Abstract
Objective
To investigate the effect of silencing GDP dissociation inhibitor 2
(GDI2) on colorectal cancer development and possible mechanisms based
on transcriptomic analysis.
Methods
The differences in the expression levels of GDI2 in normal colorectal
tissues and tumor tissues of colorectal cancer (CRC) patients were
detected. The correlation of GDI2 expression levels with survival and
clinical characteristics of CRC patients was analyzed. The effects of
GDI2 expression levels on the biological functions of CRC cells were
examined by CCK-8 assay, plate clone formation assay, wound healing
assay, and Transwell assay. The effect of GDI2 on the proliferation and
growth of xenograft tumors was investigated by a xenograft tumor model
of CRC in nude mice. Based on transcriptomics, we explored the possible
mechanisms and associated pathways of the effect of silencing GDI2 on
CRC cells. Cellular experiments and Western blot assays were performed
to verify the potential mechanisms and related pathway of GDI2 action
on CRC.
Results
The expression levels of GDI2 in CRC tissues and cells were higher than
those in normal tissues and cells. The expression level of GDI2
correlated with clinical characteristics such as lymphatic metastasis,
tumor stage, tumor volume, and lymphocyte count. Silencing of GDI2
reduced the proliferative activity and migration and invasion ability
of CRC cells, as well as inhibited the proliferation of CRC xenograft
tumors. The differentially expressed genes were significantly enriched
in biological processes such as cell cycle arrest and the p53 signaling
pathway after GDI2 silencing. The percentage of G0/G1 phase cells in
CRC cells was increased after silencing GDI2 as verified by flow
cytometry. RAB5A was highly associated with the p53 pathway and could
interact with TP53 via the ZFYVE20 protein. The mutual binding between
GDI2 protein and RAB5A protein was verified by immunoprecipitation
assay. Silencing GDI2 while overexpressing RAB5A reversed the reduced
proliferation, migration, and invasion ability as well as cell cycle
arrest of CRC cells. Meanwhile, the addition of p53 signaling pathway
inhibitor Pifithrin-α (PFT-α) also reversed the biological effects of
silencing GDI2 on CRC cells. The p-p21 and p-p53 protein expression
levels were significantly greater in the sh-GDI2 group than in the
sh-NC group. However, the p-p21 and p-p53 protein expression levels
were reduced after silencing GDI2 while overexpressing RAB5A.
Conclusion
Silencing GDI2 activates the p53 signaling pathway by regulating RAB5A
expression levels, which in turn induces cell cycle arrest and
ultimately affects the proliferative activity, migration, and invasive
ability of CRC cells.
Keywords: Colorectal cancer, GDI2, Transcriptomics, p53 signaling
pathway
1. Introduction
Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal
tract originating from the colon or rectum and is the second leading
cause of cancer deaths worldwide [[37]1]. CRC develops slowly, has a
complex and diverse pathogenesis, and can be caused by a variety of
risk factors, including personal dietary habits, environmental factors,
and genetic family history [[38]2]. Common clinical symptoms include
blood in the stool, iron deficiency anemia, abdominal pain, weight
loss, and loss of appetite. Currently, the clinical treatments for CRC
include surgical resection, radiotherapy, chemotherapy, and
immunotherapy. The predominant tool is surgical resection, but the
therapeutic efficacy is unsatisfactory, especially for patients
diagnosed with stage 4 CRC, with a 5-year survival rate of less than
10 % [[39]3]. In recent years, approximately 20 % of new CRC cases have
been observed to involve metastatic disease [[40]4]. For patients with
metastatic disease, radiotherapy is generally the preferred treatment,
but there are limited therapeutic drugs available for targeting tumors
in clinical practice [[41]5]. Although radiotherapy can effectively
reduce the risk of CRC recurrence and increase overall survival rates,
it can also have significant adverse effects, such as oral ulcers and
gastrointestinal reactions [[42]6]. The prognosis of CRC is related to
the stage at diagnosis, with less than a 10 % survival rate when
distant metastases occur. Therefore, it is crucial to develop reliable
biomarkers with the ability to predict CRC metastasis.
GDP dissociation inhibitor (GDI) is a small GTP-binding protein in the
Ras superfamily that regulates GDP-GTP exchange among RAb family
members and affects the vesicular transport of substances between
organelles [[43]7]. The identified forms of GDI include GDI1 and GDI2.
Specifically, the GDI2 gene, located at 10p15.1, consists of 76910
nucleotides and encodes a protein called GDI2 with a molecular weight
of 51 KD(8). The GDI2 gene is upregulated in various cancers and can
regulate biological functions, including tumor cell proliferation,
apoptosis, migration, and cell metabolism [[44][9], [45][10],
[46][11]]. Our previous study showed that dimethylhydrazine
(DMH)-induced CRC rats exhibited enhanced mRNA expression levels of
GDI2 [[47]12]. However, the specific role of GDI2 in CRC development
and its mechanisms remain unclear and warrant further investigation.
To investigate the role of GDI2 in CRC development, we conducted a
series of in vitro cellular experiments and in vivo experiments based
on previous studies. We aimed to elucidate its mechanism of action and
related signaling pathways through transcriptome analysis, expecting to
provide potential tumor markers for the prevention and treatment of
CRC.
2. Materials and methods
2.1. Cell culture and transfection
NCM460, HCT116, and SW1116 cell lines were purchased from the Cell Bank
of the Chinese Academy of Medical Sciences (Shanghai, China). Cells
were cultured at 37 °C in DMEM medium (Four Seasons Biological Company,
Hangzhou, China). Logarithmic growth stage HCT116 cells and
SW1116 cells were used and transfected with short hairpin RNA (shRNA)or
shRNA-GDI2. The three shRNA sequences targeting GDI2 (sh-GDI2#1,
sh-GDI2#2, and sh-GDI2#3) and the negative control shRNA (sh-NC) were
produced by GenePharma (Shanghai, China). Cells were transfected with
Lipofectamine® 2000 transfection reagent (Invitrogen, USA). The
sequence with the best inhibition rate was screened using quantitative
real-time RT-PCR (qRT-PCR) and Western blot. Overexpression of RAB5A
was performed by qRT-PCR of the complete coding fragment of RAB5A, and
the fragment was ligated into the pcDNA3.1 vector to construct
pcDNA3.1-RAB5A. Transfection of empty pcDNA3.1 was used as a negative
control. For sh-GDI2+PFT-α group and PFT-α group cells, PFT-α (SY1065,
Biolab, China) was dissolved in dimethyl sulfoxide (DMSO) before
pro-use and needed to be added to the culture plate about 4 h before
transfection.
