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Abstract

Objective

To investigate the mRNA and protein levels of calpain small subunit-1 (Capn4) in human clear cell renal cell carcinoma (ccRCC), to analyse the relationship between Capn4 mRNA level and pathological stage of ccRCC, and to examine the potential of Capn4 as a prognostic factor in ccRCC.

Methods

mRNA and protein levels of Capn4 were measured in pairs of tumour tissues and matched adjacent nontumour tissue obtained from patients with ccRCC by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and Western blotting, respectively. Associations of the mRNA level of Capn4 with pathological tumour stage and the overall survival of ccRCC patients were also analysed.

Results

Capn4 mRNA and protein level were significantly higher in ccRCC tumour tissues compared with adjacent nontumour tissues as assessed by qRT-PCR and Western blotting, respectively. Higher Capn4 mRNA levels were observed in patients with more advanced pathological stage of ccRCC and were also associated with decreased overall survival of patients with ccRCC.

Conclusions

The findings from this study indicate that Capn4 has the potential to be an independent prognostic indicator for ccRCC.

Keywords

Renal cell carcinoma calpain calpain small subunit-1 prognosis gene expression kidney

Introduction

Renal cell carcinoma (RCC, also known as hypernephroma) is a common solid lesion that develops in the lining of adult kidney tubules.¹ It represents 2–3% of all adult cancers and approximately 80% of primary renal cancers.² RCC is the third most prevalent urological cancer after prostate and bladder cancers and the mortality rate is >40%.³ It has been estimated that 65 150 new cases of renal cancer will be diagnosed in 2013, resulting in approximately 13 680 deaths,⁴ and the vast majority of these cases will be caused by RCC. Five-year survival rates for each stage of RCC have been estimated to be: stage I, 93%; stage II, 65%; stage III, 53%; stage IV, 27%.⁵ Since RCC represents a heterogeneous group of tumours, consisting of clear cell, papillary, chromophobe, collecting duct and unclassified tumours, it poses a considerable clinical challenge.⁶ Clear cell RCC (ccRCC) is the most common subtype (75–80%) of RCC.² It is associated with unpredictable clinical outcome because it can demonstrate long-term survival in the presence of metastases, late recurrence and spontaneous regression.^5,7 Several different parameters including tumour grade, DNA ploidy and nuclear morphometry have been employed to predict prognosis of RCC but a reliable marker has not yet been established.^7–8 There is a clinical need to identify prognostic biomarkers with high specificity and sensitivity for RCC in order to improve the understanding of its molecular pathogenesis and facilitate the discovery of novel therapeutic targets.

The calpains are a highly conserved family of calcium-dependent, non-lysosomal cysteine proteases that catalyse regulated proteolysis of many specific substrates.⁹ The mammalian calpains consist of 14 family members, of which micro (µ)-calpain and milli (m)-calpain are the two most extensively studied isoforms.^10–12 Both µ-calpain and m-calpain are heterodimers with a distinct catalytic subunit of 80 kDa (calpain-1 large subunit, encoded by CAPN1 for µ-calpain and calpain-2 large subunit, encoded by CAPN2 for m-calpain) and a common regulatory subunit of 28 kDa (calpain small subunit-1 [Capn4], encoded by CAPN4).¹³ Within the calpain family, some isoforms are ubiquitously expressed, such as µ-calpain and m-calpain, while others have a more restricted expression: for example, calpain-8 and calpain-9 are exclusively expressed in the stomach mucosa and digestive tract.¹³ Although the exact physiological roles of the calpain isoforms remain unclear, they have been implicated in various cellular processes such as cell motility,¹⁴ programmed cell death¹⁵ and cell cycle progression.^16,17 It has been suggested that cellular transformation leads to enhanced calpain activity, which promotes local tumour invasion and peripheral metastasis through the proteolysis of local adhesion proteins including focal adhesion kinase and talin.^13,18–20 The regulatory subunit, Capn4, plays a critical role in maintaining calpain activity; Capn4 ablation in Capn4-knockout mice is lethal at the embryonic stage, with animals demonstrating no detectable calpain activity.^21,22 In addition, recent studies have revealed that Capn4 is increased in both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) and is associated with poor prognosis; Capn4 knockdown inhibits the migration and invasion abilities of both HCC and ICC cells.^23–25 The role of Capn4 in RCC has not, as yet, been extensively studied.

The present study was undertaken to examine the mRNA and protein levels of Capn4 in clinical ccRCC tissues obtained from patients undergoing partial or radical nephrectomy. In addition, the relationship between Capn4 levels and clinicopathological features of ccRCC were examined and the prognostic value of Capn4 for post-resection survival in ccRCC patients was determined.

