Colon cancer has already had a significant impact on public health.¹ The latest statistical results showed that new cases and deaths of colon cancer patients in 2020 ranked fifth and fourth respectively among 36 types of cancers.² In China, the morbidity of colon cancer ranks third among gastrointestinal malignancies.³ The occurrence of colon cancer is a complex process, including genetic and epigenetic changes.⁴ Although there has been great progress in surgical resection, radiotherapy and chemotherapy, immunotherapy and other aspects, about 30% of patients with colon cancer have dismal prognosis, which is ascribable to recurrence or metastasis.^5,6 In addition, changes in lifestyle and dietary structure have caused an increase in new cases of colon cancer year by year and gradually become younger in China.³ It is vital to clarify molecular mechanisms of tumorigenesis and progression, to find out new molecular markers for early diagnosis, prognosis, and therapeutic evaluation.

Long noncoding RNA (lncRNA) does not have protein encoding ability due to lacking an open reading frame.⁷ lncRNAs participate in the modulation of chromatin dynamics, gene expression, cell differentiation, and development,⁸ whose expression and dysfunction can result in tumors.⁹ Therefore, lncRNAs, as novel cancer biomarkers and therapeutic targets, have a strong application prospect.

It has been reported that many lncRNA abnormalities are closely related to the progression of colon cancer. lncRNA HOXB-AS3 expression was low in colorectal cancer (CRC) patients and could suppress colon cancer malignant phenotype in vivo and in vitro.¹⁰ CYTOR is dramatically increased in colon cancer and stimulates an epithelial-mesenchymal transition (EMT) program and enhances metastasis properties of cells via interacting with β-catenin.¹¹ Overexpressed CALIC can associate with RNA-binding protein hnRNP-L, while CALIC/hnRNP-L complex fosters tyrosine kinase receptor AXL to boost the metastatic potential of colon cancer cells.¹² We found that DUXAP8 was markedly upregulated in colon cancer through bioinformatics analysis. DUXAP8, located in 22q11.1 and a 2107bp RNA, was demonstrated to be dysregulated in various tumors.^13-15 For example, Meng et al.¹⁶ found that DUXAP8 could facilitate proliferation and hamper apoptosis of ovarian cancer cells via binding microRNA-590-5p. DUXAP8 fosters cell growth by hindering KLF2 expression in human hepatocellular carcinoma.¹⁷ These suggest that DUXAP8 is signally related to cancer development. However, its role and mechanisms in colon cancer progression are unknown.

microRNAs are intimately involved in tumor cell behaviors via modulating oncogenes or tumor repressor genes.¹⁸ microRNA-378a is a key modulator of energy and glucose homeostasis, spotlighting it as a possible microRNA for metabolic dysregulation improvement.¹⁹ Xu et al.²⁰ have revealed that microRNA-378a-3p makes ovarian cancer cells sensitive to cisplatin via MAPK1/GRB2. Guo et al.²¹ have revealed that microRNA-378a-3p represses cell proliferation and migration of glioblastoma multiforme via binding tetraspanin 17. microRNA-378a-3p is down-regulated in CRC cells and its expression can inhibit the growth of CRC cells by suppressing the expression of certain genes (lncRNAs MALAT1 and NEAT1, FOXQ1, and ODC1).^22,23 Although these studies have been conducted, the potential role of microRNA-378a-3p in the progression of colon cancer is still worthy of further exploration. Therefore, understanding how microRNA-378a-3p plays a major regulatory role in cancer progression and metastasis could open up significant innovative areas for therapy in colon cancer.

Forkhead box Q1 (FOXQ1) is a forkhead box transcription factor that plays a crucial role in development and hair follicle differentiation. FOXQ1 has been reported to impact various cancer progressions. For instance, in triple-negative breast cancer, FOXQ1 recruits the MLL/KMT2 histone methyltransferase complex to activate transcription, promoting EMT process and facilitating tumor cell metastasis.²⁴ FOXQ1 is upregulated in ovarian cancer, where it promotes tumor progression both in vivo and in vitro, and this promoting effect is achieved through the regulation of the WNT/β-catenin signaling pathway.²⁵ Moreover, FOXQ1 overexpression activates LDHA transcription, enhancing aerobic glycolysis and promoting pancreatic cancer cell proliferation, tumor stemness, invasion, and metastasis.²⁶ Given the role of FOXQ1 in cancer progression, it is necessary to investigate its impact on the biological functions of colon cancer.

Through exploring the downstream pathway of DUXAP8, we identified its potential crucial regulatory role as a competing endogenous RNA (ceRNA) in the lncRNA-miRNA-mRNA network. However, the specific regulatory mechanisms still require further investigation. This study aimed to elucidate the role of DUXAP8 and its downstream regulatory factors in CRC, along with their molecular mechanisms, starting from the perspective of ceRNA. This research aimed to provide new insights for the diagnosis and treatment of CRC.

