miR‑4306 inhibits the malignant behaviors of colorectal cancer by regulating lncRNA FoxD2‑AS1.

MicroRNA (miR)‑4306 and FoxD2‑adjacent opposite strand RNA 1 (FOXD2‑AS1) are cancer‑related genes involved in tumor progression. However, the potential functional roles of miR‑4306 and FoxD2‑AS1 in colorectal cancer (CRC) development remain unknown. The present study aimed to investigate the biological functions and the molecular mechanisms of miR‑4306 and FoxD2‑AS1 in CRC. Reverse transcription‑quantitative PCR analysis was performed to determine the expression levels of FoxD2‑AS1 and miR‑4306 in CRC tissues and cell lines. Functional experiments, including Cell Counting Kit‑8, colony formation, cell cycle assays and western blotting, were conducted to examine the effects of FoxD2‑AS1 and miR‑4306 on the malignant behaviors of CRC cells. In addition, the relationship between FoxD2‑AS1 and miR‑4306 was assessed using a dual‑luciferase reporter assay and Pearson's correlation analysis. Compared with normal samples and cells, FoxD2‑AS1 expression was increased and miR‑4306 expression was decreased in CRC tissues and cells. Functional experiments demonstrated that silencing FoxD2‑AS1 inhibited proliferation and induced cell arrest at G0/G1 phase in CRC cells, while the overexpression of FoxD2‑AS1 showed opposite results. Ki‑67 and proliferating cell nuclear antigen expression levels were decreased after transfection with small interfering RNA FoxD2‑AS1, but were increased after transfection with FoxD2‑AS1 overexpression plasmid. Furthermore, investigations into the underling mechanism revealed that FoxD2‑AS1 functioned as a molecular sponge of miR‑4306. The inhibitory effects of FoxD2‑AS1 silencing on CRC progression were reversed by miR‑4306 knockdown. Collectively, the present study demonstrated that FoxD2‑AS1 functioned as an oncogene in CRC progression, and that miR‑4306 could inhibit the malignant behaviors of CRC by regulating FoxD2‑AS1. Thus, the current study provided a promising therapeutic target for CRC treatment.

Introduction

Colorectal cancer (CRC) is the second most frequent malignant tumor, with ~1.2 million new cases diagnosed and 70,000 deaths worldwide each year (1–3). The development of the diagnosis and treatment of CRC has doubled the 5-year survival rate of patients with CRC (4,5). Examining new treatment methods and providing further understanding on molecular mechanisms of CRC metastasis are currently the key to improving CRC diagnosis and effective treatment.

Data accumulated from genome-wide and transcriptome studies has revealed that most human genomes encode a large number of non-coding RNAs (6). Long non-coding RNA (lncRNAs) and microRNA (miRNAs/miRs), as the main components of non-coding RNAs, are closely involved in cancer development (7,8). lncRNAs are defined as RNA molecules with a length of >200 nucleotides without a protein-coding ability. Accumulating evidence has suggested that lncRNAs widely participate in a series of cellular processes such as proliferation, immune response and invasion (9–11). In addition, the changes of the expression levels of lncRNAs are closely associated with cancer progression (12,13). Thus, lncRNAs show great potential to serve as novel biomarkers for cancer treatment (14–16). A previous study reported that lncRNA fer-1 like family member 4 inhibited the proliferation of endometrial carcinoma cells by regulating PTEN (17), while Wu et al (18) observed that lncRNA metastasis associated lung adenocarcinoma transcript 1 promoted colon cancer progression by targeting the miR-129-5p/high mobility group box 1 axis. In CRC, our previous study examined the expression and functional role of some lncRNAs. For instance, RUNX family transcription factor 2/lncRNA-PVT1 oncogene (PVT1)/miR-455 was shown to be involved in CRC progression (19), and it was observed that prostate cancer-upregulated long non-coding RNA 1 induced CRC progression by regulating the PI3K/AKT pathway (20). Moreover, it was reported that lncRNA upregulated in hepatocellular carcinoma (URHC) could affect the proliferation and apoptosis of CRC cells (21).