Materials and methods

Tissue samples

Renal cell carcinoma tissue and adjacent nontumour tissue (≥5 mm from the carcinoma tissue) were harvested from patients undergoing partial or radical nephrectomy at the Department of Urology, the Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China between October 2000 and September 2006. No further inclusion or exclusion criteria were defined for this study. Portions of the tissue samples were immediately sent for pathological inspection to confirm the presence (tumour tissue containing >80% tumour cells) or absence (normal tissue) of ccRCC. Staging of ccRCC was performed using the American Joint Committee on Cancer (AJCC) staging manual.²⁶ The remainder of the tissue samples was immediately frozen in liquid nitrogen and stored for a mean of 6 months prior to analysis.

The study protocol was approved by the ethics committee of the Third Affiliated Hospital of Soochow University and all participating patients provided written informed consent.

RNA isolation and quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

Total RNA was isolated from ccRCC tissues and adjacent nontumour tissues (50–100 mg tissue) using TRIzol® reagent (Invitrogen Corp., Carlsbad, CA, USA). qRT-PCR was performed as described previously.²⁷ Briefly, reverse transcription was performed using SuperScript™ II RT (Invitrogen Corp.) as per the manufacturer’s protocol. TaqMan® gene expression assays (Applied Biosystems, Foster City, CA, USA) were used to quantify Capn4 mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control. The primer sequences for qRT–PCR were as follows (Invitrogen Corp.): CAPN4 forward, 5′-TCCGACGCTACTCAGATGAAAGT-3′ and reverse, 5′-GATTTGTCCAGTGCCATCTTTGT-3′; GAPDH forward, 5′-TCCACCACCCTGTTGCTGTA-3′ and reverse, 5′-ACCACAGTCCATGCCATCAC-3′. The PCR reactions were performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in duplicate in a 10 µl volume containing 5 µl Universal PCR Master Mix (Applied Biosystems), 0.5 µl TaqMan® assay and 4.5 µl diluted cDNA (50 ng reverse-transcribed RNA). PCR cycling conditions were 50 ℃ for 2 min, 95 ℃ for 10 min and 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. PCR products were visualized on 1.2% agarose, purified and then verified by sequencing. The relative expression level of Capn4 mRNA was normalized with GAPDH expression level and calculated using the 2^−ΔΔCt method.²⁸

Western blot analysis

Protein was extracted from frozen ccRCC tissues and adjacent nontumour tissues using the Whole Protein Extraction Kit (Fermentas Life Science, Glen Burnie, MD, USA) as per the manufacturer’s instructions. The microplate bicinchoninic assay (BCA) protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used according to the manufacturer’s instructions to determine protein concentration. Western blot analysis was carried out as described previously.²⁹ In brief, 30 µg of protein per lane was separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Bio-Rad) at 4 ℃ at 100 V for 1 h. Membranes were blocked in 5% skimmed milk at room temperature for 2 h before incubation with a polyclonal rabbit anti-human antibody against Capn4 (1:1000; Abcam®, Cambridge, MA, USA) at room temperature for 2 h. Membranes were washed three times for 10 min each wash with Tris-buffered saline with Tween-20 (137 mM sodium chloride, 20 mM Tris and 0.1% Tween-20) at room temperature before incubation with goat anti-rabbit immunoglobulin-G antibody conjugated with horseradish peroxidase (1:5000; Invitrogen Corp.) at room temperature for 1 h. The membranes were then washed three times as before. GAPDH (1:5000; Abcam®) was used as a loading control. The immune complex was detected using the SuperSignal West Pico kit (Thermo Fisher Scientific Inc.) as per the manufacturer’s instructions. To quantify protein levels of Capn4, the band intensities of Capn4 protein were measured using Image Lab™ Software (Biorad, Hercules, CA, USA) and normalized with the corresponding GAPDH band intensities. All experiments were performed in triplicate.

Statistical analyses

All statistical analyses were carried out using SPSS® statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Paired samples t-test was performed to analyse significant differences between the ccRCC and adjacent nontumour tissue. Spearman’s rank correlation test was carried out to determine the relationship between Capn4 expression level and tumour progression status. Capn4 mRNA levels were considered to be significantly increased if the calculated fold-change was more than 0.5 in tumour tissue compared with the matched adjacent nontumour tissue. The overall survival rates were calculated by the Kaplan–Meier method , and the difference in survival was compared with the log-rank test. The Cox proportional hazards regression model was used for univariate and multivariate analyses to assess the effects of the clinicopathological variables and Capn4 mRNA expression levels on overall survival. A P-value of <0.05 was considered statistically significant.