1. Bioinformatics analysis

Colon cancer-related htseq-count expression dataset (465 tumor and 41 normal) and isoform expression quantification data (447 tumor and 8 normal) were obtained from TCGA database. Differential analysis was conducted on messenger RNA (mRNA) data in htseq-Count dataset and microRNA data in isoform expression quantification data by using R language “edgeR” package (|logFC|>2, adjusted p-value <0.05), with normal samples as the control.

starBase database was utilized to identify the target microRNAs of DUXAP8. The obtained target microRNAs were intersected with the differentially down-regulated microRNAs and expression correlation analysis was conducted to find out potential target microRNA. Through starBase and miRDB databases (http://mirdb.org/miRDB/index.html), target mRNAs of the potential microRNA were identified. The obtained mRNAs were intersected with the differentially upregulated mRNAs and expression correlation analysis was conducted to determine the potential mRNA.

2. Clinical samples

In this study, tumor tissue and corresponding paracancerous tissue (2 cm from the edge of the cancer tissue) of 45 patients with colon cancer in Renmin Hospital, Hubei University of Medicine from April 1, 2023, to September 31, 2023, were collected, all of which were surgically removed and independently identified by two pathologists. None of the patients received chemotherapy or radiotherapy before surgery. Tumor tissue was quickly put into an RNA preservation solution after isolation. The acquisition of the samples was approved by the Ethics Committee of Renmin Hospital, Hubei University of Medicine (approval number: SYSRMYY-031). Conforming to the ethical and legal norms, patients were treated anonymously, so patient consent was not required.

3. Cell culture

Human colon epithelial cell line FHC (BNCC281458) and colon cancer cell lines SW1116 (BNCC100262), LoVo (BNCC338601), SW480 (BNCC100604), HCT116 (BNCC287750) were all obtained from Bena Culture Collection (Beijing, China). All cell lines were maintained in DMEM medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) plus 10% FBS (Hyclone; Thermo Fisher Scientific Inc.) and 100 U/mL streptomycin/penicillin (Gibco; Thermo Fisher Scientific Inc.). Cells were kept at 37^℃ in an atmosphere with 5% CO₂. The medium was changed periodically at intervals of 2 to 3 days.

4. Cell transfection

Lentivirus packaging vectors pcDNA3.1-DUXAP8 (oe-DUXAP8) and pcDNA3.1-NC (oe-NC), sh-DUXAP8 and sh-NC were synthesized from Invitrogen (Carlsbad, CA, USA). microRNA-378a-3p mimic, mimic normal control (NC), microRNA-378a-3p inhibitor, inhibitor NC, sh-FOXQ1 and sh-NC were obtained from GenePharma (Shanghai, China). When colon cancer cell lines SW1116 and HCT116 grew into logarithmic phase, cell suspension was collected and inoculated into 6-well plates (5×10⁴ cells/well) and maintained in fresh medium. Cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific Inc.) and cultured in corresponding medium (5% CO₂, 37°C) for later use.

5. qRT-PCR assay

Total RNA was extracted from colonic cancer cells, and the extraction concentration was measured by the NanoDrop 2000 system (Thermo Fisher Scientific Inc.). Complementary DNA was synthesized by reverse transcription through Transcriptor Universal cDNA Master (Roche, Basel, Switzerland). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted with SYBR Green (Takara, Kusatsu, Japan) using ABI 7500 Real-Time PCR system (Applied Biosystem; Thermo Fisher Scientific Inc.). GAPDH was utilized as internal reference for DUXAP8 and FOXQ1 while U6 was used as endogenous reference for microRNA-378a-3p. In the results, 2^-ΔΔCt value was used for quantification and normalization control. Primer sequences are shown in Table 1.

Table 1 . Primer Sequence.

Gene	Sequence
DUXAP8	F: 5’-GAGAAGCAGTGGTGGGTTCC-3’
	R: 5’-GAGCAACACAGATGAACCGC-3’
FOXQ1	F: 5’-GATTTCTTGCTATTGACCGATGC-3’
	R: 5’-CTAATAAAGCTGTAGCCCGTTGC-3’
GAPDH	F: 5’-TGCACCACCAACTGCTTAGC-3’
	R: 5’-TGCACCACCAACTGCTTAGC-3’
microRNA-378a-3p	F: 5’-ACUGGACUUGGAGUCAGAAGG-3’
U6	F: 5’-CTCGCTTCGGCAGCACA-3’
	R: 5’-AACGCTTCACGAATTTGCGT-3’

6. Western blot assay

Reagent RIPA (Invitrogen) was utilized to extract protein from tissue samples and cells, and BCA kit (Beyotime, Shanghai, China) was chosen for measuring protein concentrations. Then, equal amounts of proteins were electrophoresed by SDS-PAGE and transferred onto PVDF membranes (Amersham; Thermo Fisher Scientific Inc.). The membranes were sealed in 5% skim milk at room temperature for 1 hour, and later incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit polyclonal antibodies, including FOXQ1 antibody (ab51340, 1:1000, Abcam, UK), cyclin D1 (ab16663, 1:200, Abcam), E-cadherin (ab227639, 1:25, Abcam), vimentin (ab137321, 1:1000, Abcam), N-cadherin (ab76011, 1:5000, Abcam), c-myc (ab32072, 1:1000, Abcam), β-catenin (ab224803, 1:400, Abcam), and GAPDH antibody (ab9485, 1:2500, Abcam). Then, the membranes were rinsed with TBST, and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG H&L (ab6721, 1:10000, Abcam) at room temperature for 1 hour. Next, TBST was utilized to rinse the membrane three times again, and an optical luminescence instrument (Amersham Imager 600; GE Healthcare, Boston, MA, USA) was used to scan and develop protein bands.

