PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemness

Abstract

Background: Our previous study revealed that PLAGL2 or POFUT1 can promote tumorigenesis and maintain significant positive correlations in colorectal cancer (CRC). However, the mechanism leading to the co-expression and the underlying functional and biological implications remain unclear.

Methods: Clinical tumor tissues and TCGA dataset were utilized to analyze the co-expression of PLAGL2 and POFUT1. Luciferase reporter assays, specially made bidirectional promoter vectors and ectopic expression of 3'UTR were employed to study the mechanisms of co-expression. In vitro and in vivo assays were performed to further confirm the oncogenic function of both. The sphere formation assay, immunofluorescence, Western blot and qRT-PCR were performed to investigate the effect of both genes in colorectal cancer stem cells (CSCs).

Findings: PLAGL2 and POFUT1 maintained co-expression in CRC (r = 0.91, p < .0001). An evolutionarily conserved bidirectional promoter, rather than post-transcriptional regulation by competing endogenous RNAs, caused the co-expression of PLAGL2 and POFUT1 in CRC. The bidirectional gene pair PLAGL2/POFUT1 was subverted in CRC and acted synergistically to promote colorectal tumorigenesis by maintaining stemness of colorectal cancer stem cells through the Wnt and Notch pathways. Finally, PLAGL2 and POFUT1 share transcription factor binding sites, and introducing mutations into promoter regions with shared transcription regulatory elements led to a decrease in the PLAGL2/POFUT1 promoter activity in both directions.

Interpretation: Our team identified for the first time a bidirectional promoter pair oncogene, PLAGL2-POFUT1, in CRC. The two genes synergistically promote the progression of CRC and affect the characteristics of CSCs, which can offer promising intervention targets for clinicians and researchers. FUND: National Nature Science Foundation of China, the Hunan province projects of Postgraduate Independent Exploration and Innovation of Central South University.

Keywords: Bidirectional promoter; Cancer stem cell; Co-expression; Colorectal cancer; PLAGL2; POFUT1.

Fig. 1

PLAGL2 and POFUT1 are positively correlated in human tumors. (a) The TCGA dataset showed that PLAGL2 was co-expressed with POFUT1 in 32types of human cancer tissues; the Pearson correlation coefficient in colorectal tissues ranked first and second. The abbreviated names of all cancers are listed here; full names and co-expression analysis can be found in Supplemental Table 4 or TCGA website (https://cancergenome.nih.gov/) and ChIPBase v2.0 (http://rna.sysu.edu.cn/chipbase/). (b) PLAGL2 co-expressed with POFUT1 in 292 colorectal tumor tissues (GEO: GSE14333) and (c) 65 rectal tumor tissues (GEO: GSE 20842) and (d) 111 colorectal tumor tissues (GEO: GSE20916). (e) Correlation of PLAGL2 and POFUT1 mRNA levels analyzed by RT-PCR in 21 colorectal cancer tissues. (f) Correlation of PLAGL2 and POFUT1 mRNA levels analyzed by RT-PCR in 5 colorectal cancer cells. (g) Seven pairs of colorectal carcinoma tissues and adjacent normal tissues were randomly selected for Western blot analyses. (odd numbers represent paired colorectal cancer tissues and even numbers represent paired adjacent tissues of colorectal cancer) (h) Correlation of PLAGL2 and POFUT1 protein levels analyzed by Western blot in 7 pairs of colorectal carcinoma tissues and adjacent normal tissues.

Fig. 2

Human PLAGL2 and POFUT1 genes are concertedly regulated by a genuine bidirectional promoter. (a) Structure of human PLAGL2 and POFUT1 genes (derived from UCSC Genome Browser on Human) and schematic representation of an intervening region between both genes. The bent arrows indicate the transcription direction and PLAGL2 located on complementary strand. (b) Schematic representation of the ntervening gene fragment between both genes being cloned into the promoterless pGL3-Basic vector upstream of the luciferase coding region in two orientations. (c) and (d) Promoter activity of the POFUT1-PLAGL2 intergenic region (179 bp is flanked by parts of the first exons of both genes, 89 bp is the putative bidirectional promoter) in HCT116 and SW-480 cells transfected with promoterless pGL3-Basic vector. PGL3-PO plasmid contains the POFUT1/PLAGL2 promoter region in the POFUT1 orientation, and PGL3-PL contains the same region in the PLAGL2 orientation. (e) Fluorescence microscopy analysis of simultaneous expression of the fluorescent proteins mCherry (red) and EGFP (green) driven by the 179 bp POFUT1/PLAGL2 promoter region in HCT116 cells. pUC57 represents HCT116 cells transfected with an empty pUC57 vector, and p-mC and p-EG represent control vectors, which contain both reporter genes but with the unidirectional promoter. E-p-m represent the POFUT1/PLAGL2 promoter region cloned into pUC57 vector containing reporter genes of mCherry and EGFP (scale bar:50 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

