ABSTRACT
Objective:
To screen key
genes of renal clear
cell carcinoma based on
bioinformatics methods, identify possible
microRNA (
miRNA)-
mRNA action axis, and explore the expression of related
genes in
clear cell renal cell carcinoma tissues and
cells.
Methods:
Gene expression profiles of GSE40435 and GSE71302 datasets were obtained from the
Gene Expression Omnibus (GEO) database. TCGA-KIRC datasets were obtained from The
Cancer Genome Atlas (TCGA) database. R
software was used to identify the differentially expressed
mRNA and
miRNA, and the functional enrichment
analysis was performed. STRING database and Cytoscape
software were used to perform the
protein interaction
analysis. The
prognosis-related differentially expressed
miRNA was evaluated by the Oncomir database. The potential targeted
genes regulated by
miRNA were determined by using TargetScan and miRDB targeted
gene prediction tools. The
tissue samples and clinicopathological features of 34
patients with
clear cell renal cell carcinoma in the First
Hospital of Shanxi Medical
University from June to December 2021 were collected, and normal renal
cell line 293T and
clear cell renal cell carcinoma cell line 786O were selected. The real-
time fluorescence quantitative
polymerase chain reaction (qRT-
PCR), was used to detect the relative expression of
genes;
Western blotting and immunohistochemical
staining were used to detect the expression levels of the targeted
proteins. The dual
luciferase reporter gene assay was carried out to verify the targeting relationship between
genes.
Results:
A total of 1 351 differentially expressed
mRNA and 50 differentially expressed
miRNA were screened and identified. The result of functional enrichment
analysis suggested that the
fatty acid metabolism pathway and
xenobiotic metabolism pathway were suppressed in
clear cell renal cell carcinoma, while the
apoptosis and
immune response pathways were activated.
Protein interaction
analysis suggested that the
signal transduction and
protein ubiquitination pathways might
play a key
role in
clear cell renal cell carcinoma. The
screening results showed that
miRNA-224-5p (miR-224-5p) was most closely associated with
clear cell renal cell carcinoma progression and was highly expressed in
tumor tissues, and its
prognosis-related target
gene was NEDD4L. The relative expression of NEDD4L
mRNA in
clear cell renal cell carcinoma tissues and paraneoplastic
tissues were 0.138±0.103 and 1.000±0.026 ( t = 46.23, P < 0.05), and the relative expression of miR-224-5p was 1.000±0.043 and 0.129±0.108 ( t = 45.28, P < 0.05). The differences of NEDD4L
mRNA and miR-224-5p expressions in different grades and stages of
clear cell renal cell carcinoma tissues were statistically significant (all P < 0.05). The expression of NEDD4L
protein was decreased in
clear cell renal cell carcinoma. The relative expression of NEDD4L
gene in 293T and 786O
cells were 1.000±0.125 and 0.210±0.044 ( t = 17.52, P < 0.05); the relative expressions of miR-224-5p
gene were 0.209±0.049 and 1.000±0.234 ( t = 10.61, P < 0.05). The relative expressions of NEDD4L
mRNA in
miRNA mimic group and negative
control group were 0.236±0.062 and 1.000±0.024, and the difference was statistically significant ( t = 43.56, P < 0.05). NEDD4L
protein expression was reduced in the
miRNA mimic group. Dual
luciferase reporter gene assay suggested that NEDD4L was a direct target
gene of miR-224-5p.
Conclusions:
In
clear cell renal cell carcinoma, miR-224-5p targets and regulates NEDD4L expression, and this mechanism may be related to
carcinogenesis and progression of
clear cell renal cell carcinoma.