7. lncRNA localization assay

Cytoplasmic and Nuclear RNA of cell lines SW1116 and HCT116 were separated and isolated by Cytoplasmic & Nuclear RNA Purification Kit (Amyjet, Wuhan, China). qRT-PCR was used to detect DUXAP8 level in cytoplasm and nucleus. GAPDH and U6 were utilized as cytoplasmic and nuclear markers, respectively.

8. Dual-luciferase reporter gene

Targeted binding of DUXAP8 to microRNA-378a-3p and microRNA-378a-3p to FOXQ1 were validated by dual-luciferase assay. The part of DUXAP8 and FOXQ1 with binding sites of microRNA-378a-3p was inserted into the downstream pmiRGLO (Promega, Madison, WI, USA) luciferase vectors. Then, the binding sites were treated with site-directed mutagenesis method with QuickChange site-directed mutagenesis kit (Strategene, La Jolla, CA, USA). After that, luciferase reporter plasmids wild-type (WT)-DUXAP8, mutant (MUT)-DUXAP8, WT-FOXQ1 and MUT-FOXQ1 were constructed. Subsequently, plasmids were co-transfected into cell lines SW1116 and HCT116 with microRNA-378a-3p mimic or mimic NC, respectively. A dual-luciferase assay kit (Promega) was utilized for luciferase activity measurement.

9. RIP assay

Magna RNA immunoprecipitation (RIP) RNA-binding protein immunoprecipitation kit (Millipore, Burlington, MI, USA) was utilized for RIP assay. The colon cancer cell lines needed for the assay were harvested and added with RIP lysis buffer for 30 minutes. Then cell extracts were maintained with RIP buffer containing magnetic beads, and Ago2 antibody (ab32381, 1:500, Abcam) was added as the experimental group, and normal rabbit IgG (ab6721, 1:2000, Abcam) was added as the NC group. Finally, the samples were cultured with protease K, immunoprecipitated RNA was purified, and qRT-PCR was used for detection and analysis.

10. RNA pull down

To investigate the interaction between DUXAP8, microRNA-378a-3p, and the FOXQ1 gene, we performed RNA pull-down experiments for validation. Biotin-labeled microRNA-378a-3p probes were transfected into SW1116 and HCT116 cells. After 48 h of transfection, the cells were lysed using cell lysis buffer. Dynabead^TM M-280 Streptavidin Magnetic Beads (Invitrogen) were added to the lysate to pull down the biotin-labeled microRNA-378a-3p/DUXAP8 or microRNA-378a-3p/FOXQ1 complexes. The enrichment levels of DUXAP8 or FOXQ1 in the co-deposited complexes were detected using qRT-PCR.

11. MTT assay

MTT assay was utilized to analyze the viability of colon cancer cells. After colon cancer cells (5×10⁴ cells/well) were inoculated in 96-well plates for 0, 24, 48, and 72 hours, respectively, 10 μL MTT (Sigma-Aldrich, St. Louis, MO, USA) was dropped into each well. After 4 to 6 hours of incubation, the medium was discarded, and then 200 μL DMSO was added to each well and cells were incubated in the dark for a while. The proliferation curves were determined by computing relative absorbance value at 490 nm with the Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).

12. Colony formation assay

After transfection, SW1116 and HCT116 cells were inoculated into 6-well plates with 1×10³ cells/well for culture in a 5% CO₂ incubator at 37°C. After 2 weeks of culture, cell colonies were rinsed twice with PBS, fixed with 4% paraformaldehyde and stained with 2% crystal violet solution for 20 minutes. Colonies formed in the plate were imaged and the number was counted under a microscope.

13. Scratch healing assay

The cells (5×10⁵ cells/well) were seeded in 6-well plates. When the cell coverage reached 80%, the monolayer of cells was gently scraped with the tip of a 200 μL pipette. The culture plates were gently rinsed twice to discard the isolated cells. Finally, the renewing medium allowed cells to grow again for 48 hours. At 0 and 48 hours, cell migration was photographed under the microscope. Scratch healing rate=(0 hours scratch width–48 hours scratch width)/0 hours scratch width×100%.

14. Transwell assay

Cell migratory and invasive abilities were identified by using Transwell chambers with the upper chambers uncoated or precoated with matrix gel (BD Biosciences, Franklin Lakes, NJ, USA). About 2.5×10⁴ colon cancer cells were added into a 24-well Transwell upper insert with a lower chamber plus 700 μL DMEM with 10% FBS as a chemoattractant. At 36 hours after incubation, cells that did not pass through membrane were swabbed off with a cotton swab, and the left ones were fixed with 4% paraformaldehyde for 30 minutes and stained with crystal violet. Cells in each field of view were calculated and photographed under a microscope.

