
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

59575
10.1038/s41598-024-59575-6
Article
The up-regulation of SYNCRIP promotes the proliferation and tumorigenesis via DNMT3A/p16 in colorectal cancer
Li Chenglong
Lu Tailiang
Chen Hongxi
Yu Zhige yuzhige1977@sina.com

Chen Chaowu dr.chencw@hunnu.edu.cn

https://ror.org/03wwr4r78 grid.477407.7 0000 0004 1806 9292 Department of Gastrointestinal Surgery, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, 410005 Hunan Province China
16 9 2024
16 9 2024
2024
14 2157014 9 2023
12 4 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This 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/.
Heterogeneous nuclear ribonucleoproteins (hnRNPs), a group of proteins that control gene expression, have been implicated in many post-transcriptional processes. SYNCRIP (also known as hnRNP Q), a subtype of hnRNPs, has been reported to be involved in mRNA splicing and translation. In addition, the deregulation of SYNCRIP was found in colorectal cancer (CRC). However, the role of SYNCRIP in regulating CRC growth remains largely unknown. Here, we found that SYNCRIP was highly expressed in colorectal cancer by analyzing TCGA and GEPIA database. Furthermore, we confirmed the expression of SYNCRIP expression in CRC tumor and CRC cell lines. Functionally, SYNCRIP depletion using shRNA in CRC cell lines (SW480 and HCT 116) resulted in increased caspase3/7 activity and decreased cell proliferation, as well as migration. Meanwhile, overexpression of SYNCRIP showed opposite results. Mechanistically, SYNCRIP regulated the expression of DNA methyltransferases (DNMT) 3A, but not DNMT1 or DNMT3B, which affected the expression of tumor suppressor, p16. More importantly, our in vivo experiments showed that SYNCRIP depletion significantly inhibited colorectal tumor growth. Taken all together, our results suggest SYNCRIP as a potent therapeutic target in colorectal cancer.

Keywords

Colorectal cancer
hnRNPs
DNA methyltransferases
Tumor suppressor
Tumor growth
Subject terms

Oncogenes
Colon cancer
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Colorectal cancer (CRC) is one of the most frequent malignant tumors worldwide and is the second leading cause of cancer-related deaths1. CRC is a multifactorial disease, and the risk factors include environmental, genetic, and biochemical factors, such as smoking, alcohol, age, and obesity2,3. The identification of diagnostic biomarkers and targeted therapy remain the major challenge of CRC treatment. Recently, significant progress has been made in the targeted therapy of CRC; the drugs targeting EGFR, PD-1/PD-L1, BRAF, CTLA-4, and NTRK have been approved to treat CRC patients and achieved great benefits4,5. However, despite the advances in therapeutic plans, the five-year survival of CRC patients remains low. Thus, it is urgent to understand the fundamental mechanism of tumorigenesis and identify novel effective therapeutic targets.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large group of RNA-binding proteins that regulate multiple posttranscriptional events, such as alternative splicing, mRNA stabilization, RNA degradation and translational regulation6. The hnRNPs family members share general features and similar protein structure, consisting of at least one RNA-binding domain combines with an RGG box or acidic domain for protein–protein interaction or RNA binding7. Considering their key function in the cellular nucleic acid metabolism, the role of hnRNPs in regulating gene expression has gained more and more interest in disease research. Aberrant hnRNPs expression can cause a wide variety of diseases, including cancer. In lung cancer, hnRNP A1 knockdown inhibited lung adenocarcinoma cell proliferation through cell cycle arrest at G0/G1 phase8. In addition, hnRNP K promoted the progression of lung cancer by inhibiting the p53‐dependent signaling pathway9. In colorectal cancer, hnRNPA1 is augmented and stabilizeoncogenic mRNAs, such as cyclin D1 and c-Myc10,11. Also, the expression of hnRNP K is correlated with the prognosis of CRC12. HnRNP G, which is specifically expressed in the testis, expresses in colorectal cancer cells, and accelerates cell growth mediating ZDHHC11 mRNA stabilization13. More importantly, a study constructed siRNAs targeting 20 representative hnRNPs and discovered that all these hnRNPs promoted the progression of CRC14. hnRNP Q (also called SYNCRIP) is an AU-rich RNA-binding protein and have multifunction in regulating mRNAs, including mRNA splicing, mRNA editing, transport, turnover, and translation regulation15–17. Two studies showed that one of the hnRNP Q isoforms, hnRNP Q1, contributes to cell proliferation and tumorigenesis in colorectal cancer18,19. However, the mechanism remains largely unknown.

DNA methylation is one of the predominant epigenetic modifications in mammals and is controlled by DNA methyltransferases (DNMTs)20. In mammals, three catalytically active DNMTs (DNMT1, DNMT3A and DNMT3B) have been identified21. Elevated levels of DNMT1, DNMT3A and DNMT3B have been reported in various tumors, including hepatic, prostate, colorectal, and breast cancers22–25. Recently, several studies have shown that dysregulation of DNMT3A leads to oncogenesis, including colorectal cancer26,27. However, the regulatory mechanism of DNMT3A expression in CRC has not been well-defined and needs further investigation. In the present study, we confirmed the function of SYNCRIP in regulating colorectal cancer cells growth in vitro and in vivo and proved that the expression of DNMT3A was regulated by SYNCRIP, which suggested SYNCRIP as a potential therapeutic target for colorectal cancer.

Materials and methods

Antibodies

Antibodies against SYNCRIP (#PA5-50986) were obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies against DNMT1 (#5032) and GAPDH (#5174) were obtained from Cell Signaling (MA, USA); Antibodies against DNMT3A (#20954-1-AP), DNMT3B (#26971-1-AP), and p16 (#10883-1-AP) were obtained from Proteintech (IL, USA).

Cell culture

Human colorectal cancer cell lines SW480, SW620, HT29, HCT8, and HCT116 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) FBS (Gibco, Waltham, MA). Human colon mucosal epithelial cell line NCM460 were purchased from INCELL (TX, USA) and cultured in DMEM (Gibco, Waltham, MA) supplemented with 10% (v/v) FBS (Gibco, Waltham, MA) and penicillin/streptomycin.

Short hairpin RNA (shRNA) knockdown

SYNCRIP shRNAs and DNMT3A were purchased from Sigma-Aldrich (MO, USA). The shRNA sequences were as follow: SYNCRIP shRNA1: 5′-GCAAAGTAACAGAGGGTCTTA-3′; SYNCRIP shRNA2: 5′-GTATGACGATTACTACTATTA-3′; DNMT3A shRNA1: 5′-CCACCAGAAGAAGAGAAGAAT-3′; shRNA plasmids were co-transfected with packaging constructs according to the manufacture’s instruction to package the lentiviruses. The virus particles were collected 48 h after transfection. SW480 and HCT116 cells were infected with lentivirus mixed with 8 μg/ml polybrene. Stable clones were established by puromycin (1 mg/mL) selection.