The lncRNA FoxD2-adjacent opposite strand RNA 1 (FoxD2-AS1) is highly expressed in numerous cancer types and serves a vital role in tumor progression (22). FoxD2-AS1 has been shown to be involved in gastric cancer development by modulating the miR-185-5p/cyclin D2 axis. Moreover, Yang et al (23) reported that FoxD2-AS1 acted as an oncogene by regulating the epithelial-mesenchymal transition and Notch pathway in CRC. It has also been revealed that FoxD2-AS1 accelerates gemcitabine resistance by sponging miR-143 in bladder cancer (24). More importantly, in CRC, FoxD2-AS1 contributes to cell proliferation via an interaction with miR-185-5p (25). FoxD2-AS1 also promotes the migration and invasion of CRC cells (26).

miRNAs are involved in the development of numerous types of cancer and are regarded as new targets of cancer treatment. It has been reported that miR-29a inhibits cervical cancer cell proliferation and migration by targeting the cell division cycle 42/p21 (RAC) activated kinase 1 pathway (27). Liu and Sun (28) observed that miR-25 was an oncogene involved the progression of non-small cell lung cancer (NSCLC) by targeting cadherin 1. Furthermore, miR-4306 acts as a tumor suppressor in triple-negative breast cancer (TNBC) and is a potential therapeutic target for TNBC treatment (29). However, whether miR-4306 also exerts an anticancer role in colon cancer remains unknown.

Thus, we hypothesized that the FoxD2-AS1/miR-4306 axis may have a pivotal function in CRC progression. At present, the detailed effects and potential mechanism of FoxD2-AS1 sponging miR-4306 in CRC are yet to be elucidated. Therefore, the current study investigated the role of the FoxD2-AS1/miR-4306 axis and the potential mechanism in the pathogenesis of CRC.

Materials and methods

Tissue samples

Freshly dissected CRC tissues and paired adjacent normal tissues (5 cm from the tumoral margins) were collected from 40 patients (22 male and 18 female; age range, 20–84 years) in Shenzhen Longgang Central Hospital between July 2017 and December 2018. The patients have provided written informed consent and the study was approved by the Research Ethics Committee of Shenzhen Longgang Central Hospital (approval no. SL2017061125). In addition, FoxD2-AS1 expression in CRC and normal tissues was determined from The Cancer Genome Atlas using GEPIA2 (http://gepia.cancer-pku.cn/index.html).

Cell lines and cell culture

CRC cell lines (HCT116, SW-620, LOVO, HCT-15 and SW480) and normal epithelial cell lines, CCD-18Co, were procured from the American Type Culture Collection (ATCC). HCT116 cells (cat. no. CCL-247) were maintained in McCoy's 5a medium (cat. no. 30-2007; ATCC); SW-620 (cat. no. CCL-227) and SW480 cells (cat. no. CCL-228) were grown in Leibovitz's L-15 medium (cat. no. 30-2008; ATCC); LOVO cells (cat. no. CCL-229) were maintained in F-12K medium (cat. no. 30-2004; ATCC); HCT-15 cells (cat. no. CCL-225) were cultured in RPMI-1640 medium (cat. no. 30-2001; ATCC); and CCD-18Co cells (cat. no. CRL-1459) were maintained in Eagle's Minimum (cat. no. 30-2003; ATCC). All cells were cultured in media supplemented with 10% FBS (cat. no. 16000044; Gibco; Thermo Fisher Scientific, Inc.) in a humidified containing 5% CO2 at 37°C.

Cell transfection

HCT116 and LOVO cells (1×105) were seeded into 6-well plates and cultured to 60–70% confluence. Specific small interfering (si)RNAs against FoxD2-AS1 (siFoxD2-AS1, 5′-GCGAAGAGUACGUUGUAUTT-3′), corresponding si-negative control (NC) (5′-UUCUCCGAACGUGUCACGUTT-3′), pcDNA3.1-FoxD2-AS1, FoxD2-AS1 NC (pcDNA3.1 empty vector), miR-4306 inhibitor (I; 5′-UACUGCCUUUCUCUCCA-3′), miR-4306 inhibitor control (IC; 5′-CAGUACUUUUGUGUAGUACAAA-3′), miR-4306 mimic (M; 5′-UGGAGAGAAAGGCAGUA-3′) or miR-4306 mimic control (MC; 5′-UUUGUACUACACAAAAGUACUG-3′) were designed by Shanghai GeneChem Co., Ltd. Cell were transfected with 20 nM siFoxD2-AS1 or siNC, or 100 nM I, M, IC and MC, or 100 ng pcDNA3.1-FoxD2-AS1 and pcDNA3.1 empty vector using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.), according to manufacturer's instructions. After 48 h of transfection at 37°C, the cells were harvested and used for subsequent experiments.