15. Data analysis

Each assay was done at least three times to avoid systematic errors, and the data were presented as the mean± standard deviation. Statistical methods included the Student t-test, one-way analysis of variance, chi-square test, Kaplan-Meier test, log-rank test, Pearson correlation coefficient analysis, etc. Statistical analysis was done on SPSS 22.0 software (IBM Corp., Armonk, NY, USA) or GraphPad Prism 6.0 (San Diego, CA, USA). p<0.05 indicated huge statistical significance.

1. lncRNA DUXAP8 expression is upregulated in colon cancer and associated with poor prognosis

Analysis of colon cancer data from the TCGA database, we focused on the upregulated lncRNAs given. lncRNA DUXAP8, a dramatically increased lncRNA in colon cancer tissues, was selected for investigation (Fig. 1A). lncRNA DUXAP8 level in four colon cancer cell lines was noticeably higher than that in normal cells (Fig. 1B). In colon cancer cells, lncRNA DUXAP8 level was relatively high in HCT116 cells and relatively low in SW116 cells. Therefore, HCT116 and SW116 cell lines were chosen for subsequent experiments. lncRNA DUXAP8 was conspicuously overexpressed in colon cancer tissues in comparison to adjacent normal tissues (Fig. 1C). Combined with TCGA clinical staging data, we found that the tumor stage was proportional to DUXAP8 expression (Fig. 1D), and the patients with increased lncRNA DUXAP8 expression levels obtained a short overall survival (Fig. 1E). The correlation between clinicopathological features of colon cancer patients and lncRNA DUXAP8 level was depicted in Table 2. As shown in Table 2, the chi-square test results displayed that elevation of DUXAP8 level was remarkably associated with lymph node metastasis and TNM staging, which further suggested that lncRNA DUXAP8 was a major prognosticator for colon cancer patients. Nuclear/cytoplasmic RNA fractionation assay proved that lncRNA DUXAP8 was mainly distributed in the cytoplasm (Fig. 1F), which provided the possibility for DUXAP8 to sponge microRNA. These findings implied that lncRNA DUXAP8 was increased in colon cancer and that increased lncRNA DUXAP8 was implicated in dismal prognosis in colon cancer.

Figure 1. The expression of the long noncoding RNA (lncRNA) DUXAP8 is upregulated in colon cancer and its expression is associated with poor outcomes. (A) Bioinformatics analysis of DUXAP8 expression in colon cancer and normal tissues. (B, C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results showing lncRNA DUXAP8 expression at the cell and tissue levels. (D) Correlation between DUXAP8 expression and tumor stage in colon cancer patients. (E) Kaplan-Meier analysis showing the correlation of lncRNA DUXAP8 expression and colon cancer patient overall survival. (F) lncRNA DUXAP8 expression in subcellular fractions of SW1116 and HCT116 cells assessed via qRT-PCR. *p<0.05.

Table 2 . Relationship between the Expression of DUXAP8 and Clinicopathological Parameters in Colon Cancer.

Parameter	Group	Total	DUXAP8 expression		p-value
			High	Low
Sex	Male	26	16	10	0.102
	Female	19	7	12
Age	<60 yr	33	15	18	0.208
	≥60 yr	12	8	4
Tumor size	<5 cm	27	12	15	0.273
	≥5 cm	18	11	7
Local invasion	T1-T2	29	13	16	0.256
	T3-T4	16	10	6
Histological grade	Well and moderately	23	13	10	0.458
	Poorly	22	10	12
Lymph node metastasis	Negative	28	10	18	0.008
	Positive	17	13	4
TNM stage	I–II	26	9	17	0.010
	III–IV	19	14	5