Plasmid transfection

DNMT3A overexpression plasmid was purchased from Origene (#RC208192, MD USA). Briefly, SW480 and HCT116 cells were plated onto 6 well plate, and grew to 80% ~ 90% confluency. 2 μg empty vector or DNMT3A plasmid were transfected using Lipofectamine 2000 (Invitrogen, MA, USA) according to the manufacturer’s instructions. The cells were harvested after transfection for 24 h.

Caspase-3/7 activity assay

Caspase-3/7 activity was measured using Apo-ONE™ Homogeneous Caspase-3/7 Assay (Promega Corporation, Madison, WI, USA) according to the protocol provided by the manufacturer.

Cell proliferation and survival assay

Cell proliferation was assayed using Cell Counting Kit-8 assay (CCK8) kit following the manufacture’s instruction (Jiangsu KeyGENBioTECH Corp., Ltd, China). 3000 SW480 and HCT116 cells were seeded in 96-well plate after infected with SYNCRIP shRNA or transfected with SYNCRIP plasmid. At the indicated time, 10 μl CCK8 reagents were added into each well and incubated at 37 ◦C for 2 h, and then the absorbance at 450 nm was recorded.

Cell survival was assessed using trypan blue staining. The dead cells were blue stained and counted manually using hemocytometer.

Migration assay

Migration assay was conducted using 8 μm transwell inserts (Corning, NY, USA). Briefly, 1 × 105 SW480 and HCT116 cells infected with SYNCRIP shRNA or transfected with SYNCRIP plasmid in 100 μl serum free medium was added into the inserts and then placed in 24-well plate that fulfilled with 600 μl complete growth medium. The inserts were washed with PBS, fixed with 4% formaldehyde, and stained with 0.5% crystal violet after 24 h incubation at 37 °C. Five randomly fields were acquired using microscope.

Wound healing assay

5 × 105 SW480 cells infected with SYNCRIP shRNA or transfected with SYNCRIP plasmid were seeded 6-well plate; the cells were scratched with a 1 ml pipette tip after 100% confluent. The images were then captured using a microscope equipped with a digital camera. The images were recorded again after 24 h incubation at 37 °C.

RNA isolation and quantitative reverse‐transcription PCR (qRT‐PCR)

Total RNA was isolated with TRIzol (Vazyme, Nanjing, China) following the manufacturer's instructions and the cDNA was synthesized using script cDNA synthesis kit (Bio-Rad, CA, USA). Quantitative PCR was performed using SYBR Premix Ex TaqII (TaKaRa, Japan) on CFX96 real-time PCR detection system (Bio-Rad, USA) with the following primers: SYNCRIP, forward: 5′-TGAGTGGTAAAGTCAAGGTCTGG-3′, reverse: 5′-GGCAAGGTTGCGTACAAACAGC-3′; DNMT1, forward: 5′-AGGTGGAGAGTTATGACGAGGC-3′, reverse: 5′-GGTAGAATGCCTGATGGTCTGC-3′; DNMT3A, forward: 5′-CCTCTTCGTTGGAGGAATGTGC-3′, reverse: 5′-GTTTCCGCACATGAGCACCTCA-3′; DNMT3B, forward: 5′-TAACAACGGCAAAGACCGAGGG-3′, reverse: 5′-TCCTGCCACAAGACAAACAGCC-3′; p16, forward: 5′-CTCGTGCTGATGCTACTGAGGA-3′, reverse: 5′-GGTCGGCGCAGTTGGGCTCC-3′; GAPDH, forward: 5′-AATCCCATCACCATCTTCCAG-3′, reverse: 5′-AAATGAGCCCCAGCCTTC-3′. Values for each gene were normalized to the expression of GAPDH.

Western blot analysis

Total protein was extracted from SW480 and HCT116 cells using RIPA buffer (Invitrogen, MA, USA) supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, MO, USA) and quantified using the BCA detecting kit according to the manufacturer's instructions. A total of 20 μg proteins were subjected to 10% SDS-PAGE gel separation, transferred to a nitrocellulose membrane, and blocked with 5% milk for 1 h at room temperature. The membranes were cut based on the molecular weight before incubation with the primary antibodies at 4 °C overnight. After three times wash with PBST, the membrane was incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, MA, USA), and the images were captured with the ChemiDoc Imaging system (Bio-Rad, CA, USA). The densitometric quantification was performed using Image J software.

In-vivo xenograft experiment

5.0 × 106 SW480 cells infected with scr or SYNCRIP shRNA in 50 µl PBS were mixed with equal volume of matrigel. The mixture was then subcutaneously injected in the flank of six-weeks-old male NOD/SCID (The Jackson Laboratory, Stock No: 001303) mice. The tumor size was measured at the indicated times after injection. Tumor size was calculated by 0.5 × (long diameter) × (short diameter)2. The mice were euthanized by cervical dislocation and tumor weight was measured after 4 weeks. All animal experiments were conducted according to the NIH Guide for the Care and Use of Laboratory Animals.

All procedures agreed with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approval was obtained by the Ethics Committee of the Hunan Normal University. All procedures performed in our research followed the recommendations in the ARRIVE guidelines.

Database data

The SYNCRIP genetic alterations in colorectal cancer patients were obtained by searching SYNCRIP in cBioPortal database (www.cbioportal.org)28. The expression of SYNCRIP in colorectal tumor and normal tissue, different tumor types, and different disease stages was obtained by searching SYNCRIP in GEPIA (Gene Expression Profiling Interactive Analysis) database (http://gepia.cancer-pku.cn)29. The correlations analysis between SYNCRIP and DNMT3A, SYNCRIP and p16 were performed using Pearson correlation based on TCGA Tumor in GEPIA database.

Statistical analysis

Data were presented as mean ± SD from three independent experiments. P value was determined using paired Student’s t-test, and a P value ˂ 0.05 was considered statistically significant. SPSS version 20.0 (SPSS, Inc) was used to perform the statistical analysis.

Results

SYNCRIP was up-regulated in human colorectal cancer

To explore the potential function of SYNCRIP in colorectal cancer, we first analyzed the expression of SYNCRIP using publicly available database TCGA (The Cancer Genome Atlas) and GEPIA (Gene Expression Profiling Interactive Analysis). In TCGA database, the ratio of SYNCRIP genetic alterations in colorectal cancer patients was around 3% (Fig. 1A). More importantly, the expression level of SYNCRIP in colorectal tumor (n = 275) is significantly higher than in normal tissue (n = 349) (Fig. 1B,C. However, no significant difference of SYNCRIP expression was observed between different stages (Stage I-Stage IV) (Fig. 1D), which suggested that SYNCRIP is not one of the stage-specific differentially expressed genes. Furthermore, the CRC patients with higher SYNCRIP expression showed lower overall survival rate (Fig. 1E).Figure 1 Database analysis of SYNCRIP expression in colorectal cancer. (A) TCGA database showed gene alteration frequency of SYNCRIP in colorectal cancer. (B, C) Expression level of SYNCRIP in tumor is higher than in normal tissue, shown by GEPIA database. (D) Expression level of SYNCRIP in different stage of colorectal cancer. (E) The overall survival rate of patients with high or low level of SYNCRIP.