Reverse transcription-quantitative (RT-qPCR) assay

TRIzol® reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.) was used to extract the total RNA from tissues and cells, and RNA was reverse-transcribed to cDNA using a RT kit (cat. no. D7168M; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The RT-qPCR experiment was conducting using SYBR Green qPCR Master Mixes (cat. no. 4312704; Thermo Fisher Scientific, Inc.), and miRNA expression was detected using a miRcute miRNA qPCR kit (cat. no. FP401; Tiangen Biotech Co., Ltd.) in fluorescent qPCR 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following the conditions: Initial denaturation at 95°C for 15 min, followed by 40 cycles at 94°C for 20 sec, at 60°C for 30 sec, at 70°C for 1 min, and a final extension at 70°C for 5 min. GAPDH and U6 were internal references. The 2−ΔΔCq method (30) was used to calculate the fold changes of RNA expression. Specific primer sequences are listed in Table I.

Table I.

Primer sequence.

Gene	Primer sequences
FoxD2-AS1	Forward: 5′-CACTGAGGGACAGCCAAGA-3′
	Reverse: 5′-GGCGGCGTGTAATTGGTA-3′
miR-4306	Forward: 5′-ATCGAGCTCACATGATCGTGCGCTCCTGCAAGTG-3′
	Reverse: 5′-ACTCTCGAGGCATCTCAGAGTGTTGCTATGGTGA-3′
GAPDH	Forward: 5′-TATGATGATATCAAGAGGGTAGT-3′
	Reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′
U6	Forward: 5′-CTCGCTTCGGCAGCACA-3′
	Reverse: 5′-AACGCTTCACGAATTTGCGT-3′

miR, microRNA; FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1.

Western blot analysis

Total protein from CRC cells was extracted using RIPA buffer (Invitrogen; Thermo Fisher Scientific, Inc.), followed by the determination of protein concentration using a BCA kit (Beyotime Institute of Biotechnology). The samples were isolated on 10% SDS-PAGE and transferred into the PVDF membranes. Subsequently, the membranes were blocked with 5% non-fat milk for 1 h at room temperature and probed with primary antibody overnight at 4°C. The primary antibodies (all from Abcam) were Ki-67 (1:1,000; 359 kDa; cat. no. ab92742), proliferating cell nuclear antigen (PCNA; 1:1,000; 29 kDa; cat. no. ab18197) and GAPDH (1:2,000; 36 kDa; cat. no. ab8245). Finally, the membrane was probed with HRP-conjugated secondary antibodies (cat. no. ab6728; 1:2,000; Abcam; cat. no. ab205718; 1:2,000; Abcam) for 2 h at room temperature after being washed with TBS-0.05% Tween 20 three times. The bands were visualized using an ECL reagent (Beckman Coulter, Inc.) and were semi-quantified using ImageJ software (version 1.42; National Institutes of Health). Relative protein expression was analyzed with GAPDH as an internal reference.

Cell Counting Kit-8 (CCK-8) analysis

Cell viability was detected using a CCK-8 kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, the cells (1×105) were seeded into 96-well plates and maintained for 24 and 48 h, then processed with 10 µl CCK-8 reagent and incubated for additional 4 h at room temperature. Finally, optical density was measured at wavelength of 450 nm using a microplate reader (BMG Labtech GmbH). The experiment was conducted in triplicate.

Colony formation analysis

Colony formation analysis was performed to determine the function of FoxD2-AS1 in the proliferation of HCT116 and LOVO cells. Briefly, the cells (200 cells/well) were added into 6-well plates and maintained for 2 weeks. The cells were then fixed in 4% paraformaldehyde at room temperature for 15 min, and stained with crystal violet for another 30 min at room temperature. Finally, the numbers of colonies were counted under a light microscope (magnification, ×10; Olympus Corporation).

Flow cytometry analysis

HCT116 and LOVO cells (1×105) were trypsinized, dispersed into cell suspension and centrifuged at 1,000 × g for 10 min after a 48-h transfection. The harvested cells were fixed in 70% ethanol at 4°C overnight. The cell cycle was analyzed by staining the cells with 1% PI containing RNAase for 30 min at 4°C. The cell cycle distribution was analyzed using a flow cytometer (FACScan™; BD Biosciences). The data were analyzed by FlowJo v10 software (Tree Star, Inc.).