2.2. CRC tissue specimens
Patients diagnosed with CRC at the Affiliated Hospital of Guangdong
Medical University between January 2022 and February 2022 were
collected through the case system of the inpatient department of the
Affiliated Hospital of Guangdong Medical University. CRC tumor tissues
from CRC patients were taken as the experimental group, and normal
tissues adjacent to the cancer from the same patients were used as the
control group for subsequent experimental analysis. All patients
included in the study were diagnosed by cytological or
histopathological methods, and all patients had not received
chemotherapy or radiotherapy. All participating patients were informed
and signed an informed consent form before the study. The study
protocol was approved by the Ethics Committee of the Medical Center of
the Affiliated Hospital of Guangdong Medical University (No.
YJYS2022237).
2.3. Bioinformatics data analysis
Download RNAseq data and clinical information in grade 3 HTSeq-FPKM
format from the TCGA website for the Colon Cancer (COAD) and Rectal
Cancer (READ) programs. Meanwhile, normal control cases from the GTEx
database were downloaded from UCSC XENA and processed uniformly by the
Toil program. The level 3 RNAseq data from TCGA and GTEx in the format
of HTSeq-FPKM were converted to TPM for subsequent analysis. Referring
to the study by Liu [[48]13], we tested whether the GDI2 gene was
correlated with the clinical data. Clinical data and TCGA data were
first processed, including age, gender, lymphatic invasion, Tumor Node
Metastasis (TNM) stage, tumor stage, presence of metastasis, lymphocyte
count, and tumor volume size. Differences in GDI2 gene expression
between the control group and the cancer group were compared. Clinical
symptoms were combined with gene and gene expression after all
treatments. The gene risk values and clinical traits were correlated by
the beeswarm package in R language. The correlation between genes and
different clinical traits was determined based on the magnitude of the
P-value. The clinical prognosis of GDI2 regarding overall survival
(OS), progression-free survival (PFS), disease-free survival (DFS), and
disease-specific survival (DSS) in CRC patients was assessed using the
Survminer package concerning the experimental approach of Liu [[49]14].
Survival curves were constructed using the Kaplan-Meier method. P
values and hazard ratios (HR) with 95 % confidence intervals (CI) were
derived by log-rank test and univariate Cox regression [[50]15].
P < 0.05 was considered statistically significant.
2.4. qRT-PCR
The primers were designed according to the sequences of related genes
included in GenBank and synthesized and purified by Shanghai Sankyo
Biotechnology Co. The sequences of GDI2 primers are as follows,
forward: 5′-ATTCCACAGAACCAAGTCAATCGA-3′, reverse:
5′-CCTCTCAGCTCCTTGGTTTCC-3'. The GAPDH primer sequences were as
follows, forward: 5′-GGGCTGCTTTTAACTCTGGT-3′, reverse:
5′-TGGCAGGTTTTTTTCTAGACGG-3'. Total RNA was extracted from HCT116 and
SW1116 cells. cDNA, SYBR Green PCR Master Mix (Applied Biosystems,
Foster City, CA), and gene-specific primers were used for qRT-PCR.
Reaction conditions: 95 °C for 30 s, 95 °C for 10 s, 72 °C for 15 s, 40
cycles. Using the 2^−ΔΔCT method, the relative expression of each
target gene was calculated.
2.5. CCK-8 assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8)
assay. CRC cells were seeded into 96-well plates for 24 h. Then cells
were incubated in 10 % CCK-8 solution for 1 h, and the absorbance value
was measured at 450 nm using a microplate reader. Each experiment was
performed in triplicate.
2.6. Plate clone formation assay
HCT116 and SW1116 cells at the logarithmic growth stage were
transfected for 48 h and then inoculated into pore plates after
re-suspension. When visible clones are observed, the culture is
terminated. Fixed with pure ethanol, and stained with 0.1 % crystal
violet, images were collected, and the clones were counted directly
with the naked eye.
2.7. Wound healing assay
HCT116 and SW1116 cells were collected and seeded into six-well plates.
After the cell density reached the whole plate, the scratch wounds were
made by dragging a 200-μl pipette tip across the monolayer. Samples
were taken at 0 h,4 h,16 h, and 24 h time points, and the state of
wound healing in each well was followed under a 50 × microscope at each
horizontal straight-line marker.
2.8. Transwell assay
HCT116 and SW1116 cells at the logarithmic growth stage were taken, set
up in groups, and transfected or administered. Dilute BD matrigel and
serum-free medium at a ratio of 1:8 and add to the upper chamber of the
transwell for incubation. Serum-free medium was added to the upper
chamber. Pre-chilled ethanol was fixed and 0.1 % crystal violet was
used for staining. Finally, the chambers were removed, properly
air-dried, and photographed for counting under the microscope.
2.9. Tumor xenograft assay
The experimental animals used in this experiment were 4-week-old BALB/c
nude mice, purchased from Chengdu Dashuo Experimental Animal Co. The
experimental production license number was SCXK(Chuan)2020-030. CRC
cell line SW1116 was grown to logarithmic phase, digested, resuspended,
and all adjusted and diluted to a concentration of 3 × 10^6/mL. CRC
cells of the sh-NC group and the sh-GDI2 group were grown
subcutaneously on both sides of nude mice, respectively. SW1116 cells
of the sh-NC group were injected on the left side and SW1116 cells of
the sh-GDI2 group were injected on the right side. Six transplanted
tumors were formed on each side, for a total of 12 tumors. The long and
short diameters of the tumors were measured with vernier calipers and
recorded on days 7, 14, 21, and 28 from the beginning of inoculation,
respectively. Tumor volume = π/6 × a × b^2 (a = length, b = wide).
After 28 days, the nude mice were removed from the neck and executed,
and the subcutaneous tumors were separated and weighed. Tumor tissues
were stored in a −80 °C refrigerator and used for subsequent studies.
Animal experiments were conducted in strict compliance with the Guide
for the Management and Use of Laboratory Animals and were approved by
the Experimental Animal Ethics Committee of Guangdong Medical
University (No. GDY2202366).