PLAGL2 and POFUT1 maintenance of co-expression may not be through the 3′-untranslated regions of both genes. (a) Schematic outlining the two fragments PLAGL2 3’ UTR1 and 3’UTR2 used for overexpression and luciferase experiments. (b) qRT-PCR of lentivirus-mediated PLAGL2 3’UTR overexpression, and the POFUT1 and PLAGL2 expression in response to overexpression of PLAGL2 3’UTR in HCT-116 and SW620 cells. (c) WB showing POFUT1 protein in response to overexpression of PLAGL2 3’UTRs in SW620 and HCT-116 cells. (d) Quantification of (C). (e) Luciferase activity in SW620 cells cotransfected with luciferase-PLAGL2 3’UTR2 reporter construct and ShRNA against POFUT1. (f) qRT-PCR in lentivirus-mediated PLAGL2 and POFUT1 overexpression and cross regulation in HT-29 and HCT-116 cells. (g) Western blot in lentivirus-mediated PLAGL2 and POFUT1 overexpression and cross regulation in HT-29 and HCT-116 cells. EV: empty vector.

Fig. 4

PLAGL2 and POFUT1 owned overexpression driven by CNA and combined PLAGL2 /POFUT1 inhibition have greater effects on opposing tumors. (a) AccuCopy copy number analysis of copy number of PLAGL2 (including PLAGL2–1 and PLAGL2–2) and POFUT1 (including POFUT1–1 and POFUT1–2) in 14 pair colorectal tissues. (b and c) The correlation between copy number value of PLAGL2–2 and PLAGL2 mRNA expression and POFUT1–2 and POFUT1 mRNA expression. (d) Comparison of PLAGL2 and POFUT1 protein expression between CRC tissues without CNA (Amp. (−)) and CRC tissues with amplified (Amp (+)). PL: PLAGL2; PO: POFUT1; GA: GAPDH. (e) Quantification of (d). (f) The EdU proliferation assay was performed after shRNA-mediated suppression of POFUT1 or/and PLAGL2 in SW480 cells; shown here are the results for the ShPLAGL2–1 and ShPOFUT1–1 sequences (scale bar:50 μM). (g) Quantification of (B). (h) Suppressing PLAGL2 or/and POFUT1 expression reduce anchorage-independent growth in HCT116 cells, shown here are the results for the ShPLAGL2–1 and ShPOFUT1–1 sequences. (scale bar:200 μm (left), 100 μm (right)); d: day. (i) Quantification of (D), upper showing average number of colonies after cultivation for 10 days and down showing relative size of colonies after cultivation for 20 days; the shNC group (NC) was set to 1. (j) Representative images of invasion assay showing control group (NC) and shRNA-mediated suppression of POFUT1 or/and PLAGL2 in SW480 and HCT116 cells; shown here are the results for the ShPLAGL2–1 and ShPOFUT1–1 sequences (scale bar:50 μm). (k) Quantification of (j) (l and m) shRNA-mediated suppression of POFUT1 or/and PLAGL2 can decelerated growth of HCT116 xenografts in nude mice (n = 5). (n and o) Representative macroscopic appearances of liver metastasis are shown (some tumor nodules were indicated by black arrows). (HCT116 cells was used in this assay). (scale bar:100 μm) (n = 5). Data in (d)–(o) presented as violin plots from at least two independent experiments with triplicates. Each plot represents the mean from one independent experiment with triplicates. *p < .05, **p < .01. The *at the vertex of the bar graph is compared to the control group.

