Eukaryotic translation initiation factor 3 subunit G promotes human colorectal cancer

In this study, we investigated the role of eukaryotic translation initiation factor 3 subunit G (EIF3G) in colorectal cancer. Immunohistochemical analysis showed higher EIF3G expression in stage IV human colorectal cancer tissues than in adjacent normal tissues (P<0.01). EIF3G short hairpin RNA (shRNA) knockdown in HCT116 colon cancer cells reduced proliferation and increased apoptosis as compared to control. EIF3G knockdown also increased autophagy and reduced mTOR signaling, as evidenced by low phospho-AKT, phospho-S6K and phospho4EBP1 levels. Functional experiments indicated that overexpression of EIF3G promoted HCT-116 cells proliferation, migration and xenograft tumor growth. Finally, we observed lower xenograft tumor weights and volumes with EIF3Gsilenced HCT116 cells than with control cells. These findings demonstrate that EIF3G promotes colon cancer growth and is a potential therapeutic target.


Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth leading cause of cancer mortality in the worldwide [1,2].The survival rate for patients with CRC depends on the stage at diagnosis.Although localized and regional stage patients with CRC have 5-year survival rates of 90% and 70%, respectively, patients with regional and distant metastatic stage CRC have very low survival rates of 13% [3].Liver is the most common site of CRC metastasis since majority of the intestinal mesenteric drainage enters the hepatic portal venous system.More than 50% of patients with CRC develop liver metastatic disease, which leads to death of more than two thirds of these patients [4,5].Therefore, novel effective therapies for CRC are urgently needed.Previous studies indicated that somatic mutations in KRAS, BRAF, PIK3CA, AKT1, PTEN, NRAS, and TGFBR2 genes are common in CRC and have been used as prognostic biomarkers or therapeutic targets [6][7][8][9][10][11][12].However, the exact mechanisms underlying the onset of colorectal cancer are still unknown.
Eukaryotic translation initiation factors (EIFs) regulate cellular protein synthesis at the level of mRNA translation initiation [13].EIF3 is the most complex translation initiation factor that mediates mRNA binding to the 40S ribosomal subunit [14].In most multicellular eukaryotes, EIF3 is composed of 13 subunits, named eIF3a-eIF3m [15][16][17].Differential expression of EIF3 subunits are frequently observed in many human cancers [18,19].Moreover, overexpression of some EIF subunits drives de novo holocomplex formation, which results in modest increase in protein synthesis along with cell transformation [20].However, the specific mechanisms related to transformation are unknown.
The eukaryotic translation initiation factor 3 subunit G (EIF3G) binds to the eIF3 holo-complex via EIF3E.The presence of EIF3G and EIF3E distinguishes a sub-complex that translates a restricted set of mRNAs from another complex, which contains the full complement of EIF3 subunits and promotes the translation of all mRNAs [21,22].Recently, EIF3G has been implicated in the development and progression of several tumors.It is involved in caspase-Am J Transl Res 2019;11(2):612-623 mediated apoptosis in breast cancer [23], ovarian cancer [24], and prostate cancer cells [25], thereby suggesting an oncogenic role for EIF3G in cancer.However, the mechanisms by which EIF3G regulated the apoptosis and autophagy in CRC remain unclear.Therefore, in this study, we explored the role of EIF3G in colorectal cells and pathways involved in its regulation.

Immunohistochemistry
Immunohistochemical staining of human colon cancer tissue specimens collected from Liaocheng People's Hospital were carried out as previously described (25).In brief, the specimens were first deparaffinized and subjected to heat-induced antigen retrieval with citrate buf-ing; and score 3, strong staining.The scoring for positive staining was as follows: score 0, no staining; score 1, 0-20% staining; score 2, 21-60% staining; and score 3, 61-100% staining.Total score was the sum of scores for staining intensity and the proportion of positive staining.A total score of 0 indicated negative; score 1, weak; score 2-4, moderate and score 5-6, strong.

EIF3G stably overexpressed HCT116 cell line construction
In order to construct EIF3G stably overexpressed CRC cell line, the over-expression vector of EIF3G was constructed in pcDNA3.1 by Genscript (Nanjing, China).The plasmid was transfected into HCT116 cells, and selected using neomycin (1000 µg/ml) for four weeks.