2.10. Immunohistochemistry
The collected tissues were fixed in formalin for 24 h. The collected
CRC tumor tissues were then paraffin-embedded and sectioned. Before the
start of staining, the sections were subjected to xylene dewaxing,
gradient alcohol hydration, citric acid high-pressure repair, hydrogen
peroxide treatment, and BSA antigen closure in sequence. The
corresponding primary antibody (ki-67, [51]GB121141, Servicebio, 1:100;
GDI2, [52]GB121141, protein tech, 1:200) was added and incubated
overnight, followed by secondary antibody (GB23301, GB23303,
Servicebio, 1:5000) and incubated for 30 min. At the end of the
reaction, the sections were washed 3 times with PBS. Finally, freshly
prepared DAB was used for color development and hematoxylin
re-staining. The sections were dehydrated and transparent in gradient
alcohol and xylene in turn and then sealed. The sections were observed
under a microscope (BA410, McArdy Industries Ltd.) and images were
captured using a microscopic camera system. The percentage of positive
area per image (%DAB Positive Tissue) was calculated using the Halo
Data Analysis System.
2.11. Transcriptomic analysis
Total RNA was extracted from CRC cells in the sh-NC and sh-GDI2 groups,
respectively. The mRNA was enriched in Oligo dT, then fragmented and
synthesized into cDNA, ligated into an adaptor, and finally sequenced
on the Illumina platform. The transcriptome detection and analysis were
performed with the assistance of Beijing NovoTech Technology Co. The
raw data of transcriptome sequencing were filtered to obtain
high-quality data information. The genes were defined as differentially
expressed genes with |log[2]FC|≥2 and FDR<0.05. The mRNAs in the
samples were screened for differential genes. The IDs of differential
genes were queried through the Bioconductor database, and the relevant
installation packages of the Bioconductor platform were installed using
the R language. Set P = 0.05, Q = 0.05, and perform GO enrichment
analysis and KEGG enrichment analysis. Output the results and barplot
histograms were plotted and bubble plots.
2.12. Protein interaction analysis
The possible target proteins bound by the GDI2 protein were predicted
by Hitpredict online software. Based on the prediction results, the
correlation between target proteins and the p53 pathway and TP53
protein was further analyzed by the TCGA database and String database.
The binding of the target proteins to the GDI2 protein was verified in
combination with Co-Immunoprecipitation (Co-IP) assay.
Total protein samples from CRC cells were extracted and lysed, and the
total protein concentration of the lysates was measured using the BCA
protein quantification kit (P0009, Beyotime, China). To Spin columns
with End caps at the lower end, total protein was added, along with
specific antibodies (GDI2, [53]JC803862, abmart; RAB5A, #46449, CST)
and Incubation buffer. The same amount of Control IgG was added to
another group as a negative control. Incubate overnight at 4 °C. Add
Protein A sepharose beads slurry to Spin columns to precipitate the
immune complexes and incubate for 4 h with rotation. Remove the End
caps and discard the supernatant. Wash the precipitated complexes with
1 × Washing buffer. After washing, Spin columns are centrifuged in
Collection tubes and the Collection tubes and centrifugation products
are discarded. Spin columns are placed in new EP tubes to collect the
eluted product. The precipitated complexes are eluted with Elution
buffer and the product is collected by centrifugation. Finally, Alkali
neutralization buffer and 5 × Sample Buffer were added to the eluted
product and heated in a boiling water bath. The final IP samples were
separated by SDS-PAGE and the proteins were transferred to PVDF
membranes for Western blot analysis using detection antibody
hybridization and HRP-conjugated protein A secondary antibody at
dilutions of 1:1500–1:3000.
2.13. Flow cytometry
Flow cytometric analysis was performed to determine the cell cycle
phase distribution. HCT116 and SW1116 cells were inoculated on plates
and trypsin digestion was performed after PBS washing. The appropriate
amount of medium was added to become a single-cell suspension and
counted. After the cells were attached to the wall, transfection was
performed according to different groups. After 48 h of incubation, the
supernatant was aspirated and discarded to obtain cell precipitates,
which were fixed by adding 75 % pre-cooled ethanol and washed again
with PBS. The cells were stained with PI, and then 10 μg/ml of RNase A
was added for 30 min at room temperature and protected from light.
Finally, the data collected from the flow cytometer (cytoflex, Beckman,
American) were processed by ModiFit LT5.1 cell cycle analysis software.
2.14. Western blot
CRC cells were inoculated on the plate. Add RIPA cell lysis buffer to
lyse cells and centrifuge to collect protein supernatant. Add SDS
loading buffer and boil it in boiling water. Used SDS-PAGE gel
electrophoresis to separate protein, transfer it to PVDF membrane, and
then the membrane was blocked with 5 % non-fat dry milk in TBST at room
temperature. After the incubation of primary antibody (GDI2,
proteintech, 1:5000; p21, Abclonal, 1:2000; p53, Abclonal, 1:2000;
p-p21 Affinity, 1:2000; p-p53, Abclonal, 1:2000; RAB5A, CST, 1:1000;
β-actin, Abclonal, 1:50000), PBS was washed three times and secondary
antibody (AS014, abclonal, 1:5000) was added for incubation. After PBS
washed, the luminous solution was added and an ECL chemiluminescence
system was used to observe the final results.
2.15. Statistical analysis
The data were analyzed with GraphPad Prism 8 (GraphPad Software, USA)
and are presented as the means ± SD. Comparisons between the two groups
were performed using Student's t-tests or one-way ANOVA. Differences
with P-values of less than 0.05 were considered statistically
significant.
3. Results
3.1. Differential expression and impact of GDI2 in CRC
GDI2 mRNA expression in CRC tissues was analyzed using the TCGA
database. The expression of GDI2 was greater in tumor tissues of colon
cancer patients or rectal cancer patients than in normal tissues
(P < 0.05) ([54]Fig. 1A), and it is hypothesized that GDI2 plays an
important role in the proliferation and development of human CRC. The
clinical prognostic relationship between GDI2 mRNA expression levels
and overall survival (OS), progression-free survival (PFS),
disease-free survival (DFS), and disease-specific survival (DSS) of CRC
patients was examined by the GEPIA data analysis website to assess the
relationship between GDI2 expression and survival of CRC patients in
the TCGA database. The results of Kaplan-Meier survival curves showed
([55]Fig. 1B–E) that the disease-specific survival of patients with low
GDI2 expression was better than that of patients with high expression.