Fig. 5

PLAGL2 and POFUT1 can activate WNT and Notch pathways, respectively. (a)TCF reporter activity was evaluated through the β-catenin responsive TOPflash reporter in empty vector control (NC) and PLAGL2-expressing HCT116 cells or in HCT116 and SW480 cells stably transduced with lentiviral vectors carrying negative control shRNA (shNC) and PLAGL2-specific small hairpin RNAs (ShPL1 and ShPL2) *p < .05, **p < .01. (b) qRT-PCR analyses of the expression of Wnt signaling pathway related (WNT3, WNT6, WNT7B, FZD2 and FZD9) and targeted genes (MYC, CCND1, BIRC5 and MMP7) after PLAGL2 knockdown in SW620 cells. Control group set to 1 (*p < .05, ** p < .01). (c) Western blot analyses of related Wnt protein Wnt 6, downstream effectors of the Wnt signaling pathway including non-phospho (active) β-Catenin and total β-catenin, and accepted canonical targets including cyclin D1 and c-Myc in PLAGL2-silenced or PLAGL2-overexpressing CRC cells. (d) Immunofluorescence of de-phospho-β -catenin in PLAGL2-silenced SW480 cells. (e) qRT-PCR analyses of the expression of Notch signaling pathway target genes after POFUT1 knockdown in SW480 cells. Control group set to 1 (*p < .05, ** p < .01). (f) Western blot analyses of cleaved Notch1 (the Notch1 intracellular domain), and accepted canonical targets include HES1 and HES5 in POFUT1-silenced or POFUT1-overexpressing CRC cells.

Fig. 6

PLAGL2 and POFUT1 can influence stem cell-like properties and impede differentiation of CSCs. (a) qRT-PCR analyses of stem and differentiation markers after PLAGL2 and POFUT1 knockdown in SW480 cells. Control group set to 1 (*p < .05, ** p < .01). (b) Western blot analyses of stem and differentiation markers after PLAGL2 and POFUT1 knockdown in SW480 cells (*p < .05, ** p < .01). (c) The immunofluorescence assay shows the expression of stem marker CD44V6 protein after PLAGL2 and POFUT1 knockdown in SW620 cells; shown here is the result for ShPLAGL2–2 and ShPOFUT1–2 sequence (scale bar:10 μm). (d) Representative images of spheres from the SW620 and HCT116 cell lines (PLAGL2 or POFUT1 knockdown, here lists the result about ShPLAGL2–1 and ShPOFUT1–1 sequence) and HT29 cell (PLAGL2 or POFUT1 overexpressed) cultured in a serum-free medium system. (200 μm) (e) Quantification of (d) (*p < .05, ** p < .01). (f and g) Representative images showing that suppressing PLAGL2 and POFUT1 can promote differentiation (the sphere rapidly adopted a flattened cell morphology) and attenuate their spheres in the presence of medium plus 2% FBS. A reverse trend was observed in PLAGL2 and POFUT1-expressing cell (scale bar:100 μm). (h and i) shRNA-mediated suppression of POFUT1 or PLAGL2 can decelerate growth of SW620 sphere xenografts in nude mice (n = 5). Violin plots in the right panel presented quantification (*p < .05). The weight unit of the tumor is grams. (j and k) Representative macroscopic appearances of lung metastasis are shown (some tumor nodules were indicated by black arrows), representative hematoxylin-eosin stained images of lung metastasis lesions in three different groups (×100), Violin plots in right panel presented quantification. (SW620 cells dissociated from these spheres were used in this assay and *p < .05) (scale bar:100 μm).

Fig. 7

Mutations in shared transcription factor binding sites in the PLAGL2/POFUT1 bidirectional promoter region lead to decreased promoter activity in both directions. (a) Schematic outlining the 6 mutations in the 179 bp promoter sequence (more details can be found in Supplementary Table 2). (a) Schematic outlining the 6 mutations in the 179 bp promoter sequence (more details can be found in Supplementary Table 2). (b) Activity of mutated and wild-type forms of the PLAGL2/POFUT1 promoter region in HCT116 cells. (c) Fluorescence microscopy analysis of simultaneous expression of the fluorescent proteins mCherry (red) and EGFP (green) driven by the 179 bp POFUT1/PLAGL2 promoter region with or without mut1 mutation in HCT116 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Model of bidirectional gene pair PLAGL2-POFUT1 maintaining coexpression and collaboratively involved in colorectal cancer by enhancing stemness in CSCs.

Supplemental Fig. 1

Human PLAGL2 and POFUT1 genes are concertedly regulated by a genuine bidirectional promoter. (a) The 179 bp intergenic sequence between POFUT1 and PLAGL2 used in this article. The sequence on the horizontal line represents the 89 bp putative bidirectional promoter (POFUT1 orientation). (b) Simultaneous expression of the fluorescent proteins mCherry (red) and EGFP (green) driven by the 179 bp POFUT1/PLAGL2 promoter region in SW480 cells.

