The SW480 and SW620 cell lines were purchased from Procell Life Science & Technology Co., Ltd., (cat. no. CL-0223 and CL-0225, respectively) and cultured in Leibovitz's L15 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) at 37°C (100% air). The 293T cell line was purchased from Procell Life Science & Technology Co., Ltd. (cat. no. CL-0005) and cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. All cell lines were authenticated by STR authentication.

The NDRG4 lentivirus and its lentiviral vector GV358, were purchased from Shanghai GeneChem Co., Ltd. SW480 and SW620 cells were infected with lentivirus at a multiplicity of infection (MOI) of 10 and 80 for 24 h, respectively. And the stably transfected cells were selected with 3 µg/ml puromycin (Beyotime Institute of Biotechnology) for 2 weeks. In subsequent experiments, puromycin was maintained at 0.5 µg/ml. Short hairpin (sh)RNAs targeting the DDIT3 gene (shRNA-1, 5′-GATCCCTGCACCAAGCATGAACAATTCTCGAGAATTGTTCATGCTTGGTGCAGTTTTTG-3′; shRNA-2, 5′-GATCCTGAACGGCTCAAGCAGGAAATCTCGAGATTTCCTGCTTGAGCCGTTCATTTTTG-3′), and non-targeting shRNA-negative control (shRNA-NC 5′-GATCCCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) were cloned into the lentiviral vector PLVshRNA-EGFP(2A)Puro by Inovogen Biotechnology Pvt. Ltd. According to the manufacturer's protocol, 4 µg lentiviral shRNA plasmids were mixed with packaging vector (PAX2 plasmid) and envelope vector (PMD2G plasmid) at the mass ratio of 4:3:1, and subsequently transfected into 293T cells using 20 µl Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Inc.). Following incubation at 37°C with serum-free DMEM for 12 h, the 293T cells were changed to be cultured in complete DMEM at 37°C for 48 h. The lentivirus particles were subsequently collected and purified from the cell supernatants through a 0.45 µm filter, and meanwhile, the titer of the lentivirus was determined using the qPCR Lentivirus Titer kit (Applied Biological Materials, Inc.). Then SW480 cells in the logarithmic growth phase were added to the lentivirus suspension (MOI=10) and incubated at 37°C for 48 h, after which the medium was discarded and the SW480 cells were cultivated with screening Leibovitz's L15 medium containing 3 µg/ml puromycin for 2 weeks to select the positive infected cells. In subsequent experiments, they were maintained with 0.5 µg/ml puromycin.

Cells were seeded into a 96-well plate with five biological replicates per group. Before detection, 20 µl MTT solution (Beyotime Institute of Biotechnology) was added to each well. The medium was replaced with 150 µl DMSO (Amresco, LLC) after 4 h at 37°C, and the plates were then shaken for 10 min. The OD values were determined at 570 nm using a microplate reader. To investigate the effect of NDRG4 on the proliferative ability of CRC cell lines, activity was detected at 0, 1, 2, 3 and 4 days of cell culture. To determine the effects of 5-FU on cell viability, different concentrations of 5-FU (5, 10, 20, 40 and 50 µg/ml) were added to cells in the logarithmic growth phase, and the absorbance was measured 48 h after treatment.

Cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology), and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 40 µg protein per lane was separated by 10% or 15% SDS-PAGE and transferred onto PVDF membranes. The PVDF membrane was then blocked with TBST (Tween-20 at 0.1%) containing 5% skim milk at room temperature for 1.5 h. The following primary antibodies were diluted 1:1,000 in Primary Antibody Dilution Buffer (Beyotime Institute of Biotechnology): anti-NDRG4 (monoclonal, rabbit anti-human; cat. no. 9039; Cell Signaling Technology, Inc.), anti-DDIT3 (monoclonal, mouse anti-human; cat. no. 2895; Cell Signaling Technology, Inc.), anti-p-AKT (monoclonal, rabbit anti-human; cat. no. 4060; Cell Signaling Technology, Inc.), anti-AKT (monoclonal, rabbit anti-human; cat. no. 4685; Cell Signaling Technology, Inc.), anti-p-ERK (polyclonal, rabbit anti-human; cat. no. ab4819; Abcam), anti-ERK (polyclonal, rabbit anti-human; cat. no. ab17942; Abcam), anti-cleaved caspase-3 (polyclonal, rabbit anti-human; cat. no. 9661; Cell Signaling Technology, Inc.), anti-PARP (monoclonal, rabbit anti-human; cat. no. 9532; Cell Signaling Technology, Inc.), and anti-β-actin (monoclonal, mouse anti-human; cat. no. D191047; Sangon Biotech, Co., Ltd.). After incubated by the primary antibody overnight at 4°C, the membrane was washed with TBST (Tween-20 at 0.1%) at room temperature three times for 10 min each. Appropriate secondary antibodies derived from the same species as the primary antibodies (anti-rabbit IgG, HRP-linked; cat. no. 7074; Cell Signaling Technology, Inc.; and anti-mouse IgG, HRP-linked; cat. no. 7076; Cell Signaling Technology, Inc.) were diluted at 1:5,000 and added to the membranes, incubated at room temperature for 1 h. After that, the membrane was washed with TBST three times for 10 min each, and then FDbio-dura ECL Kit (Hangzhou Fude Biological Technology Co., Ltd.) was added for visualization. The blots were detected using the Tanon 550 Imaging System (Tanon Science and Technology Co., Ltd.).

The RNeasy Mini kit (Qiagen, Inc.) was used to extract total cellular RNA, from which cDNA was then synthesized using the PrimeScript RT-PCR kit (Takara Bio, Inc.) according to the manufacturers' protocols. qPCR was performed using qPCR SYBR-Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.) per the manufacturer's protocol. The thermocycling conditions were as follows: Pre-denaturation at 95°C for 5 min, then 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 20 sec. Relative mRNA expression levels were determined using the 2^−∆∆Cq method (15). The primer sequences were as follows: DDIT3 forward, 5′-GGAAACAGAGTGGTCATTCCC-3′ and reverse, 5′-CTGCTTGAGCCGTTCATTCTC-3′; CASP7 forward, 5′-AGTGACAGGTATGGGCGTTC-3′ and reverse, 5′-CGGCATTTGTATGGTCCTCTT-3′; and β-actin forward, 5′-CCTGGGCATGGAGTCCTGTG-3′ and reverse, 5′-TCTTCATTGTGCTGGGTGCC-3′. For PCR array analysis, the extracted cDNA was used for with the real-time RT² Profiler PCR Array (Qiagen, Inc.) according to the manufacturer's protocol.

TUNEL assays were performed using the One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. TUNEL regent was added to cells after 48 h of treatment with PBS or 5-FU. Each sample was observed by microscopy in five visual fields. Images of the cells were acquired using a fluorescence microscope.

Apoptosis assays were performed with cell lines using an Annexin V-FITC/PI apoptosis detection kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. The experiments were conducted 48 h after treatment with PBS or 5-FU. Flow cytometry was performed using a FC500 Flow Cytometer (Beckman Coulter Co., Ltd.), and the data were analyzed using FlowJo 10 software (FlowJo LLC).

Single cells (~200 per dish) were seeded into cell culture dishes with a diameter of 6 cm. After 18 days of culture at 37°C, visible colonies (>50 cells per colony) had formed and the culture was terminated. The medium was replaced every 3 days during the culture period. The colonies were then washed, fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 15 min and stained with crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. Finally, images of the stained colonies were captured, and the colonies were manually counted.

EdU staining was performed using the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 594 (cat. no. C0078S; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Cells in the logarithmic growth phase (~24–48 h in culture) were used for detection. Images of the cells were captured using a fluorescence microscope.

All data are expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). Student's t-test was used to analyze two independent groups. For comparisons between multiple groups, ANOVA was applied; Sidak's multiple comparisons test was used following two-way ANOVA, and Dunnett's multiple comparisons test was used following one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

SW480 and SW620 CRC cells overexpressing NDRG4 were successfully constructed through lentiviral infection. The NDRG4 protein levels of the overexpression cells were notably higher than those of their control counterparts (Fig. 1A). The MTT assay results indicated that the proliferative capacity of NDRG4-overexpressing cells was significantly lower than that of the control cells on days 1–4 of cell culture (Fig. 1B). The colony formation assay revealed that the NDRG4-overexpressing cells formed fewer visible clones than the control cells (Fig. 1C). Furthermore, EdU analysis indicted that the proliferation of NDRG4-overexpressing cells was inhibited relative to that of the control cells (Fig. 1D). These results indicate that NDRG4 inhibited CRC cell proliferation.