To further confirm the expression of SYNCRIP in CRC, we detected the mRNA and protein level of SYNCRIP in tumor tissue and adjacent normal tissues. Our immunohistochemistry staining showed that the positive rate of SYNCRIP was higher in CRC tissues than in normal tissues (Fig. 2A). In addition, we found that the mRNA and protein level of SYNCRIP was significantly higher in tumor tissue (Figs. 2B,C, S1A). We also compared the expression of SYNCRIP in normal human colon mucosal epithelial cell line (NCM460) and CRC cell lines (SW480, SW620, HT29, HCT8, and HCT116). Consistent with results from patients’ sample, SYNCRIP was highly expressed in CRC cell lines compared with normal NCM460 cell line (Figs. 2D,E, S1B).Figure 2 The expression of SYNCRIP in colorectal cancer cells. (A) Immunohistochemistry staining of SYNCRIP in CRC tumor tissue and adjacent normal tissues. (B, C) The mRNA and protein level of SYNCRIP in human colorectal cancer tissues and adjacent normal tissues was detected by QRT-PCR (B) and Western blotting (C). (D, E) The mRNA and protein level of SYNCRIP in human normal colon mucosal epithelial cell and colorectal cancer cell lines was detected by QRT-PCR (D) and Western blotting (E). *P ˂ 0.05, **P ˂ 0.01.

SYNCRIP regulated colorectal cancer cells apoptosis, proliferation, and motility

Our previous results showed highly expressed SYNCRIP in colorectal cancer cell, which suggested that SYNCRIP could play important role in CRC. To investigate the role of SYNCRIP in CRC, we knockdown SYNCRIP in SW480 and HCT116 cells using shRNA. Here, we tested two different shRNAs, and about 80% SYNCRIP was knockdown using both shRNAs in SW480 and HCT116 cells (Fig. 3A). Interestingly, SYNCRIP depletion increased the activity of caspase-3/7, which indicated that the apoptosis was increased (Fig. 3B). Furthermore, the cell proliferation and survival were also impaired after SYNCRIP depletion (Fig. 3C,D). To investigate the function of SYNCRIP on cell motility, we performed trans-well migration and wound healing assays. The motility of SW480 and HCT116 cells were inhibited in SYNCRIP shRNA infected cells compared with scramble (scr) infected cells (Figs. 3E,F, S2).Figure 3 Effect of SYNCRIP depletion on cells apoptosis, proliferation, and migration. (A) The knockdown efficiency of SYNCRIP in colorectal cancer cells were detected by QRT-PCR. **P ˂ 0.01, ***P ˂ 0.001. (B) The activity of caspase3/7 was measured after SYNCRIP knockdown. *P ˂ 0.05, **P ˂ 0.01. (C, D) The proliferation and survival of SW480 and HCT116 cells after SYNCRIP depletion were measured using CCK-8 assay (C) and trypan blue stain (D). *P ˂ 0.05, **P ˂ 0.01. (E) Transwell migration assay of SW480 and HCT116 cells after SYNCRIP knockdown. (F) Wound healing assay of SW480 cells after SYNCRIP knockdown.

Next, we wonder whether SYNCRIP overexpression has the reverse effects on cell apoptosis, proliferation, and motility. SYNCRIP plasmid and empty vector were used to transfect SW480 and HCT116 cells, about 2.5 times level of SYNCRIP was observed after SYNCRIP plasmid transfection (Fig. 4A). Not surprisingly, the activity of caspase-3/7 decreased, while the proliferation and motility increased after SYNCRIP overexpression, which indicated that SYNCRIP overexpression inhibited CRC cell apoptosis, but promoted cell proliferation and migration (Figs. 4B–F, S3).Figure 4 Effect of SYNCRIP overexpression on cells apoptosis, proliferation, and migration. (A) The overexpression efficiency of SYNCRIP in colorectal cancer cells were detected by QRT-PCR. *P ˂ 0.05, **P ˂ 0.01. (B) The activity of caspase3/7 was measured after SYNCRIP overexpression. **P ˂ 0.01. (C, D) The proliferation and survival of SW480 and HCT116 cells after SYNCRIP overexpression were measured using CCK- 8 assay (C) and trypan blue stain (D). *P ˂ 0.05, **P ˂ 0.01. (E) Transwell migration assay of SW480 and HCT116 cells after SYNCRIP overexpression. (F) Wound healing assay of SW480 cells after SYNCRIP overexpression.

SYNCRIP regulated colorectal cancer cell proliferation and motility via regulating DNMT3A expression

We then sought to investigate the mechanism of how SYNCRIP regulated the CRC cell proliferation and motility. DNMT was highly expressed in CRC and the expression level of SYNCRIP significantly correlated with the reduced survival probability30. In addition, SYNCRIP was reported to partner with Lin28a and regulate the expression of DNMT3A31. Thus, we wonder whether SYNCRIP could regulate DNMT expression in CRC cells. SYNCRIP depletion significantly decreased the mRNA and protein level of DNMT3A, but not DNMT1 or DNMT3B in both SW480 and HCT116 cells (Figs. 5A–C, S4A). On the contrary, overexpression of SYNCRIP significantly increased the expression of DNMT3A, but not DNMT1 or DNMT3B (Figs. 5D–F, S4B). More importantly, we found that the expression level of DNMT3A in colorectal tumor (n = 275) is higher than in normal tissue (n = 41) by analyzing TCGA database (Fig. 5G). The CRC patients with higher DNMT3A expression showed lower overall survival rate (Fig. S4C). Furthermore, the correlation between the expression of SYNCRIP and DNMT3A was studied by linear regression analysis of data from the TCGA database. A significant positive association between the expression of SYNCRIP and DNMT3A in CRC was identified (P = 2.6e−10), and the R2 coefficient was 0.37 (Fig. 5H), which suggested a moderate correlation between SYNCRIP and DNMT3A.Figure 5 Effect of SYNCRIP on regulating the expression of DNMT3A. (A–C) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA (SYN sh) for 72 h, the mRNA and protein level of DNMTs were detected by QRT-PCR (A, B) and Western blotting (C). *P ˂ 0.05, **P ˂ 0.01. (D–F) SW480 and HCT116 cells were transfected with empty vector (EV) and SYNCRIP plasmid, the mRNA and protein level of DNMTs were detected by QRT-PCR (D, E) and Western blotting (F). * P ˂ 0.05, ** P ˂ 0.01. (G) Expression level of DNMT3A in tumor is higher than in normal tissue, shown by GEPIA database. (H) The correlation between SYNCRIP and DNMT3A in colorectal cancer.

