(2023) 24:10
Shi et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00473-6

BMC Molecular andCell Biology

Open Access

RESEARCH

Role of the human solute carrier family 14
member 1 gene in hypoxia‑induced renal cell
carcinoma occurrence and its enlightenment
to cancer nursing
Jing Shi, Ruili Sha and Xilan Yang*

Abstract
Background Hypoxia is considered a critical contributor to renal cell carcinoma progression, including invasion andmetastasis. However, the potential mechanisms by which it promotes invasion and metastasis have not yet beenclarified. The purpose of this study was to investigate the role and mechanism of hypoxia-induced renal cell carcinoma and provide evidence-based medical proof for improvements to postoperative nursing of renal cell carcinoma
patients. A total of 64 patients with renal cell carcinoma were divided into the observation group (nursing based on
oxygen administration) and the control group (conventional nursing). Renal function indexes, serum inflammatory
factors, and tumor markers were evaluated. The human renal cell carcinoma cell line A498 under hypoxia/normoxia
was used as an experimental model in vitro and the biological characteristics and mitochondrial function of the cells
were assessed.
Results Nursing based on oxygen administration decreased the value of renal function indexes, serum inflammatory
factors, and tumor markers in renal cell carcinoma patients. Hypoxia was found to induce A498 cell invasion, migration, and the release of inflammatory cytokines, while repressing human solute carrier family 14 member 1 gene
expression. Elevated levels of solute carrier family 14 member 1 expression induced mitochondrial reactive oxygen
species accumulation, diminished the intracellular adenosine triphosphate level, and destroyed both mitochondrial
membrane potential integrity and mitochondrial morphology. Overexpression of the solute carrier family 14 member 1 gene could abolish hypoxia-induced invasion, reduce the migration of A498 cells, inhibit the hypoxia-induced
release of inflammatory cytokines, and arrest the cell cycle at the G1/S checkpoint.
Conclusions These data reveal that nursing based on oxygen administration can improve the clinical efficacy ofrenal cell carcinoma therapies, being safe and effective. The results elucidate a mechanism wherein the solute carrierfamily 14 member 1 gene participates in the occurrence and development of hypoxia-induced renal cell carcinoma ina mitochondria-dependent manner.
Keywords Human Solute Carrier Family 14 Member 1, Hypoxia, Renal Cell Carcinoma, Nursing*Correspondence:
Xilan Yang
njyyyangxilan@163.com
Department of Urology, Nanjing First Hospital, Nanjing Medical University,
Qinhuai District, 68 Changle Road, Nanjing 210012, China

Background
Renal cell carcinoma (RCC) originates from either thecancerous renal cortex or renal tubular epithelial cells,
and is the second most lethal urological malignancy,
accounting for 90–95% of kidney neoplasms. Its incidence and mortality are steadily increasing at a rate of© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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Shi et al. BMC Molecular and Cell Biology(2023) 24:10

approximately 2–3% every decade [1, 2]. There are threemain histological subtypes of RCC, including clear cell
RCC (ccRCC), papillary RCC, and chromophobe RCC,
which jointly represent over 85% of all primary renalmalignancies [3]. The treatment for renal parenchymal
tumors has changed over time, and this trend continues
today as a result of technological progress made withclinical research and improved diagnostic and therapeutic tools. Unfortunately, the traditional treatment methods of chemotherapy and radiotherapy have proven to beineffective and lead to multidrug resistance in target cells.
Although modern approaches were initially popular, suchas immunotherapy and the use of angiogenesis inhibitors, subsequent studies have shown their limited andconfounding effects. Radical nephrectomy and nephronsparing surgery are still the only effective treatments for
localized RCC, but the characteristics of RCC recurrence
and metastasis lead to a 5-year survival rate of approximately 12%, posing a great challenge to clinical therapies[4]. Therefore, the development of a new and accurate
therapeutic strategy is needed to improve the clinicaltreatment of RCC. Importantly, it must overcome theissue of RCC’s poor response to chemotherapy, radiotherapy, and immunotherapy.
Adaptation to hypoxia (≤ 2% ­O2) is involved inmany physiological and pathological conditions, suchas stroke, tissue ischemia, and cancer [5]. A recent
report indicated that long-term hypoxia is a major
driving force of cancer tumorigenesis [6]. Hypoxia
is an indicator of poor prognosis in a variety of solid
tumors, including prostate [7], cervical [8], breast[9], and head and neck cancers [10]. Hypoxia participates in epithelial-to-mesenchymal transition (EMT),
angiogenesis (vascular endothelial growth factor(VEGF)), the shift from oxidative phosphorylation
to anaerobic glycolysis and energy metabolism (glucose transporter isoform 1 (GLUT1)), the promotion of proliferation, the induction of invasion andmetastasis, and the inhibition of cancer cell apoptosis, ultimately leading to carcinogenesis [11, 12]. In
this case, the manipulation of the hypoxic microenvironment can be considered a means to prevent orreverse malignant transformation. Mitochondrial
dysfunction is an important factor that plays an activerole in the hypoxia response by regulating tumor cellproliferation, apoptosis, neovascularization, invasion, and metastasis [13]. It is therefore imperativethat normal cells remove this hypoxia-induced mitochondrial damage. However, the exact mechanism
by which hypoxia-induced mitochondrial dysfunction contributes to tumor occurrence and development only attracted interest a few years ago, andstill remains unclear. It is known that hypoxia is a