2. lncRNA DUXAP8 promotes the progression of colon cancer cells

To investigate the biological role of DUXAP8 in colon cancer cells, we transfected SW1116 and HCT116 cells with overexpressed DUXAP8 (oe-DUXAP8) and the positive control (oe-NC) or DUXAP8-shRNA (sh-DUXAP8) and the NC (sh-NC). Transfection efficiency was assayed via qRT-PCR (Fig. 2A). DUXAP8 level was markedly increased in oe-DUXAP8 group relevant to oe-NC group and it was notably decreased in sh-DUXAP8 group relevant to sh-NC group. Subsequently, MTT and colony formation assays unveiled that overexpression of DUXAP8 noticeably promoted proliferation capability of colon cancer cells (Fig. 2B and C). Scratch healing and Transwell assays revealed that enforced expression of DUXAP8 induced cell migration and invasion (Fig. 2D-F). In contrast, DUXAP8 knockdown inhibited malignant phenotype of colon cancer cells. We have confirmed a positive correlation between DUXAP8 and lymph node metastasis. To examine the effects of the DUXAP8-microRNA-378a-3p-FOXQ1 axis on the expression of EMT-associated proteins vimentin, N-cadherin, and E-cadherin, we conducted western blot experiments for validation. The western blot results showed that compared to the oe-NC group, the oe-DUXAP8 group exhibited significantly increased levels of vimentin and N-cadherin expression, while E-cadherin expression was significantly decreased. In contrast, compared to the sh-NC group, the sh-DUXAP8 group showed the opposite trend in the expression levels of these proteins (Fig. 2G and H). Cyclin D1 is an important mediator of cell cycle progression,²⁷ and DUXAP8 has been reported to activate cyclin D1 expression.²⁸ The Wnt/β-catenin signaling pathway is highly conserved in biological evolution. Previous studies have shown that FOXQ1 interacts with ANXA2 to promote Wnt/β-catenin signaling in mesenchymal stem cells, thereby promoting osteogenic differentiation.²⁹ c-myc and β-catenin are key genes in the Wnt/β-catenin signaling pathway.^30,31 The experimental results showed that compared to the oe-NC group, the oe-DUXAP8 group exhibited significantly increased levels of cyclin D1, c-myc, and β-catenin expression. In contrast, compared to the sh-NC group, the sh-DUXAP8 group showed significantly decreased levels of cyclin D1, c-myc, and β-catenin protein expression (Fig. 2G and H). Overall, DUXAP8 overexpression fostered cancer cell phenotype progression.

Figure 2. The expression of the long noncoding RNA (lncRNA) DUXAP8 promotes the proliferation of colon cancer cells. (A) Transfection efficiencies of overexpressing or silencing DUXAP8 in colon cancer cells as determined via quantitative reverse transcription polymerase chain reaction assay. (B) MTT assay was used to determine the viability of colon cancer cells. (C) A colony formation assay was performed to analyze the proliferation of colon cancer cells. (D) The migratory ability of colon cancer cells was measured in a scratch healing assay (×40). (E, F) Transwell assays were performed to investigate the migratory and invasive abilities of colon cancer cells (×100). (G, H) Western blot assays showing cyclin D1, E-cadherin, vimentin, N-cadherin, c-myc and β-catenin protein expression levels. NC, normal control. *p<0.05 versus mimic NC; ^†p<0.05 versus si-NC.

3. lncRNA DUXAP8 acts as a microRNA-378a-3p sponge in colon cancer cells

lncRNAs function as ceRNAs in affecting microRNA level and biological functions.^32,33 Based on lncRNA DUXAP8 was mainly distributed in the cytoplasm, we inspected that lncRNA DUXAP8 may be a microRNA sponge that protects microRNAs from binding with their target mRNAs. Through the intersection of targeting prediction and differentially down-regulated genes from starBase database (Supplementary Fig. 1A and B), we displayed that DUXAP8 was significantly negatively correlated with microRNA-378a-3p (Supplementary Fig. 1C). In TCGA database, microRNA-378a-3p was down-regulated in colon cancer (Supplementary Fig. 1D). Meanwhile, we verified microRNA-378a-3p level at cell level and clinical tissue level. mRNA level of microRNA-378a-3p in colon cancer cells and para-cancer tissues was remarkably lower than its expression in normal cells and paracancerous tissues, respectively (Fig. 3A and B). Correlation analysis exhibited that DUXAP8 and microRNA-378a-3p levels were negatively correlated in colon cancer tumor tissues (Fig. 3C). Therefore, we identified microRNA-378a-3p as a likely target of lncRNA DUXAP8. Subsequently, the binding sequence was predicted as depicted in Supplementary Fig. 1E. Luciferase reporter assay demonstrated that enforced microRNA-378a-3p level memorably depressed the luciferase activity of the pmirGLO-DUXAP8-WT vector but failed to impact luciferase activity of the pmirGLO-DUXAP8-MUT (Fig. 3D). The RNA pull-down experiment results showed significant enrichment of DUXAP8 in the microRNA-378a-3p group (Fig. 3E). The AGO2 immunoprecipitation assay revealed that DUXAP8 was enriched in Ago2 protein in microRNA-378a-3p mimic group (Fig. 3F), authenticating binding potential. lncRNA DUXAP8 knockdown fostered microRNA-378a-3p expression, and lncRNA DUXAP8 overexpression hampered microRNA-378a-3p expression (Fig. 3G). In conclusion, microRNA-378a-3p was a repressive target of lncRNA DUXAP8 in colon cancer.

Figure 3. DUXAP8 downregulates microRNA-378a-3p expression through adsorption. (A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to determine the microRNA-378a-3p expression level in cell lines and tissues. (C) Correlation analysis of DUXAP8 and microRNA-378a-3p expression in colon cancer tissues. (D) Targeted binding of microRNA-378a-3p to DUXAP8 as detected by dual-luciferase assay. (E) The binding relationship between microRNA-378a-3p and DUXAP8 as determined by an RNA pull-down assay. (F) Binding relationship between microRNA-378a-3p and DUXAP8 as determined by a radioimmunoprecipitation assay. (G) Impact of overexpressed/silenced DUXAP8 on microRNA-378a-3p expression in colon cells assayed through qRT-PCR. NC, normal control. *p<0.05.

