Background
Colorectal cancer (CRC) is the third most prevalent malignant tumor and one of the leading causes of cancer-related death worldwide [
1]. Despite progress in surgery, chemotherapy and radiotherapy, the prognosis of CRC is still poor due to its postoperative recurrence and metastasis. Therefore, it is urgent to explore the molecular mechanism of CRC and to develop new therapeutic targets.
Long noncoding RNAs (lncRNAs) are a class of transcribed RNAs with lengths greater than 200 nucleotides, and their protein coding ability is lost or restricted [
2]. Recent studies have shown that lncRNAs play vital roles in regulating various biological processes, such as proliferation, differentiation, apoptosis, and chemoresistance [
3]. Growing evidence has shown that the abnormal regulation or expression of lncRNAs is implicated in the tumorigenesis and progression of CRC [
4,
5]. We have reported that some lncRNAs regulate CRC growth, metastasis and chemoresistance and may be potential prognostic biomarkers or therapeutic targets [
6‐
12]. Recently, other groups also reported the regulatory roles of some lncRNAs, including ENO1-IT1 [
13], LINC00460 [
14], RAMS11 [
15] and FLANC [
16], in CRC development and progression. All these studies indicate the key regulatory roles of lncRNAs in CRC.
Small nucleolar RNAs (snoRNAs) are predominantly distributed in the nucleolus and play a role in guiding the sequence-specific chemical modification or processing of pre-ribosomal RNA [
17]. As the host genes of snoRNAs, lncRNA small nucleolar RNA host genes (SNHGs) have been shown to be abnormally expressed in multiple cancers and regulate cell proliferation, metastasis, and chemoresistance [
18,
19]. Of them, SNHG17 has been reported to be aberrantly overexpressed in multiple human cancers [
20‐
25], suggesting that SNHG17 has extensive functions and universal roles in tumorigenesis. However, the biological function and mechanism of SNHG17 in CRC remain poorly understood. In this study, we demonstrated that SNHG17 is aberrantly overexpressed in CRC and correlated with poor clinical outcomes. We also uncovered, for the first time, that SNHG17 promotes tumor growth and metastasis via two different regulatory mechanisms, SNHG17-Trim23-PES1 axis and SNHG17-miR-339-5p-FOSL2-SNHG17 positive feedback loop, suggesting that SNHG17 could be a potential therapeutic target for CRC.
Materials and methods
Clinical samples
Human primary CRC tissues and their paired adjacent noncancerous tissues (NCTs) were obtained from the Affiliated Hospital of Jiangnan University and Fudan University Shanghai Cancer Center (Supplementary Table S
1). All pathologically confirmed CRC tissue samples (cohort 1: NCTs = 51, CRC tissues = 91; cohort 2: NCTs = 107, CRC tissues = 107) were transferred to the laboratory in liquid nitrogen and stored at − 80 °C until use. Participants were excluded in the presence of any other malignancies or preoperative anti-cancer treatment. All patients signed informed consent forms, and the project was approved by the Clinical Research Ethics Committees of the participating institutions.
Cell lines and culture
Human CRC cell lines HCT116, HCT8, SW620, HT29 and LoVo were purchased from the American Type Culture Collection (ATCC). LoVo cells were cultured in F12K medium, and the other cells were maintained in DMEM containing 10% fetal bovine serum. All these cell lines were cultured in 5% CO2 at 37 °C.
Plasmids and transfection
siRNAs targeting SNHG17, PES1, and FOSL2 and miR-339-5p mimics and miR-339-5p inhibitor were ordered from Shanghai Genepharma Co., Ltd. Full-length SNHG17 was amplified and cloned into the pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS vector using ClonExpress II One Step Cloning Kit (Vazyme, China). The short hairpin SNHG17-#2 was inserted into the pLKO.1 TRC cloning vector for lentiviral packaging. The promoter of SNHG17 was amplified from HCT116 genomic DNA by PCR and cloned into the pGL3-Basic vector. The related primers are listed in Supplementary Table S
2. The transfection of these plasmids was conducted by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.
Quantitative reverse transcription PCR (RT-qPCR)
Total RNA of cells or tissue specimens was extracted using RNA isolate (Vazyme) and reverse transcribed into cDNA using a HiFiScript cDNA Synthesis Kit (CWBIO, China). Cytoplasmic and nuclear RNA isolations were performed with a PARIS Kit (Life Technologies, USA) following the manufacturer’s instructions. SNHG17 expression levels were measured by RT-qPCR using UltraSYBR Mixture (CWBIO) on a ViiA 7 Real-Time PCR System (Applied Biosystems, USA) with the program of 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression levels were calculated by the 2
-△△Ct method with ACTB as an internal control. The related primer sequences are listed in Table S
2.
Cell counting Kit-8 (CCK-8) and colony formation assays
Cell proliferation was measured by CCK-8 (Beyotime, China) and colony formation assays. For the CCK-8 assay, a total of 10 μL CCK-8 solution was added to each well of a 96-well plate at 1, 2, 3 and 4 days after transfection. The absorbance was measured at 450 nm with a microplate reader (BioTek Instruments). For the colony formation assay, approximately 800 HCT116 or 2000 LoVo cells were plated in 6-well plates and incubated at 37 °C for 2 weeks.