Dual-luciferase reporter assay

It was predicted that miR-4306 was the target of FoxD2-AS1 using StarBase (v2.0; http://starbase.sysu.edu.cn). Wild-type (WT) or mutant (MUT) 3′ untranslated regions of FoxD2-AS1 were sub-cloned into pmirGLO dual-luciferase vector (Promega Corporation) and then co-transfected into HCT116 and LOVO cells with 100 nM miR-4306 mimic or their respective MC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. The relative luciferase activities were determined using a Dual-Luciferase reporter assay system (Promega Corporaiton) 48 h after the co-transfection. The relative luciferase activities were analyzed using a GloMax® Discover Multimode microplate reader (cat. no. GM3000; Promega Corporation) and normalized to that of Renilla luciferase.

Statistical analysis

The experiments were performed at least in triplicate and the data are presented as the mean ± SD. A paired Student's t-test was used to assess significant differences between two groups. The differences among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. A Pearson test was used to analyze the correlation between FoxD2-AS1 and miR-4306. Statistical analyses were conducted using SPSS v19.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

FoxD2-AS1 expression is upregulated in CRC tissues and cell lines

FoxD2-AS1 expression in CRC and normal tissues was determined from The Cancer Genome Atlas using GEPIA2 (http://gepia.cancer-pku.cn/index.html), and it was found that FoxD2-AS1 was significantly upregulated in CRC tissues compared with non-cancerous tissues (Fig. 1A). Next, RT-qPCR analysis was performed to detect FoxD2-AS1 expression in 40 paired of CRC tissue samples, and it was observed that FoxD2-AS1 expression was upregulated in CRC tissues compared with non-cancerous tissues (Fig. 1B). Consistently, RT-qPCR results revealed that FoxD2-AS1 was upregulated in CRC cell lines (HCT116, SW-620, LOVO, HCT-15 and SW480) compared with CCD-18Co cells (Fig. 1C).

Figure 1.

FoxD2-AS1 is upregulated in CRC tissues and cell lines. (A) FoxD2-AS1 expression in CRC tissues (red) and paired normal tissues (gray) from GEPIA2 webserver. *P<0.05 vs. normal group. (B) Expression level of FoxD2-AS1 in CRC tissues and corresponding normal tissues was detected via reverse transcription-quantitative PCR (n=40). ###P<0.001 vs. Normal. (C) Expression level of FoxD2-AS1 in CRC cell lines (HCT116, SW-620, LOVO, HCT-15 and SW480) and normal epithelial cell line, CCD-18Co. The experiment was independently performed three times. ***P<0.001 vs. CCD-18Co. COAD, colon adenocarcinoma; Num(T), the number of tumor sample; Num(N), the number of normal sample; CRC, colorectal cancer; FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1.

Knocking down FoxD2-AS1 inhibits the proliferation of CRC cells

Next, the precise effects of FoxD2-AS1 on CRC were investigated, and based on the fact that FoxD2-AS1 had the highest expression in HCT116 cells and the lowest expression in LOVO cells, these cells were selected for subsequent experiments. siFoxD2-AS1 was transfected into HCT116 cells to silence the FoxD2-AS1 expression, with siNC as the control. LOVO cells were transfected with pcDNA3.1-FoxD2-AS1, and the results of RT-qPCR assay demonstrated the successful knockdown or overexpression of FoxD2-AS1 expression in HCT116 and LOVO cells (Fig. 2A).

Figure 2.

Knocking down FoxD2-AS1 suppresses the proliferation of CRC cells. (A) The relative expression level of FoxD2-AS1 in HCT116 and LOVO cells. (B) Cell Counting Kit-8 and (C) colony formation assays were used to detect the effects of FoxD2-AS1 on proliferation of CRC cells. The experiment was independently performed three times. **P<0.01, ***P<0.001 vs. siNC; ^P<0.05, ^^^P<0.001 vs. NC. CRC, colorectal cancer; FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1; NC, negative control; si, small interfering RNA; OD, optical density.