4. DUXAP8 affects the effect of microRNA-378a-3p on cell proliferation

To examine the role of DUXAP8/ microRNA-378a-3p in cell proliferation, SW1116 and HCT116 cells were transfected with oe-NC+mimic NC, oe-DUXAP8+mimic NC, oe-NC+microRNA-378a-3p mimic, oe-DUXAP8+microRNA-378a-3p mimic, respectively. Transfection efficiency of oe-DUXAP8 and microRNA-378a-3p mimics were assessed via qRT-PCR (Fig. 4A and B). DUXAP8 level was remarkably increased in the oe-DUXAP8+mimic NC group and oe-DUXAP8+microRNA-378a-3p mimic group but had no marked effect on the oe-NC+miR-378a-3p mimic group. Furthermore, miR-378a-3p level was markedly decreased in oe-DUXAP8+mimic NC group, but its level was restored to the control group level in oe-DUXAP8+microRNA-378a-3p mimic group. As can be seen from Fig. 4C and D, after overexpression of DUXAP8, colon cancer cell proliferative activity and colony-forming ability were also remarkably improved. However, under forced expression of microRNA-378a-3p, the proliferative ability of cancer cells was markedly hindered. Rescue experiments verified the functional relationship between lncRNA DUXAP8 and microRNA-378a-3p. These results suggested that DUXAP8 could affect cell proliferation of colon cancer by decreasing microRNA-378a-3p level.

Figure 4. microRNA-378a-3p participates in the modulatory effect of DUXAP8 on cell proliferation in colon cancer. (A, B) Quantitative reverse transcription polymerase chain reaction determination of DUXAP8 and microRNA-378a-3p expression in colon cancer cell lines in each transfection group (vector+oe-NC, oe-DUXAP8+oe-NC, vvector+microRNA-378a-3p mimic, and DUXAP8+microRNA-378a-3p mimic). (C) Proliferation of colon cancer cells was measured via an MTT assay. (D) Colony-forming capability of colon cancer cells as determined by a colony formation assay. NC, normal control. *p<0.05 versus vector+mimic NC; ^†p<0.05 versus DUXAP8+mimic NC.

5. microRNA-378a-3p can reverse promoting impact of DUXAP8 on cell migration and invasion in colon cancer cells

We also examined whether changes in DUXAP8 and microRNA-378a-3p levels could affect migratory and invasive capability of differently treated SW1116 and HCT116 cells. In scratch healing assay, it was revealed that migration of cells was promoted by overexpressing DUXAP8 while transfecting microRNA-378a-3p mimic could restrain cell migratory ability. In rescue assays, it was found that the concurrent forced expression of DUXAP8 and microRNA-378a-3p could largely reverse the promoted effect of overexpressed DUXAP8 on cell migration (Fig. 5A). In addition, a Transwell assay was conducted for further verification. As seen from Fig. 5B and C, high expression of DUXAP8 promoted cell migration and invasion. However, overexpression of microRNA-378a-3p could inhibit cell migration and invasion and reduce the cancer-promoting effect caused by DUXAP8. Western blot experiment results showed that high expression of DUXAP8 increased the protein expression of cyclin D1, vimentin, N-cadherin, c-myc, and β-catenin in colon cancer cells, while decreasing the protein expression of E-cadherin. Conversely, overexpression of microRNA-378a-3p decreased the protein expression of cyclin D1, vimentin, N-cadherin, c-myc, and β-catenin, while increasing the protein expression of E-cadherin. This reduced the promoting effect of DUXAP8 on these proteins (Fig. 5D). Overall, the results substantiated that DUXAP8 affected migration and invasion of colon cancer cells via sponging microRNA-378a-3p.

Figure 5. microRNA-378a-3p can reverse the effect of DUXAP8 on cell migration and invasion abilities in colon cancer. (A) Migratory ability of colon cancer cells as determined via a scratch healing assay (×40). (B, C) Migratory and invasive abilities of colon cancer cells as determined via a Transwell assay (×100). (D) Western blot assays showing cyclin D1, E-cadherin, vimentin, N-cadherin, c-myc and β-catenin protein expression levels. NC, normal control. *p<0.05 versus vector+mimic NC; ^†p<0.05 versus DUXAP8+mimic NC.

6. FOXQ1 is highly expressed in colon cancer and targeted by microRNA-378a-3p

Target gene prediction was carried out through starBase and miRDB databases. The obtained genes were intersected with differentially upregulated mRNAs, and it was found that there were three potential downstream targets of microRNA-378a-3p: CELSR3, FOXQ1, and UHRF1 (Supplementary Fig. 2A and B). As analyzed by bioinformatics, FOXQ1 level in tumor group was notably higher than that in normal group, and FOXQ1 had the most significant inverse correlation with microRNA-378a-3p (Supplementary Fig. 2C and D). By detecting FOXQ1 mRNA level in clinical tissue samples, it was confirmed that FOXQ1 level was noticeably upregulated in tumor tissues (Fig. 6A), inversely correlated with microRNA-378a-3p level, and positively associated with DUXAP8 level (Fig. 6B and C). Therefore, FOXQ1 was chosen as a possible target of microRNA-378a-3p.