Page 2 of 13

typical microenvironment characteristic of almost all
solid tumors. RCC derives from highly heterogeneous renal tubular epithelial cells and is highly sensitive to hypoxia. The hypoxic tumor environment ishighly prevalent in poorly vascularized and necrotic
RCC tumor cores [14]. It has been reported that the
hypoxia-inducible transcription factor (HIF) signaling
pathway plays a critical role in the genetic instability
and pathogenesis of RCC [15]. At present, therapeutic drugs targeting HIF, VEGF, and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin(mTOR) are important first-line treatment options
for advanced RCC patients, but only a small proportion of patients benefit from them [16]. These reports
prompted us to investigate the molecular pathways
underpinning RCC tumorigenesis to identify more
effective therapeutic targets in hypoxia-induced RCC.
The hypoxia regulates numerous target genes associated with malignant behavior, including angiogenesis, cell survival, proliferation, mitochondrial function,and metabolism. Mitochondrial function is involvedin the initiation and progression of malignant tumors
and modulates multiple processes, such as altering
glucose metabolism, destroying redox balance, producing reactive oxygen species (ROS), and damaging
the intrinsic apoptotic pathway, which plays a central role in the growth, metastasis, and tumorigenesis
of RCC [17, 18]. Urea transporter B (UT‐B), which is
encoded by the SLC14A1 (human solute carrier family 14 member 1) gene, has been verified to be widelydistributed in numerous tissues, including the kidney,
brain, heart, liver, and other tissues; it regulates theurea concentration, blood pressure, bone metabolism,
and cardiac functions [19]. A deficiency in SLC14A1
gene expression or its dysfunction contributes to thepathogenesis of diseases. For example, SLC14A1 geneknockout mice were reported to exhibit mitochondrial dysfunction and increased rates of apoptosis of
the heart [20]. The expression of the SLC14A1 gene
was also found to induce melanoma B16 cell death via
the activation of p53 and the induction of mitochondrial apoptosis [21]. Therefore, a better understandingof the mechanisms of SLC14A1 in malignancies maylead to the development of more effective and more
specific drugs. The primary objective of this study
was to investigate the role and mechanism of hypoxiainduced RCC, as well as to provide evidence-based
medical proof for ways to improve postoperative nursing in the context of this disease. We also explored theeffect of the SLC14A1 gene on mitochondrial function
in a human RCC cell line and elucidated that it participates in the occurrence and development of hypoxiainduced RCC in a mitochondria-dependent manner.

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

In summary, the findings suggest that SLC14A1 gene
expression sensitively responds to hypoxia, andhence serves as an ideal prospective target for RCC
treatment.

Page 3 of 13

Characteristics of the SLC14A1 gene in hypoxia‑associated
RCC determined by gene expression profiling interactive
analysis (GEPIA)

To investigate the effect of hypoxia on the biological characteristics of RCC, A498 cells were cultured under normoxic/hypoxic conditions. The results show that hypoxia
induced the invasion and migration of A498 cells in atime-dependent manner when compared to the normoxic
cells (Fig. 1a-b). Flow cytometry analysis indicated thathypoxia inhibited A498 cell apoptosis compared to thenormoxia (Fig. 1c). In addition, the viability of A498 cells
was significantly increased with the extension of incubation time under hypoxia (Fig. 1d). Western blot analysis
revealed that UT-B expression in A498 cells cultured
under hypoxia was markedly lower than that in the corresponding normoxic group (Fig. 1e). Moreover, hypoxia
significantly increased the production of the serum
inflammatory cytokines interleukin 6 (IL-6) and tumor
necrosis factor α (TNF-α) compared to the normoxic
group (Fig. 1f-g). Such tumor-related inflammatory factors promote the development and progression of cancer,
so hypoxia is closely associated with changes in the biological characteristics of RCC.

An interactive bodymap shows that the SLC14A1 geneis widely distributed in human tissues and organs, suchas the brain, lungs, kidneys, and bladder. It is particularly
highly expressed in the human urinary system, especiallyin the kidney and bladder. Moreover, Fig. 2a shows that
SLC14A1 gene expression in RCC tissue (red) is lower
than that in the corresponding normal renal tissue (blue).
Figure 2b illustrates its gene expression profile acrossall tumor samples and paired normal tissues, revealingthat SLC14A1 gene expression levels in kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC),
and kidney renal papillary cell carcinoma (KIRP) tissues(red) are significantly lower than that in normal renal
tissue (brown, Fig. 2b). Box plots show that SLC14A1
gene expression is significantly relatively lower in KICH,
KIRC, and KIRP tissues (Fig. 2c). The relationship of
SLC14A1 gene expression with the overall survival of
RCC was analyzed using the Kaplan–Meier plotter. HighSLC14A1 gene expression was found to prolong overall
survival to some extent in patients with RCC (LogrankP = 0.00017, Fig. 2d). Moreover, Kaplan–Meier plotting
demonstrated that the hazard ratio (HR) of the SLC14A1
gene was 0.55 (where HR < 1 is identified as a protective
factor for disease), thereby supporting it as an important
protective factor for RCC [P(HR) = 0.00021].