The CCK-8 assay revealed that knockdown of FoxD2-AS1 significantly inhibited HCT116 viability, while overexpression of FoxD2-AS1 significantly promoted LOVO cell viability (Fig. 2B). Consistently, the colony formation assay demonstrated that the proliferation of HCT116 cells was decreased by knockdown of FoxD2-AS1, and the proliferation of LOVO cells was increased by the overexpression of FoxD2-AS1 (Fig. 2C). In addition, western blotting was used to determine the protein expression levels of Ki-67 and PCNA in HCT116 and LOVO cells. As shown in Fig. 3A-D, Ki-67 and PCNA expression was decreased when HCT116 cells were transfected siFoxD2-AS1 compared with siNC group, but increased when LOVO cells were transfected FoxD2-AS1 compared with NC group. In addition, the cell cycle of CRC cells was detected via flow cytometry, and it was observed that the proportion of G0/G1 of HCT116 cells after transfection with siFoxD2-AS1 was significantly higher compared with the siNC group. Moreover, after transfection with FoxD2-AS1, the G1 phase of LOVO cells was decreased compared with NC group, indicating that knocking down FoxD2-AS1 could induce CRC cell arrest at the G0/G1 phase (Fig. 3E). These results suggested that FoxD2-AS1 exerted oncogenic roles in the proliferation of CRC cells.

Figure 3.

Knocking down FoxD2-AS1 induces the cell cycle arrest of CRC. (A) Expression levels of Ki-67 and PCNA were determined in HCT116 cells using western blotting. GAPDH was used as an internal reference. (B) Protein expression levels of Ki-67 and PCNA were semi-quantified in HCT116 cells. (C) Expression levels of Ki-67 and PCNA were determined in LOVO cells using western blotting. (D) Protein expression levels of Ki-67 and PCNA were semi-quantified in LOVO cells. (E) Flow cytometry was performed to analyze cell cycle of CRC cells. The experiment was independently performed three times. *P<0.05, **P<0.01, ***P<0.001 vs. siNC; ^P<0.05, ^^P<0.01, ^^^P<0.001 vs. NC. CRC, colorectal cancer; FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1; NC, negative control; si, small interfering RNA; PCNA, proliferating cell nuclear antigen.

FoxD2-AS1 acts as a molecular sponge of miR-4306

The potential target miRNA of FoxD2-AS1 was predicted by StarBase, which identified that FoxD2-AS1 contained complementary binding sequences of miR-4306 (Fig. 4A). A dual-luciferase reporter assay was conducted to confirm the interaction between FoxD2-AS1 and miR-4306, and it was found that the miR-4306 mimic inhibited the luciferase activity of FoxD2-AS1-WT, whereas no change was observed in the luciferase activities of HCT116 and LOVO cells in FoxD2-AS1-MUT group (Fig. 4B). In addition, miR-4306 expression was significantly downregulated in CRC tissues compared with non-tumor tissues (Fig. 4C). Furthermore, Pearson's correlation analysis revealed that FoxD2-AS1 was weakly, negatively correlated with miR-4306 expression in CRC tissues (Fig. 4D).

Figure 4.

FoxD2-AS1 acts as a molecular sponge of miR-4306. (A) Complementary site of miR-4306 in 3′ untranslated region of FoxD2-AS1 was predicted via bioinformatics analysis. (B) Dual-luciferase reporter assay was conducted to detect the luciferase activities of HCT116 and LOVO cells. (C) miR-4306 expression in CRC tissues. (D) The correlation between the expression levels of FoxD2-AS1 and miR-4306 was analyzed in CRC tissues via Pearson's correlation analysis. The experiment was independently performed three times. ###P<0.001 vs. Normal; ^^^P<0.001 vs. MC. CRC, colorectal cancer; FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1; WT, wild-type; MUT, mutant; MC, mimic control; M, mimic; miR, microRNA.

Overexpression of miR-4306 attenuates the CRC cell proliferation promoted by FoxD2-AS1

To confirm whether the biological functions of FOXD2-AS1 in CRC cells were mediated via miR-4306, miR-4306 inhibitor oligonucleotides or negative IC were transfected into FoxD2-AS1-knockdown HCT116 cells. Moreover, miR-4306 mimic oligonucleotides or negative MC were transfected into FoxD2-AS1-overexpressed LOVO cells. The results of the RT-qPCR revealed that miR-4306 mimic increased the expression level of miR-4306, and miR-4306 inhibitor transfection decreased miR-4306 expression (Fig. 5A). In addition, the miR-4306 inhibitor significantly increased FoxD2-AS1 expression, and siFoxD2-AS1 transfection decreased FoxD2-AS1 expression but had no effect on miR-4306 expression in HCT116 cells (Fig. 5B and C). After transfection with miR-4306 mimic, LOVO cells showed a higher miR-4306 expression and a lower FoxD2-AS1 expression compared with those in the MC group. Moreover, FoxD2-AS1 overexpression could significantly induce FoxD2-AS1 expression but did not affect the expression level of miR-4306 in LOVO cells in the MC + FoxD2-AS1 group (Fig. 5B and C).