Figure 6. FOXQ1 is targeted by microRNA-378a-3p in colon cancer. (A) FOXQ1 expression level in clinical colon cancer tissues as determined via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay (n=45). (B) Correlation analysis of DUXAP8 and FOXQ1 expression in colon cancer tissues. (C) Correlation analysis of microRNA-378a-3p and FOXQ1 expression in colon cancer tissues. (D) Binding relationship between microRNA-378a-3p and FOXQ1 as determined by a dual-luciferase assay. (E) Binding relationship between microRNA-378a-3p and FOXQ1 as determined by an RNA pull-down assay. (F) Binding relationship between microRNA-378a-3p and FOXQ1 as determined by a radioimmunoprecipitation (RIP) assay. (G, H) qRT-PCR assays showing the impact of microRNA-378a-3p on FOXQ1 expression. (I, J) Western blot assays showing the impact of microRNA-378a-3p on FOXQ1 protein expression. NC, normal control. *p<0.05.

According to the predicted binding sites (Supplementary Fig. 2E), dual-luciferase reporter vectors containing FOXQ1 WT or MUT 3’UTR were generated for detection of luciferase activity. The results demonstrated that enforced microRNA-378a-3p level reduced luciferase activity of the WT FOXQ1 reporter rather than MUT reporter, demonstrating that FOXQ1 was direct target of microRNA-378a-3p (Fig. 6D). The RNA pull-down experiment results showed significant enrichment of FOXQ1 in the microRNA-378a-3p group (Fig. 6E). Meanwhile, RIP assay also confirmed that FOXQ1 was enriched in Ago2 precipitate after enforcing microRNA-378a-3p level (Fig. 6F). Furthermore, mRNA and protein levels of FOXQ1 were diminished via microRNA-378a-3p overexpression and improved by down-regulated microRNA-378a-3p expression (Fig. 6G-J). These data implied that microRNA-378a-3p targeted FOXQ1.

7. DUXAP8 regulates the cellular function of colon cancer cells via microRNA-378a-3p/FOXQ1 axis

We further verified the impact of DUXAP8 on FOXQ1 level through the adsorption of microRNA-378a-3p and its ultimate impact on development of colon cancer cells. Firstly, we transfected vector+sh-NC, oe-DUXAP8+sh-NC, oe-DUXAP8+sh-FOXQ1 and obtained three groups of colon cancer cells. Then, DUXAP8, microRNA-378a-3p and FOXQ1 mRNA expression levels in cell lines of each group were assayed through qRT-PCR. Overexpression of DUXAP8 inhibited microRNA-378a-3p level, thereby reducing inhibition of microRNA-378a-3p on FOXQ1 (Fig. 7A).

Figure 7. DUXAP8 regulates the cellular function of colon cancer cells through microRNA-378a-3p/FOXQ1 axis. (A) Quantitative reverse transcription polymerase chain reaction determination of DUXAP8, microRNA-378a-3p and FOXQ1 expression in colon cancer cell line HCT116 transfection groups (vector+sh-NC, oe-DUXAP8+sh-NC and oe-DUXAP8+sh-FOXQ1). (B) Proliferation of colon cancer cells transfected with vector+sh-NC, oe-DUXAP8+sh-NC or oe-DUXAP8+sh-FOXQ1 as determined by an MTT assay. (C) Colony-forming ability of colon cancer cells as determined by a colony formation assay. (D) Scratch healing assay evaluation of the migration of colon cancer cells (×40). (E) Transwell assay evaluation of the migration and invasion abilities of colon cancer cells (×100). (F) Western blot assays showing cyclin D1, E-cadherin, vimentin, N-cadherin, c-myc and β-catenin protein expression levels. NC, normal control. *p<0.05 versus vector+sh-NC; ^†p<0.05 versus oe-DUXAP8+sh-NC.

Then, the effect of DUXAP8 and FOXQ1 on functional changes in colon cancer cells was verified. MTT and colony formation assays indicated that cell proliferation ability was promoted with overexpression of DUXAP8 but hugely restored after continuing to inhibit FOXQ1 expression (Fig. 7B and C). In addition, both scratch healing and Transwell assays further proved that further inhibition of the expression of FOXQ1 could reduce the promotion of enforced DUXAP8 level on cell migration and invasion (Fig. 7D and E). Western blot experiment results showed that overexpression of DUXAP8 promoted the protein expression of cyclin D1, vimentin, N-cadherin, c-myc, and β-catenin, while decreasing the protein expression of E-cadherin. On the other hand, overexpression of FOXQ1 reduced the promoting effect of DUXAP8 on these proteins (Fig. 7F). Hence, high expression of DUXAP8 could promote malignant behaviors of colon cancer cells, but the impact could be notably reversed after silencing downstream FOXQ1.