Results

Paired tissue samples (tumour tissue and matched adjacent nontumour tissue) were collected from 40 patients with ccRCC. Patient and tumour characteristics are shown in Table 1.

Table 1.

Demographic and tumour characteristics of 40 patients with clear cell renal cell carcinoma (ccRCC) who had tumour and adjacent nontumour tissues removed during partial or radical nephrectomy.

Patient no.	Sex	Age, years	Fuhrman grade ^a	Primary tumour stage^b
1	M	62	G2	pT1b
2	F	67	G2	pT3b
3	F	65	G3	pT4
4	M	58	G1	pT1a
5	M	58	G4	pT2b
6	M	50	G3	pT3a
7	M	63	G2	pT2b
8	F	80	G3	pT3b
9	M	59	G3	pT2b
10	F	66	G2	pT2a
11	F	52	G2	pT1b
12	F	70	G3	pT3a
13	M	47	G3	pT3a
14	M	31	G2	pT1b
15	M	38	G3	pT3a
16	M	63	G2	pT3a
17	F	63	G3	pT3b
18	M	63	G2	pT3a
19	F	47	G1	pT2a
20	F	36	G2	pT4
21	M	75	G3	pT3b
22	F	50	G2	pT3a
23	M	45	G1	pT1a
24	F	60	G2	pT3b
25	F	71	G2	pT3b
26	F	65	G3	pT1b
27	M	63	G3	pT2b
28	M	47	G2	pT3b
29	F	67	G4	pT1b
30	F	45	G3	pT3a
31	M	66	G3	pT2a
32	M	45	G1	pT1a
33	M	45	G3	pT3b
34	F	72	G4	pT4
35	M	60	G2	pT2b
36	M	71	G2	pT3b
37	F	65	G3	pT3a
38	F	63	G3	pT3b
39	F	47	G2	pT2a
40	M	66	G3	pT1b

Fuhrman grading schema of renal cell carcinoma is based on the microscopic morphology of a neoplasm with haematoxylin and eosin staining;³¹ four nuclear grades (1–4) are defined in order of increasing nuclear size, irregularity and nucleolar prominence.

pT1a: primary tumour ≤4 cm in greatest dimension, limited to the kidney; pT1b: primary tumour >4 cm but not >7 cm in greatest dimension, limited to the kidney; pT2a: tumour >7 cm but ≤10 cm in greatest dimension, limited to the kidney; pT2b: primary tumour >10 cm, limited to the kidney; pT3a: tumour grossly extends into the renal vein or its segmental, muscle-containing, branches, or tumour invades perirenal and/or renal sinus fat but not beyond Gerota’s fascia; pT3b: primary tumour grossly extends into the vena cava below the diaphragm; pT4: primary tumour invades beyond Gerota’s fascia.²⁶

As shown in Figure 1, the mRNA level of Capn4 in ccRCC tumour samples was statistically significantly higher than in nontumour tissue samples (P = 0.013).

Figure 1.

Calpain small subunit-1 (Capn4) mRNA levels in tumour tissues and adjacent nontumour tissues (NT) from 40 patients with clear cell renal cell carcinoma (ccRCC) as assessed by quantitative reverse transcription–polymerase chain reaction. ^*P = 0.013 as determined by paired sample t-test.

In all 40 paired tissue samples evaluated, the protein band intensities of Capn4 in ccRCC tumour tissue were visually stronger compared with adjacent nontumour tissues. Representative results for the first four pairs of tissue samples are shown in Figure 2a. As shown in Figure 2b, protein levels of Capn4 in ccRCC tumour tissues were significantly increased compared with levels determined in adjacent nontumour tissues (P = 0.005).

Figure 2.

Western blot analysis of: (a) calpain small subunit (Capn4) protein in tumour tissues (T) and adjacent nontumour tissues (N) from the first four patients with clear cell renal cell carcinoma (ccRCC); (b) densitometric analysis of the relative Capn4 protein levels (Capn4/GAPDH) in ccRCC tissues and nontumour tissues (NT). GADPH, glyceraldehyde-3-phosphate dehydrogenase. ^*P = 0.005 as determined by paired sample t-test.

Capn4 mRNA levels were correlated with the presence of ccRCC and tumour stage: Capn4 mRNA was increased in 77.5% (31/40) of patients with ccRCC. High levels of Capn4 mRNA were observed in 61.1% (11/18) of patients with low primary tumour stages (pT1 + pT2) and 90.9% (20/22) of patients with high primary tumour stages (pT3 + pT4) (Figure 3).

Figure 3.