There was a significant difference between the progression-free
survival of the two groups of patients (P < 0.05).
Fig. 1.
[56]Fig. 1
[57]Open in a new tab
Analysis of GDI2 expression in CRC.
A: TCGA database analysis of GDI2 mRNA expression differences in tumor
tissues and paracancerous tissues of CRC patients. B-E: KM survival
curves of GDI2 mRNA and OS, DFS, DSS, and PFS in TCGA data. Different
groups were tested by log-rank. HR (High groups) represents the risk
coefficient of the high expression group relative to low expression
group samples, if HR > 1 means the mRNA is a risk factor, if HR < 1
means the mRNA is a protective factor. 95 % CL represents the HR
confidence interval. F: qRT-PCR to detect GDI2 mRNA expression levels
in normal tissues adjacent to cancer and CRC tissues. G-H:
Immunohistochemical detection of GDI2 expression levels in normal
tissues adjacent to cancer and CRC tissues ( × 20). ∗P < 0.05,
∗∗P < 0.01 compared with normal tissues next to cancer. I-O: TCGA
database to analyze the correlation between GDI2 expression levels and
age, gender, lymphatic count, tumor stage, M stage, T stage, and N
stage of CRC patients. P-R: Clinical information to analyze the
correlation between GDI2 expression level and the presence of distant
metastasis, lymphocyte count, and tumor volume size in CRC patients.
GDI2 expression levels in clinical samples were detected by qRT-PCR and
immunohistochemical assays. The qRT-PCR results showed ([58]Fig. 1F)
that the mRNA expression level of GDI2 in CRC tumor tissues was higher
than that in normal tissues adjacent to the cancer (P < 0.01). In the
results of immunohistochemistry experiments ([59]Fig. 1G and H), the
expression of GDI2 in tumor tissues was significantly higher than that
in normal tissues adjacent to cancer (P < 0.05). The correlation
between GDI2 expression levels and clinicopathological indices of CRC
patients was analyzed by the TCGA database and clinical information.
Among them, the results of TCGA data analysis showed ([60]Fig. 1I–O)
that the high expression of GDI2 was not significantly correlated with
age (P = 0.13), gender (P = 0.24), lymphatic count (P > 0.05) and
T-stage (P > 0.05). However, there were significant correlations with
lymphatic metastasis (P = 0.00014), M0/M1 stage (P = 0.047), N0/N1
(P = 0.011) stage, and tumor stage (P < 0.05). The results of clinical
information analysis showed ([61]Fig. 1P–R) that the expression level
of GDI2 was significantly correlated with the presence of metastasis
(P = 0.0121), lymphocyte count (P = 0.0018), and tumor volume size
(P < 0.05).
3.2. Silencing GDI2 inhibits the proliferation, migration, and invasion of
CRC cells
The difference in mRNA expression of GDI2 in CRC cells and normal
colorectal epithelial cells was determined by qRT-PCR assay. The
results showed that the mRNA expression levels of GDI2 in CRC cells
(HCT116 and SW1116) were both higher than those in normal colonic
epithelial cells NCM460 (P < 0.05) ([62]Fig. 2A). In order to
successfully investigate the effect of GDI2 on the development of CRC
cells subsequently, HCT116 cells and SW1116 cells were used to screen
the most suitable shRNA for subsequent study by silencing the
expression of GDI2 mRNA in the cells. In CRC cells, the GDI2 mRNA
expression level and protein expression level of cells in the sh-GDI2#2
group were lower than those in sh-GDI2#1 and sh-GDI2#3 groups, and
significantly lower than those in the sh-NC group (P < 0.01) ([63]Fig.
2B–D). Therefore, GDI2#2 was selected as the most suitable shRNA for
the follow-up experiments.
Fig. 2.
[64]Fig. 2
[65]Open in a new tab
Effect of GDI2 silencing on proliferation, migration, and invasion of
CRC.
A: qRT-PCR to detect the difference of GDI2 mRNA expression in normal
colonic epithelial cells and CRC cells. B: qRT-PCR to detect GDI2 mRNA
expression levels in CRC cells after treatment with different shRNA
sequences. C-D: Western blot detection of GDI2 protein expression
levels in CRC cells after treatment with different shRNA sequences. E:
CCK-8 assay to detect the effect of silencing GDI2 on the proliferation
of CRC cells. F-G: Plate clone formation assay to detect the ability of
CRC cells to form clones. H-I: The wound healing assay was used to
detect the migration ability of CRC cells at 0 h, 4 h, 16 h, and 24 h
( × 50). J-K: Transwell invasion assay to detect the invasive ability
of CRC cells ( × 40). Compared with the sh-NC group, ∗P < 0.05,
∗∗P < 0.01.
In the CCK-8 assay, the OD450 values of cells in the sh-GDI2 group were
all significantly lower than those of CRC cells in the sh-NC group
(P < 0.01) ([66]Fig. 2E). Silencing of GDI2 mRNA was able to reduce the
proliferative activity of CRC cells HCT116 and SW1116 cells. The
results of the plate clone formations assay are shown in [67]Fig. 2F
and G. The number of clone formation in the sh-GDI2 group was smaller
than that in the sh-NC group for both HCT116 cells or SW1116 cells
(P < 0.01). The results of wound healing experiments are shown in
[68]Fig. 2H and I. Compared with the cells in the sh-NC group, the mean
migration distance of both HCT116 cells and SW1116 cells in the sh-GDI2
group was significantly reduced (P < 0.05, P < 0.01) in 4 h,16 h, and
24 h, and the migration ability was decreased. The number of invasions
of HCT116 and SW1116 cells was also significantly reduced (P < 0.01)
compared to the sh-NC group after silencing GDI2 mRNA ([69]Fig. 2J and
K), and the invasion ability of CRC cells was diminished. The above
results demonstrated that silencing GDI2 inhibited the proliferative
activity, migration, and invasive ability of CRC cells.