Effect of oxygen treatment on the clinical efficacy of RCC
patients

SLC14A1 gene expression induced mitochondrial
dysfunction in A498 cells

It is known that hypoxia is a significant characteristic of
RCC patients. Therefore, this study aimed to explore theeffect of oxygen treatment on the clinical efficacy of RCC
patients. The results show that the observation group had
significantly decreased values for biochemical indexes
compared to the control group, as indicated by decreased
levels of the renal function indexes blood urea nitrogen(BUN) and creatinine (Cr), the serum inflammatory factors
IL-6 and TNF-α, and the tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 50 (CA50)(t = 2.213, 2.490, 2.035, 2.500, 2.281, and 2.282, respectively, all P < 0.05, Table 1). Moreover, the observationgroup had fewer postoperative complications (total incidence) and a lower incidence of RCC metastasis and recurrence compared to the control group (t = 4.267, 5.851, and4.267, respectively, all P < 0.05, Table 2). The present study
also demonstrated that the intervention of oxygen treatment significantly alleviated the anxiety and depression of
RCC patients, while nursing satisfaction was also significantly improved compared to the corresponding control
group (t = 6.478, 5.107, and 3.925, respectively, all P < 0.05,
Table 3).

This study aimed to explore the effect of increased
SLC14A1 gene expression on mitochondrial function in
A498 cells. As shown in Fig. 3a and b, treatment withthe overexpression plasmid pc-SLC14A1 significantly
increased mitochondrial ROS generation and destroyed
mitochondrial membrane potential (ΔΨm) integrity
compared to treatment with the empty plasmid (pc-NC)(P < 0.01). Meanwhile, observations of representative
mitochondrial morphology indicated that overexpressionof the SLC14A1 gene (pc-SLC14A1) caused the mitochondria to become swollen and vacuolated with cristae
breakage and disappearance (Fig. 3c). Additionally, thepc-SLC14A1 treatment significantly decreased the intracellular adenosine triphosphate (ATP) level relative tothat of the pc-NC group (Fig. 3d).

Results
Effect of normoxic/hypoxic conditions on the biologicalcharacteristics of A498 cells

Effect of SLC14A1 gene expression on hypoxia‑induced
biological characteristics of A498 cells

To evaluate whether SLC14A1 gene expression plays animportant role in hypoxia-regulated apoptosis in A498
cells, the apoptotic level of cells was detected by annexin
V- fluorescein isothiocyanate (FITC)/ propidium iodide

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

Page 4 of 13

Fig. 1 Effect of hypoxia/normoxia on the biological characteristics of A498 cells. A498 cells were cultured at 37 °C in a humidified incubator with
5% ­CO2, 2% ­O2, and 93% ­N2 (hypoxia) or with 5% C
­ O2 and 95% O­ 2 (normoxia) for 24 and 48 h. a The invasion of A498 cells was detected by thetranswell assay. b The migration of A498 cells was examined by the wound healing assay. c The apoptosis of A498 cells was measured by flow
cytometry. d The viability of A498 cells was assessed by the CCK-8 assay. e Expression of the UT-B protein was detected by western blot. Full-length
blots/gels are presented in Supplementary file 5. The levels of IL-6 (f) and TNF-α (g) in the culture supernatant were determined by a Beckman
Coulter AU5800 automatic biochemical analyzer. Each bar represents the median expression obtained from 3 independent experiments. *P < 0.05,**P < 0.01 vs the corresponding normoxic group

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

Table 1 Comparison of clinical index ( x ± s)

Page 5 of 13

Groupsn

BUN(mmol/L)Cr(μmol/L)

IL-6(pg/mL)TNF-α(pg/mL)

CEA(μg/L)CA50(U/mL)

Observation group32

10.62 ± 1.49*

112.47 ± 7.33*

6.25 ± 0.67*

52.13 ± 6.23*

4.26 ± 0.85*

14.75 ± 0.86*

Control group

32

t

-

2.213

11.53 ± 1.78

2.490

119.97 ± 15.38

2.035

6.57 ± 0.63

2.500

56.31 ± 7.11

2.281

4.81 ± 1.07

2.282

15.4 ± 1.39

P

-

0.031

0.015

0.046

0.015

0.026

0.026

Note: *, P < 0.05 vs control group

Table 2 Comparison of postoperative complications, metastasis and recurrence between the two groups (%)
Groupsn

Incision infection

Bleeding

Urinary tract
infection

Total incidence

Recurrence

Metastasis

Observation group32

1(3.10)1(3.10)2(6.30)4(12.5)*3(9.40)*2(6.30)*Control group32

3(9.40)4(12.5)4(12.5)

11(34.4)

11(34.4)8(25.0)

X2

-

1.067

1.953

0.736

4.267

5.851

4.267

P

-

0.302

0.162

0.391

0.039

0.016

0.039

Note: *, P < 0.05 vs control group

Table 3 Comparison of incidence of anxiety and depression andsatisfaction between the two groups (%)
Groupsnanxietydepression

Treatmentsatisfaction

Observation group32

8 (25.0)*

10 (31.3)*

27 (84.4)*

Control group32

18 (56.3)

19 (59.4)

20 (62.5)

2

X

-

6.478

5.107

3.925

P

-

0.011

0.024

0.048

Note: *, P < 0.05 vs control group(PI) staining. As shown in Fig. 4a, the hypoxia + pcSLC14A1 treatment significantly increased the number
of apoptotic cells relative to the hypoxia-alone treatment(P < 0.05). There was no significant change in the number of apoptotic cells between the hypoxia-alone treatment and hypoxia + pc-NC treatment (P > 0.05). The cell
cycle distribution was calculated by flow cytometry with
PI staining. There was a significantly increased incidenceof the G0/G1 phase of cells following the hypoxia + pcSLC14A1 treatment, while that of the S phase decreased
markedly compared to the hypoxia-alone treatment(P < 0.05), indicating that A498 cells were arrested in
G1/S checkpoint under SLC14A1 overexpression. The cell
cycle distribution showed no significant changes between
the hypoxia-alone group and the hypoxia + pc-NC group(P > 0.05, Fig. 4b). The hypoxia-alone treatment induced
the invasion and migration of cells, but these changes
were markedly abolished when combined with the pcSLC14A1 treatment (hypoxia + pc-SLC14A1 group),
and the change was significant (P < 0.05). There wasno significant change in the invasion and migration of

