Background
Colorectal cancer (CRC) is the third most common malignant carcinoma and the second leading cause of cancer-related death worldwide. The incidence of CRC is continually increasing, and it is estimated that approximately 1.9 million new CRC cases emerged and 935,000 deaths occurred in 2020 [
1]. The molecular pathogenesis of CRC has not been fully elucidated, and limited success has been achieved in improving the survival of CRC patients. Thus, constant efforts are required to elucidate the underlying molecular mechanisms and identify novel therapeutic targets.
Current progress in cancer transcriptomics has demonstrated that many cancer-related genes are noncoding RNAs (ncRNAs). As a major member of the ncRNA family, long ncRNAs (lncRNAs) have gained widespread attention. Gradually accumulating evidence has shown that lncRNAs participate in various physiological and pathological processes by regulating protein-protein, RNA-protein or protein-DNA interactions, as well as by sponging miRNAs [
2]. Some lncRNAs have been shown to contribute to CRC development and could be used as biomarkers for cancer diagnostics and therapy [
3‐
9].
We extensively analyzed the differentially expressed lncRNAs in the CRC genome and characterized a series of CRC-related lncRNAs, including FEZF1-AS1, LINC00152 ( CYTOR ), MCM3AP-AS1, SLCO4A1-AS1, SNHG6, SNHG15, SHNG17 and UCA1 [
7‐
15]. For example, we showed that FEZF1-AS1 promotes CRC tumorigenesis and progression by regulating PKM2/STAT3 signaling and glycolysis [
7]. In addition, we revealed that CYTOR drives CRC progression by interacting with NCL and Sam68 [
12].
In this study, we identified a novel transcript of DLGAP1-AS2 that is significantly upregulated in CRC and is associated with the malignant features and prognosis of CRC. Functional and mechanistic studies revealed that DLGAP1-AS2 promotes CRC tumorigenesis and progression by enhancing Trim21-mediated ubiquitination and degradation of Elongin A (ELOA). Furthermore, ELOA directly binds to the promoter of LHPP and increases its expression, thus activating LHPP-mediated suppression of the AKT signaling pathway. In addition, we also demonstrated that cleavage and polyadenylation specificity factor (CPSF2) and cleavage stimulation factor (CSTF3) bind to DLGAP1-AS2 and synergistically increase its stability in CRC cells. Our study uncover a previously unknown regulatory axis of DLGAP1-AS2/Trim21/ELOA/LHPP in CRC and highlight that manipulation of this signaling axis may be a novel strategy for the treatment of CRC and other tumors.
Methods
Cell lines
The CRC cell lines Caco-2, DLD1, HCT116, HCT8, HT29, LoVo, RKO and SW480 were purchased from ATCC and cultured following their instructions. These cells were characterized by Genewiz, Inc.(China) using short tandem repeat markers and were confirmed to be mycoplasma-free.
Clinical samples
Human primary CRC tissues and their paired adjacent noncancerous tissues (NCTs) were collected from Affiliated Hospital of Jiangnan University with informed consent. The study was approved by the Clinical Research Ethics Committees of Affiliated Hospital of Jiangnan University, and written informed consent was obtained from all patients.
RNA-sequence analyses
The total RNA samples were purified from CRC and NCTs using TRIzol (Invitrogen, USA) and were treated with Ribo-off rRNA Depletion Kit (Vazyme, China). These treated RNA samples were subjected to RNA-seq library construction using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme). These constructed libraries were then sequenced by Illumina sequencing platform on a 150 bp paired-end run. Sequencing reads were aligned using the spliced read aligner HISAT2, with Genome Reference Consortium GRCh38 as the reference genome. Annotations of lncRNAs and mRNAs in the human genome were retrieved from the GENCODE (v25) database. In addition, the expression profiling data and the relevant clinical information of several CRC cohorts were downloaded from TCGA (
https://portal.gdc.cancer.gov/) and GEO (
http://www.ncbi.nlm.nih.gov/geo).