3.3. Silencing GDI2 inhibits CRC xenograft tumor proliferation
The tumor diameter of transplanted tumors in nude mice was measured on
days 7, 14, 21, and 28 after inoculation with CRC cells SW1116. At the
end of the experiment, the mice were sacrificed using the cervical
dislocation method and executed and all subcutaneous transplanted
tumors were photographed as in [70]Fig. 3A. The volume and mass of the
transplanted tumors in the sh-GDI2 group were significantly smaller
than those in the sh-NC group. The tumor volume was calculated based on
the tumor diameter, and the growth curve of the transplanted tumors was
plotted in [71]Fig. 3B. It can be seen that the volume growth trend of
transplanted tumor in the sh-GDI2 group of nude mice was lower compared
with that of the transplanted tumor in the sh-NC group (P < 0.05). The
results of the statistical analysis of the final transplanted tumor
weight are shown in [72]Fig. 3C. The tumor weight of the transplanted
tumor in the sh-NC group was (0.589 ± 0.168) g, and the tumor weight of
the transplant tumor in the sh-GDI2 group was (0.342 ± 0.083) g, which
was lower than the sh-NC group (P < 0.05). It is suggested that
silencing the GDI2 mRNA has an inhibitory effect on the growth of CRC
xenograft tumors.
Fig. 3.
[73]Fig. 3
[74]Open in a new tab
Effect of silencing GDI2 on CRC xenograft tumors.
A: Tumor plot of dissected xenograft tumors. B: Growth curves of CRC
transplant tumors during the trial. C: Weight of xenograft tumors. D:
Immunohistochemical detection of ki-67 expression level of tumors
( × 10). E: qRT-PCR detection of GDI2 mRNA expression level of tumors.
F: Western blot detection of GDI2 protein expression of tumors.
Compared with the sh-NC group, ∗P < 0.05, ∗∗P < 0.01.
The expression levels of ki-67 in xenograft tumors were detected by
immunohistochemical staining ([75]Fig. 3D). The expression level of
ki-67 in the tumors of the sh-GDI2 group was remarkably lower than that
of the sh-NC group (P < 0.01), which means that the proliferation
ability of the tumors was reduced. The results of qRT-PCR experiments
and Western blot experiments are shown in [76]Fig. 3E and F. The mRNA
expression level of GDI2 in transplanted tumors of sh-GDI2 group was
lower than that of transplanted tumors of the sh-NC group (P < 0.05),
and the protein expression level was significantly lower than that of
the sh-NC group (P < 0.01). In conclusion, silencing the GDI2 mRNA
inhibited the proliferation development of CRC tumors in vivo.
3.4. Silencing of GDI2 induces cell cycle arrest in CRC
The possible mechanism of GDI2 gene action in CRC cells was analyzed
based on transcriptomics. The distribution of differentially expressed
genes was reflected by the volcano plot ([77]Fig. 4A). The total number
of differentially expressed genes obtained was 855, with 448
up-regulated genes and 407 down-regulated genes. [78]Fig. 4B shows the
top ten down-regulated differentially expressed genes and the top ten
up-regulated differentially expressed genes. C1-C3 are the control CRC
cells, T1-T3 are the sh-GDI2 group CRC cells, and the vertical
coordinates indicate the normalized values of differential genes FPKM.
Among the down-regulated differential genes, ADRM1 and ITGA5 were
associated with CRC. Among the up-regulated differential genes, TXNRD1,
EIF2S2, PEG10, and RB1 were associated with CRC.
Fig. 4.
[79]Fig. 4
[80]Open in a new tab
Exploration of the mechanism by which silencing of GDI2 affects CRC.
A: Volcano plot of differentially expressed genes after silencing GDI2
gene in CRC cells (red shows up-regulated genes, green shows
down-regulated genes). B: Heat map of Top10 down-regulated differential
genes and Top10 up-regulated differential genes. C: GO functional
enrichment analysis of differentially expressed genes histogram. D:
Flow cytometry detection of CRC cell cycle arrest after silencing GDI2.
To investigate the biological processes involved in GDI2 in CRC cells,
GO functional enrichment analysis was performed on differentially
expressed genes. From the results of the GO enrichment analysis, the
most significant 30 items were selected to draw a bar graph for display
([81]Fig. 4C). Among them, the significantly differentially expressed
genes were mainly involved in RNA splicing, signal transduction by p53
class mediator, cadherin, ubiquitin-like protein ligase binding, G1/S
transition of the mitotic cell cycle, DNA integrity, and other
biological processes. This suggests that GDI2 may be involved in
regulating the development of CRC through the above biological
processes. To verify the effect of GDI2 expression on the cell cycle,
the cell cycle phase distribution of each group was detected by flow
cytometry, and the analysis results were shown in [82]Table 1,
[83]Table 2, and [84]Fig. 4D. Compared with the sh-NC group, the
percentage of G0/G1 phase cells in CRC cells in the sh-GDI2 group was
increased (P < 0.01). Meanwhile, the percentage of S-phase cells in CRC
was significantly decreased in the sh-GDI2 group (P < 0.01). It
demonstrated that CRC cells were significantly stalled in the G1 phase
after silencing GDI2, which was consistent with the results of GO
functional enrichment analysis.
Table 1.
Effect of different groups on the cell cycle of HCT116 cells(
[MATH: x‾
:MATH]
±standard deviation; n = 3).
Group G0/G1 phase G2/M phase S phase
sh-NC 18.66 ± 1.07 9.38 ± 0.62 71.96 ± 0.61
sh-GDI2 44.04 ± 1.52[85]^a 6.30 ± 2.19 49.66 ± 1.27[86]^a
[87]Open in a new tab
Note: Compared with sh-NC group.
^a
P < 0.01.
Table 2.
Effect of different groups on the cell cycle of SW1116 cells(
[MATH: x‾
:MATH]
±standard deviation; n = 3).
Group G0/G1 phase G2/M phase S phase
sh-NC 27.47 ± 0.89 6.73 ± 0.48 65.79 ± 0.97
sh-GDI2 44.04 ± 0.64[88]^a 8.76 ± 0.47[89]^a 47.18 ± 1.10[90]^a
[91]Open in a new tab
Note: Compared with sh-NC group.
^a
P < 0.01.