A498 cells between the hypoxia-alone treatment andhypoxia + pc-NC treatment group (P > 0.05, Fig. 4c-d).
Next, we determined the role of SLC14A1 gene expression in inflammatory cytokine production in A498 cells.
The hypoxia-induced elevations of IL-6 and TNF-α levels
observed in the hypoxia-alone group were suppressed by
the pc-SLC14A1 treatment in the hypoxia + pc-SLC14A1
group (P < 0.05). The IL-6 and TNF-α levels showed nosignificant differences between the hypoxia-alone groupand the hypoxia + pc-NC group (P > 0.05, Fig. 4e-f ).

Discussion
Hypoxia, a typical microenvironment characteristic, leads to tumor development and metastasis. In the
hypoxic microenvironment, cytokines are secreted inprimary tumor cells and macrophages accumulate underaltered gene expression [22]. The imbalance between
oxygen consumption and oxygen supply is a crucial eventat all stages of cancer development and is commonly
associated with molecular and cellular characteristics
of cancer, including mutagenesis and impaired DNA
repair, resistance to radio- and chemo-therapy, EMT,
and cancer metabolic reprogramming [23–25]. Hypoxia
escalates tumor growth, inflammation, and immunosuppression, thereby transforming the tumor from benign
to invasive [26]. Recent studies have profiled the related
hypoxia- and immune system-related gene expression
changes, which may be used to optimize prognostic risk
stratification and individual therapy in triple-negative
breast cancer patients [27]. Another study indicated thatthe hypoxia-induced RNA miR-3677-3p enhanced theproliferation, migration, and invasion of hepatocellularShi et al. BMC Molecular and Cell Biology(2023) 24:10

Page 6 of 13

Fig. 2 Expression characteristics of the SLC14A1 gene in hypoxia-associated RCC using Gene Expression Profiling Interactive Analysis (GEPIA).
a The median expression of tumor (red) and normal (blue) samples in the bodymap. b The gene expression profile across all tumor samples andpaired normal tissues (bar plot). The height of each bar represents the median expression of a certain tumor type or normal tissue. KICH: kidney
chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; c SLC14A1 gene expression in box plot, including
66 cases of KICH with 53 paired normal tissues, 523 cases of KIRC with 100 paired normal tissues, and 286 cases of KIRP with 60 paired of normal
tissues. Data were obtained from the Genotype-Tissue Expression (GTEx) project and are presented as the mean ± quartile values. *P < 0.05 by the
unpaired t-test. d Relationship of SLC14A1 gene expression with the overall survival of 515 RCC patients, as obtained by the online Kaplan–Meiersurvival plotter

carcinoma cells by inhibiting sirtuin 5 [28]. Moreover,the exosome-mediated transfer of miR-193a-3p, miR210-3p, and miR-5100 (which are derived from hypoxic
bone marrow-derived mesenchymal stem cells) has beenshown to induce lung cancer cell invasion by modulating signal transducer and activator of transcription 3(STAT3) signaling pathway [29].
Our results are consistent with those obtained by
the previous literature, namely that hypoxia promotes
RCC cell viability, invasion, and migration in a timedependent manner, while inhibiting RCC cell apoptosis. It is a generally accepted concept that hypoxia can
induce inflammation, and the findings herein indeed
indicate that hypoxia significantly induced the releaseof the serum inflammatory cytokines IL-6 and TNF-αin RCC cells. Thus, tumor hypoxia is a primary deleterious factor involved in malignant progression and hence
represents a viable target for cancer treatment. Thereis convincing evidence to indicate that well-oxygenated cells (approximately 20–21% oxygen, 160 mmHg)
are more sensitive to chemotherapy, radiotherapy, andimmunotherapy; whereas hypoxic cells (approximately
2.6% oxygen, 20 mmHg) readily develop drug resistanceand lead to poor treatment response [30]. Our research
explored the effect of oxygen inhalation on the clinicalefficacy of RCC patients. The results indicate that theoxygen administration intervention group had significantly decreased values of renal function indexes, serum