Western blot
Total protein was extracted from HCT116 cells with Sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris-HCl pH 6.8, 5 mM EDTA, 2% SDS and 5% glycine).Protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher, MA, USA).Equal amounts (30 μg) of protein from each sample were seperated in a 10% polyacrylamide SDS gel at 80V for 2 h.The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA) at 200 mA for 3 h.The membranes were blocked for 1 h with 5% non-fat milk in 1X Tris-buffered Saline with Tween 20 (TBST, 20 mM Tris pH 7.6, 150 mM NaCl and 0.05% Tween-20) followed by primary antibodies at 4°C overnight.Then, the membranes were washed thrice with 1XTBST and incubated for 2 h at room temperature with horseradish peroxidase (HRP)conjugated goat anti-rabbit secondary antibody (Santa Cruz, dilution 1:5000).The primary antibodies included rabbit anti human EIF3G

Flow cytometry
Apoptosis was measured in control and EIF3G shRNA transfected HCT116 cells with Annexin V-FITC and propidium iodide (PI) using the Dead Cell Apoptosis Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and then assessed by flow cytometry using the FACS Calibur (BD Biosciences).In brief, cells were harvested at 80% confluence after trypsinization and washed with ice-cold PBS.Then, the cells were fixed in 70% cold ethanol, centrifuged, washed with 1X PBS washes and stained with Annexin V/PI solution for 30 min at room temperature.The cells were analyzed by flow cytometry.FACS plots were analyzed to determine percent early apoptotic (AnnexinV + PI -), late apoptotic/secondary necrotic (AnnexinV + PI + ), and necrotic (AnnexinV - PI + ) cells.

Monodansylcadaverine (MDC) staining
The auto fluorescent agent, monodansylcadaverine (MDC), was used to analyze the autophagic process.After culturing for three days, autophagy was induced in HCT116 cells by amino acid starvation for 24 h.Then, the cells were incubated with 0.05 mM MDC in PBS at 37°C for 10 minutes.After incubation, the cells were washed thrice with PBS and immediately observed under a fluorescence microscope (Olympus, Tokyo, Japan).The cells were assessed by flow cytometry and MDC stained cells were quantified.

Statistical analysis
Statistical analysis was performed with the SPSS 11.0 software (SPSS Inc., Chicago, IL, USA).The immunohistochemical data was analyzed by the Kruskal-Wallis nonparametric oneway ANOVA followed by the Nemenyi pairwise multiple tests.Data were expressed as mean ± SD and were analyzed by one-way analysis of variance followed by the Student-Neuman-Keul's test.P<0.05 was considered statistically significant.

Ethics statement
Informed consent was obtained from all patients, and experiments were performed in

EIF3G is highly expressed in colorectal cancer tissues
Immunohistochemical analysis of EIF3G staining in colorectal cancer tissue specimens showed no correlation between EIF3G expression and different cancer stages (Table 1, P=0.286).However, EIF3G staining gradually increased from stages I to IV and was the strongest in stage IV, which is highly metastatic.Furthermore, as shown in Figure 1A, EIF3G was mainly localized to the cytoplasm, and its expression was higher in tumor tissues than in adjacent normal tissues of stages III and IV.Quantitative RT-PCR and western blot analysis demonstrated high EIF3G mRNA and protein expression in tumor tissues than in adjacent tissues, respectively (Figure 1B and 1C).

EIF3G knockdown efficiency in HCT116 cells by shRNAs
Next, we compared EIF3G knock down efficiency in HCT116 cells transfected with 4 different EIF3G shRNAs (1-4) and 1 control scramble shRNA.QRT-PCR and western blot analysis revealed that EIF3G mRNA and protein levels were significantly decreased by shRNAs 2 and 4 than shRNAs 1, 3 and scramble, respectively (Figure 2A and 2B).

EIF3G silencing suppresses HCT116 cell proliferation
Next, we analyzed the consequences of EIF3G knockdown in HCT116 cells.As shown in Figure 3A, there were no changes in cell morphology due to EIF3G knockdown.However, EIF3G shRNA2 reduced cell density after 72 h.CCK-8 cell proliferation assay demonstrated that EIF3G silencing decreased HCT116 growth rate (Figure 3B).This suggested that EIF3G promoted colorectal cell proliferation.

EIF3G silencing promotes HCT116 cell apoptosis
We analyzed apoptosis by flow cytometry of AnnexinV and PI double stained control and EIF3G silenced HCT116 cells.EIF3G silenced HCT116 cells showed increased early and late apoptosis than controls (Figure 4A and 4B).This was further confirmed by increased cleaved caspase-3 in EIF3G silenced HCT116 cells than in controls (Figure 4C and 4D).We have repeated these experiments using another CRC SW480 cell lines.The results were consistent with data using HCT-116 (Figure S1A-D).

EIF3G silencing induces HCT116 cell autophagy
Next, we analyzed the effect of EIF3G silencing on autophagy in HCT116 cells by monodansylcadaverine (MDC) staining.EIF3G silenced HCT116 cells demonstrated autophagosome staining under fluorescence microscopy (Figure 5A).Flow cytometry analysis of MDC stained cells demonstrated increased autophagy in EIF3G silenced HCT116 cells than in controls EIF3G in CRC, we stably overexpressed EIF3G in HCT116 cells by transfecting EIF3G overexpression plasmid (Figure 7A).The EIF3G protein levels in stably EIF3G overexpressed HCT116 or control cells were detected by western blotting (Figure 7B).The results of CCK-8 assay indicated that EIF3G overexpression drastically promoted HCT116 cell proliferation (Figure 7C).In addition, transwell migration assays showed that EIF3G overexpression significantly enhanced HCT116 cell migration (Figure 5B).Western blot analysis demonstrated that downregulation of EIF3G reduced phosphorylated Akt, p70 S6K and 4E-BP1 levels (Figure 5C-F).These data demonstrated that down-regulation of EIF3G induced HCT116 cell autophagy by inhibiting the mTOR signaling pathway.