Quantitative RT-PCR(qRT-PCR)
Total RNA was reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (CWBIO, China). Gene expression levels were measured by qRT-PCR using Ultra SYBR Mixture (Vazyme, China). The relative gene expression levels were normalized to those of β-actin and calculated using the 2
−△△Ct method. The sequences of the related primers are listed in Tab.S
1.
Vector constructs and siRNA
The DLGAP1-AS2 sequence was cloned into the lentiviral expression vector pLenti-EF1a -F2A-Puro-CMV-MCS. The CPSF2 and ELOA sequences were cloned into the expression vector pRK7-Flag. The CSTF3 sequence or Trim21 sequence was cloned into the expression vector PCMV5-HA or pcDNA3.1-Myc, respectively. SiRNAs targeting DLGAP1-AS2, CPSF2, CSTF3, ELOA and Trim21 were purchased from GenePharma (China). The validated shRNA sequence of DLGAP1-AS2 or ELOA was synthesized and cloned into the pLKO.1 lentiviral expression vector. The promoter of LHPP was amplified from human genomic DNA by PCR and cloned into the pGL3-Basic vector. The related sequences are listed in Tab.S
2-
3.
Cell proliferation and colony formation assays
Cell viability was measured with the Cell Counting Kit 8 (CCK8, Beyotime, China) according to the manufacturer’s instructions. For the colony formation assay, 800 to 1500 CRC cells were seeded into each well of a 6-well plate and maintained in medium containing 10% FBS for 10–15 days. The colonies were fixed with methanol, stained with 0.1% crystal violet and counted using an inverted microscope. Each experiment was repeated at least three times.
Invasion and migration assays
Migration and invasion assays were performed in Transwell chambers (Corning, USA) according to the manufacturer’s instructions.
In vivo assays
Male athymic BALB/c nude mice were purchased from the Shanghai Animal Center, Chinese Academy of Sciences and maintained under specific pathogen-free conditions at Jiangnan University. Nude mice, aged 5 weeks old, randomly divided into different groups (n = 5 for each group) were injected subcutaneously with 0.1 ml of a cell suspension containing 2 × 106 CRC cells. The tumor size was measured, and the tumor volume was calculated according to the formula volume = length×width2 × 0.5. For the in vivo metastasis model, 2 × 106 CRC cells were injected into 7-week-old male BALB/c nude mice randomly divided into different groups (n = 5 for each group) by tail vein. Five weeks after injection, the lung nodules in the mice were observed to measure the capability of the cells to form metastatic tumors. All animal experiments were performed in accordance with the relevant institutional and national guidelines and the regulations of Jiangnan University Medical Experimental Animal Care Commission (JN.No20190615b0320925).
RNA pull-down assays and mass spectrometry analyses
RNA pull-down assays were performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. The RNA pull-down samples were separated by gel electrophoresis and visualized with silver staining. Specific bands were excised for proteomics screening by mass spectrometry analyses and retrieved from the Human Protein Reference Database (
http://www.hprd.org/). The primers for DLGAP1-AS2 and its deletion fragments for in vitro transcription are provided in Tab.S
4.
RNA immunoprecipitation (RIP) assays
RIP assays were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. Cell lysates were incubated overnight at 4 °C with magnetic beads conjugated to anti-CPSF2, anti-CSTF3, anti-ELOA or anti-IgG. The RIP samples were subjected to RNA extraction and subsequent RT-PCR analyses to detect the abundance of DLGAP1-AS2. Information about the antibodies is listed in Tab.S
5.
Immunoblotting analyses
Cells were lysed in lysis buffer (Beyotime) with protease inhibitor cocktail treatment (Roche, USA). The protein extracts were then separated by SDS–PAGE and transferred to PVDF membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies at 4 °C overnight and were then incubated with a peroxidase conjugated secondary antibody (1:5000, Thermo Fisher) for 1 h at room temperature. Finally, the membranes were visualized with ECL substrate (Vazyme).
Immunoprecipitation (IP) assay
The indicated plasmids were transfected into HCT116 cells and lysed in IP buffer (Beyotime) containing protease inhibitors. The supernatants were incubated with the beads overnight at 4 °C with gentle rotation. The beads with the attached immune complexes were washed three times and analyzed by immunoblotting with the indicated antibodies.