3.5. Silencing GDI2 targets binding to RAB5A protein to inhibit CRC
The results of KEGG pathway enrichment analysis showed ([92]Fig. 5A)
that upregulated differentially expressed genes were significantly
enriched in tight junctions and cellular senescence pathways, and
significantly enriched in the p53 signaling pathway. It is suggested
that the p53 signaling pathway is one of the key pathways of GDI2
affecting the development of CRC cells. The Hitpredict software was
used to predict the possible target proteins bound by the GDI2 protein
and to investigate the relationship between target proteins and p53 to
further validate the effect of GDI2 on the p53 signaling pathway.
Hitpredict software predicted 130 target proteins, among which the 20
proteins with the highest interaction scores are shown in [93]Fig. 5B.
The RAB5A protein had the highest interaction score, so RAB5A was
selected for the follow-up study. Spearman correlation analysis between
RAB5A and p53 pathway score was performed according to the TCGA
database. The results showed P = 0.0001, and there may be a high
correlation between RAB5A and the p53 pathway ([94]Fig. 5C). The
interaction between TP53 and RAB5A was analyzed by the String database
([95]Fig. 5D), and the results showed that TP53 could interact with
RAB5A protein through ZFYVE20.
Fig. 5.
[96]Fig. 5
[97]Open in a new tab
Effect of overexpression of RAB5A on CRC when GDI2 was silenced.
A: Scatter plot of KEGG pathway enrichment analysis of upregulated
differentially expressed genes. B: Results predicted by Hitpredict. C:
Correlation between RAB5A protein and p53 pathway analyzed by TCGA
database. D: Protein interaction network analysis of RAB5A and TP53 by
String database. E: Immunoprecipitation assay to verify the presence of
GDI2 protein binding to RAB5A. F: WB detection of the effect of
silencing GDI2 on RAB5A protein expression. G: Flow cytometry to detect
the effect of overexpression of RAB5A on cell cycle when GDI2 was
silenced. H: CCK-8 assay to detect the effect of overexpression of
RAB5A on cell cycle when GDI2 was silenced. I: Wound healing assay to
detect the effect of overexpression of RAB5A on CRC cell migration when
GDI2 was silenced ( × 50) (0 h, 4 h, 16 h, and 24 h). J: Transwell
invasion assay to detect the effect of overexpression of RAB5A on CRC
cell invasion when GDI2 was silenced ( × 40).
Compared with the sh-NC group, ∗P < 0.05, ∗∗P < 0.01; compared with the
sh-GDI2 group, ^#P < 0.05, ^##P < 0.01.
The binding of GDI2 to RAB5A was verified by Co-IP assay. The results
showed that RAB5A protein was present in the enriched GDI2 protein and
GDI2 protein was also present in the enriched RAB5A protein in both
HCT116 cells and SW1116 cells ([98]Fig. 5E). There was mutual binding
between the GDI2 protein and RAB5A protein. The results of Western blot
analysis demonstrated that the expression levels of the RAB5A protein
in HCT116 cells and SW1116 cells were lower than those in the sh-NC
group after silencing GDI2 (P < 0.05) ([99]Fig. 5F).
Cells were divided into the sh-NC group, sh-GDI2 group,
sh-GDI2+pcDNA-NC group, and sh-GDI2+pcDNA-RAB5A group to detect the
effects of overexpression of RAB5A on cell cycle, cell proliferation,
migration and invasion when GDI2 was silenced. The percentage of cells
stalled in the G0/G1 phase was compared between groups ([100]Table 3,
[101]Table 4, and [102]Fig. 5G). The percentage of CRC cells stalled at
the G0/G1 phase was significantly increased in the sh-GDI2 group
compared with the sh-NC group (P < 0.01). Compared with the sh-GDI2
group, there was no significant change in the sh-GDI2+pcDNA-NC group
(P > 0.05), and the percentage of cells in the G0/G1 phase was
significantly lower in the sh-GDI2+pcDNA-RAB5A group (P < 0.01).
Overexpression of RAB5A inhibited the cell cycle arrest induced by
silencing GDI2.
Table 3.
Effect of different groups on the cell cycle of HCT116 cells(
[MATH: x‾
:MATH]
±standard deviation; n = 3).
Group G0/G1 phase G2/M phase S phase
sh-NC 19.96 ± 1.31 5.71 ± 2.67 74.33 ± 1.65
sh-GDI2 41.56 ± 0.50∗∗ 7.64 ± 1.29 50.80 ± 1.01∗∗
sh-GDI2+pcDNA-NC 41.54 ± 0.54 7.69 ± 0.42 50.77 ± 0.67
sh-GDI2+pcDNA-RAB5A 28.36 ± 0.43^## 14.73 ± 1.10^## 56.91 ± 1.10^##
[103]Open in a new tab
Note: Compared with sh-NC group, ∗P < 0.05, ∗∗P < 0.01; compared with
sh-GDI2 group, ^#P < 0.05, ^##P < 0.01.
Table 4.
Effect of different groups on the cell cycle of SW1116 cells(
[MATH: x‾
:MATH]
±standard deviation; n = 3).
Group G0/G1 phase G2/M phase S phase
sh-NC 29.44 ± 0.37 5.98 ± 0.99 64.58 ± 0.62
sh-GDI2 45.68 ± 0.65∗∗ 8.50 ± 0.22 45.82 ± 0.80∗∗
sh-GDI2+pcDNA-NC 45.18 ± 0.83 7.22 ± 1.19 47.60 ± 1.57
sh-GDI2+pcDNA-RAB5A 38.05 ± 0.23^## 7.10 ± 0.89 54.85 ± 0.68^##
[104]Open in a new tab
Note: Compared with sh-NC group, ∗P < 0.05, ∗∗P < 0.01; compared with
sh-GDI2 group, ^#P < 0.05, ^##P < 0.01.
The results of the CCK-8 assay showed ([105]Fig. 5H) that the OD values
of CRC cells in the sh-GDI2 group were all lower compared with the
sh-NC group (P < 0.01). Cell proliferation activity was significantly
increased in the sh-GDI2+pcDNA-RAB5A group compared with the sh-GDI2
group (P < 0.01). In the results of the wound healing assay ([106]Fig.
5I), the healing distance of CRC cells in the sh-GDI2+pcDNA-RAB5A group
was significantly increased compared with the sh-GDI2 group (P < 0.05,
P < 0.01) at 4 h and 24 h. The changes in cell invasion ability were
detected by Transwell assay ([107]Fig. 5J). The invasion ability of
HCT166 cells and SW1116 cells in the sh-GDI2+pcDNA-RAB5A group was
significantly higher than that in the sh-GDI2 group (P < 0.05,
P < 0.01). Silencing of GDI2 inhibited the proliferation, migration and
invasion of CRC cells and induced cell cycle arrest, while
overexpression of RAB5A increased the proliferation, migration, and
invasion ability of CRC cells and inhibited cell cycle arrest.
