Downregulated circular RNA itchy E3 ubiquitin protein ligase correlates with advanced pathologic T stage,high lymph node metastasis risk and poor survivals in prostate cancer patients

Abstract

OBJECTIVE:

This study aimed to explore the correlation of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) with pathological features as well as its predictive value on prognosis in prostate cancer patients.

METHODS:

Three hundred and twenty-four patients with prostate cancer underwent radical prostatectomy were consecutively included in this retrospective study, and patients’ specimens of tumor as well as paired adjacent tissues were collected for detection of circ-ITCH expression. Patients’ baseline characteristics and survival data were obtained from follow-up records, and correlation of circ-ITCH with patients’ pathological features and survival was determined.

RESULTS:

Circ-ITCH expression was decreased in tumor tissue compared with paired adjacent tissues ( $P<$ 0.001), and it presented with good value in distinguishing tumor tissues from paired adjacent tissues with area under curve (AUC) of 0.812 (95%CI: 0.780–0.845). Circ-ITCH low expression was associated with advanced pathologic T stage ( $P=$ 0.002) and high risk of lymph node metastasis ( $P=$ 0.047) in prostate cancer patients. As for prognosis, patients with circ-ITCH high expression had longer disease-free survival (DFS) ( $P<$ 0.001) and overall survival (OS) ( $P<$ 0.001). Additionally, circ-ITCH (high vs. low) independently predicted better survival, while Gleason score ( $>$ 7 vs. $\leqslant$ 7) and surgical margin status (positive vs. negative) independently predicted worse survival in prostate patients underwent radical prostatectomy.

CONCLUSIONS:

Circ-ITCH is downregulated in prostate cancer tissues, and its low expression correlates with advanced pathologic T stage, high lymph mode metastasis risk and poor survival in prostate cancer patients underwent radical prostatectomy.

Keywords

Circ-ITCH prostate cancer expression pathological features prognosis

1. Introduction

Prostate cancer is the most prevalent and the third death-leading malignancy among males accounting for 13.5% of all cancer cases and 6.7% of cancer deaths worldwide [1]. In Eastern Asia, the incidence of prostate cancer is 23.9 per 100,000 and the mortality is about 14.8 per 100,000, and its prevalence is constantly increasing in China largely accounting for the change in lifestyle and dietary structure [1, 2]. Modern diagnosis approaches (such as biomarker screening using prostate-specific antigen (PSA) and imageological examinations) allow early determination as well as proper staging of prostate cancer, while despite the success in diagnosis, the emergence of disease recurrence and metastasis is still very common, which leads to unfavorable prognosis in prostate cancer patients [3, 4, 5, 6]. Recently, understanding about the pathogenesis of prostate cancer is getting deeper with growing functional researches, while there is still a substantial gap in our knowledge about therapeutically reliable biomarkers for disease risk and prognosis in prostate cancer patients [7, 8]. Therefore, exploring novel biomarkers is greatly needed for improvement of diagnostic and prognostic forecasting in prostate cancer as well as benefiting patients’ survival.

Circular RNAs (circRNAs) are defined as a class of RNAs that form covalently closed loops, which make them stable in structure and resistant to degradation [9]. In recent years, thousands of circRNAs have been newly identified and some of them play vital roles in biological processes involved in the development of human cancers, and a number of them have been reported to participate in pathogenesis of prostate cancer [10, 11]. CircRNA itchy E3 ubiquitin protein ligase (circ-ITCH) is coded from ITCH gene and shares micro RNA (miRNA) binding sites with 3’ translated region of ITCH gene, which makes it a sponge for miRNAs [12]. The anti-tumor effect of circ-ITCH has been discovered in various cancers including bladder cancer, hepatocellular carcinoma, lung cancer and esophageal squamous cell carcinoma [12, 13, 14, 15]. However, the role of circ-ITCH in prostate cancer still remains uninvestigated. Therefore, this study aimed to explore the correlation of circ-ITCH with pathological features and its predictive value on prognosis in prostate cancer patients underwent radical prostatectomy.

2. Methods

2.1 Patients

From Jan 2013 to Dec 2016, 324 patients with prostate cancer underwent radical prostatectomy in Huangshi Central Hospital were consecutively included in this retrospective study. The inclusion criteria were: (1) histopathologically confirmed prostate cancer; (2) age above 18 years old; (3) radical prostatectomy, and fresh-frozen tumor tissue and paired adjacent tissue were reserved and available; (4) clinicopathologic data and follow-up records were complete and accessible. The exclusion criteria included: (1) underwent neoadjuvant therapy; (2) with incomplete medical records; (3) missing follow-up records; (4) complicated with other solid tumors or had a history of other malignancies. The study protocol was approved by the Institutional Review Board of Huangshi Central Hospital, and written informed consents were obtained from all patients or their guardians.