Figure 5.

miR-4306 overexpression attenuates the colorectal cancer cell proliferation promoted by FoxD2-AS1. (A) Expression level of miR-4306 in HCT116 and LOVO cells was detected using RT-qPCR analysis. Expression levels of (B) miR-4306 and (C) FoxD2-AS1 were detected using RT-qPCR analysis. (D) Cell Counting Kit-8 and (E and F) colony formation assays were used to examine the effects of miR-4306 and FoxD2-AS1 on CRC cell proliferation. The experiment was independently performed three times. ▲▲▲P<0.001 vs. IC; &&&P<0.001 vs. MC; *P<0.05, **P<0.01, ***P<0.001 vs. IC + siNC; ^P<0.05, ^^^P<0.001 vs. IC + siFoxD2-AS1; #P<0.05, ###P<0.001 vs. I + siNC; ξP<0.05, ξξξP<0.001 vs. MC + NC; ΔP<0.05, ΔΔΔP<0.001 vs. MC + FoxD2-AS1; †P<0.05, †††P<0.001 vs. M + NC. FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1; MC, mimic control; M, mimic; miR, microRNA; NC, negative control; si, small interfering RNA; I, inhibitor; IC, inhibitor control; OD, optical density; RT-qPCR, reverse transcription-quantitative PCR.

Rescue experiments were conducted to further verify whether FoxD2-AS1 exerted its effects in CRC via miR-4306. The CCK-8 assay demonstrated that HCT116 cell proliferation inhibited by silencing FoxD2-AS1 was induced by miR-4306 inhibitor compared with the IC + siNC group (Fig. 5D). In addition, compared with MC + NC group, the proliferation of LOVO cells was enhanced after the transfection of FoxD2-AS1 and was decreased by co-transfected with miR-4306 mimic and FoxD2-AS1 (Fig. 5D). Similar results were obtained from the colony formation assay (Fig. 5E and F).

Western blot analysis was conducted to assess the protein expression levels of factors associated with cell proliferation, and the results demonstrated that Ki-67 and PCNA expression was abrogated by siFoxD2-AS1, and subsequent inhibition of miR-4306 restored Ki-67 and PCNA expression in HCT116 cells in comparison with the IC + siNC group. LOVO cells transfected with FoxD2-AS1 and miR-4306 mimic showed opposite results (Fig. 6A-C). Flow cytometry was used to evaluate cell cycle of CRC cells. siFoxD2-AS1-transfected HCT116 cells showed more cells at G0/G1 phase, while this effect was weakened by transfection with miR-4306 inhibitor, as compared with the cells transfected with IC + siNC (Fig. 6D and E). LOVO cells overexpressing FoxD2-AS1 had more cells in the S phase, and this effect was weakened by transfection with the miR-4306 mimic (Fig. 6D and E). These results indicated that miR-4306 exerted an inhibitory effect on CRC malignant behaviors via the regulation of FoxD2-AS1.

Figure 6.

miR-4306 overexpression attenuates the colorectal cancer cell proliferation promoted by FoxD2-AS1 in HCT116 and LOVO cells. (A) Expression levels of Ki-67 and PCNA were detected by western blotting in (B) HCT116 and (C) LOVO cells. GAPDH was used as an internal reference. (D) Flow cytometry was performed to analyze (E) cell cycle of CRC cells. The experiment was independently performed three times. *P<0.05, ***P<0.001 vs. IC + siNC; ^P<0.05, ^^^P<0.001 vs. IC + siFoxD2-AS1; #P<0.05, ##P<0.01 vs. I + siNC; ξP<0.05, ξξP<0.01, ξξξP<0.001 vs. MC + NC; ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. MC + FoxD2-AS1; †P<0.05, †††P<0.001 vs. M + NC. FoxD2-AS1, long non-coding RNA FoxD2-adjacent opposite strand RNA 1; MC, mimic control; M, mimic; miR, microRNA; NC, negative control; si, small interfering RNA; I, inhibitor; IC, inhibitor control; PCNA, proliferating cell nuclear antigen.