3.6. Silencing GDI2 inhibits CRC through activation of p53 signaling pathway
CRC cells, HCT116 and SW1116, were divided into sh-NC, sh-GDI2,
sh-GDI2+PFT-α, and PFT-α groups, respectively. The expression levels of
p53 pathway-related proteins in CRC cells in the sh-GDI2 group were
similar to those in the sh-NC group, and the expression levels of p-p21
and p-p53 proteins were significantly higher than those in the sh-NC
group (P < 0.01). It indicates that silencing GDI2 mediates the
activation of the p53 signaling pathway. Compared with the sh-GDI2
group, the p-p21 and p-p53 protein expressions in the sh-GDI2+PTF-α
group were significantly downregulated (P < 0.05), and the p53 pathway
inhibitor inhibited the activation of the p53 pathway by silencing GDI2
([108]Fig. 6A and B).
Fig. 6.
[109]Fig. 6
[110]Open in a new tab
Silencing of GDI2 via p53 pathway affects CRC cells.
A: WB detection of p21, p53, p-p21, and p-p53 protein expression in
HCT116 cells. B: WB detection of p21, p53, p-p21, and p-p53 protein
expression in SW1116 cells. C: CCK-8 assay to detect proliferative
activity of CRC cells. D-E: Plate clone formation assay to detect CRC
cell clone formation ability. F: The wound healing assay was used to
detect the migration ability of CRC cells at 0 h, 4 h, 16 h, and 24 h
( × 50). G: Transwell assay to detect the invasive ability of CRC cells
( × 40). H: WB assay to detect the effect of overexpression of RAB5A on
p53 pathway protein expression in HCT116 cells when GDI2 was silenced.
I: WB detection of the effect of overexpression of RAB5A on p53 pathway
protein expression in SW1116 cells upon silencing of GDI2.
Compared with the sh-NC group, ∗P < 0.05, ∗∗P < 0.01; compared with the
sh-GDI2 group, ^#P < 0.05, ^##P < 0.01.
The results of CCK-8 experiments were shown in [111]Fig. 6C. The OD450
values of CRC cells in the sh-GDI2 group were significantly decreased
compared with the sh-NC group (P < 0.01). The OD450 values of
HCT116 cells and SW1116 cells in the sh-GDI2+PFT-α group were increased
compared with cells in the sh-GDI2 group (P < 0.05, P < 0.01). The
results of the plate formation clone assay were shown in [112]Fig. 6D
and E, in which the number of clone formation in CRC cells in the
sh-GDI2 group was significantly less than that in the sh-NC group
(P < 0.01). Both HCT116 cells and SW1116 cells in the sh-GDI2+PFT-α
group formed significantly more clones compared with cells in the
sh-GDI2 group (P < 0.01). The results showed that at 16 h and 24 h, the
mean migration distance of CRC cells in the sh-GDI2+PFT-α group was
significantly greater than that of CRC cells in the sh-NC group
(P < 0.05, P < 0.01) ([113]Fig. 6F). In addition, the results of the
Transwell invasion assay showed ([114]Fig. 6G) that the number of CRC
cells crossing Transwell chambers was significantly increased in the
sh-GDI2+PFT-α group compared with cells in the sh-GDI2 group (P < 0.05,
P < 0.01). After silencing the GDI2 gene, the proliferation, migration,
and invasion ability of CRC cells were reduced, but the addition of the
p53 pathway inhibitor restored the proliferation, migration, and
invasion ability of CRC cells.
Western blot detected the effect of overexpression of RAB5A on the
expression level of p53 pathway-related proteins upon silencing of GDI2
([115]Fig. 6H and I). Compared with the sh-GDI2 group, RAB5A protein
expression levels were significantly higher in the sh-GDI2+pcDNA-RAB5A
group (P < 0.01, P < 0.05). The expression levels of p-p21 and p-p53
proteins were significantly increased in the sh-GDI2 group compared
with the sh-NC group (P < 0.01). The expression levels of p-p21 and
p-p53 proteins were decreased in cells of the sh-GDI2+pcDNA-RAB5A group
compared with the sh-GDI2 group (P < 0.05). Silencing of GDI2 mediated
the activation of the p53 pathway, while overexpression of RAB5A
conversely decreased p53 pathway activity, and overexpression of RAB5A
exerted the same effect as the p53 pathway inhibitor.
4. Discussion
CRC is one of the most common malignancies. The prognostic outcome of
CRC often correlates with the stage at which it is diagnosed, with
survival rates higher for early diagnosis than for advanced stages, and
much lower for patients who develop distant metastatic stages
[[116][16], [117][17], [118][18]]. Although great progress has been
made in recent years in the screening and treatment of CRC, the
incidence, prevalence, and mortality of CRC remain high [[119]19].
Therefore, the search for new CRC biomarkers and therapeutic targets is
crucial to improve the prognosis and enhance the clinical management of
CRC.
GDI2 belongs to a small family of chaperone proteins that are mainly
expressed in hematopoietic, endothelial, and epithelial cells
[[120]20,[121]21]. Abnormal expression of the GDI2 gene has been
demonstrated in many cancer types. In recent studies in prostate
cancer, GDI2 expression was found to be upregulated in prostate cancer
cells and tissues. Knockdown of GDI2 inhibits cell proliferation but
promotes apoptosis [[122]8]. In thyroid cancer cells, microRNA-15b-5p
can inhibit cell proliferation and invasion by targeting GDI2, which is
associated with poor prognosis in thyroid cancer patients and is
negatively regulated by miR-15b-5p [[123]22]. The differential
expression of GDI2 in gastric and hepatocellular carcinoma tissues
provides potential insights for identifying biomarkers, predicting
diagnosis, and assessing prognosis in gastric and hepatocellular
carcinoma patients [[124]9,[125]23]. However, the expression profile
and functional role of GDI2 in CRC have not been investigated. CRC
possesses the common biological characteristics of most malignant
tumors, including malignant proliferation, invasion, migration, and
high levels of angiogenesis [[126]24].In this study, we first examined
clinical specimens by qRT-PCR assay and immunohistochemical assay, and
based on bioinformatics analysis of the TCGA database, we investigated
the expression differences and effects of GDI2 in CRC tissues. Our
study demonstrated that silencing GDI2 in CRC cells reduces their
proliferative activity, migration, and invasive capabilities as well as
in vivo tumor growth inhibition. In addition, the possible mechanism of
the effect of silencing GDI2 on CRC was further explored by
transcriptomics.