Since both the PI3K/AKT and ERK signaling pathways are associated with cellular proliferation (16), the levels of AKT and ERK phosphorylation can be measured to reflect their degrees of activation. Western blotting revealed decreased levels of p-AKT and p-ERK in NDRG4-overexpressing cells (Fig. 1E), indicating that NDRG4 inhibited the activation of the PI3K/AKT and ERK signaling pathways.

MTT assays were used to assess cell viability after 48 h of culture with different concentrations of 5-FU. The overexpression of NDRG4 significantly decreased the viability of cells treated with 5-FU at five different concentrations (Fig. 2A). Moreover, the survival rates of NDRG4-overexpressing SW480 and SW620 cells were decreased most significantly following treatment with 40 and 10 µg/ml 5-FU, respectively. Therefore, subsequent experiments were carried out using these two concentrations of 5-FU. Flow cytometry was performed to detect apoptosis, which showed that NDRG4 overexpression increased the rates of SW480 and SW620 apoptosis induced by 5-FU, compared with those of the control cells; measurements were based on the percentage of annexin-V-positive cells (Q2 + Q3), and two-way ANOVA revealed that the interaction between NDRG4 and 5-FU was statistically significant (Fig. 2B). Subsequently, a TUNEL apoptosis assay was conducted, and the experimental results confirmed that NDRG4 overexpression increased the apoptotic rate induced by 5-FU, compared with that of control cells (Fig. 2C and D). Furthermore, expression of the apoptosis-associated molecules cleaved caspase-3 (C-caspase-3) and poly-ADP-ribose polymerase (PARP, the cleaved substrate of caspase) was also examined. The expression level of C-caspase-3 was increased in NDRG4-overexpressing cells compared with the control cells, indicating an increase in cellular apoptosis. After adding 5-FU, the expression of C-caspase-3 was notably increased, and the difference between NDRG4-overexpressing cells and control cells was still apparent. Meanwhile, NDRG4 overexpression and 5-FU treatment also increased PARP expression (Fig. 2E). These results suggest that NDRG4 promoted the apoptosis of CRC cells induced by 5-FU.

To investigate the molecular mechanism by which NDRG4 inhibits CRC cells, a PCR array experiment was conducted using SW480 cells to identify genes with considerable fold changes between NDRG4-overexpressing cells and control cells (Fig. 3A). Then, apoptosis-related genes, such as CASP7 and DDIT3, were selected for qPCR verification. DDIT3 exhibited the greatest differential expression between NDRG4-overexpressing cells and control cells (Fig. 3B). The western blot results also confirmed that DDIT3 was expressed at higher levels in NDRG4-overexpressing SW480 and SW620 cells than in their control counterparts (Fig. 3C). These results indicate that NDRG4 upregulated DDIT3 expression in CRC cells.

To further confirm whether the promotional effect of NDRG4 on 5-FU-induced CRC cell apoptosis was associated with the increase in DDIT3 expression, two shRNAs were designed to target the DDIT3 gene, and transfected into SW480 cells, which showed a successful decrease in DDIT3 mRNA expression (Fig. S1), and then into NDRG4-overexpressing SW480 cells to verify the gene silencing effect (Fig. 4A); further experiments were performed with shRNA-2, which exhibited the most prominent gene-silencing effect. The expression levels of the apoptosis-related proteins C-caspase-3 and PARP were decreased following DDIT3-knockdown in SW480 NDRG4-overexpressing cells. Silencing DDIT3 reduced the increase in C-caspase-3 and PARP expression induced by 5-FU treatment (Fig. 4B). Subsequently, SW480 NDRG4-overexpressing cells were analyzed by flow cytometry, revealing that DDIT3-knockdown resulted in a decreased apoptotic rate compared with that of control cells, and that the decreasing trend was more apparent after the addition of 5-FU (Fig. 4C and D). These results indicate that DDIT3 plays an important role in the NDRG4-mediated promotion of CRC cell apoptosis induced by 5-FU.