Supplemental Fig. 2

Combined PLAGL2 /POFUT1 inhibition have greater effects on opposing tumor. (a and b) HCT116 and SW480 were stably transduced with lentiviral vectors carrying PLAGL2 or /and POFUT1-specific small hairpin RNAs. The control group contained lentiviral vectors carrying negative control shRNA (shNC). Silencing of both or single genes was confirmed by RT-PCR (a) and Western blot analysis (b). (c) The EdU proliferation assay was performed after shRNA-mediated suppression of POFUT1 or/and PLAGL2 in SW480 cells. Violin plots in the right panel present quantification. Shown here are the results for the ShPLAGL2–2 and ShPOFUT1–2 sequences (scale bar:50 μM). (d) Suppressing PLAGL2 or/and POFUT1 expression reduces anchorage-independent growth in HCT116 cells. Violin plots in the right panel present quantification. Shown here are the results for the ShPLAGL2–2 and ShPOFUT1–2 sequences (scale bar:100 μm); d: day. (e) Representative images of invasion assays showing HCT116 and SW480 cells stably transduced with lentiviral vectors carrying PLAGL2 or /and POFUT1-specific small hairpin RNAs after 36–72 h induction with 10% FBS followed by crystal violet (0.1%) staining. Violin plots in the right panel presented quantification. Shown here are the results for the ShPLAGL2–2 and ShPOFUT1–2 sequences.

Supplemental Fig. 3

Representative images of invasion assays showing control group (NC) and shRNA-mediated suppression of POFUT1 or/and PLAGL2 in SW480 and HCT116 cells. (scale bar:200 μm).

Supplemental Fig. 4

PLAGL2 and POFUT1 can influence stem cell-like properties and impede differentiation of CSCs. (a) The immunofluorescence assay shows the expression of stem marker CD44V6 protein after PLAGL2 and POFUT1 knockdown in SW620 cells; shown here are the results for the ShPLAGL2–1 and ShPOFUT1–1 sequences (scale bar:50 μm). (b) Representative images of spheres from the SW620 and HCT116 cell lines (PLAGL2 or POFUT1 knockdown; shown here are the results for the ShPLAGL2–2 and ShPOFUT1–2 sequences) cultured in a serum-free medium system (200 μm). (c) Quantification of (b) (*p < .05, **p < .01). (d) Representative images showing that suppressing PLAGL2 and POFUT1 can promote differentiation (the sphere rapidly adopted a flattened cell morphology) and attenuate their spheres in the presence of medium plus 2% FBS (scale bar:100 μm). (e) Representative images of spheres from the HT29 cell line (PLAGL2 or/and POFUT1 knockdown) cultured in a serum-free medium system; shown here are the results for the ShPLAGL2–1 and ShPOFUT1–1 sequences (200 μm). (f) Quantification of (e) (*p < .05, **p < .01, ***p < .001).

Supplemental Fig. 5

PLAGL2 and POFUT1 share transcription factor binding sites in the highly evolutionarily conserved bidirectional promoter region. (a) The 179 bp promoter region is highly evolutionarily conserved using the UCSC Genome Browser and Ensembl genome browser 96(more DNA sequence details can be found in Supplemental Table 6). The consensus sequence was generated using WebLogo (http://weblogo.berkeley.edu/). (b) From top to bottom: (top) schematic figure of the 179 bp sequence that contained the 89 bp putative bidirectional promoter flanked by parts of the first exons of PLAGL2 and POFUT1. The sequence marked by red colour and horizontal lines representes 6 mutation regions (more details on mutations can be found in Supplemental Table 2). (middle) The 101 transcription factor biding sites predicted by The MatInspector program are depicted according to the binding site on the 179 bp. The horizontal axis represented the 179 bp, and the Y-axis representes the 101 transcription factors biding to the179bp. The red line indicates the binding site from start position to end position of each transcription factor. The rectangular boxes of different colors in the figure indicate the six regions in which the transcription factors mainly bind, such as gray, which represents the binding sites of transcription factors such as EGR1, ZNF263 and Sp4 (bottom). Based on the six regions in which the transcription factors are mainly bound, we constructed six mutants, mut1–6. Some representative transcription factors were predicted by MatInspector and JASPAR 2018 (http://jaspar.genereg.net/analysis).