Since we have found that SYNCRIP regulated the expression of DNMT3A, we wonder whether SYNCRIP regulated CRC cell proliferation and motility via DNMT3A. We knockdown SYNCRIP using shRNA followed with or without DNMT3A overexpression in SW480 and HCT116 cells (Figs. 6A, S5A). Consistent with our previous finding (Fig. 3), SYNCRIP depletion significantly inhibited CRC cell proliferation and motility. However, DNMT3A overexpression blocked the inhibitory effect of SYNCRIP depletion (Figs. 6B–E, S5B,C), which suggested that SYNCRIP regulated CRC cell proliferation and motility via DNMT3A.Figure 6 SYNCRIP regulated colorectal cancer cell growth and migration via DNMT3A. (A) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the protein level of DNMT3A was detected by Western blotting. (B, C) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the proliferation and survival of SW480 and HCT116 cells were measured using CCK-8 assay (B) and trypan blue stain (C). ** P ˂ 0.01. (D, E) SW480 and HCT116 cells were infected with scr or SYNCRIP shRNA followed with or without DNMT3A plasmid, the motility of SW480 and HCT116 cells were analyzed using migration assay (D) and wound healing assay (E).

SYNCRIP regulated the expression of p16 via regulating DNMT3A expression

DNMTs could suppress the expression of tumor suppressor genes in human cancer cells32. P16 is one of the most common studied tumor suppressor genes, and is regulated by DNMT3s in cervical cancer33. To confirm that DNMT3A could regulate the expression of p16 in colorectal cancer, we knockdown the DNMT3A in SW480 and HCT116 cells. The protein and mRNA level increased after DNMT3A depletion (Figs. 7A,B, S6A). Not surprisingly, the expression of p16 increased after SYNCRIP depletion. However, DNMT3A overexpression blocked the effect of SYNCRIP depletion on p16 expression, which indicated that SYNCRIP regulated the expression of p16 via DNMT3A (Figs. 7C,D, S6B). Furthermore, the linear regression analysis of data from the TCGA database indicated negative correlation between SYNCRIP and p16 (Fig. 7E).Figure 7 SYNCRIP regulated the expression of p16 via DNMT3A. (A, B) SW480 and HCT116 cells were infected with scr or DNMT3A shRNA for 72 h, the protein and mRNA level of p16 were detected by Western blotting (A) and QRT-PCR (B). **P ˂ 0.01. (C, D) SW480 and HCT116 cells were infected with scr or SYNCRIP (shRNA) shRNA followed with or without DNMT3A plasmid, the protein and mRNA level of p16 were detected by Western blotting (C) and QRT-PCR (D). **P ˂ 0.01. (E) The correlation between SYNCRIP and p16 in colorectal cancer.

SYNCRIP regulated colorectal cancer cell growth in vivo

To further confirm SYNCRIP as a therapeutic target, we tested the function of SYNCRIP on colorectal tumor cell growth in a mouse xenograft model. SW480 cells were infected with scramble or SYNCRIP shRNA before injected into NOD/SCID mice frank. At the end of the experiments (4 weeks), the mice were euthanized, and the weight of tumor was analyzed. As shown in Fig. 8A–D, the tumor size and weight were significantly decreased in SYNCRIP depletion group compared with control group. Moreover, immunohistochemical staining showed that the cell proliferation indicator Ki67 of tumors was decreased in the SYNCRIP depletion group. The effect of SYNCRIP depletion on DNMT3A, DNMT3B, and p16 expression were evaluated. As shown in Fig. 8E and F, the protein level of DNMT3A and p16 were lower, while p16 level is higher in SYNCRIP depletion group compared with scramble control group.Figure 8 Effects of SYNCRIP depletion on tumor growth in vivo. (A) Typical photos of tumors on day 28 from scr and on shRNA groups. (B) HE and Ki67 staining of sections from xenograft tumors. (C, D) SYNCRIP depletion significantly decreased the tumor size (C) and weight (D). **P ˂ 0.01, ***P ˂ 0.001. (E, F) Protein level of SYNCRIP and p16 in 3 representative tumors from each group were analyzed by western blotting (E) and quantified by densitometry analysis (F). *P ˂ 0.05, **P ˂ 0.01.

Discussion

As a group of RNA-binding proteins (RBPs), hnRNPs are important for gene expression regulation and many of the hnRNPs are keystones in tumor development. The functions of hnRNPs rely on their cellular localization. Most of the hnRNPs are predominantly present in the nucleus and possess a conventional nuclear localization signal6. hnRNP proteins undergo several post-translational modifications, and such changes regulate their subcellular localization and biological activity. Reported post-translational modifications on hnRNPs include phosphorylation, simulation, ubiquitination, and methylation34. For example, MAPK/ERK phosphorylated hnRNP K, which in turn change its cellular localization and ability to silence mRNA translation35. Except post-translational modification, the expression of SYNCRIP could be regulated by oncogenic transcription factor, such as c-Myc. C-Myc upregulates the expression of hnRNPs, which ensuring a high PKM2/PKM1 ratio in cancer cells36. Another oncoprotein, KITENIN promotes aerobic glycolysis by upregulating c-myc/hnRNP axis in colorectal cancer37. In addition, lncNT5E enhanced the activity of SYNCRIP promoter to promote its transcription in pancreatic cancer38. The downstream targets of hnRNPs include multiple oncogenes. The hnRNP A1 has been associated with tumor cell growth and metastasis in several different cancer types. Several oncogenes, including Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS) and a splice variant of Recepteur d’Origine Nantais (ΔRON), have been identified as direct targets of hnRNP A139. hnRNP C was also reported to regulate another oncogene named breast cancer (BRCA)40. Its role in gene expression regulation, either on transcriptional or translational level, makes hnRNP E one of the best-studied hnRNP in cancer research. hnRNP E1 together with hnRNP E2 regulated the half-life of p21WAF and therefore regulate the cell cycle in a p53-independent manner41. hnRNP E1 could also stabilized the mRNA of p63, another transcription factor and a p53 family protein42. Unlike other hnRNPs, hnRNP Q (also known as SYNCRIP) was less associated with cancer. However, two studies showed that one of the hnRNP Q isoform, hnRNP Q1, is involved in tumorigenesis via regulating cell cycle-related genes, such as Aurora-A in colorectal cancer18,19, which suggested SYNCRIP as a key player in colorectal cancer development and pathology. We first analyzed the expression of SYNCRIP in TCGA and GEPIA database and found that the expression of SYNCRIP is significantly higher in CRC tumor compared with normal tissue. These finding was further confirmed by comparing the expression of SYNCRIP from tumor tissue and adjacent normal tissues, normal human colon mucosal epithelial cell line and CRC cell lines. More importantly, SYNCRIP depletion inhibited CRC cell growth and migration, whereas SYNCRIP overexpression promoted CRC cell growth and migration. These results suggested SYNCRIP as an oncogene in colorectal cancer.