EIF3G silencing inhibits HCT116 xenograft tumor growth in nude mice
Next, we investigated the effect of EIF3G

Discussion
EIF3G belongs to the EIF3 family and is involved in transcriptional initiation.However, it has also been implicated in other roles.For example, it inhibits HIV replication and is cleaved by HIV-1 PR [26].Recent studies have indicated that aberrant expression of EIF3G may be critical to many human diseases including cancers.In the present study, we demonstrated gradual increase in EIF3G expression with increasing colorectal cancer stages (I to IV).EIF3G was mainly localized to the cytoplasm, and its expression was higher in tumor tissues than in adjacent normal tissues of stages III and IV.Apoptosis or other types of cell death mechanisms are activated under prolonged stress [28].Moreover, autophagy leads to autophagic cell death (ACD) [29].Furthermore, the autophagic roles of caspases and apoptotic functions of autophagy-related proteins (ATGs) overlap.
According to the present results, deregulation of EIF3G inhibited the phosphorylation of Akt, p70 S6K and 4E-BP1, which led to increased HCT116 cell apoptosis and autophagy.This suggests a role of EIF3G in the crosstalk between apoptosis and autophagy.These data also indicate that down-regulation of EIF3G induces HCT116 cell autophagy via inhibition of mTOR signaling pathway.However, the detailed interaction between apoptosis and autophagy needs to be investigated further.
In conclusion, our findings indicated that EI3FG was upregulated in colorectal cancer patient's tumor tissues, promotes CRC cell proliferation, apoptosis, autophagy and migration in vitro, and CRC tumor growth in vivo.Our study demonstrates that EIF3G plays an important role in

Figure 1 .
Figure 1.Immunochemistry of EIF3G in stage IV colorectal cancer.A. Three representative images showing immunohistochemical staining of EIF3G in stage IV colorectal cancer patient tumor and adjacent normal tissues (magnification, ×200).The brown dots represent EIF3G staining and the blue dots represent nuclear staining by hematoxylin.B. Quantitative RT-PCR analysis showing EIF3G mRNA expression in tumor and adjacent normal tissues from CRC patients (n=35).C. Western blot analysis showing EIF3G protein expression in tumor and adjacent normal tissues from CRC patients.D. Quantitative analysis of relative EIF3G protein expression in tumor and adjacent normal tissues from CRC patients.**P<0.01compared with the adjacent group.
according to manufacturer's protocol.Briefly, experimental and control HCT116 cells were seeded in 96-well plate at a density of 3000 cells per well and cultured for 3 days.Then, the cells were further incubated for another 2 hrs with 1/10 th volume of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt solution.The plate was read in a microplate reader at 450 nm.Cell proliferation was also detected using Ethynyl deoxyuridine (EdU) Kit (Thermo Fisher, MA, USA), according to the manufacturer's protocol.All experiments were performed in triplicates.

Figure 6 .
Figure 6.EIF3G silencing inhibits HCT16 xenograft growth in nude mice. A. Xenograft tumor volume from control and EIF3G transfected HCT116 cells at various time points (days 1-21).EIF3G silenced HCT116 cells show decreased tumor volume from days 14 to 21 than control shRNA transfected HCT116 cells (n=5).B. Photographs showing xenograft tumors from control and EIF3G shRNA transfected HCT116 cells on day 21 in nude mice.C. Bar graph shows mean xenograft tumor weight on day 21 from control and EIF3G shRNA transfected HCT116 cells (n=3).D. Analysis of body weight of nude mice xenografted with control and EIF3G shRNA transfected HCT116 cells at various time points (days 1-21; n=5).E. Representative EIF3G protein levels in xenograft tumor tissues from control and EIF3G shRNA transfected HCT116 cells with β-actin as internal control.F. Quantitative analysis of EIF3G proteins expression in xenograft tumors derived from control and EIF3G shRNA transfected HCT116 cells (n=3).**P<0.01compared with the normal control group.

Figure 7 .
Figure 7. EIF3G overexpression promotes HCT116 cell proliferation and migration.A. EIF3G expression levels in EIF3G stably overexpressed and control HCT116 cells were analyzed by qRT-PCR (n=3).B. EIF3G protein levels in EIF3G stably overexpressed and control HCT116 cells were detected by western blotting.C. CCK8 cell proliferation analysis of EIF3G stably overexpressed and control HCT116 cells (n=3).D and E. Transwell migration assays analysis of the migrated cell numbers of EIF3G stably expressed and control HCT116 cells per field after 48 h incubation (n=3).**P<0.01compared with the pcDNA3.1 group.

Table 1 .
The expression of EIF3G in CRC patients' tissues (IHC)