Background
Liver tumor initiating cells (TICs), also termed as liver cancer stem cells (CSCs), are the reason of liver tumorigenesis, metastasis, drug resistance and relapse [
1]. Because these cells are in quiescent state and highly express many drug pumps (including ABCG2), liver TICs can survive during drug treatment, and initiate new tumors once drugs are removed [
2]. Liver TICs have self-renewal and differentiate capacities, and can form many cell types along with tumorigenesis [
3]. Based these specific characteristics, several functional systems have been developed for liver TICs, including sphere formation, side population, colony formation, transwell assay and tumor initiation assay [
4‐
6]. Recent years, various surface markers of liver TICs were found, including CD13, CD133, EPCAM, CD24 and CD90 [
7‐
10]. Some transcription factors were also found to participate in the self-renewal regulation of liver TICs, including Sox4, Zic2 and Oct4 [
6,
11]. Although the importance of liver TICs is well-known, the self-renewal regulation of liver TICs are still unclear.
The activation of liver TICs is finely regulated. Wnt/β-catenin, Notch, Hedgehog signaling pathways play critical roles in liver TICs [
12,
13]. G protein coupled receptors (GPCR) are critical modulators in signal transduction [
14]. GPCRs play critical roles in many disease, including tumorigenesis, conferring GPCRs the most widely-used drug-targets [
15]. While, the exact roles of GPCRs in liver TIC self-renewal are still elusive. Here we performed an unbiased screening and found GPR107 was highly expressed in liver cancer and liver TICs. GPR107 plays an essential role in the internalization of pseudomonas aeruginosa exotoxin A and can interacts with neuronostatin [
16,
17]. The role of GPR107 in tumorigenesis hasn’t been reported. Here, we found GPR107 was required for the self-renewal of liver TICs and liver tumorigenesis.
Long noncoding RNAs (LncRNAs) are RNA molecules longer than 200 nt but have no coding potential [
18]. LncRNAs were considered as byproductions of transcription process. Recently, the biological roles of lncRNAs are emerging. LncRNAs play critical roles in immunological regulation [
19], development [
20], tumorigenesis [
21] and so on. LncRNAs exert their roles through binding with remodeling complex and recruiting them to the promoter of target genes, and also can interact with proteins to modulate their stabilities or activities [
22]. According to the relative location between lncRNAs and their target genes, two kinds of function pattern of lncRNAs have been discovered:
in cis and
in trans [
23]. As one of the common mechanisms, many lncRNAs regulate the transcription activity of nearby genes through recruiting chromatin remodeling complex [
24]. Here we found a lncRNA was located near from
GPR107 locus, and required for the transcription initiation of GPR107.
Methods
Reagents
Anti-β-actin (cat. no. A1978) and DAPI (cat. no. 28718–90-3) were from Sigma-Aldrich. Anti-GPR107 (25076–1-AP) antibody was from Proteintech Company. Anti-SRCAP (cat. no. PAB22228) antibody was from abnova. Phycoerythrin (PE)-conjugated CD133 (cat. no. 130098826) was from Miltenyi Biotec. The Chemiluminescent Nucleic Acid Detection Module (cat. no. 89880) and LightShift Chemiluminescent RNA EMSA kit (cat. no. 20158) were from Thermo Scientific. Biotin RNA Labeling Mix (cat. no. 11685597910) and T7 RNA polymerase (cat. no. 10881767001) were from Roche Life Science.
For liver TIC sphere formation assay, 5000 HCC cells were cultured in sphere formation medium (1 × B27, 1 × N2, 20 ng/ml EGF, 20 ng/ml bFGF, in DMEM/F12) and seeded into Ultra Low Attachment six-well plates (cat. no. 3471, Corning). Two weeks later, typical images were taken and sphere numbers were counted. Sphere initiating ratios (%) = Sphere numbers/5000 × 100%.