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

Page 7 of 13

Fig. 3 Effect of SLC14A1 overexpression on mitochondrial dysfunction in A498 cells. A498 cells were transfected with either the pc-SLC14A1 vector(2.5 μg/ml) or pcDNA3.1 null vector (pc-NC, 2.5 μg/ml) for 48 h. a The fluorescent probe MitoSOX™ Red was used to assess mitochondrial ROS
level. b The JC-1 probe was used to determine changes in the mitochondrial membrane potential. c Observation of A498 cell morphology was
performed using electron microscopy (× 5,200). Red arrows indicate abnormal mitochondria structure, including mitochondrial swelling andrupture. Scale bar = 1 μm. d The ATP level of A498 cells was evaluated using a luciferase-based assay kit. Results are presented as the mean ± SD
from 3 independent experiments. **P < 0.01 vs the pc-NC groupinflammatory factors, and tumor markers. Moreover, theoxygen administration group had fewer postoperativecomplications and lower incidence rates of RCC metastasis and recurrence. As a result, the intervention of oxygen
administration significantly alleviated the anxiety anddepression of RCC patients, while nursing satisfaction
was significantly improved. The above data suggest that
nursing intervention based on oxygen administration can
improve the clinical efficacy of RCC therapies, comprising a safe and effective nursing intervention method.
Our data show that hypoxia-regulated UT-B expression in cultured A498 cells significantly lower than thatof the normoxia, so its encoding gene, SLC14A1, may beclosely associated with hypoxia-induced RCC development, and the underlying mechanism is worth pursuing.
The SLC14A1 gene is located on chromosome 18q12.1–
21.1 and encodes the human UT-B protein [31], a membrane channel protein that plays a crucial role in thepassive transport of urea [32]. It was recently reported
that the SLC14A1 gene is associated with human malignancies, exhibiting downregulated expression in lungadenocarcinoma specimens, while its elevated expression was found to inhibit lung squamous cell carcinoma
colony formation [33]. Another study similarly indicatedthat SLC14A1 expression in malignant prostate cancer
tissues was significantly low, being inversely proportional
to both the clinical grade and stage [34]. It has beenreported that several single nucleotide polymorphisms(SNPs) within the SLC14A1 gene (such as rs1058396,
rs2298720, rs11877062, and rs17674580) are strongly correlated with bladder cancer [35–38]. The Gene Expression Profiling Interactive Analysis (GEPIA) databaseshows that SLC14A1 gene expression levels in KICH,
KIRC, and KIRP tissues are significantly lower than thatin corresponding normal renal tissue. A Kaplan–Meierplot of data obtained from Fig. 2d indicated that high
SLC14A1 gene expression prolongs overall survival tosome extent in patients with RCC. Our experiments also
demonstrated that enhanced SLC14A1 gene expression
induced mitochondrial dysfunction, including increased
mitochondrial ROS generation, diminished intracellular
ATP level, and the destruction of both ΔΨm integrity andShi et al. BMC Molecular and Cell Biology(2023) 24:10

Page 8 of 13

Fig. 4 Effect of SLC14A1 overexpression on hypoxia-induced biological characteristics of A498 cells. A498 cells were transfected with either the
pc-SLC14A1 vector (2.5 μg/ml) or pcDNA3.1 null vector (pc-NC, 2.5 μg/ml) and then cultured at 37 °C in a humidified incubator with 5% C
­ O2, 2% ­O2,
and 93% ­N2 (hypoxia) for 48 h. a The apoptosis of A498 cells was measured by flow cytometry. b The distribution of the cell cycle was calculated byflow cytometry. (c) The invasion of A498 cells was assessed by the transwell assay. d The migration of A498 cells was analyzed by the wound healing
assay. The levels of IL-6 e and TNF-α f in the cell culture supernatant were measured by the Beckman Coulter AU5800 automatic biochemical
analyzer. n = 3, **P < 0.01, *P < 0.05, #P > 0.05 versus the hypoxia-alone group

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

mitochondrial morphology. Therefore, the SLC14A1 geneappears to participate in the development and occurrence of RCC by regulating mitochondrial function.
Recent studies have explored the mechanisms andpathways of hypoxia in tumor progression and metastasis, such as canonical HIF signaling, mitochondrial
function, the unfolded protein response (UPR), andmethylation changes [39, 40]. ROS generation is responsible for causing progressive mitochondrial dysfunction
and cell apoptosis [41, 42]. In our study, mitochondrial
ROS accumulation was significantly enhanced in A498
cells transfected with the overexpression plasmid pcSLC14A1; the toxic effect of ROS generation may be a
crucial cause of RCC cell apoptosis. Moreover, the present study further confirmed that SLC14A1 overexpression abolished the hypoxia-induced invasion andmigration of A498 cells, and moreover, the cell cycle wasarrested at the G1/S checkpoint. In addition, SLC14A1
overexpression enhanced ROS accumulation, which inturn triggered A498 cell apoptosis. Hypoxia-induced elevations in the levels of IL-6 and TNF-α were suppressedby SLC14A1 overexpression via a mitochondria-dependent pathway.

Conclusion
These findings indicate that oxygen administration topatients does transiently relieve tumor hypoxia, andas a result, improves therapeutic outcomes. Our findings show that hypoxia induces changes in gene expression, such as the inhibition of SLC14A1 gene expression,
which has many important downstream cellular andphysiological effects in RCC. Accordingly, enhancing
the expression of the SLC14A1 gene led to mitochondrial ROS accumulation, ΔΨm disruption, and abnormal mitochondrial morphology, which could abolish the
hypoxia-induced invasion and migration of A498 cells
and inhibit the hypoxia-induced release of inflammatory
cytokines. These findings support a mechanism wherein
the SLC14A1 gene plays a crucial role in hypoxia-induced
RCC in a mitochondria-dependent manner.
Our experimental work showed that SLC14A1 gene
expression is an important factor involved in hypoxiainduced RCC development and progression, but evidence
from large cohort studies on its expression and association with clinical outcomes is lacking. Further researchis therefore required to accurately analyze the effects of
SLC14A1 gene expression on each aspect of tumor progression (including proliferation, invasion, angiogenesis,
and metastasis) to guide novel therapeutic strategies.
Of note, the specific molecular mechanism by which
hypoxia regulates the SLC14A1 transcription level isstill elusive and merits further investigation. Existingstudies on therapeutic strategies for RCC have obvious