Discussion

lncRNAs have been increasingly discovered to participate in the progression of CRC by regulating cell behaviors, indicating that lncRNAs may be important diagnostic and therapeutic targets of CRC (31–33). It has been reported that lncRNA CRC metastasis-suppressed lncRNA suppressed the invasion and migration of CRC cells by targeting high mobility group box 2 (HMGB2) (34). Shang et al (35) also revealed that silencing of lncRNA PVT1 inhibited cell proliferation and invasion by regulating miR-214-3p of CRC cells.

The present study identified FoxD2-AS1 as a novel and key CRC-associated lncRNA. FoxD2-AS1 promoted the progression of glioma by modulating the miR-185-5P/HMGA2 axis (36). Furthermore, FoxD2-AS1 confers cisplatin resistance of NSCLC by regulating the miR-185-5p/SIX homeobox 1 axis (37), while the upregulation of FoxD2-AS1 affects cell proliferation of esophageal squamous cell carcinoma (38). These findings suggested the important regulatory role of FoxD2-AS1 in cancer progression. The present study aimed to determine the oncogenic role of FoxD2-AS1 in CRC by acting as a sponge to miRNA.

The present study found that the expression level of FoxD2-AS1 was upregulated in CRC tissues and cell lines, which was consistent with the findings of a previous study (23). The biological function and regulatory mechanism of FoxD2-AS1 in CRC were examined via functional assays. Notably, the current study identified that silencing FoxD2-AS1 expression inhibited cell proliferation, as well as significantly decreased Ki-67 and PCNA expression and colony formation and promoted cell arrest of HCT116 cells at G0/G1 phase. Moreover, as shown by the notably increased Ki-67 and PCNA expression, FoxD2-AS1 overexpression markedly promoted cell proliferation and colony formation and reduced LOVO cell arrest at G0/G1 phase.

miRNAs are validated important regulators in affecting the expression of multiple genes at the post-transcriptional level, and lncRNAs serve as miRNA sponges at the post-transcriptional level in the progression of various types of cancer, including in CRC (39–42). The interaction between lncRNAs and miRNAs is closely associated with tumorigenesis and cancer progression. The present study hypothesized that FoxD2-AS1 may also serve as a miRNA sponge in CRC tumorigenesis and development. Bioinformatics analysis identified that FoxD2-AS1 contained several target binding sites for miR-4306. A recent study reported that downregulation of miR-4306 served as a new therapeutic target for TNBC (29). The current study observed that miR-4306 expression in CRC tissues was lower compared with that in normal tissues. A dual-luciferase reporter assay was conducted to confirm the regulation of FoxD2-AS1 on miR-4306, and it was found that FoxD2-AS1 could directly bind to miR-4306. In addition, the expression levels of FoxD2-AS1 and miR-4306 may be negatively correlated in CRC tissue samples. It was demonstrated that knocking down miR-4306 partly reversed the inhibitory effect of knockdown of FoxD2-AS1 on the proliferation, colony formation and cell cycle of CRC cells. The regulatory mechanism of miR-4306 in cancer is complex. It may not only inhibit the malignant behavior of CRC by downregulating the expression of FoxD2-AS1, but also regulate other lncRNAs to exert its role. For example, LINC0095 promotes tumorigenesis and metastasis in osteosarcoma by competitively inhibiting miR-4306 expression (43). Thus, whether miR-4306 could also serve a role in CRC by regulating LINC0095 needs to be further investigated. The clinical sample size in the present study was limited, and the sample size should be expanded in future studies to further analyze the correlation between the expression levels of FoxD2-AS1 and miR-4306.

In conclusion, the present study demonstrated that FoxD2-AS1 was an oncogene, and that FoxD2-AS1 knockdown contributed to the inhibition of CRC cell proliferation via its interaction with miR-4306. Overexpression of miR-4306 could inhibit the expression level of FoxD2-AS1 and further suppressed the proliferation of CRC cells. In vitro experiments confirmed that miR-4306 could inhibit CRC cell proliferation by regulating FoxD2-AS1, which supported the anti-oncogenic role of miR-4306 in CRC tumorigenesis. Collectively, the current study provides an effective target for the treatment of patients with CRC.