Accumulating evidence suggests that aberrant DNA methylation was a driver of many human malignant tumor types43,44. DNA methylation is an epigenetic modification typically occurred in the CpG islands of the gene promoter region and is catalyzed by a family of DNA methyltransferases (DNMTs). This modification will generate highly stable and transmittable gene silencing45. There are three subtypes of DNMTs in mammals, DNMT1, DNMT3A, and DNMT3B. Among the three DNMTs, DNMT1 prefers to methylate hemi-methylated DNA to maintain the existing DNA methylation, while DNMT3A and DNMT3B are de novo methyltransferases that apply to unmethylated DNA, and are critical enzymes during embryogenesis and pathogenesis46. Many studies have shown that dysregulation of DNMT3A leads to oncogenesis in several cancer types, including gastric cancer, hepatocellular cancer, and colorectal cancer26,27,47. To investigate the mechanism of how SYNCRIP regulated the CRC cell proliferation and motility, we investigated the effect of SYNCRIP on DNMTs expression. Surprisingly, SYNCRIP only regulated the expression of DNMT3A, but not DNMT1 and DNMT3B. Also, a significant positive correlation between the expression of SYNCRIP and DNMT3A in CRC was identified. More importantly, DNMT3A overexpression blocked the inhibitory effect of SYNCRIP depletion on CRC cell growth and migration, which suggested that SYNCRIP regulated CRC cell tumorigenesis via DNMT3A. However, the mechanism of how SYNCRIP regulates the expression of DNMT3A remains unclear. Indeed, an important binding partner of SYNCRIP, Lin28a, was proved to bind Dnmt3a mRNA and regulate the expression of DNMT3A protein31. More importantly, the activity of Lin28a is dependent on its binding partner, such as SYNCRIP31, which suggested that SYNCRIP could regulate the expression of DNMT3A through Lin28a.

The incidence of colorectal cancer (CRC) is increasing globally and has an extremely high mortality rate. Activation of oncogenes and inactivation of tumor suppressor genes contribute to the manipulation of CRC malignant phenotypes. P16 is one of the most common studied tumor suppressor genes. The promoter regions of p16 are often methylated, which decreases the levels of p16. Previous studies have demonstrated that p16 promoter hypermethylation was found in 26% of CRC cases48. In cervical cancer, DNMT3A and DNMT3B regulated the methylation of p16 promoter, and affected its expression33. Thus, we wonder whether DNMT3A could regulate p16 expression in CRC cells. We found that DNMT3A depletion increased the expression of p16, which showed same results as SYNCRIP depletion. More importantly, DNMT3A overexpression blocked the effect of SYNCRIP depletion on p16 expression, which indicated that SYNCRIP regulated p16 expression via DNMT3A.

Taken all together, we investigated the important role of SYNCRIP in regulating colorectal cancer growth and motility in the present study. We found that SYNCRIP regulated the expression of DNMT3A, which then methylated the promoter of tumor suppressor-p16 and inhibited its expression. Our results suggested SYNCRIP as a potential prognostic biomarker and therapeutic target in colorectal cancer.

Supplementary Information

Supplementary Figures.

Supplementary Information 1.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-59575-6.