Calpain small subunit (Capn4) mRNA levels in tumour tissue from 40 patients with renal cell carcinoma according to primary tumour stage. pT1: primary tumour ≤7 cm in greatest dimension, limited to the kidney; pT2: primary tumour >7 cm in greatest dimension, limited to the kidney; pT3: primary tumour extends into major veins or perinephric tissues but not into the ipsilateral adrenal gland and not beyond Gerota’s fascia; pT4: primary tumour invades beyond Gerota’s fascia including contiguous extension into the ipsilateral adrenal gland.²⁶

Kaplan–Meier analysis revealed that higher levels of Capn4 mRNA in ccRCC tissues were significantly associated with decreased overall survival (P = 0.006; Figure 4).

Figure 4.

Kaplan–Meir curves demonstrating overall survival of 40 patients with renal cell carcinoma following partial or radical nephrectomy according to mRNA levels of calpain small subunit (Capn4) in tumour tissues. P = 0.006 high versus low Capn4 mRNA levels as determined by paired samples t-test.

Univariate analysis using a Cox proportional hazards model to evaluate the potential of using Capn4 mRNA expression level as a prognostic marker for ccRCC patients after surgery showed that both Capn4 overexpression (P = 0.009) and primary tumour stage (P = 0.010) were the prime variables for ccRCC prognosis (Table 2). After adjusting for clinicopathologic variables, Capn4 overexpression (P = 0.013) and primary tumour stage (P = 0.008) remained significantly correlated with the prognosis of ccRCC patients (Table 2).

Table 2.

Univariate and multivariate analyses of different clinicopathological variables and Capn4 expression status as predictors for overall survival of clear cell renal cell carcinoma

Variable	Univariate analysis		Multivariate analysis
Variable	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (<60 vs ≥60 years)	1.016 (0.989–1.029)	0.136	1.281 (0.976–1.289)	0.142
Sex (male vs female)	1.012 (0.982–1.032)	0.121	1.001 (0.979–1.019)	0.120
Fuhrman grade (G1/2 vs G3/4)	1.423 (0.467–4.223)	0.070	1.236 (0.524–4.653)	0.080
Primary tumour stage (pT1/2 vs pT3/4)	0.610 (0.408–0.911)	0.010	0.550 (0.373–0.824)	0.008
Capn4 expression (low vs high)	1.715 (1.158–2.534)	0.009	1.666 (1.117–2.452)	0.013

CI, confidence interval; HR, hazard ratio.

Discussion

Renal cell carcinoma is the most common malignant primary tumour in human kidney cancer. Improvements in disease therapy have led to disease remission in many patients but the overall prognosis remains unsatisfactory. One of the essential factors governing prognosis in patients with RCC is the tumour stage at the time of diagnosis. Patients with RCC who have tumours confined to the kidney have the best prognosis.³⁰ There is an important clinical need to identify biomarkers that can assist in the diagnosis of RCC and predict disease prognosis.

Calpains are a family of intracellular cysteine proteases, with both ubiquitous and tissue-specific isoforms.¹³ The prototypical calpain is composed of an isoform-specific large subunit and a common small subunit (Capn4).¹³ Capn4 was dramatically increased in metastatic tissues compared with nonmetastatic tissues from patients with HCC in a previous study.²³ It was also shown that increases in Capn4 were correlated with the invasive phenotype of HCC and that Capn4 may represent a potential prognostic factor for tumour recurrence and survival in patients with HCC.²³ In addition, previous studies have demonstrated the ectopic expression of Capn4 in patients with ICC, a primary malignant liver neoplasm secondary to HCC, and the association of increased Capn4 with lymphatic metastasis and tumour-node-metastasis stage of ICC.²⁵ The mechanisms by which Capn4 promotes metastasis and invasion in liver neoplasms has been studied; it has been demonstrated that Capn4 and osteopontin interact with each other and promote cell migration through the nuclear factor-κB pathway.²⁴

The present study investigated the role of Capn4 in ccRCC. Compared with matched adjacent nontumour tissues, the levels of Capn4 mRNA and protein were aberrantly high in the tumour tissue of patients with ccRCC. Furthermore, Capn4 mRNA levels appeared to be correlated with the pathological stage of ccRCC, with higher Capn4 levels observed in patients with high primary tumour stages compared with patients with low primary tumour stages. In addition, higher levels of Capn4 mRNA were found to be associated with decreased overall survival in patients with ccRCC. Both univariate and multivariate analysis indicated that Capn4 expression status is an independent prognostic indicator for ccRCC. These data are consistent with previous findings in other tumour types^23–25 and indicate that Capn4 could be a potential prognostic marker in ccRCC.