Primary cell isolation and culture
Primary HCC cells were obtained from hepatocellular carcinoma patients, and the information of HCC samples used in this research was listed in Table
1. For primary cell isolation, a portion of excised tumor was incubated in Hanks balanced salt solution (HBSS; Gibco) and transported to the laboratory on ice. Then the samples were cut into small fragments, and digested in HBSS containing 0.03% pronase, 0.05% type IV collagenase, and 0.01% deoxyribonuclease for 30 min at 37 °C. The sample was filtered through 100-μm-nylon filter and centrifuged for 2 min at 50 x g in 4 °C. HCC primary cells were in precipitation, and cell survival & purification were examined. For sphere formation, 5000 primary cells were seeded into sphere formation medium and cultured in Ultra Low Attachment six-well plate. For liver TIC enrichment, HCC primary cells were incubated with CD133 antibody for FACS.
Table 1
Characteristics of HCC samples
Age | 61 | 72 | 53 | 74 | 63 |
Sex | Female | Male | Male | Male | Female |
HBsAg | + | + | + | + | + |
Serum α-fetoprotein | 5.8 ng/ml | 2182 | 3168 | 439 | 1216 |
Tumor bulk used (mm) | 7.2 × 6.3 × 5.2 | 5.8 × 4.2 × 3.9 | 9.2 × 8.3 × 7.5 | 7.9 × 5.3 × 4.8 | 6.9 × 6.1 × 4.2 |
Cirrhosis | No | No | Yes | No | Yes |
Vascular invasion | No | No | Yes | Yes | No |
Stages | T1 | T2 | T3 | T3 | T2 |
Sphere formation | + |
–
| + | + | + |
CD133+ cells | + | + |
–
| + | + |
Liver TIC sorting
For liver TIC sorting, primary cells were incubated in PBS at 5000 cells/μl, supplementary with 2% FBS. Then Phycoerythrin (PE)-conjugated anti-CD133 antibody or control IgG were added (1:100). The samples were placed in 4 °C for 30 min, and then liver TICs were isolated by FACS. BD FACSAria was used for sorting and CD133+ cells were liver TICs.
Fluorescence in situ hybridization (FISH)
For FISH assay, fluorescence-conjugated lncGPR107 probes were generated according to the manual of Biosearch Technologies. HCC samples, liver TICs or oncospheres were treated in a non-denaturing condition, followed by hybridization with lncGPR107 probes. After RNA hybridization, then corresponding antibodies (anti-CD133, Zic2, SRCAP) or fluorescence-conjugated antibodies were added for protein observation. Finally samples were incubated in 4′,6-diamidino-2-phenylindole (DAPI, 1:1000) for 5 min’ counterstain, and observed by confocal microscopy (FV1000, Olympus). All experiments were performed according to manuals of Biosearch Technologies.
RNA pulldown
For RNA pulldown assays, biotin-labeled lncGPR107 or fragments were obtained by in vitro transcription and biotin RNA labeling mix (Roche). Then the labeled RNAs were incubated with liver oncosphere lysate for six hours. Streptavidin beads were added for another three hours’ incubation. The samples were washed for three times (five minutes once), and then incubated in loading buffer and boiled at 100 °C for 10 min. Finally the samples were subjected into SDS–PAGE. For protein identification, specific bands enriched by lncGPR107 were identified by mass spectrometry (LTQ Orbitrap XL). For Western blot, the samples in SDS-PAGE were transferred onto NC membrane and detected by antibodies.
Chromatin immunoprecipitation (ChIP)
ChIP assays were completed according to Upstate Biotechnology. Briefly, lncGPR107 silenced or lncGPR107 overexpressing spheres were fixed with 1% formaldehyde for 15 min’ crosslink, and crushed in SDS lysis buffer. The samples were then treated by ultrasonic for shearing DNA. SRCAP, H3K4Me3, RNA polymerase II or control IgG antibodies were added into samples for 6 h’ incubation. Then beads containing sonicated salmon sperm (Upstate Biotechnology) were added into samples for DNA enrichment. DNA segment enrichment of GPR107 promoter was examined by real-time PCR.