Page 9 of 13

limitations, such as small sample sizes, further research
will be required to collect data from a larger RCC cohort.
A therapeutic strategy for RCC should be supported byhigh-quality, evidence-based medicine, in the future, alarge-scale, multicenter, randomized controlled clinical
trial must be conducted to verify the effectiveness of oxygen administration to RCC patients. We will share andrecommend this strategy in medical practice guidelines
on a global platform to ensure future guidelines adhere to
the principles of evidence-based medicine.
The development of specific agents for targeting
hypoxia and its related pathways will also be crucial.
These will have the potential to provide innovative cancer therapies that can enhance antitumor immunity andovercome the barriers of treatment resistance, tumor
tolerance, and escape from immune surveillance. Thereexists the question of whether it will be possible to use
therapeutic targets derived from the hypoxic tumor
microenvironment and its related pathways as new therapeutic schemes for cancer immunotherapy, this deserves
further investigation. Integrating the manipulation of
hypoxic stress into cancer immunotherapy may leadto more durable and effective cancer immunotherapy
approaches in the future.

Methods
Patient recruitment

The present study was based on the principles of the
Declaration of Helsinki. This study was approved by the
Medical Ethics Committee of Nanjing Hospital Affiliated to Nanjing Medical University (Date: 2016.11.17/No.
2016–227). The written informed consent of each participant was obtained. A total of 64 patients with RCC diagnosed at Nanjing Hospital Affiliated to Nanjing Medical
University between Jan 2017 and Jan 2020 were recruited
into the study cohort. Among them, there were 32 casesin the control group (15 females and 17 males), in which,the average age was 52.1 years old, and the histologicaltype was as follows: 5 cases of KICH, 19 cases of KIRC,
and 8 cases of KIRP. There were 32 cases in the observation group (18 females and 14 males), in which, the average age was 51.9 years old, and the histological type was
as follows: 4 cases of KICH, 21 cases of KIRC, and 7 casesof KIRP. The inclusion criteria were as follows: ① RCC
histological diagnosis was based on both hematoxylin
and eosin (H&E) staining and immunochemistry results;② the clinical data of patients were complete; ③ thepatients underwent radical nephrectomy. The exclusion
criteria were as follows: ① patients with other malignant tumors; ② patients with severe postoperative complications caused by uncontrollable basic diseases; ③patients with incomplete clinical case data. There was no

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

significant difference in patients’ clinical and pathologicalcharacteristics between these two groups.
Nursing intervention

The control group patients received conventional nursing
care, including as preoperative psychological intervention, postoperative anti-infection, complication care, andrehabilitation training. Using conventional nursing careas a reference, the observation group (evidence-based
nursing model group) implemented the evidence-based
nursing model for the patients. Namely, the PubMed,
EMBASE, and Cochrane Library databases were searched
for entries from Jan 2010 to Jan 2020 for literature
regarding difficulties in clinical practice following RCC
operation, and a systematic evaluation and meta-analysis
were employed to guide the nursing model for RCC postoperative complications. From this, an evidence-based
nursing model was developed and used in the present
study, which employed the following: ①oxygen inhalation, involving monitoring the blood oxygen saturation(> 90%), oxygenation index (> 300 mmHg), blood lactic
acid concentration, and evaluating the degree of hypoxia
in patients; ② local oxygen therapy, whereby the surgical wound was treated with local oxygen therapy for
90 min/d for 5 consecutive days and 2 intermittent days,
while the wound pressure was maintained at 3 kPa during the care process; ③ complications during the nursing
intervention: a common complication after RCC operation is anastomotic leakage with abdominal infection,
and an evidence-based nursing model must judge theoccurrence of anastomotic bleeding and abdominal leakage according to the patient’s abdominal pain, abdominal
distension, blood pressure, body temperature, and drainage fluid characteristics.
Clinical index observation

The venous blood from RCC patients was collected. Samples from patients with jaundice, hemolysis, and highlipids were not included. Renal function indexes (BUN
and Cr) and serum inflammatory factors (IL-6 and TNFα) were detected by the Beckman Coulter AU5800 automatic biochemical analyzer. Tumor markers (CEA andCA50) were measured by the Roche Cobas e601 automatic electrochemiluminescence immunoanalyzer. The
Self-Rating Anxiety Scale (SAS, > 50 for anxiety) and
Self-Rating Depression Scale (SDS, > 53 for depression)
were used to evaluate the psychological status of RCC
patients. Nursing satisfaction was scored as follows: satisfied, 4 points; somewhat satisfied, 3 points; dissatisfied, 2points; very dissatisfied, 1 point. The nursing satisfaction
rate was calculated as the sum of the given answers ‘satisfied’ and ‘somewhat satisfied’ over the total number of
responses.