Author contributions

Chenglong Li, Zhige Yu, and Chaowu Chen contributed to the conception and design of the study. Chenglong Li, Tailiang Lu and Hongxi Chen performed the statistical analysis, the experimental operation and organized the experimental data. Chenglong Li and Chaowu Chen wrote the first draft of the manuscript. Tailiang Lu and Hongxi Chen contributed to data collection and some of the experiments.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Siegel RL Miller KD Fuchs HE Jemal A Cancer statistics, 2022 CA Cancer J. Clin. 2022 72 7 33 10.3322/caac.21708 35020204
Siegel, R. L., Miller, K. D., Fuchs, H. E. & Jemal, A. Cancer statistics, 2022. CA Cancer J. Clin. 72, 7–33 (2022).35020204 10.3322/caac.21708
2. Thanikachalam K Khan G Colorectal cancer and nutrition Nutrients 2019 11 1 10 10.3390/nu11010164
Thanikachalam, K. & Khan, G. Colorectal cancer and nutrition. Nutrients 11, 1–10 (2019).10.3390/nu11010164
3. Buccafusca G Proserpio I Tralongo AC Rametta Giuliano S Tralongo P Early colorectal cancer: Diagnosis, treatment and survivorship care Crit. Rev. Oncol. Hematol. 2019 136 20 30 10.1016/j.critrevonc.2019.01.023 30878125
Buccafusca, G., Proserpio, I., Tralongo, A. C., Rametta Giuliano, S. & Tralongo, P. Early colorectal cancer: Diagnosis, treatment and survivorship care. Crit. Rev. Oncol. Hematol. 136, 20–30 (2019).30878125 10.1016/j.critrevonc.2019.01.023
4. Vasaikar S Huang C Wang X Petyuk VA Savage SR Wen B Dou Y Zhang Y Shi Z Arshad OA Gritsenko MA Zimmerman LJ McDermott JE Clauss TR Moore RJ Zhao R Monroe ME Wang YT Chambers MC Slebos RJC Lau KS Mo Q Ding L Ellis M Thiagarajan M Kinsinger CR Rodriguez H Smith RD Rodland KD Liebler DC Liu T Zhang B Clinical Proteomic Tumor Analysis Proteogenomic analysis of human colon cancer reveals new therapeutic opportunities Cell 2019 177 1035 1049 10.1016/j.cell.2019.03.030 31031003
Vasaikar, S. et al. Proteogenomic analysis of human colon cancer reveals new therapeutic opportunities. Cell 177, 1035–1049 (2019).31031003 10.1016/j.cell.2019.03.030
5. Boman BM Huang E Human colon cancer stem cells: A new paradigm in gastrointestinal oncology J. Clin. Oncol. 2008 26 2828 2838 10.1200/JCO.2008.17.6941 18539961
Boman, B. M. & Huang, E. Human colon cancer stem cells: A new paradigm in gastrointestinal oncology. J. Clin. Oncol. 26, 2828–2838 (2008).18539961 10.1200/JCO.2008.17.6941
6. Geuens T Bouhy D Timmerman V The hnRNP family: Insights into their role in health and disease Hum. Genet. 2016 135 851 867 10.1007/s00439-016-1683-5 27215579
Geuens, T., Bouhy, D. & Timmerman, V. The hnRNP family: Insights into their role in health and disease. Hum. Genet. 135, 851–867 (2016).27215579 10.1007/s00439-016-1683-5
7. Chaudhury A Chander P Howe PH Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles RNA 2010 16 1449 1462 10.1261/rna.2254110 20584894
Chaudhury, A., Chander, P. & Howe, P. H. Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1’s multifunctional regulatory roles. RNA 16, 1449–1462 (2010).20584894 10.1261/rna.2254110
8. Liu X Zhou Y Lou Y Zhong H Knockdown of HNRNPA1 inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at G0/G1 phase Gene 2016 576 791 797 10.1016/j.gene.2015.11.009 26581508
Liu, X., Zhou, Y., Lou, Y. & Zhong, H. Knockdown of HNRNPA1 inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at G0/G1 phase. Gene 576, 791–797 (2016).26581508 10.1016/j.gene.2015.11.009
9. Li M Yang X Zhang G Wang L Zhu Z Zhang W Huang H Gao R Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway Thorac. Cancer 2022 13 1311 1321 10.1111/1759-7714.14387 35352475
Li, M. et al. Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway. Thorac. Cancer 13, 1311–1321 (2022).35352475 10.1111/1759-7714.14387
10. Ushigome M Ubagai T Fukuda H Tsuchiya N Sugimura T Takatsuka J Nakagama H Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers Int. J. Oncol. 2005 26 635 640 15703818
Ushigome, M. et al. Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers. Int. J. Oncol. 26, 635–640 (2005).15703818
11. Jo OD Martin J Bernath A Masri J Lichtenstein A Gera J Heterogeneous nuclear ribonucleoprotein A1 regulates cyclin D1 and c-myc internal ribosome entry site function through Akt signaling J. Biol. Chem. 2008 283 23274 23287 10.1074/jbc.M801185200 18562319
Jo, O. D. et al. Heterogeneous nuclear ribonucleoprotein A1 regulates cyclin D1 and c-myc internal ribosome entry site function through Akt signaling. J. Biol. Chem. 283, 23274–23287 (2008).18562319 10.1074/jbc.M801185200
12. Carpenter B McKay M Dundas SR Lawrie LC Telfer C Murray GI Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer Br. J. Cancer 2006 95 921 927 10.1038/sj.bjc.6603349 16953238
Carpenter, B. et al. Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer. Br. J. Cancer 95, 921–927 (2006).16953238 10.1038/sj.bjc.6603349
13. Murakami Y Konishi H Fujiya M Takahashi K Ando K Ueno N Kashima S Moriichi K Tanabe H Okumura T Testis-specific hnRNP is expressed in colorectal cancer cells and accelerates cell growth mediating ZDHHC11 mRNA stabilization Cancer Med. 2022 11 3643 10.1002/cam4.4738 35384384
Murakami, Y. et al. Testis-specific hnRNP is expressed in colorectal cancer cells and accelerates cell growth mediating ZDHHC11 mRNA stabilization. Cancer Med. 11, 3643 (2022).35384384 10.1002/cam4.4738
14. Konishi H Fujiya M Kashima S Sakatani A Dokoshi T Ando K Ueno N Iwama T Moriichi K Tanaka H Okumura T A tumor-specific modulation of heterogeneous ribonucleoprotein A0 promotes excessive mitosis and growth in colorectal cancer cells Cell Death Dis. 2020 11 245 10.1038/s41419-020-2439-7 32303675
Konishi, H. et al. A tumor-specific modulation of heterogeneous ribonucleoprotein A0 promotes excessive mitosis and growth in colorectal cancer cells. Cell Death Dis. 11, 245 (2020).32303675 10.1038/s41419-020-2439-7
15. Bannai H Fukatsu K Mizutani A Natsume T Iemura S Ikegami T Inoue T Mikoshiba K An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites J. Biol. Chem. 2004 279 53427 53434 10.1074/jbc.M409732200 15475564
Bannai, H. et al. An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites. J. Biol. Chem. 279, 53427–53434 (2004).15475564 10.1074/jbc.M409732200
16. Kim DY Kim W Lee KH Kim SH Lee HR Kim HJ Jung Y Choi JH Kim KT hnRNP Q regulates translation of p53 in normal and stress conditions Cell Death Differ. 2013 20 226 234 10.1038/cdd.2012.109 22935615
Kim, D. Y. et al. hnRNP Q regulates translation of p53 in normal and stress conditions. Cell Death Differ. 20, 226–234 (2013).22935615 10.1038/cdd.2012.109
