CREB3-mediated upregulation of MIR210HG transcription enhances proliferation in colon cancer cells

Abstract

Background: Understanding the regulatory mechanisms behind colon cancer (CC) pathogenesis is crucial for developing effective therapeutic strategies. Long non-coding RNA (lncRNA) MIR210HG has been implicated in various cancers, including CC, where it may play a role in tumor progression. Additionally, the transcription factor cyclic adenosine monophosphate-responsive element-binding protein 3 (CREB3) has been suggested to regulate lncRNA expression, but its role in CC remains unclear. This study investigates the CREB3-MIR210HG regulatory axis, focusing on how this interaction influences CC cell proliferation and its potential as a therapeutic target for cancer treatment.

Methods: To explore the CREB3-MIR210HG axis, bioinformatics analysis was conducted to identify the MIR210HG promoter and predict potential transcription factor binding sites. Expression levels of CREB3 and MIR210HG were analyzed using The Cancer Genome Atlas (TCGA) database, which provided a broader understanding of their correlation in human CC samples. Additionally, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed in SW480 CC cells to validate these findings at the cellular level. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) experiments were employed to confirm the binding of CREB3 to the MIR210HG promoter, providing direct evidence of their interaction. Finally, functional assays, including cell proliferation assays and knockdown experiments, were carried out to assess the impact of MIR210HG on CREB3-induced proliferation in CC cells.

Results: Bioinformatics and experimental analysis revealed that CREB3 directly binds to the MIR210HG promoter, leading to significant upregulation of MIR210HG transcription in CC cells. Data from TCGA and RT-qPCR analyses showed a positive correlation between CREB3 and MIR210HG expression in CC tissues, supporting the hypothesis of a regulatory link. Functional assays demonstrated that overexpression of CREB3 enhanced CC cell proliferation, while silencing MIR210HG reversed this effect, indicating that MIR210HG mediates CREB3-induced proliferation. These results suggest that the CREB3-MIR210HG axis plays a critical role in CC progression.

Conclusions: This study highlights the CREB3-MIR210HG axis as a pivotal mechanism driving CC cell proliferation. Targeting this regulatory pathway may provide a novel therapeutic strategy for CC treatment, with the potential for developing lncRNA-based therapies aimed at inhibiting this axis to slow down tumor growth and progression.

Keywords: MIR210HG; Transcription regulation; colon cancer (CC); cyclic adenosine monophosphate-responsive element-binding protein 3 (CREB3); long non-coding RNA (lncRNA).

Figure 1

Expression and regulatory association of MIR210HG and CREB3 in CC. (A) Differential expression analysis of MIR210HG in CC samples stratified by CREB3 expression. Significant upregulation of MIR210HG was observed in the high CREB3 expression cohort. (B) Co-expression correlation analysis between CREB3 and MIR210HG using the starBase v3.0 platform. A significant positive correlation was found in CC samples (P<0.05), suggesting an interactive and regulatory association between CREB3 and MIR210HG relevant to the molecular pathology of the disease. The blue points represent the data points showing the co-expression correlation, and the red line indicates the fitted linear trend, highlighting the positive correlation between the two genes. (C,D) Quantification of MIR210HG expression in SW480 and SW620 cells following experimental manipulations of CREB3 expression, including overexpression and knockdown. Data from RT-qPCR analysis revealed that MIR210HG levels positively correlated with CREB3 expression, while CREB3 suppression resulted in decreased MIR210HG mRNA. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CC, colon cancer; COAD, colon adenocarcinoma; CREB3, cyclic adenosine monophosphate-responsive element-binding protein 3; FPKM, fragments per kilobase of transcript per million mapped reads; LV-CREB3, cells transfected with CREB3 overexpression lentivirus; LV, lentivirus; LV-NC, cells transfected with negative control lentivirus; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sh-CREB3, short hairpin RNA targeting CREB3; sh-NC, short hairpin RNA negative control.

Figure 2

Effect of various fragments of MIR210HG promoter on the expression of a luciferase reporter gene. 293T cells were transfected with PRL-TK luciferase reporter gene constructs along with MIR210HG-WT1, WT2, WT3, or WT4 promoter fragments. Transfection with WT1 showed similar luciferase activity compared to the control group pGL3-basic + PRL-TK, while WT2, WT3, and WT4 transfections exhibited a significant increase in luciferase activity relative to the control. ns, not significant; ****, P<0.0001.

Figure 3

Verification analysis of CREB3 protein expression and chromatin fragmentation. (A) Western blot analysis using CREB3 antibody to confirm the expression of CREB3 protein. Total protein loaded was 50 μg, with the CREB3 antibody diluted at 1:1,000 and detected using enhanced sensitivity exposure for 10 seconds. (B) Western blot analysis using GAPDH antibody as a loading control. The antibody was diluted at 1:5,000 and detected using standard exposure for 2 min. (C) Assessment of chromatin fragmentation. Electrophoresis results indicate that the size of fragmented DNA ranged from 100 to 500 bp, meeting the requirements for subsequent ChIP enrichment experiments. (D) ChIP-qPCR results for specific target sites of MIR210HG. The bar graphs represent the enrichment of six primer sets [1–6] targeting two distinct regions of MIR210HG. Primers 1, 2, and 3 correspond to target site 1, while primers 4, 5, and 6 correspond to target site 2. Compared to the IgG control group, primers 1, 2, and 3 targeting site 1, as well as primers 4 and 5 targeting site 2, exhibit significant enrichment, with primers 3 and 5 showing the most pronounced enrichment effects. ns, not significant; *, P<0.05; ***, P<0.001. bp, base pairs; CREB3, cyclic adenosine monophosphate-responsive element-binding protein 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (internal control/housekeeping gene); IgG, immunoglobulin G; IP, immunoprecipitation; KDa, kilodaltons; M, molecular weight marker.

Figure 4

CREB3 modulates MIR210HG expression and cellular proliferation in CC cells. (A) RT-qPCR analysis depicting the alteration in MIR210HG expression levels in SW480 and SW620 cell lines. The cell lines include those overexpressing CREB3, those with combined overexpression of CREB3 and knockdown of MIR210HG, and a control group. Notably, upregulation of MIR210HG was observed following CREB3 overexpression, which was subsequently restored to normal levels upon MIR210HG knockdown. (B) CCK-8 assay indicating the impact of MIR210HG silencing on the proliferative capabilities of CC cells induced by CREB3 overexpression. The silencing of MIR210HG partially mitigates the increased proliferation mediated by CREB3, underscoring the crucial role of CREB3 in transcriptionally activating MIR210HG and promoting cellular proliferation in SW480 and SW620 cells. (C) EdU incorporation assay providing further evidence of the impact of CREB3 overexpression and MIR210HG silencing on cellular proliferation in SW480 and SW620 cells. CREB3 overexpression significantly increased the proportion of EdU-positive cells, indicating enhanced proliferation, whereas MIR210HG knockdown mitigated this effect. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ASO, antisense oligonucleotide; CREB3, cyclic adenosine monophosphate-responsive element-binding protein 3; DAPI, 4’,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2’-deoxyuridine (click chemistry-based thymidine analogue for labeling proliferating cells); LV, lentivirus; LV-CREB3, cells transfected with CREB3 overexpression lentivirus; LV-NC, cells transfected with negative control lentivirus; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.