Two sgRNA target DNA sequences located in exon 2 of TRIM21 were designed using the CRISPR library and inserted into pLentiCRISPR (Trim21-gRNA-pLentiCRISPR). The plasmids were transfected into HCT116 cells to generate a Trim21 knockout (KO) cell line according to the manufacturer´s instructions. Cells were treated with puromycin and separated into single cell to form colonies. Those colonies were harvested and verified for Trim21 KO by Sanger sequencing and western blotting.
Immunohistochemistry(IHC) staining
ELOA protein levels in CRC tissues were determined by IHC. IHC staining was performed on 4-mm sections of paraffin-embedded tissue samples. Briefly, the slides were incubated with an anti-ELOA antibody (Santa, 1:200) at 4 °C overnight. The subsequent steps were performed using the GT Vision III Detection System/Mo&Rb (GeneTech, China). The slides were concurrently examined and scored by two blinded pathologists.
Chromatin immunoprecipitation(ChIP)-on-chip arrays and data analyses
The protein-DNA complexes were precipitated using an antibody against ELOA or IgG. The ChIP samples were then amplified, labeled and hybridized to Nimblegen human 720 K RefSeq promoter arrays (Roche Nimblegen, USA). The significant peak regions with false discovery rates ≤ 0.05 were mapped to the nearest genes. The fasta sequences were extracted from the promoters of the differentially expressed genes (DEGs) from ChIP data. The MEME-ChIP database and Markov model were used to search the candidate motif sites of ELOA in the promoters of the potential ELOA target genes.
Dual luciferase reporter assays
Luciferase reporter plasmids were co-transfected with ELOA expression plasmids. The luciferase activities of these cells were detected at 48 h after transfection using the Dual-Luciferase® Reporter Assay System (Beyotime).
Statistical analyses
The data are presented as the mean ± standard deviation. The statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, USA) and SPSS 20.0 (SPSS Inc., USA), and p < 0.05 was considered to be statistically significant.
Discussion
The identified number of lncRNA increased rapidly with the progress of RNA sequencing technology. In our previous studies, we screened and identified several tumor-related lncRNAs in CRC using microarrays [
7‐
15]. In this study, we further screened CRC-related lncRNAs using next-generation sequencing and identified a cancer-promoting lncRNA, DLGAP1-AS2, is upregulated in many types of cancers.
DLGAP1-AS2 is located at chromosome 18p11.31, and a total of three transcripts of DLGAP1-AS2 have been included in GENECODE or GenBank. In this study, we identified a novel DLGAP1-AS2 transcript that was the predominant transcript in CRC and other types of cancer cells. Although the expression levels of the other two transcripts of DLGAP1-AS2 ((NR_119377.1 and ENST00000572856.1) were much lower than that of the identified novel transcript in most cancer cells checked, our preliminary functional investigations showed that they also showed cancer-promoting functions in CRC cells (data not shown), suggesting that they may also play a role under specific psychological and pathological conditions.
Based on multiple CRC cohorts, we showed that DLGAP1-AS2 is a promising prognostic biomarker for CRC. Functionally, we demonstrated that DLGAP1-AS2 has strong oncogenic activity by promoting CRC tumorigenicity and progression. Recent studies also reported the upregulation and prognostic value of DLGAP1-AS2 in several cancer types. Similar tumor-promoting functions have also been observed in multiple cancer types, including rectal cancer [
19‐
26]. For example, a recent study conducted by Zeng et al. reported that DLGAP1-AS2 promotes the radioresistance in rectal cancer stem cells through regulating the E2F1-CD151 axis [
27]. These findings indicate that DLGAP1-AS2 is a potential oncogene, and may act as a pancancer therapeutic target and biomarker. However, the detailed functions and mechanisms of DLGAP1-AS2 in CRC were still unclear.