2.2 Data collection

Patients’ age, level of prostate-specific antigen (PSA) before surgery, Gleason score, pathologic T stage, lymph node metastasis and surgical margin status were collected from medical records. All patients were followed up until 2018/6/30, and the survival data were obtained from follow-up records, which were used to calculate disease-free survival (DFS) and overall survival (OS). The DFS was defined as the duration from partial remission (PR) to recurrence or progression of disease or death; the OS was defined as the time interval from PR to death.

2.3 Detection of circ-ITCH in tumor and paired adjacent tissue

Fresh prostate tissues were procured immediately after surgery, cut into the size of 1 cm ${}^{2}\times$ 0.5 cm pieces, snap-frozen in liquid nitrogen and stored at $-$ 80 ${}^{\circ}$ C. For the detection of circ-ITCH in tumor and paired adjacent tissue, patients’ specimens were collected from the storage compartment of the hospital with permission. A diagnostic hematoxylin (H) and eosin (E) stained section was prepared to verify tumor content and margin status and to identify areas of tumor tissue and adjacent normal tissue. The H & E slides were read by the pathologist to ensure more than 70% tumor cells in specimens and the defined area of the tumor tissue was outlined on each slide (the area of adjacent tissue was no detectable cancer cells). Then the H & E stained slides were used as a template and corresponding frozen sections were superimposed on these. Defined area of the tumor cells was excised and used as tumor tissue, and the remnant section containing no tumor cells was served as paired adjacent tissue. Then total RNA extractions were carried out using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations and underwent Quibit (Invitrogen, USA) for quality control. One $\mu$ g of sample was used for reverse transcription of cDNA synthesis, which was performed using PrimeScript ${}^{\text{TM}}$ RT reagent Kit (TAKARA, Kusatsu, Japan) for detection of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 1 ug of sample was used for digestion of linear RNAs using RNase R (Epicentre, WI, USA), then underwent reverse transcription of cDNA synthesis using PrimeScript ${}^{\text{TM}}$ RT reagent Kit (TAKARA, Kusatsu, Japan) for circ-ITCH detection. qPCR with SYBR Green kit (TAKARA, Kusatsu, Japan) was subsequently performed. Melting curve was used for analysis and Circ-ITCH relative expression was calculated by the method of 2 ${}^{-\Delta\Delta\text{Ct}}$ (2 {[mean ct of circ-ITCH-mean ct of GAPDH]-[mean ct of control-mean ct of GAPDH]}). GAPDH was used as internal reference, and the primers used in the study were as follows: circ-ITCH, forward: TCCTCTTGCCTCAGCC TTCTC, reverse: GCAGGTGCCTCTAATCCCATCT; GAPDH, forward: CATGAGAAGTATGACAACAGC CT, reverse: AGTCCTTCCACGATACCAAAGT.

2.4 Statistical analysis

All statistical analyses were performed with the use of SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.00 (GraphPad Software, La Jolla, CA, USA). Normal distributed continuous variable was presented as mean value $\pm$ standard deviation, skewed distributed continuous variable was presented as median (25 ${}^{\text{th}}$ –75 ${}^{\text{th}}$ quantiles), and the categorized variable was presented as count (percentage). Comparison of circ-ITCH relative expression between tumor tissue and paired adjacent tissue was determined by Wilcoxon signed-rank sum test. The log-rank test was applied to check the dependence of patients’ survival on single variables or on combinations of variables. The Kaplan Meier curve was used to represent patients’ survival. The Receiver-operating characteristic (ROC) curve and the area under the ROC curve (AUC) were used to assess the ability of circ-ITCH relative expression to discriminate between tumor tissue and adjacent tissue. Univariate and multivariate Cox’s proportional hazards regression analyses were performed to determine the factors affecting DFS and OS. $P$ value $<$ 0.05 was considered significant.

Figure 1.

Study flow.

3. Results

3.1 Study flow

Six hundred and eight prostate cancer patients who underwent radical prostatectomy were screened for eligibility, while 261 patients were excluded including 123 patients without fresh-frozen tumor tissue and paired adjacent tissue, 98 patients with incomplete clinicopathological data or missing follow-up records, 28 patients underwent neoadjuvant therapy and 12 patients complicated with other solid tumors or had a history of other malignancies (Fig. 1). Three hundred and forty-seven patients were eligible, whereas 23 of them who could not be contacted to obtain informed consents were excluded. Finally, 324 patients were included in the analysis.