17. Lyabin DN Nigmatullina LF Doronin AN Eliseeva IA Ovchinnikov LP Identification of proteins specifically interacting with YB-1 mRNA 3′ UTR and the effect of hnRNP Q on YB-1 mRNA translation Biochemistry 2013 78 651 659 23980891
Lyabin, D. N., Nigmatullina, L. F., Doronin, A. N., Eliseeva, I. A. & Ovchinnikov, L. P. Identification of proteins specifically interacting with YB-1 mRNA 3′ UTR and the effect of hnRNP Q on YB-1 mRNA translation. Biochemistry 78, 651–659 (2013).23980891
18. Lai CH Huang YC Lee JC Tseng JT Chang KC Chen YJ Ding NJ Huang PH Chang WC Lin BW Chen RY Wang YC Lai YC Hung LY Translational upregulation of Aurora-A by hnRNP Q1 contributes to cell proliferation and tumorigenesis in colorectal cancer Cell Death Dis. 2017 8 e2555 10.1038/cddis.2016.479 28079881
Lai, C. H. et al. Translational upregulation of Aurora-A by hnRNP Q1 contributes to cell proliferation and tumorigenesis in colorectal cancer. Cell Death Dis. 8, e2555 (2017).28079881 10.1038/cddis.2016.479
19. Wang YC Chang KC Lin BW Lee JC Lai CH Lin LJ Yen Y Lin CS Yang SJ Lin PC Lee CT Hung LY The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes Exp. Mol. Med. 2018 50 1 14 30504803
Wang, Y. C. et al. The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes. Exp. Mol. Med. 50, 1–14 (2018).30504803
20. Kohler F Rodriguez-Paredes M DNA methylation in epidermal differentiation, aging, and cancer J. Invest. Dermatol. 2020 140 38 47 10.1016/j.jid.2019.05.011 31427190
Kohler, F. & Rodriguez-Paredes, M. DNA methylation in epidermal differentiation, aging, and cancer. J. Invest. Dermatol. 140, 38–47 (2020).31427190 10.1016/j.jid.2019.05.011
21. Menezo Y Clement P Clement A Elder K Methylation: An ineluctable biochemical and physiological process essential to the transmission of life Int. J. Mol. Sci. 2020 21 9311 10.3390/ijms21239311 33297303
Menezo, Y., Clement, P., Clement, A. & Elder, K. Methylation: An ineluctable biochemical and physiological process essential to the transmission of life. Int. J. Mol. Sci. 21, 9311 (2020).33297303 10.3390/ijms21239311
22. Hassouna MM Naguib M Radwan EM Abdel-Samiee M Estaphan S Abdelsameea E DNA methyltransferases as potential biomarkers for HCV related hepatocellular carcinoma Asian Pac. J. Cancer Prev. 2020 21 3357 3363 10.31557/APJCP.2020.21.11.3357 33247696
Hassouna, M. M. et al. DNA methyltransferases as potential biomarkers for HCV related hepatocellular carcinoma. Asian Pac. J. Cancer Prev. 21, 3357–3363 (2020).33247696 10.31557/APJCP.2020.21.11.3357
23. Zhu A Hopkins KM Friedman RA Bernstock JD Broustas CG Lieberman HB DNMT1 and DNMT3B regulate tumorigenicity of human prostate cancer cells by controlling RAD9 expression through targeted methylation Carcinogenesis 2021 42 220 231 10.1093/carcin/bgaa088 32780107
Zhu, A. et al. DNMT1 and DNMT3B regulate tumorigenicity of human prostate cancer cells by controlling RAD9 expression through targeted methylation. Carcinogenesis 42, 220–231 (2021).32780107 10.1093/carcin/bgaa088
24. Cervena K Siskova A Buchler T Vodicka P Vymetalkova V Methylation-based therapies for colorectal cancer Cells 2020 9 1540 10.3390/cells9061540 32599894
Cervena, K., Siskova, A., Buchler, T., Vodicka, P. & Vymetalkova, V. Methylation-based therapies for colorectal cancer. Cells 9, 1540 (2020).32599894 10.3390/cells9061540
25. Liu H Song Y Qiu H Liu Y Luo K Yi Y Jiang G Lu M Zhang Z Yin J Zeng S Chen X Deng M Jia X Gu Y Chen D Zheng G He Z Downregulation of FOXO3a by DNMT1 promotes breast cancer stem cell properties and tumorigenesis Cell Death Differ. 2020 27 966 983 10.1038/s41418-019-0389-3 31296961
Liu, H. et al. Downregulation of FOXO3a by DNMT1 promotes breast cancer stem cell properties and tumorigenesis. Cell Death Differ. 27, 966–983 (2020).31296961 10.1038/s41418-019-0389-3
26. Zhou Y Yang Z Zhang H Li H Zhang M Wang H Zhang M Qiu P Zhang R Liu J DNMT3A facilitates colorectal cancer progression via regulating DAB2IP mediated MEK/ERK activation Biochim. Biophys. Acta Mol. Basis Dis. 2022 1868 166353 10.1016/j.bbadis.2022.166353 35063646
Zhou, Y. et al. DNMT3A facilitates colorectal cancer progression via regulating DAB2IP mediated MEK/ERK activation. Biochim. Biophys. Acta Mol. Basis Dis. 1868, 166353 (2022).35063646 10.1016/j.bbadis.2022.166353
27. Zhang X Yang Y Zhang W Huang K Xu L Shahid N Pan Y Xu C Jiao X Yang K Downregulation of MiR-1538 promotes proliferation and metastasis of colorectal cancer by targeting DNMT3A Biochem. Biophys. Res. Commun. 2022 609 119 126 10.1016/j.bbrc.2022.04.006 35429679
Zhang, X. et al. Downregulation of MiR-1538 promotes proliferation and metastasis of colorectal cancer by targeting DNMT3A. Biochem. Biophys. Res. Commun. 609, 119–126 (2022).35429679 10.1016/j.bbrc.2022.04.006
28. Cerami E The cBio cancer genomics portal: An open platform for exploring multidimensional cancer genomics data Cancer Discov. 2012 2 401 404 10.1158/2159-8290.CD-12-0095 22588877
Cerami, E. et al. The cBio cancer genomics portal: An open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).22588877 10.1158/2159-8290.CD-12-0095
29. Tang Z Li C Kang B Gao G Li C Zhang Z GEPIA: A web server for cancer and normal gene expression profiling and interactive analyses Nucleic Acids Res. 2017 45 W98 w102 10.1093/nar/gkx247 28407145
Tang, Z. et al. GEPIA: A web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 45, W98-w102 (2017).28407145 10.1093/nar/gkx247
30. Meng X Liu N Jia Y Gong K Zhang J Zhang W Shao G Dang T DNMT3B expression might contribute to abnormal methylation of RASSF1A in lager colorectal adenomatous polyps Gastroenterol. Res. Pract. 2020 2020 1798729 10.1155/2020/1798729 33061956
Meng, X. et al. DNMT3B expression might contribute to abnormal methylation of RASSF1A in lager colorectal adenomatous polyps. Gastroenterol. Res. Pract. 2020, 1798729 (2020).33061956 10.1155/2020/1798729
31. Parisi S Castaldo D Piscitelli S D'Ambrosio C Divisato G Passaro F Avolio R Castellucci A Gianfico P Masullo M Scaloni A Russo T Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression Sci. Rep. 2021 11 2345 10.1038/s41598-021-81429-8 33504840
Parisi, S. et al. Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression. Sci. Rep. 11, 2345 (2021).33504840 10.1038/s41598-021-81429-8
32. Kedhari Sundaram M Hussain A Haque S Raina R Afroze N Quercetin modifies 5′CpG promoter methylation and reactivates various tumor suppressor genes by modulating epigenetic marks in human cervical cancer cells J. Cell Biochem. 2019 120 18357 18369 10.1002/jcb.29147 31172592