Page 10 of 13

Postoperative follow‑up

RCC patients were rechecked every 3 months after theiroperation, whereby each underwent a routine blood test,
routine urine test, biochemical test, chest radiograph,and abdominal and urinary ultrasound test. If there wassuspicion of RCC recurrence, urinary computed tomography (CT), chest CT, and a bone scan were conducted.
Cell culture

The human RCC cell line A498 was obtained from the
Cell Bank of China Union Medical University (Beijing,
China). A498 cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM, CAT#: 01–050-1A, Life Technologies, Grand Island, NY, USA) supplemented with
10% heat-inactivated fetal bovine serum (FBS, CAT#:
04–127-1A, Life Technologies, Grand Island, NY, USA)and 100 μg/ml penicillin/streptomycin (CAT#: PSF-1,
Life Technologies, Grand Island, NY, USA), then maintained at 37 °C in constant 5% ­CO2 and 90–100% humidified atmosphere. The A498 cells underwent annual shorttandem Repeat (STR) profiling analysis, population doubling time and morphology for genetic confirmation.
A498 cells in the logarithmic growth phase were divided
into two groups: ① the hypoxic condition group at 37 °C,
5% ­CO2, 2% O
­ 2, and 93% ­N2; ② the normoxic conditiongroup at 37 °C, 5% ­CO2, and 95% ­O2. All experiments
were performed with early-passage cells (passages 4–10).
SLC14A1 plasmid construction and transfection.

SLC14A1 cDNA was subcloned into a pcDNA3.1 mammalian expression vector (CAT#: V79520, Invitrogen
Life Technologies, Carlsbad, CA, USA). The primer
sequences of the pcDNA3.1-SLC14A1 (pc-SLC14A1)
plasmid were as follows: SLC14A1-sense, 5’-CCA GTG
GGA GTT GGT CAG AT-3’; SLC14A1-antisense,5’-GTT GAA ACC CCA GAG TCC AA-3′. The reconstituted pc-SLC14A1 plasmid and empty plasmid (pc-NC)
were transfected into A498 cells. Briefly, 2.5 μg of either
the pc-SLC14A1 plasmid or pc-NC plasmid was mixed
with 8 μL of lipofectamine 2000 (CAT#: 11,668,019, Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated with A498 cells for 6 h. The cells were then cultured
in DMEM containing 20% FBS for another 48 h.
Western blot analysis

A498 cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of cellular protein were loaded per lane, subjected to 12%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred
to a polyvinyl difluoride (PVDF) membrane (CAT#:GVWP02500, Millipore, Billerica, MA, USA). The membrane was then blocked with 5% skimmed milk for 1 h

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

Page 11 of 13

and immunoblotted with primary antibodies specificto SLC14A1 (1: 100 dilution, CAT#: AV48116; SigmaAldrich, St. Louis, MO, USA) and actin (1: 5,000; CAT#:A5441; Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. The blot was then incubated with goat anti-rabbit
IgG horseradish peroxidase-conjugated secondary antibody (1: 5,000; CAT#: SA00001-2; ProteinTech Group,
Chicago, IL, USA). An enhanced chemiluminescence(ECL) western detection system (CAT#: 7003; Cell Signaling Technology, Beverly, MA, USA) was used to detect
immunoreactive bands.

Instrument Inc., Winooski, VT, USA). The ATP level was
determined according to the standard curve.

Electron microscopy

Cell apoptosis and cell cycle assay

A498 cells were harvested in 1 m­ m3 blocks and immersed
in 3% glutaraldehyde solution for 2 h. The samples were
post-fixed in 1% osmium tetroxide ­(OsO4; CAT#: 18,459;
Ted Pella, Redding, CA, USA) for 2 h, dehydrated inascending alcohols, and then embedded in Eponate 12
resin (CAT#: 18,005; Ted Pella, Redding, CA, USA).
Ultrathin (60 nm) sections were cut and post-stainedwith 5% uranyl acetate (CAT#: 19,481; Ted Pella, Redding, CA, USA) and lead citrate (CAT#: 19,312; Ted Pella,
Redding, CA, USA). The mitochondrial ultrastructure of
A498 cells was observed under a transmission electron
microscope (JEM 1011, Japan).
Measurement of mitochondrial ROS generation

The mitochondrial ROS level in A498 cells was analyzed by
the MitoSOX™ Red mitochondrial superoxide indicator. In
brief, A498 cells were subjected to different treatments andincubated with MitoSOX™ Red (5 µM, CAT#: M36008,
Invitrogen Life Technologies, Carlsbad, CA, USA) in thedark at 37 °C for 20 min. The fluorescence intensity wasevaluated using a flow cytometer (BD FACSCalibur).
Determination of ΔΨm

Changes in the ΔΨm of A498 cells were measured using
the fluorescent cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethyl-imidacarbocyanine iodide (JC-1). Briefly, A498
cells were subjected to various treatments and then incubated with JC‑1 (20 µg/mL, CAT#: T3168, Invitrogen Life
Technologies, Carlsbad, CA, USA) in the dark at 37 °C for
15 min. The fluorescence intensity was evaluated using an
epifluorescence microscope (Nikon Corp., Tokyo, Japan).
Measurement of ATP level

A luciferase-based assay kit (CAT#: S0026; Beyotime,
Shanghai, China) was used to determine the intracellular ATP content. A498 cells were lysed with ATP lysis
buffer and centrifuged at 12,000 g for 8 min at 4 °C. The
resulting supernatant and ATP-detection working solution were then mixed before luminescence was measured
using a BioTek Synergy H­ 1 Microplate Reader (BioTek