ASO treatment
For HCC propagation, primary HCC cells were injected into BALB/c nude mice for tumor propagation. Six weeks later, tumors were obtained for another round tumor propagation (if necessary) until the cells were enough for experiments in 18 mice. For each mice, 1 × 106 cells were used for subcutaneous injection. When tumors were reached to 300 mm3 (about three weeks), mice were randomly assigned into three groups (six mice per group). LncGPR107 ASOs or control ASO were injected into tumors every 4 days. Three weeks after ASO intratumoral injection, tumors were obtained, weighted and used for further experiments. GPR107 antibody was used for immunohistochemistry, Ki67 and CD133 antibodies were used for FACS.
GPCR expression analysis
For gene expression analysis, GSE14520 was selected because it contained a large number of HCC samples with detailed clinical information, and download form NCBI database. R language and Bioconductor methods were utilized for background correction, normalization, calculation and annotation. Gene lists and expression levels generated by R3.1.0 were used for further analyses. For G protein coupled receptors (GPCRs) analysis, GPCRs list was download from DAVID website. GPCRs were listed according to expression changes in liver tumorigenesis. The expression levels in every samples were normalized to the average expression of peri-tumor samples, and shown as heatmap.
Tumor propagation and initiating assay
For tumor propagation, 1 × 10
6 lncGPR107 silenced and control cells were subcutaneously injected into 6-week-old BALB/c nude mice. One month later, tumors were selected and tumor weight was examined. For tumor initiation, 10, 1 × 10
2, 1 × 10
3, 1 × 10
4 and 1 × 10
5 lncGPR107 silenced and control cells were subcutaneously injected into 6-week-old BALB/c nude mice for 3 months’ tumor formation. Tumor formation was observed for tumor-free ratios. Tumor initiating ratios were calculated by Extreme Limiting Dilution Analysis (ELDA) (
http://bioinf.wehi.edu.au/software/elda/) [
25].
Immunohistochemistry
For immunohistochemistry, formalin-fixed liver cancer sections were treated with xylene and graded alcohols (100, 90% and then 70%) for hydratization. Then the samples were incubated 15 min in 3% H2O2, followed by 15 min’s boiling in citrate buffer. The tissues were incubaed with primary antibody (GPR107, 1:100) and HRP-conjugated secondary antibody. Finally the sections were counterstained with hematoxylin and dehydrated in graded alcohols, followed by observation with Nikon-EclipseTi microscopy.
Realtime PCR
Total RNA was obtained from primary samples or oncospherss by standard TRIZOL method. Briefly, 1 ml TriZol was added into samples for dissolution through 5 min’s incubation. 200ul chloroform was then added into samples and mixed vigorously, followed by separation with 12,000 g centrifugation. The RNA components in supernatant were extracted with isopropanol and then washed with 75% ethanol. RNA pallets were dissolved with RNase-free H2O. RT-PCR (revers-transcription PCR) was performed to generate cDNA from RNA, and reatime PCR was performed to examine RNA expression with ABI7300. The primer sequences for realtime PCR were: GPR107, 5’-GGGGCAGTGTCATTTCAGTTT-3′, 5’-CTGAGAGGTAGCTGTCAGGAT-3′. lncGPR107, 5’-GGCTCAGCTACTGAAGCCGG-3′, 5’-AGGGGCCTGGCTGTCCTGCT-3′.
Western blot
Liver cancer or spheres were crushed with RIPA buffer (0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, 0.1% SDS and 50 mM Tris, 150 mM NaCl, pH 8.0) and subjected into SDS–PAGE. The protein samples were transferred onto NC membrane (Beyotime Biotechnology), followed by incubation with primary antibodies and HRP-conjugated secondary antibodies for visualization.
RNA pulldown
Biotin-labeled lncGPR107 transcript was obtained with in vitro transcription (Roche), and incubated with oncosphere lysate. Then streptavidin conjugated beads were used for pulldown and enriched components were subjected into SDS–PAGE, followed by mass spectra or Western blot analyses.