Transcriptome analysis showed that silencing of GDI2 resulted in a
total of 855 differential genes, of which 448 differential genes were
upregulated and 407 differential genes. GO functional enrichment
analysis showed that differentially expressed genes were significantly
enriched in the biological process of cell cycle arrest. The cell cycle
is usually divided into G1, S, G2, and M phases. Cell cycle blocks can
be divided into three categories, including G1 phase DNA damage
checkpoints, S phase DNA damage checkpoints, and G2 phase DNA damage
checkpoints [[127]25]. When DNA damage occurs in cells in the G1 phase,
the G1 phase checkpoint prevents the initiation of the DNA replication
phase so that the cells do not enter the S phase, thus preventing the
damaged DNA from replicating [[128]26]. In a study by Yong Jungkang
[[129]25], MHY2245 can trigger apoptosis by inducing cell cycle arrest
and exhibits anti-CRC effects associated with DNA damage response. A
study by Muhammad Akhtar Ali demonstrated that the deletion of the
transcription factor regulator ZBEED6 reduced the growth rate of CRC
cells HCT116 by affecting the cell cycle [[130]27]. Transcriptome
analysis revealed that GDI2 silencing leads to cell cycle arrest,
particularly at the G1 phase, which is consistent with the observed
inhibition of CRC cell proliferation and is associated with DNA damage
response.
In the results of the KEGG pathway enrichment analysis, up-regulated
differentially expressed genes were significantly enriched in the p53
signaling pathway, and silencing GDI2 may mediate the activation of the
p53 signaling pathway in CRC cells. To further explore the mechanism of
action of GDI2 activation of the p53 pathway, the target protein RAB5A,
which binds to GDI2, was predicted and analyzed by Hitpredict online
software, and the relationship between RAB5A and p53 was investigated.
RAB5A belongs to the Ras family of G proteins, which mainly regulates
vesicle transport [[131]28] and is involved in the development of many
human cancers, which can promote tumor proliferation and distant
metastasis, including breast, liver, lung, pancreatic, and ovarian
cancers [[132]29,[133]30]. RAB5A and GDI2 protein existed to bind each
other and there was a high correlation between RAB5A and p53 pathway
activation. The higher the level of RAB5A expression, the lower the p53
pathway score. The p53 signaling pathway was found to be upregulated
upon GDI2 silencing, with RAB5A, a G protein, predicted to mediate this
effect. Western blot results showed that the silencing of GDI2
expression was also followed by a decrease in RAB5A expression level
and an increase in p53 pathway activity. RAB5A's role in vesicle
transport and cancer progression was highlighted, and its expression
inversely correlated with p53 pathway activity.
Further analysis highlighted the p53 signaling pathway as a key
mediator of GDI2's effects in CRC. p53 oncoprotein is activated in
response to various stress signals and coordinates many downstream
responses, such as DNA repair, cell cycle arrest, and cellular
senescence [[134]31].
The role of p53 in cell cycle arrest has been extensively studied. For
example, activation of the ATM/ATR pathway by mouse embryonic
fibroblasts exposed to DNA damage leads to activation of p53 and
subsequent G1 arrest [[135]32]. p53 induces G1 arrest mainly through
transactivation of p21Waf1/Cip1, a cell cycle protein-dependent kinase
inhibitor, and targeted disruption of the p21Waf1/Cip1 gene impairs the
G1/S checkpoint in MEFs. Both p53 and p21 are important oncogenes in
vivo and are involved in some cell cycle regulation through
post-translational modifications such as phosphorylation and
ubiquitination [[136]33]. Lacroix's study showed that p53 and p21 in
the p53 signaling pathway are biologically active after phosphorylation
and affect tumor cell proliferation, invasion and cell cycle, and other
biological processes [[137]34]. Western blot experiment showed that
silencing GDI2 was found to activate the p53 pathway, potentially
through the regulation of RAB5A, a protein that interacts with GDI2.
This activation leads to increased phosphorylation of p53 and p21,
crucial for cell cycle regulation and tumor suppression. In conclusion,
our findings suggest that GDI2 is a promising biomarker and therapeutic
target for CRC. Its upregulation in tumor tissues and its role in
modulating the p53 pathway offers new avenues for improving CRC
diagnosis and treatment strategies. Further research is needed to fully
understand the mechanisms by which GDI2 influences CRC progression and
to validate its potential as a clinical tool.
Funding statement
None.
Ethics approval and informed consent
Written informed consent was obtained from all donors who provided
samples. The trial was approved by the Ethics Committee of the Medical
Center of the Affiliated Hospital of Guangdong Medical University (No.
YJYS2022237). All animal experimental procedures were reviewed and
approved by the Experimental Animal Ethics Committee of Guangdong
Medical University (No. GDY2202366).
Data availability statement
The datasets used and/or analyzed during the present study are
available from the corresponding author upon reasonable request.
CRediT authorship contribution statement
Wen-Ting Ou: Writing – review & editing, Software, Resources, Project
administration, Funding acquisition, Formal analysis. Rong-Jian Tan:
Writing – original draft, Software, Methodology, Investigation, Data
curation. Jia-Wei Zhai: Writing – original draft, Project
administration, Methodology, Data curation. Li-Jun Sun: Project
administration, Methodology, Data curation. Fei-Peng Xu: Software,
Project administration, Methodology, Funding acquisition, Formal
analysis. Xian-Jin Huang: Methodology, Investigation, Data curation.
Zhen-Hao Quan: Project administration, Methodology, Data curation.
Cai-Jin Zhou: Writing – review & editing, Supervision, Software,
Resources, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Footnotes
^Appendix A
Supplementary data to this article can be found online at
[138]https://doi.org/10.1016/j.heliyon.2024.e37770.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
Multimedia component 1
[139]mmc1.zip^ (12.7MB, zip)
References