NDRG1 Inhibition of Tumor Progression in Caco2 Cells

Background: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Invasiveness and migration are the main cause of death, and so there is a need to nd a sensitive, reliable molecular marker that can predict the migration of colorectal cancer at an early stage. NDRG1 (N-myc Downstream Regulated Gene 1) has been reported to be a multifunctional gene that has a strong relationship with tumor invasion and migration, but theories about the current role of NDRG1 in colorectal cancer remains to be conclusively determined. Methods and Results: Through lentivirus infection and CRISPR/Cas9 methods, respectively, we established that NDRG1 stably overexpressed and knocked out Caco2 cell lines. CCK8(Cell Counting kit 8) data showed that NDRG1 inhibited Caco2 proliferation. Flow cytometry further conrmed that the cell cycle can be arrested at the G1/S phase when NDRG1 overexpresses, while the number of G2 phase cells signicantly increased after NDRG1 was knocked out. This means that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase. Our data also demonstrated that NDRG1 promotes early cell apoptosis. The strength of invasion and migration was decreased when NDRG1 overexpressed. Conclusions: Our results underline that NDRG1 inhibits tumor progression in Caco2 cells. These ndings may provide a new potential therapeutic strategy for the treatment of CRC.


Introduction
Among new cancer cases in the world in 2018, colorectal cancer accounted for 10.2% of new cancer cases and 9.2% of cancer deaths. In general, colorectal cancer has become the third most common cancer [1]. Invasiveness and migration are the two malignant features of colorectal cancer that are the main causes of death. Research has shown that the invasion and migration of colorectal cancer is associated with genes as well as miRNA [2]. However, early, sensitive biological indicators that can predict the migration of colorectal cancer at an early stage have not yet been discovered.
NDRG1 is a multifunctional gene. Recent studies have shown that NDRG1 is related to tumor invasion and migration [3], apoptosis [4], tumor cell proliferation [5], drug response, and the drug resistance of tumor cells [6]. Its expression in tumors is tissue-speci c. It acts as a tumor suppressing gene in prostate cancer [7] and ovarian cancer [8], but in lung cancer and esophagus squamous cell carcinoma NDRG1 promotes tumor development [9]. The current role of NDRG1 in colorectal cancer is also not conclusively known. Many researchers have found that NDRG1 inhibits tumor invasion and migration in colorectal cancer [10][11][12], while Wang [13] and Shah [14] found that NDRG1 promoted the development of colorectal cancer. Koshiji [15] found that the expression of NDRG1 differed with race and pathological stage in colorectal cancer patients.
To further investigate the role of NDRG1 in the development of colorectal cancer, we conducted the experiments described below aimed at establishing an early diagnosis and prognosis estimation for colorectal cancer migration, thus providing new targets for molecular therapy for colorectal cancer.

Material And Methods
Cell culture A Caco2 colorectal cancer cell line was purchased from the Kunming Cell Bank of the Kunming Institute of Zoology, Chinese Academy of Sciences. The Caco2 cell line was derived from a 72-year-old male Caucasian colorectal adenocarcinoma. The 293T cells were provided by the Kunming Institute of Zoology, Chinese Academy of Sciences. The medium required for Caco2 and 293T cells was a DMEM medium ( Dulbecco's Minimum Essential Medium ,Gbico, USA) containing 10% FBS(Fetal Bovine Serum ,Gbico, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. All cell lines were incubated at 37 °C in a humidi ed atmosphere with 5% CO 2 .

