17	− 2.483	0
ILMN_2326737	PPIE	PPIE	6.061	2.363	0
ILMN_1800131	LOC652826	LOC652826	6.011	2.061	0
ILMN_1788108	TXNDC5	TXNDC5	− 5.947	− 2	0
ILMN_2332105	WRNIP1	WRNIP1	− 5.922	− 2.222	0
ILMN_1687887	PSMC4	PSMC4	5.851	2.251	0
ILMN_1685798	MAGEA6	MAGEA6	5.832	2.07	0
ILMN_1744765	KRT4	KRT4	− 5.788	− 3.158	0
ILMN_3308295	MIR205	MIR205	5.548	2.083	0
ILMN_3204734	LOC100134648	LOC100134648	5.337	2.551	0
ILMN_1766762	DYNLRB1	DYNLRB1	5.207	2.955	0
ILMN_1732074	LOC648210	LOC648210	5.099	2.925	0
ILMN_2261076	NEDD9	NEDD9	− 5.097	− 2.074	0
ILMN_1681301	AIM2	AIM2	5.083	2.042	0
ILMN_2371169	ZYX	ZYX	− 5.063	− 2.451	0
ILMN_2174127	DCBLD2	DCBLD2	− 5.038	− 2.605	0
ILMN_1696187	PYGL	PYGL	− 5.02	− 2.216	0
ILMN_1690259	RAE1	RAE1	4.98	2.121	0
ILMN_1680246	MAT2B	MAT2B	4.961	3.137	0
ILMN_1798454	MAD2L1BP	MAD2L1BP	4.925	2.136	0
ILMN_1711702	CLEC2D	CLEC2D	4.921	2.116	0
ILMN_1753449	CST1	CST1	4.802	2.783	0
ILMN_1746465	FJX1	FJX1	− 4.758	− 2.225	0
ILMN_1715175	MET	MET	− 4.688	− 2.751	0
ILMN_1795342	MLPH	MLPH	4.626	2.108	0
ILMN_1703108	UBE2L6	UBE2L6	4.599	2.721	0
ILMN_2129572	F3	F3	− 4.593	− 2.65	0
ILMN_1660345	NGRN	NGRN	4.578	2.387	0
ILMN_1658053	DYNLRB1	DYNLRB1	4.504	3.005	0
ILMN_2150856	SERPINB2	SERPINB2	− 4.471	− 2.41	0
ILMN_1664543	IFIT3	IFIT3	4.47	2.218	0
ILMN_1766650	FOXA1	FOXA1	− 4.469	− 2.072	0
ILMN_1829845	HS.553301	HS.553301	4.408	3.363	0
ILMN_3231944	LOC100130516	LOC100130516	− 4.399	− 6.137	0
ILMN_1784602	CDKN1A	CDKN1A	4.381	2.092	0
ILMN_1768470	EIF4G1	EIF4G1	− 4.297	− 2.061	0
ILMN_2405233	FAM133B	FAM133B	− 4.278	− 2.054	0
ILMN_2148527	H19	H19	− 4.25	− 7.3	0
ILMN_1756071	MFGE8	MFGE8	4.125	3.204	0
ILMN_1739645	ANLN	ANLN	− 4.096	-2.037	0
ILMN_3215206	LOC100133836	LOC100133836	4.001	2.078	0
ILMN_1673880	EFEMP1	EFEMP1	3.963	2.289	0
ILMN_2073604	EBP	EBP	3.916	2.207	0
ILMN_1777765	C12orf10	C12ORF10	3.909	2.098	0
ILMN_2239754	IFIT3	IFIT3	3.879	4.952	0
ILMN_1774077	GBP2	GBP2	3.856	3.317	0
ILMN_2279635	EIF4G2	EIF4G2	3.832	2.469	0

Table 2.

Gene ontology enrichment analysis of the most differentially regulated transcripts between the wild type BxPC3 cells and the β-catenin deficient clones #4 and #111 (average).

The GO analysis was performed using the top 85 most regulated transcripts from Table 1.

Category	Term	Count	%	Genes/transcripts	Fold enrichment	Bonferroni
GOTERM_BP_2	GO:0007155 ~ cell adhesion	9	1.4	DCBLD2, LGALS3BP, FAT1, NEDD9, COL12A1, SCARB1, MFGE8, ZYX, MUC4	3.26	0.46
GOTERM_BP_2	GO:0008037 ~ cell recognition	3	0.5	SCARB1, MFGE8, SEMA3A	13.83	0.90
GOTERM_BP_2	GO:0040008 ~ regulation of growth	5	0.8	DCBLD2, CDKN1A, NEDD9, SEMA3A, IGFBP5	3.72	0.99
GOTERM_BP_2	GO:0065008 ~ regulation of biological quality	11	1.8	DCBLD2, UGT1A10, CDKN1A, ANXA8, PYGL, TXNDC5, F3, FOXA1, MT2A, SCARB1, SEMA3A	1.90	1.00
GOTERM_BP_2	GO:0006950 ~ response to stress	12	1.9	DCBLD2, UGT1A10, CDKN1A, TMEM173, LGALS3BP, ANXA8, F3, WRNIP1, GAGE12I, SERPINB2, SCARB1, PTTG1	1.81	1.00
GOTERM_BP_2	GO:0009605 ~ response to external stimulus	8	1.3	DCBLD2, UGT1A10, CDKN1A, ANXA8, F3, SERPINB2, SCARB1, SEMA3A	2.22	1.00
GOTERM_BP_2	GO:0042445 ~ hormone metabolic process	3	0.5	UGT1A10, FOXA1, SCARB1	7.18	1.00
GOTERM_BP_2	GO:0022402 ~ cell cycle process	6	1.0	EIF4G2, CDKN1A, PSMC4, NEDD9, ANLN, PTTG1, LOC652826	2.69	1.00
GOTERM_BP_2	GO:0045926 ~ negative regulation of growth	3	0.5	DCBLD2, CDKN1A, SEMA3A	6.91	1.00
GOTERM_BP_2	GO:0032879 ~ regulation of localization	6	1.0	F3, SCARB1, MFGE8, SEMA3A, IGFBP5, MYCBP2	2.49	1.00
GOTERM_BP_2	GO:0044419 ~ interspecies interaction between organisms	4	0.6	EIF4G1, SCARB1, MFGE8, ZYX	3.58	1.00

Discussion

We describe the dataset from the transcriptome analysis comparing wild type and β-catenin deficient BxPC-3 cells. In this analysis 85 transcripts were identified to be the most differentially regulated between the two groups. GO term enrichment analysis of the transcripts identified “cell adhesion” as the GO term that was most significantly enriched for. These results together with the rest the data from the previous published article [1] points towards a central role of β-catenin in enabling cell-cell contacts in BxPC3 cells.