Statistical analysis
One-tailed Student’s t tests were used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. P < 0.05 was defined to be statistically significant. All experiments were repeated at least three times.
Discussion
With tumor initiation and self-renewal capacities, liver TICs (liver CSCs) are the origin of liver tumor formation, metastasis, relapse and drug resistance [
3]. Several signaling pathways have been reported in the self-renewal regulation of liver TICs, including Wnt/β-catenin, Notch, Hedgehog and Yap1 signaling [
12,
13,
31‐
33]. However, the role of G-protein-coupled receptor (GPCR) signaling in liver TICs hasn’t been explored. Here, we analyzed the expression level of GPCRs using online-available HCC dataset and found GPR107 was the most highly expressed GPCRs along with liver tumorigenesis. Moreover, GPR107 is required for the self-renewal of liver TIC, revealing the relationship of liver TICs and GPCR signaling. Besides GPR107, several other GPRs, including OPN3, GPR88, LGR5, PTH2R, ACKR3 F2RL1, GABBR2, PTCFT and GPR5B, are also highly expressed in liver cancers (Fig.
1a). As one of the most important pathways in signal transduction, it is possible that more than one GPCR member participate in the self-renewal of liver TIC and liver tumorigenesis. Here, we revealed that GPR107 and lncGRP107 was highly expressed in liver cancer, especially in liver TICs (Fig.
1 and Fig.
2). In peri-tumor, the expression levels of GPR107 and lncGRP107 were extremely low, and very few cells showed modest expression of GPR107 and lncGRP107 (Figs.
1f and
2d). These GPR107 and lncGRP107 expressed cells may also have self-renewal and propagating capacities. Some groups identified liver progenitor cells in normal tissue, with self-renewal and differentiating capacities [
34,
35]. It’s an interesting issue to investigate whether liver progenitor cells also show GPR107/lncGRP107 expression and the potential role of GPR107/lncGRP107 in liver progenitor cells.
Recently, GPCR signaling was found to be related to NFkB signaling [
36] and Yap/Taz signaling [
37], which regulated the activation of liver TICs. NFkB and Yap signaling pathways also participated in liver tumorigenesis [
31]. Here we found GPR107 was overexpressed in and required for liver TICs, how GPR107 functions is still unclear. It’ll be interesting to detect the role of GPR107 in the activation of NFkB signaling and Yap/Taz signaling.
LncRNAs were recognized as the byproduct of RNA polymerase II, while, their critical roles emerge recently. Several lncRNAs regulate the transcription activation
in cis. For example, lncTCF7 recruits SWI/SNF complex to activate TCF7 expression, and finally drives the self-renewal of liver TICs [
24]. Our results provided another example for
in cis regulation of lncRNA. Interestingly, lncGPR107 also recruited chromatin remodeling complex to the promoter of target gene. Through this regulation mechanism, lncGPR107 was co-expressed with GPR107 in liver TICs. LncGPR107 participated in the self-renewal of liver TICs through facilitating the transcriptional activation of GPR107. Through loss-of-function assays, GPR107 was also involved in the self-renewal of liver TICs.
To some extent, tumorigenesis is a process of chromatin remodeling [
38]. Chromatin remodeling is mediated by chromatin remodeling complexes, including SWI/SNF, NURD, NURF, PRC1/2 and SRCAP complexes [
39‐
41]. Usually, the key components of many chromatin remodeling complexes are dys-regulated along with tumorigenesis. For example, two components of SWI/SNF (BRG1 and BRM) were differently expressed during liver tumorigenesis. BRG1 was highly expressed and BRM was lowly expressed in liver cancer and liver TICs [
31]. EZH2, the core component of PRC2 complex, was also highly expressed in liver tumorigenesis [
32]. Here, we found SRCAP was required for GPR107 expression, and the binding of SRCAP complex and
GPR107 promoter is lncGPR107-dependent.
Acknowledgements
The authors thank Min Liang (Department of Abdominal Oncology, The Fifth Affiliated Hospital of Guangzhou Medical University) for experimental supports.