Establishment of cell lines
The lentiviral plasmid used for NDRG1 overexpression was GV358-NDRG1, which was purchased from Genechem (Shanghai,China). The plasmid vector used for NDRG1 knockout was pL-CRISPR.EFS.GFP, which was provided by the Kunming Institute of Zoology, Chinese Academy of Sciences. We designed three sgRNAs which were both located in exon 3 of the NDRG1 gene: sgRNA1, sgRNA2,sgRNA3 (Table 1), inserting these three sgRNAs into pL -CRISPR.EFS.GFP plasmid, respectively. All constructed plasmids were identi ed by sequencing. The GV358 and pL-CRISPR.EFS.GFP empty plasmids were used as a control. The plasmid and its corresponding packaging plasmid were co-transfected into 293T cells for virus packaging, and then the cells were infected with virus. Finally, they were sorted by ow cytometry.
The monoclonal cells of NDRG1 knockout were isolated by ow cytometry sorting. Then the monoclonal cells were expanded and cultured to extract the DNA; the third exon of NDRG1 was ampli ed by PCR(Polymerase Chain Reaction). The PCR product was sequenced and identi ed, the successfully identi ed cells were subjected to a T-A clone, and T-A clone product was also sent for sequencing. (All the primers used are shown in Table 1 High-e ciency RIPA lysate extracted the total cellular protein and quanti ed protein using the BCA method. 80 µg protein was separated by electrophoresis on a 10%SDS-polyacrylamide gel. The proteins were electrotransferred from the gel to the PVDF membrane. The membrane was blocked with 5% non-fat milk solution for 2 h. The antibody rabbit, anti-human NDRG1 monoclonal antibody (1:500, cat.no. 9485S;CST Co., Ltd), β-tublin (1:5000, cat.no. 6046; Abcam Co., Ltd) was incubated for 1 h, and then incubated at 4 °C overnight. The next day, the corresponding secondary antibody (NDRG1 1:500, β-tublin 1:10000, cat.no. AS014; Abclonal Co., Ltd) was incubated for 1 h at room temperature. The membrane was washed and underwent detection by the enhanced ECL detection system.
Cell counting kit-8 (CCK-8) assay GV358-control, GV358-NDRG1, pL-control, and pL-NDRG1-knockout cells were seeded in 96-well plates at 2 × 10 3 cells per well for the CCK-8 cell proliferation assay (CCK8 kit, Beyotime, Co., Ltd), then cultured for 1, 2, 3, and 4 days, respectively. According to the manufacturer's instructions, the cells were incubated with the CCK-8 reagent at 37 °C for 1 hour, with the absorbance of each sample scanned on a microplate reader equipped to read absorbance values at 450 nm.

Assessment of cell cycle by ow cytometric analysis
For cell cycle synchronization, 5 × 10 5 cells were plated in 6-well plates. After the cells were completely adherent, the cells were cultured in the serum-free medium for 24 h, and then cultured in a serum medium for 24 h. The cells were collected and underwent xing with pre-cooled 75% ethanol at 4 °C for 14 ~ 24 h. After xation, PI and RNaseA were added to the stain cells for 30 min in the dark, then detected by ow cytometry.

Assessment of apoptosis by ow cytometric analysis
The cells were trypsinized without EDTA then washed twice with Cell Staining Buffer and resuspended with Annexin V Binding Buffer to adjust the cell concentration to (0.25-1.0) x 10 7 /mL. 100 µL of the cell suspension was taken into a new 1.5 mL centrifuge tube and 5 µL of APC Annexin V and 10 µL of Propidium Iodide Solution were added, then incubated at room temperature for 15 min in the dark and tested by ow cytometry.

24-transwell for invasion and migration
The invasion experiment was done with the Chamber Matrigel Invasion 24-well DO (cat. no. 354480; Biocoat Co., Ltd) and the migration experiment was done with a Transwell Chamber (cat. no. 3422; Corning Co., Ltd). All experimental steps were carried out according to the instructions. The cells inoculated were 1 × 10 5 , and the lower chamber was added with a DMEM medium containing 20% FBS. After 48 hours, the chambers were xed in 4% paraformaldehyde, stained with 0.1% crystal violet, and a number that passed through the microscope was observed. Then, the cells stained by crystal violet were eluted with 33% acetic acid, and the absorbance at 570 nm of the eluate was measured by a microplate reader.

Statistical analysis
The data were statistically analyzed using the GraphPad Prism 7.0. The Student's t test was used for statistical analysis. Data are presented as a means ± standard deviation. P < 0.05 was considered a statistically signi cant difference.