Transwell assays
Approximately 1 × 105 HCT116 cells or 1.5 × 105 LoVo cells were added to the upper compartment of a Transwell chamber (Corning, USA). After 24 h of incubation, cells on the lower surface were fixed with 10% formaldehyde for half an hour and then stained with crystal violet for observation. In the invasion assay, Matrigel (BD, USA) was used to coat the Transwell chamber before the experiments.
Xenograft mouse model
A total of 2.0 × 106 SNHG17-overexpressing HCT116 cells or 3.5 × 106 SNHG17-depleted LoVo cells and their respective control cells were subcutaneously injected into the different flanks of 4-week-old male BALB/c nude mice (Shanghai SLAC Laboratory Animal, China) (randomly divided into 2 groups, n = 5 for each group). The tumor size was measured every 3 days after the tumors became visible. For the in vivo metastasis model, 1.5 × 106 SNHG17-overexpressing HCT116 cells or SNHG17-depleted LoVo cells were injected into 7-week-old male BALB/c nude mice (n = 5 for each group) via the tail vein. Five weeks after injection, the lung nodules of mice were observed to assess tumor metastasis. All animal experiments were approved by the Clinical Research Ethics Committees of Affiliated Hospital of Jiangnan University.
Fluorescence in situ hybridization (FISH)
SNHG17 and 18S FISH probes were purchased from RiboBio. A FISH kit was employed to detect the signals of the probes according to the manufacturer’s protocol (RiboBio, China). The cells were fixed with 4% polyoxymethylene and incubated with permeabilizing solution (0.5% Triton X-100 diluted in PBS) at 4 °C for 5 min. After washing three times with PBS, the cells were treated with prehybridization buffer at 37 °C for 30 min. Then, a 20 μM probe mixture diluted in hybridization buffer was incubated with the cells overnight at 37 °C. Images of cells were captured after staining with DAPI dye for 10 min using an OLYMPUS DP80-Cellsens microscopic imaging system.
Western blotting
The cells were lysed in RIPA buffer (Beyotime) supplemented with protease inhibitor cocktail (MCE, USA), and the obtained proteins were then separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). After being blocked in 5% skimmed milk powder, the membranes were incubated with primary antibodies against PES1 (1:5000, Proteintech, USA), FOSL2 (1:1000, Cell Signaling Technology, USA), and GAPDH (1:5000, ABclonal, China) overnight at 4 °C. The protein band intensity was detected using a ChemiDOCTMXRS+ imaging system (BIO-RAD, USA).
RNA pull-down assay
RNA pull-down assays were performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions as we previously described [
8]. Detailed information regarding the primers used for in vitro transcription is depicted in Supplementary Table S
2.
RNA immunoprecipitation (RIP)
The EZ-Magna RIP kit (Millipore) was used for RIP assays according to the manufacturer’s instructions. HCT116 cell lysates were incubated overnight at 4 °C in RIP buffer containing magnetic beads conjugated to anti-PES1 or anti-IgG as a control. The lysates were then treated with proteinase K buffer, followed by RNA extraction. Finally, the purified RNA was examined by RT-qPCR to detect the abundance of SNHG17.
Dual luciferase reporter assay
Luciferase reporter vectors containing SNHG17 and FOSL2 with wild-type (WT) or mutated (MUT) miR-339-5p binding sites were constructed. Luciferase reporter plasmids were cotransfected with miR-339-5p mimics or miR-NC mimics into 293 T and HCT116 cells by Lipofectamine 2000. The luciferase activities of these cells were detected at 48 h after transfection using a Dual-Luciferase Reporter Assay System (Beyotime).
Chromatin immunoprecipitation (ChIP)
CRC cells were preserved with formaldehyde and fixated for 10 min to produce DNA-protein cross-links. Then, ChIP assays were performed using ChIP assay kits (Beyotime) according to the manufacturer’s instructions. The cell lysates were sonicated to produce chromatin fragments of 200–400 bp, which were immunoprecipitated with FOSL2 (Cell Signaling Technology) or IgG (Beyotime) antibodies. Precipitated chromatin DNA was recovered and analyzed by PCR. The primers used for the promoters are listed in Supplementary Table S
2.
Immunohistochemistry (IHC)
The slides of the tissue microarray were incubated with the primary antibody for PES1 (1:100, Proteintech) or FOSL2 (1:100, Cell Signaling Technology) overnight at 4 °C. IHC was performed as we described previously [
8].
Statistical analyses
The data were analyzed by GraphPad Prism version 8.0 (GraphPad Prism) and SPSS 20 software (SPSS). All results are presented as the mean ± SD. Student’s t test and χ2 test were used to assess the significance of differences between groups. The differences in survival rates were determined with the Kaplan-Meier method and compared with the log-rank test. P < 0.05 was considered to indicate statistical significance.