Kedhari Sundaram, M., Hussain, A., Haque, S., Raina, R. & Afroze, N. Quercetin modifies 5′CpG promoter methylation and reactivates various tumor suppressor genes by modulating epigenetic marks in human cervical cancer cells. J. Cell Biochem. 120, 18357–18369 (2019).31172592 10.1002/jcb.29147
33. Wang A Xu Q Sha R Bao T Xi X Guo G MicroRNA-29a inhibits cell proliferation and arrests cell cycle by modulating p16 methylation in cervical cancer Oncol. Lett. 2021 21 272 10.3892/ol.2021.12533 33717269
Wang, A. et al. MicroRNA-29a inhibits cell proliferation and arrests cell cycle by modulating p16 methylation in cervical cancer. Oncol. Lett. 21, 272 (2021).33717269 10.3892/ol.2021.12533
34. Chaudhury A Chander P Howe PH Heterogeneous nuclear ribonucleo proteins (hnRNPs) in cellular processes: Focus on hnRNP E1’s multifunctional regulatory roles RNA 2010 16 1449 1462 10.1261/rna.2254110 20584894
Chaudhury, A., Chander, P. & Howe, P. H. Heterogeneous nuclear ribonucleo proteins (hnRNPs) in cellular processes: Focus on hnRNP E1’s multifunctional regulatory roles. RNA 16, 1449–1462 (2010).20584894 10.1261/rna.2254110
35. Habelhah H Shah K Huang L Ostareck-Lederer A Burlingame AL Shokat KM Hentze MW Ronai Z ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation Nat. Cell Biol. 2001 3 325 330 10.1038/35060131 11231586
Habelhah, H. et al. ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation. Nat. Cell Biol. 3, 325–330 (2001).11231586 10.1038/35060131
36. David CJ Chen M Assanah M Canoll P Manley JL HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer Nature 2010 463 364 368 10.1038/nature08697 20010808
David, C. J., Chen, M., Assanah, M., Canoll, P. & Manley, J. L. HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature 463, 364–368 (2010).20010808 10.1038/nature08697
37. Varlı M Kim SJ Noh MG Kim YG Ha HH Kim KK Kim H KITENIN promotes aerobic glycolusis through PKM2 induction by upregulating the c-Myc/hnRNPs axis in colorectal cancer Cell Biosci. 2023 13 146 10.1186/s13578-023-01089-1 37553596
Varlı, M. et al. KITENIN promotes aerobic glycolusis through PKM2 induction by upregulating the c-Myc/hnRNPs axis in colorectal cancer. Cell Biosci. 13, 146 (2023).37553596 10.1186/s13578-023-01089-1
38. Zhang P Cao M Zhang Y Xu L Meng F Wu X Xia T Chen Q Shi G Wu P Chen L Lu Z Yin J Cai B Cao S Miao Y Jiang K A novel antisense lncRNA NT5E promotes progression by modulating the expression of SYNCRIP and predicts a poor prognosis in pancreatic cancer J. Cell Mol. Med. 2020 24 10898 10912 10.1111/jcmm.15718 32770626
Zhang, P. et al. A novel antisense lncRNA NT5E promotes progression by modulating the expression of SYNCRIP and predicts a poor prognosis in pancreatic cancer. J. Cell Mol. Med. 24, 10898–10912 (2020).32770626 10.1111/jcmm.15718
39. Jean-Philippe J Paz S Caputi M hnRNP A1: The Swiss army knife of gene expression Int. J. Mol. Sci. 2013 14 18999 19024 10.3390/ijms140918999 24065100
Jean-Philippe, J., Paz, S. & Caputi, M. hnRNP A1: The Swiss army knife of gene expression. Int. J. Mol. Sci. 14, 18999–19024 (2013).24065100 10.3390/ijms140918999
40. Anantha RW Alcivar AL Ma J Cai H Simhadri S Ule J Konig J Xia B Requirement of heterogeneous nuclear ribonucleoprotein C for BRCA gene expression and homologous recombination PLoS ONE 2013 8 e61368 10.1371/journal.pone.0061368 23585894
Anantha, R. W. et al. Requirement of heterogeneous nuclear ribonucleoprotein C for BRCA gene expression and homologous recombination. PLoS ONE 8, e61368 (2013).23585894 10.1371/journal.pone.0061368
41. Waggoner SA Johannes GJ Liebhaber SA Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest J. Biol. Chem. 2009 284 9039 9049 10.1074/jbc.M806986200 19211566
Waggoner, S. A., Johannes, G. J. & Liebhaber, S. A. Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest. J. Biol. Chem. 284, 9039–9049 (2009).19211566 10.1074/jbc.M806986200
42. Cho SJ Jung YS Chen X Poly (C)-binding protein 1 regulates p63 expression through mRNA stability PLoS ONE 2013 8 e71724 10.1371/journal.pone.0071724 23940783
Cho, S. J., Jung, Y. S. & Chen, X. Poly (C)-binding protein 1 regulates p63 expression through mRNA stability. PLoS ONE 8, e71724 (2013).23940783 10.1371/journal.pone.0071724
43. Meir Z Mukamel Z Chomsky E Lifshitz A Tanay A Single-cell analysis of clonal maintenance of transcriptional and epigenetic states in cancer cells Nat. Genet. 2020 52 709 718 10.1038/s41588-020-0645-y 32601473
Meir, Z., Mukamel, Z., Chomsky, E., Lifshitz, A. & Tanay, A. Single-cell analysis of clonal maintenance of transcriptional and epigenetic states in cancer cells. Nat. Genet. 52, 709–718 (2020).32601473 10.1038/s41588-020-0645-y
44. Phillips RE Soshnev AA Allis CD Epigenomic reprogramming as a driver of malignant glioma Cancer Cell 2020 38 647 660 10.1016/j.ccell.2020.08.008 32916125
Phillips, R. E., Soshnev, A. A. & Allis, C. D. Epigenomic reprogramming as a driver of malignant glioma. Cancer Cell 38, 647–660 (2020).32916125 10.1016/j.ccell.2020.08.008
45. Jeltsch A Jurkowska RZ Allosteric control of mammalian DNA methyltransferases: A new regulatory paradigm Nucleic Acids Res. 2016 44 8556 8575 10.1093/nar/gkw723 27521372
Jeltsch, A. & Jurkowska, R. Z. Allosteric control of mammalian DNA methyltransferases: A new regulatory paradigm. Nucleic Acids Res. 44, 8556–8575 (2016).27521372 10.1093/nar/gkw723
46. Masalmeh RHA Taglini F Rubio-Ramon C Musialik KI Higham J Davidson-Smith H Kafetzopoulos I Pawlicka KP Finan HM Clark R Wills J Finch AJ Murphy L Sproul D De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG islands Nat. Commun. 2021 12 694 10.1038/s41467-020-20716-w 33514701
Masalmeh, R. H. A. et al. De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG islands. Nat. Commun. 12, 694 (2021).33514701 10.1038/s41467-020-20716-w
47. Sun W Ma G Zhang L Wang P Zhang N Wu Z Dong Y Cai F Chen L Liu H Liang H Deng J DNMT3A-mediated silence in ADAMTS9 expression is restored by RNF180 to inhibit viability and motility in gastric cancer cells Cell Death Dis. 2021 12 428 10.1038/s41419-021-03628-5 33931579
Sun, W. et al. DNMT3A-mediated silence in ADAMTS9 expression is restored by RNF180 to inhibit viability and motility in gastric cancer cells. Cell Death Dis. 12, 428 (2021).33931579 10.1038/s41419-021-03628-5
48. Sugara M Chowdappa R Kumar KVV Gawari R Swamy SN Kumar SS Aberrant promoter hypermethylation of p16 and RASSF1a genes in colorectal cancer: Significance in young patients Indian J. Surg. Oncol. 2021 12 454 459 10.1007/s13193-021-01325-5 34658570
Sugara, M. et al. Aberrant promoter hypermethylation of p16 and RASSF1a genes in colorectal cancer: Significance in young patients. Indian J. Surg. Oncol. 12, 454–459 (2021).34658570 10.1007/s13193-021-01325-5