Flow cytometry sorting of cells
After the virus infected the cells, we sorted the positive cells (GFP positive) by sorting ow cytometry. The results showed that the positive rates of GV358-control cells, GV358-NDRG1 cells, pL-control cells, and pL-NDRG1-knockout cells were 58.3%, 54.1%, 0.506%, and 0.461%, respectively (Fig. 1A-D). For knockout cells, we used sorting ow cytometry for monoclonal cells with an e ciency of 4.73% (Fig. 2).

Identi cation of gene knockout cell sequences
After the monoclonal cells were expanded and cultured, the DNA of the cells was extracted, and the third exon of NDRG1 was ampli ed. The product was sent for sequencing. The sequencing result showed that a DNA strand nick was generated on the third exon of NDRG1, then an A base was inserted. An analysis of the NDRG1 sequence in which the A base was inserted was performed using SnapGene software, and it was found that a TGA stop codon was formed after the inserted A base, thereby terminating the translation of NDRG1. The ampli ed exon of NDRG1 was identi ed by T-A clone and sent to sequencing again. The sequencing results were consistent with the previous results (Fig. 3).
Veri cation of NDRG1 stable overexpression and knockout of the Caco2 cell line After sorting GFP-positive cells by ow cytometry and sequencing identi cation, we detected the changes of NDRG1 mRNA and protein expression levels by QPCR and Western Blotting. The relative expression level of NDRG1 mRNA of GV358-NDRG1 cells increased about three-fold compared with the control group (P=0.0006).In the pL-NDRG1-knockout cells,NDRG1 mRNA levels were clearly diminished relative to the controls (P 0.0001) (Fig. 4A). Western Blotting results revealed that the protein expression level in the overexpression cells rose approximately doubled(P 0.0001). After NDRG1 gene knockout, the level reduced signi cantly (P 0.0001) (Fig. 4B, Fig. 4C, Fig. 4D). All the cells were expanded (Fig. 5).
NDRG1 arrested the cell cycle in the G1/S phase, which inhibited the proliferation of Caco2 cells In different tumor cells, NDRG1 promoted or inhibited the proliferation of tumor cells. To con rm the role of NDRG1 in Caco2 cells, we detected cell proliferation by CCK8 and ow cytometry for the cell cycle. As shown in the gure (Fig. 6), the relative proliferative activity of GV358-NDRG1 cells was decreased at 48 h, 72 h and 96 h compared with the GV358-Control cells (P<0.001) (Fig. 6A, Fig. 6B), while pL-NDRG1knockout cells were higher than pL-control cells at 24 h, 48 h, 72 h, and 96 h (P<0.01) Fig. 6C, Fig. 6D), which indicates that overexpression of NDRG1 could inhibit the proliferation of Caco2 cells. Flow cytometry may illuminate the mechanism of these. The proportion of G1 phase cells was signi cantly increased (P<0.0001) (Fig. 6E, Fig. 6F, Fig. 6G) after overexpression of NDRG1, while G2 phase cells were increased after NDRG1 knockout (P<0.0001) (Fig. 6H, Fig. 6I, Fig. 6J). The above results showed that NDRG1 inhibits the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.

NDRG1 inhibited metastasis and invasion of Caco2 cells
NDRG1 was also involved in tumor invasion and metastasis. Metastasis ability is also called exercise ability. Invasion and metastasis were two complementary processes. Metastasis ability can be regarded as the basis of tumor cell invasion. Thus, we also detected changes in the invasion and metastasis ability of cells through the 24-Transwell chamber. The number of cells passing through the chamber decreased after overexpression of NDRG1 (P=0.0001) but increased after NDRG1 knockout (P=0.0001). These results indicate that NDRG1 inhibited metastasis and invasion of Caco2 cells (Fig. 7).
NDRG1 promoted early apoptosis of Caco2 cells.
In order to further investigate the effect of NDRG1 on Caco2 cells, we used ow cytometry to detect cell apoptosis. The early apoptosis of the cells was increased after the overexpression of NDRG1 (P=0.0002) (Fig. 8A, Fig. 8B, Fig. 8C), and the rate of pL-NDRG1-knockout cells was decreased compared with pLcontrol cells (P=0.0013) (Fig. 8D, Fig. 8E, Fig. 8F), indicating that NDRG1 promoted early apoptosis of Caco2 cells.