Discussion
CRC is one of the most common malignant tumors of the digestive system. LncRNAs can act as oncogenes or tumor suppressors, and their dysregulation is highly associated with the development and progression of CRC. SNHG17 was initially confirmed to be highly expressed in CRC, and then its overexpression was observed in a variety of cancers. Recent studies have indicated the oncogenic role of SNHG17 in several cancer types, including CRC. For example, recent studies showed that SNHG17 promotes tumor cell proliferation and invasion in tongue squamous cell carcinoma and CRC by sponging miR-23a-3p and miR-876, respectively [
25,
27]. In addition, SNHG17 aggravates prostate cancer progression by positively regulating its homolog SNORA71B [
28]. All these data suggest that SNHG17 is a pan-cancer oncogene and may be a promising therapeutic target across cancers.
In this study, we confirmed that SNHG17 is significantly upregulated in CRC tissues and was associated with poor survival in multiple CRC cohorts. Functionally, SNHG17 can promote CRC proliferation and metastasis. SNHG17 exerts tumor-promoting functions by two different mechanisms. We demonstrated that PES1 is a SNHG17-associated protein. PES1 is highly evolutionarily conserved and is involved in ribosome biogenesis and cell proliferation [
29]. The ribosome is an important organ that is responsible for protein synthesis. To meet the needs for the continuous growth of tumor cells, it is necessary to increase ribosome biogenesis to maintain high protein synthesis efficiency. Therefore, increased ribosome biogenesis is an important feature of cancer cells [
30]. Wang et al. reported that PES1 promotes tumorigenesis in hepatocellular carcinoma by regulating the PI3K/AKT pathway [
31]. It has been reported that PES1 interacts with BRD4 to enhance c-Myc expression, thereby promoting cell growth and cell resistance to extra-terminal inhibitors in pancreatic cancer [
32]. In addition, a recent study revealed that PES1 facilitates telomerase assembly and negatively correlates with senescence in cancer cells, suggesting that PES1 is a promising target for cancer therapy [
33]. All these data suggest that PES1 plays an oncogenic role in various cancers. In this study, we showed that SNHG17 binds to PES1 and increases its protein expression in CRC cells. Mechanistically, SNHG17 interacts with PES1 to inhibit the Trim23-mediated ubiquitination of PES1, resulting in increased PES1 stability and enhanced tumor growth and metastasis. Clinical analyses revealed that PES1 is significantly upregulated in CRC tissues and predicts a poor prognosis. These findings suggest that targeting the SNHG17-PES1 regulatory axis is a promising strategy for CRC treatment.
SNHG17 is mainly located in cytoplasm. Most cytoplasmic lncRNAs act as miRNA sponges and are involved in miRNA-mediated posttranscriptional regulation. SNHG17 has already been confirmed to facilitate cell growth by modulating the miR-384/ELF1 axis in oral squamous cell carcinoma [
34]. SNHG17 can promote tumor-like behavior in hepatocellular carcinoma cells via miR-3180-3p/RFX1 [
35]. These results suggest that binding with miRNAs is one of the most important mechanisms of SNHG17 in cancer. In addition to the SNHG17-Trim23-PES1 mechanism, we also verified that SNHG17 could bind with miR-339-5p and inhibit its function. Previous studies have demonstrated that exosome-derived miR-339-5p mediates radiosensitivity by inhibiting Cdc25A [
36]. MiR-339-5p has also been reported to inhibit glycolysis and colon cancer growth by reducing PKM2 expression through hnRNPA1 and PTBP1 [
37]. In addition, we confirmed the tumor-suppressive effect of miR-339-5p in CRC and found that it was inhibited by SNHG17.
We further searched for the targets of SNHG17/miR-339-5p and revealed that FOSL2 is a novel functional target of miR-339-5p. Subsequent functional experiments confirmed that SNHG17 regulates CRC development and progression by competitively sponging miR-339-5p and restoring the activity of FOSL2. FOSL2 and the oncoproteins Fos and Jun belongs to the activator protein 1 transcription factor family that transactivates the transcription of its downstream targets. Aberrantly increased expression of FOSL2 has been documented in many cancer types, including CRC. Previous studies have shown that FOSL2 promotes the proliferation, migration, and invasion of various cancers, including non-small-cell lung cancer [
38], hepatocellular carcinoma [
39], and CRC [
40]. We also observed that FOSL2 is upregulated and positively correlates with SNHG17 expression in CRC tissues, which is positively associated with and predicts poor clinical outcomes. Interestingly, we identified a putative binding site of FOSL2 at the promoter of SNHG17 and uncovered that FOSL2 transcriptionally activates SNHG17 expression by binding to this site. Collectively, our data revealed a positive feedback loop of SNHG17-miR-339-5p-FOSL2-SNHG17 in CRC and suggest that targeting this loop might be a promising strategy for CRC therapy.
Conclusion
Overall, our study uncovered two different molecular mechanisms by which SNHG17 promotes CRC development and progression. SNHG17, miR-339-5p, PES1, FOSL2 and their downstream targets form a complicated regulatory network that contributes to colorectal tumorigenesis and metastasis. These data may inspire the development of conceptually novel cancer therapeutics.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.