Cell viability assay

The Cell Counting Kit-8 (CCK-8; CAT#: CK04; Dojindo,
Kyushu Island, Japan) was employed to evaluate A498 cell
viability. Briefly, A498 cells underwent various treatments
before being incubated with CCK-8 solution (10 μL/100μL) at 37 °C for 2 h. The absorbance was then assessedusing a BioTek Synergy H­ 1 Microplate Reader (BioTek
Instrument Inc., Winooski, VT, USA) at 450 nm.
Annexin V-FITC/PI staining (CAT#: A211-01/02;
Vazyme Biotech Co., Ltd., Nanjing, China) was used toevaluate A498 cell apoptosis. A498 cells underwent various treatments before being harvested and resuspended
with 500 μL of binding buffer. Subsequently, the A498
cells were stained with 5 μL of Annexin V and 10 μL of PI(CAT#: A211-01/02; Vazyme Biotech Co., Ltd., Nanjing,
China) for 30 min in the dark. Then, apoptotic cells weredetected using a flow cytometer (BD FACSCalibur).
Cell cycle assay

A498 cells underwent various treatments before being
harvested and fixed in 70% ethanol for 18 h. The A498
cells were then stained with 50 mg/mL PI (CAT#: A21101/02; Vazyme Biotech Co., Ltd., Nanjing, China) for
30 min at 37 °C. Finally, the cell cycle distribution wasdetermined using a flow cytometer (BD FACSCalibur).
Cell invasion assay

A transwell chamber (CAT#: CLS3422; Corning Incorporated, Corning, NY, USA) was employed to assess theinvasive ability of A498 cells. In brief, starved A498 cells(5 × ­106/mL) were seeded into the top chamber (pore size,
8 μm), while 500 μL of DMEM containing 20% FBS was
filled into the lower chamber. Following incubation for theindicated time, the invasive cells were fixed and stainedwith 0.1% crystal violet (CAT#: C8470; Solarbio Science
Technology Co., Ltd., Beijing, China). The invasive abilityof A498 cells was then evaluated under the microscope(× 200 magnification, Olympus, Tokyo, Japan).
Cell migration assay

The wound healing assay was used to estimate the migratory ability of A498 cells. In brief, A498 cells were passaged to attain 80–90% confluence and were thenscratched in a straight line on the surface of the monolayer cells using a 200 µl tip (at time point 0). After incubation durations of 24 h and 48 h, the healing width of
the wound was surveyed with an inverted optical microscope (× 200 magnification, Olympus, Tokyo, Japan).

Shi et al. BMC Molecular and Cell Biology(2023) 24:10

Statistical analysis

Statistical analysis was conducted using SPSS software
version 13.0 (SPSS Inc. Chicago, IL, USA). The data were
presented as the mean ± standard deviation (SD). Comparisons between groups were conducted the Student’s
t-test if the data were normally distributed, otherwise,
the Wilcoxon rank-sum test was performed. P < 0.05 was
set to indicate statistical significance.
Abbreviations
RCC​	Renal cell carcinoma
ccRCC​	Clear cell RCC​
EMT	Epithelial-to-mesenchymal transition
VEGF	Vascular endothelial growth factor
GLUT1	Glucose transporter isoform 1
HIF	Hypoxia-inducible transcription factor
PI3K	Phosphatidylinositol 3-kinase
mTOR	Mammalian target of rapamycin
ROS	Reactive oxygen species
UT‐B	Urea transporter B
SLC14A1	Solute carrier family 14 member 1
BUN	Urea nitrogen
Cr	Creatinine
IL-6	Interleukin 6
TNF-α	Tumor necrosis factor α
CEA	Carcinoembryonic antigen
CA50	Carbohydrate antigen 50
KICH	Kidney Chromophobe
KIRC	Kidney renal clear cell carcinoma
KIRP	Kidney renal papillary cell carcinomaHR	Hazard ratioΔΨm	Mitochondrial membrane potentialATP	Adenosine triphosphatePI	Propidium iodide
STAT3	Signal transducer and activator of transcription 3
SNPs	Single nucleotide polymorphisms
GEPIA	Gene Expression Profiling Interactive Analysis
UPR	Unfolded protein response
H&E	Hematoxylin and eosinSAS	Self rating Anxiety Cale
SDS	Self rating Depression Scale
CT	Computed tomography
DMEM	Dulbecco’s modified Eagle’s medium
FBS	Fetal bovine serum
RIPA	Radio immuno precipitation assay
SDS-PAGE	Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
PVDF	Polyvinyl difluoride
ECL	Enhanced chemiluminescence
OsO4	Osmium tetroxide
JC-1	Fluorescent cationic dye 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide
CCK-8	Cells Counting Kit-8
FITC	Fluorescein isothiocyanate
SD	Standard deviation
GTEx	Genotype-Tissue Expression
STR	Short tandem Repeat

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​023-​00473-6.
Additional file 1: Supplementary Figures
Acknowledgements
Not applicable.

Page 12 of 13

Informed consent
Informed consent was obtained from all individual participants includedin the
study.
Authors’ contributions
XLY made substantial contributions to the conception and design of thestudy. JS drafted the manuscript and revised it critically for important intellectual content. RLS have agreed to be accountable for all aspects of the workin ensuring that questions related to the accuracy or integrity of any part of
the work are appropriately investigated and resolved. All authors read andapproved the final manuscript.
Funding
This study was supported by grants from the National Natural Science Foundation of China (Contract grant number: No. 82102717, 82272671).
Availability of data and materials
The datasets generated during and/or analyzed during the current study areavailable from the corresponding author on reasonable request.

Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of Helsinki. The research protocol was approved by the Medical Ethics Committeeof Medical Ethics Committee of Nanjing Hospital Affiliated to Nanjing Medical
University. Informed consent was obtained from all individual participants
included in the study.
Consent for publication
Not applicable.
Conpeting of interests
The authors declare that they have no competing interests.
Received: 3 November 2022 Accepted: 13 March 2023

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