Discussion
According to the 2018 Global Cancer Statistics Report, the third most common cancer is colorectal cancer [1], thus presenting a serious threat to people's life and health. Recent studies have shown that an excessive intake of red meat and processed meat increases the risk of colorectal cancer [16][17][18]. A large alcohol intake is also a risk factor [17,18], and a high-fat diet may also be related to colorectal cancer [19].
Invasiveness and metastasis are synergistic results of a variety of factors that promote the transfer mechanism of tumor cells in response to changes in the living environment. Thus, the search for colorectal cancer migration suppressor genes is particularly important in current research. Further research on migration-associated genes in colorectal cancer cells will guide the early diagnosis of colorectal cancer, predicting early migration of cancer, which is of signi cance for reducing the mortality of patients with colorectal cancer.
Recent studies have shown that NDRG1 may promote or inhibit the development of tumors, but its function in tumors is highly controversial. Some scholars believe that NDRG1 promotes the progression of liver cancer [31][32][33], lung cancer [34][35][36], bladder cancer [37], and gastric cancer [38,39]. However, other researchers have found that NDRG1 inhibits the development of prostate cancer [7,40], nasopharyngeal carcinoma [41], oropharyngeal squamous cell carcinoma [42], and ovarian cancer [8]. The role of NDRG1 in colorectal cancer is also not conclusively understood. Nathalie [43] found that NDRG1 mRNA levels were signi cantly reduced in colorectal cancer tissues. Wang [10] showed that silencing NDRG1 expression in colorectal cancer cells increased cell growth, invasion, and migration. Mi [11] also found that NDRG1 promoted epithelial-mesenchymal transition, invasion, and migration of colorectal cancer cells. These studies indicate that NDRG1 is a positive regulator in colorectal cancer. Interestingly, Koshiji [15] found that the expression of NDRG1 was different in colorectal cancer patients of different races and at different pathological stages, which resulted in different clinical outcomes. Some research also supports the premise that NDRG1 is a transfer-promoting gene [13,44]. Why does NDRG1 have different effects in different tumor cells? In addition to the above factors, it may be related to speci c cell types -pleiotropic functions of genes. We established that NDRG1 stably overexpressed and knocked out the Caco2 cell line, then measured changes in cell proliferation ability, cell cycle, apoptosis, invasion, and migration ability. The results show that NDRG1 can arrest the cell cycle in the G1/S phase, slowing the proliferation rate of the cells. In addition, the overexpression of NDRG1 promoted the early apoptosis of Caco2 cells, and NDRG1 knockout inhibited the early apoptosis of Caco2 cells. In this experiment, we used a 24-Transwell chamber to detect the invasion and migration of cells at 48 h. The results show that NDRG1 overexpression inhibited cell invasion and migration, while cell invasion and migration were promoted after knockout. In conclusion, NDRG1 is a tumor suppressor in Caco2 cells.
The speci c role of NDRG1 in colorectal cancer is currently the subject of controversy. To further clarify the mechanism by which NDRG1 inhibits tumorigenesis, we are proceeding to observe the signal pathway and the abilities of NDRG1 in nude mice. This preliminary basic research is useful in nding new targets for the molecular therapy of colorectal cancer, providing a greater theoretical basis for future clinical applications.

Conclusion
In conclusion, we conducted analyses on the effect of NDRG1 on the Caco2 cell line. Through the analyses on the NDRG1 on the biological behaviors of Caco2 cell line, we found that NDRG1 would reduce the proliferation, apoptosis, migration and invasion abilities of Caco2 cell line. NDRG1 may have potential application value as a molecular biological index to predict the early invasion and metastasis of colorectal cancer.    Experiments were repeated three times.