LncRNAs typically exert their biological functions through physical interactions with DNA, RNA, or proteins [
28]. Little was known about the molecular mechanisms of DLGAP1-AS2 in cancers. Limited studies reported that DLGAP1-AS2 sponges and inhibits the activities of miR-503 or miR-505. In addition, a recent study reported that DLGAP1-AS2 binds to the transcription factor Six3 and inhibits its occupancy in Wnt1 promoter, resulting in the activation of Wnt/β-catenin signaling in gastric cancer cells. In view of relative low abundance of miR-503 and miR-505 and mainly cytoplasm location of DLGAP1-AS2 in CRC cells, we speculated that there are other mechanisms mediating the cancer-promoting functions of DLGAP1-AS2 in CRC.
In this study, we identified CPSF2, CSTF3 and ELOA as bona fide interacting partners of DLGAP1-AS2. ELOA was originally identified as a transcriptionally active subunit of RNA polymerase II (Pol II) transcription factor Elongin (SIII), which stimulates the overall rate of Pol II elongation through direct interactions with the enzyme [
29,
30]. Elongin is composed of ELOA and a heterodimeric submodule comprised of Elongin B and C proteins, which bind to a short ELOA sequence motif referred to as the BC box that has potent transcriptional regulation activity [
31]. In addition to being a part of SIII, ELOA also functions as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitinates the RPB1 subunit of Pol II in response to DNA damage.
Although Elongins B and C have been well characterized in tumorigenesis, little is known about the role of ELOA in tumorigenesis and how its activity is regulated. By a series of experimental validations, we identified ELOA as a key downstream target of DLGAP1-AS2.Here, we revealed that DLGAP1-AS2 enhances ELOA ubiquitination and degradation by promoting the binding of the E3 ligase Trim21 to ELOA, revealing a novel regulatory mechanism for ELOA at the posttranslational level. Using a series of in vitro and in vivo assays, we revealed, for the first time, the inhibitory functions of ELOA in CRC growth and metastasis, discovering a novel role of ELOA in tumorigenesis and progression.
By expression profile analyses, we demonstrated that DLGAP1-AS2 and ELOA share highly similar regulatory effects on the patterns of signaling pathways. Moreover, rescue assays also confirmed that ELOA is a downstream target of DLGAP1-AS2. As a family of transcription elongation factors, ELOA has been shown to play a critical role in optimal gene induction under stress conditions or developmental stimuli [
32]. Little is known about the downstream targets of ELOA in cancer cells. We reveled ELOA as a novel transcription regulator for LHPP, a newly identified tumor suppressor [
16]. LHPP impedes tumor proliferation and metastasis and inhibits cancer progression through the PI3K/AKT pathway by regulating the phosphorylated AKT at Ser473 in multiple cancer types [
33‐
38]. In this study, for the first time, we provided evidence that ELOA directly binds to the specific promoter region of LHPP and promotes its transcription, resulting in increased LHPP expression and decreased AKT activity.
Although several studies have reported aberrant upregulation of DLAGP1-AS2 in human cancers, its underlying mechanism is unclear. CPSF2 and CSTF3 reported to be members of the cytoplasmic polyadenylation element(CPF) family, which are parts of a multi protein complex essential for the formation of the mRNA 3’ end-processing machinery [
39]. CPSF2 is the 100 kDa subunit of CPSF, which consists of four polypeptides. CPSF2 recognizes the AAUAAA polyadenylation signal, which determines the site of 3’-end cleavage interactions within pre mRNAs and plays a key role in nuclear export, translational initiation and transcript stability [
40]. Whereas CSTF3 binds GU-rich sequences located downstream from the cleavage site AAUAAA and adds a poly(A) tail for polyadenylation, resulting in increased RNA stability [
41]. Here, we proved that CPSF2 and CSTF3 bind to DLGAP1-AS2 and enhance its stability, thereby inhibiting the ELOA/LHPP axis in CRC. Our data reveal a posttranscriptional regulatory mechanism that, at least partially, mediates the upregulation of DLGAP1-AS2 in CRC. However, how CPSF2 and CSTF3 regulate the stability of DLGAP1-AS2 is not clear, and whether the regulatory mechanism could be validated in other cancer types also remains to be elucidated.
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