Many studies disclosed that lncRNAs play a pivotal modulatory role in the malignant progression of varying tumors like colon cancer.^34-36 Since lncRNAs are differentially expressed in tumors, they can also be utilized as tumor diagnostic markers and therapeutic targets in the future.^37,38 It has been demonstrated that a stroma-related lncRNA panel can be employed for the prediction of recurrence and adjuvant chemotherapy, which benefits patients with early-stage colon cancer.³⁹ Therefore, to help patients with colon cancer better, we probed into the functional mechanism of lncRNAs in colon cancer to find the key lncRNA and clarify its regulatory mechanism.

In this study, lncRNA DUXAP8 was confirmed to play a vital role in colon cancer cells based on differential analysis in TCGA and data from in vitro experiments. DUXAP8, located in 22q11.1 and a 2107-bp RNA, is dysregulated in several tumors.^13,17,40 DUXAP8 is a sponge of cancer repressor microRNA-422a in hepatocellular carcinoma, enhancing PDK2 level and functioning as an oncogenic lncRNA.¹⁵ Increased DUXAP8 promotes the development and progression of clear cell renal cell carcinoma and serves as a marker for poor prognosis.⁴⁰ In this research, DUXAP8 was upregulated in colon cancer. In combination with clinical information of patients, we displayed that DUXAP8 level was markedly correlated with lymph node metastasis and TNM staging, which also displayed that DUXAP8 may be a biomarker for colon cancer diagnosis.

To demonstrate the potential mechanism of DUXAP8 in colon cancer, nucleoplasmic separation confirmed that DUXAP8 was abundant in the cytoplasm, providing a basis for its role as a molecular sponge. With the help of bioinformatics, we explored a microRNA with high binding potential for DUXAP8: microRNA-378a-3p and confirmed their binding relationship through dual-luciferase reporter gene and RIP assays. Many studies displayed that microRNA-378a-3p played a regulatory role in cancers such as lung cancer,⁴¹ breast cancer,⁴² ovarian cancer,²⁰ and pancreatic cancer.⁴³ The data suggested that microRNA-378a-3p was notably reduced in colon cancer tissues and cells. Enforced DUXAP8 level could markedly down-regulate microRNA-378a-3p level and affect malignant behaviors of cancer cells. It is believed that this was probably caused by the massive sponge of microRNA-378a-3p by DUXAP8 in the cytoplasm.

As small RNAs, microRNAs are not able to encode proteins, which usually interact with the 3’ UTRs of downstream target genes to degrade target mRNA or inhibit its translation to participate in post-transcriptional gene control.⁴⁴ Therefore, we predicted that FOXQ1 was likely to be a downstream target of microRNA-378a-3p through starBase and miRDB databases. We further confirmed the binding relationship of microRNA-378a-3p and FOXQ1, and ability of microRNA-378a-3p to down-regulate FOXQ1 level through experiments. FOXQ1 serves as a transcription factor.⁴⁵ FOX family members are vital in cancer occurrence and progression by regulating cell cycle, DNA damage repair, and cancer stem cell properties.⁴⁶ For example, FOXQ1 is increased in gastric cancer cells while microRNA-519 can down-regulate its expression to inhibit EMT and biological behavior of gastric cancer cells.⁴⁷ FOXQ1 is increased in colon cancer tissues and cell lines and facilitates cell proliferation via down-regulating CDK6.⁴⁸ We also verified increased FOXQ1 levels in colon cancer and demonstrated that silencing of FOXQ1 markedly rescued promotion of overexpressed DUXAP8 on cell malignant behaviors. This also indicated that the regulatory axis in which they were involved could influence functions of colon cancer cells.

In summary, we identified lncRNA DUXAP8 as an oncogene in colon cancer, and a high lncRNA DUXAP8 level was correlated with cancer metastasis and dismal prognosis. lncRNA DUXAP8 as a sponge for microRNA-378a-3p attenuated repressive impact of microRNA-378a-3p on FOXQ1. Our results bolster a better understanding of role of lncRNA DUXAP8 in colon cancer progression and bring new insight into developing possible therapeutic targets for colon cancer. However, several limitations exist in the study. For example, we have not yet confirmed through in vivo experiments that lncRNA DUXAP8 facilitates the malignant progression of colon cancer via microRNA-378a-3p/FOXQ1 axis. In the follow-up study, we will explore the mechanism of downstream signaling pathways regulated by the lncRNA DUXAP8/microRNA-378a-3p/FOXQ1 axis in colon cancer progression.

No potential conflict of interest relevant to this article was reported.

Study concept and design: R.S., J.J. Data acquisition: Y.W. Data analysis and interpretation: J.J. Drafting of the manuscript: R.S. Critical revision of the manuscript for important intellectual content: R.S. Statistical analysis: J.J. Administrative, technical, or material support, study supervision: Y.W. Approval of final manuscript: all authors.

The data and materials in the current study are available from the corresponding author on reasonable request.

Supplementary materials can be accessed at https://doi.org/10.5009/gnl240178.