In conclusion, the present study has shown for the first time that Capn4 mRNA and protein are increased in ccRCC tissues and that higher Capn4 levels are associated with advanced clinical tumour stage and shorter overall survival. The precise role of Capn4 in ccRCC development and progression, however, remains to be elucidated and further investigations in cell and animal models are in progress.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

Preparation of this manuscript was supported by Changzhou Health Research Program, grant no. CE20125025.

References

Rini

Campbell

Escudier

. Renal cell carcinoma. Lancet 2009; 373: 1119–1132.

Cairns

. Renal cell carcinoma. Cancer Biomark 2010; 9: 461–473.

Bukowski

. Prognostic factors for survival in metastatic renal cell carcinoma: update 2008. Cancer 2009; 115(10 suppl): 2273–2281.

Siegel

Naishadham

Jemal

. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11–30.

Cohen

McGovern

. Renal-cell carcinoma. N Engl J Med 2005; 353: 2477–2490.

Störkel

Eble

Adlakha

. Classification of renal cell carcinoma: Workgroup No. 1. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997; 80: 987–989.

Zimmermann

Woenckhaus

Pietschmann

. Expression of annexin II in conventional renal cell carcinoma is correlated with Fuhrman grade and clinical outcome. Virchows Arch 2004; 445: 368–374.

Yaycioglu

Eskicorapci

Karabulut

. A preoperative prognostic model predicting recurrence-free survival for patients with kidney cancer. Jpn J Clin Oncol 2013; 43: 63–68.

Hanna

Campbell

Davies

. Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin. Nature 2008; 456: 409–412.

10.

Croall

DeMartino

. Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev 1991; 71: 813–847.

11.

Goll

Thompson

. The calpain system. Physiol Rev 2003; 83: 731–801.

12.

Sorimachi

Hata

Ono

. Expanding members and roles of the calpain superfamily and their genetically modified animals. Exp Anim 2010; 59: 549–566.

13.

Storr

Carragher

Frame

. The calpain system and cancer. Nat Rev Cancer 2011; 11: 364–374.

14.

Glading

Lauffenburger

Wells

. Cutting to the chase: calpain proteases in cell motility. Trends Cell Biol 2002; 12: 46–54.

15.

Tan

De Veyra

. Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem 2006; 281: 17689–17698.

16.

Carragher

Westhoff

Riley

. v-Src-induced modulation of the calpain-calpastatin proteolytic system regulates transformation. Mol Cell Biol 2002; 22: 257–269.

17.

Wang

Rosales

Magliocco

. Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain. Oncogene 2003; 22: 769–774.

18.

Beckerle

Burridge

DeMartino

. Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion. Cell 1987; 51: 569–577.

19.

Franco

Rodgers

Perrin

. Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 2004; 6: 977–983.

20.

Liu

Schnellmann

. Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death. J Pharmacol Exp Ther 2003; 304: 63–70.

21.

Dutt

Croall

Arthur

. m-Calpain is required for preimplantation embryonic development in mice. BMC Dev Biol 2006; 6: 3–3.

22.

Arthur

Elce

Hegadorn

. Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division. Mol Cell Biol 2000; 20: 4474–4481.

23.

Bai

Dai

Zhou

. Capn4 overexpression underlies tumor invasion and metastasis after liver transplantation for hepatocellular carcinoma. Hepatology 2009; 49: 460–470.

24.

Zhang

You

Wang

. Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. PLoS One 2012; 7: e31458–e31458.

25.

Zhang

Bai

Huang

. Prognostic significance of Capn4 overexpression in intrahepatic cholangiocarcinoma. PLoS One 2013; 8: e54619–e54619.

26.

Edge

Byrd

Compton

. AJCC Cancer Staging Manual, 7th ed. New York: Springer, 2010, pp. 479–489.

27.

Zhong

Qian

Hua

. Effects of feeding with corn gluten meal on trypsin activity and mRNA expression in Fugu obscurus. Fish Physiol Biochem 2011; 37: 453–460.

28.

Livak

Schmittgen

. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods 2001; 25: 402–408.

29.

Qian

Zhao

. Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells. Int J Biochem Cell Biol 2013; 45: 724–735.

30.

Chin

Lam

Figlin

. Surveillance strategies for renal cell carcinoma patients following nephrectomy. Rev Urol 2006; 8: 1–7.

31.

Fuhrman

Lasky

Limas

. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982; 6: 655–663.

Capn4 mRNA level is correlated with tumour progression and clinical outcome in clear cell renal cell carcinoma

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Tissue samples

RNA isolation and quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

Western blot analysis

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interest

Funding

References