Table 1
Baseline characteristics of patients with prostate cancer

Characteristics	Prostate cancer patients
	( $N=$ 324)
Age (years)	63	(56–69)
PSA ( $n$ /%)
$\leqslant$ 10 ng/ml	97	(29.9)
10–20 ng/ml	157	(48.5)
$\geqslant$ 20 ng/ml	70	(21.6)
Gleason score ( $n$ /%)
$\leqslant$ 6	77	(23.8)
7	168	(51.8)
$\geqslant$ 8	79	(24.4)
Pathologic T stage ( $n$ /%)
pT2a	98	(30.3)
pT2b	55	(17.0)
pT2c	46	(14.2)
pT3a	63	(19.4)
pT3b	62	(19.1)
Lymph node metastasis ( $n$ /%)
No	234	(72.2)
Yes	90	(27.8)
Surgical margin status ( $n$ /%)
Negative	230	(71.0)
Positive	94	(29.0)

Data were presented as mean value $\pm$ standard deviation or count (percentage). PSA, prostate-specific antigen.

Figure 2.

Circ-ITCH relative expression in tumor tissue and paired adjacent tissue in prostate cancer patients. Circ-ITCH relative expression was downregulated in tumor tissues compared with paired adjacent tissues (A). ROC curve displayed the good value of circ-ITCH in distinguishing tumor tissue and paired adjacent tissue (B). Comparison of circ-ITCH relative expression between tumor tissue and paired adjacent tissue was determined by Wilcoxon signed-rank sum test, and the value of circ-ITCH in distinguishing tumor tissue and paired adjacent tissue was assessed by ROC curve. $P<$ 0.05 was considered significant. Circ-ITCH, circular RNA Itchy E3 ubiquitin protein ligase; ROC curve, Receiver-operating characteristic curve.

3.2 Patients’ baseline characteristics

Three hundred and twenty-four prostate cancer patients with median age of 63 (56–69) years were included in the analysis (Table 1). Among whom, 97 (29.9%), 157 (48.5%) and 70 (21.6%) of patients were with PSA $\leqslant$ 10 ng/ml, PSA 10–20 ng/ml and PSA $\geqslant$ 20 ng/ml respectively, and the number of patients with Gleason score $\leqslant$ 6, 7 and $\geqslant$ 8 were 77 (23.8%), 168 (51.8%) and 79 (24.4%) respectively. As for pathologic T staging, the number of patients with pT2a, pT2b, pT2c, pT3a and pT3b were 98 (30.3%), 55 (17.0%), 46 (14.2%), 63 (19.4%) and 62 (19.1%) respectively. Other baseline characteristics were listed in Table 1.

3.3 Circ-ITCH expression in tumor tissue and paired adjacent tissue

Circ-ITCH relative expression in tumor tissue (0.764 (0.387–1.381)) was lower compared with paired adjacent tissue (2.576 (1.242–4.455)) ( $P<$ 0.001) (Fig. 2A). In addition, ROC curve illustrated that circ-ITCH presented good value in distinguishing tumor tissue from paired adjacent tissue with area under curve (AUC) being 0.812 (95% CI: 0.780–0.845) in prostate cancer patients (Fig. 2B). The values for sensitivity and specificity were 88.3% and 61.7% respectively at the best cut-off point were the largest sum of sensitivity and specificity occurred.

Table 2
Correlation of circ-ITCH relative expression with patients’ characteristics

Characteristics	Circ-ITCH low	Circ-ITCH high	$P$ value
	expression	expression
	( $N=$ 162)	( $N=$ 162)
Age			0.369
$\leqslant$ 60 years	65 (47.1)	73 (52.9)
$>$ 60 years	97 (52.2)	89 (47.8)
PSA			0.115
$\leqslant$ 10 ng/ml	55 (56.7)	42 (43.3)
$>$ 10 ng/ml	107 (47.1)	120 (52.9)
Gleason score ( $n$ /%)			0.897
$\leqslant$ 7	122 (49.8)	123 (50.2)
$>$ 7	40 (50.6)	39 (49.4)
Pathologic T stage			0.002
pT2	86 (43.2)	113 (56.8)
pT3	76 (60.8)	49 (39.2)
Lymph node metastasis			0.047
No	109 (46.6)	125 (53.4)
Yes	53 (58.9)	37 (41.1)
Surgical margin status			0.624
Negative	117 (50.9)	113 (49.1)
Positive	45 (47.9)	49 (52.1)

Data were presented as count (percentage). Comparison was determined by Chi-square test. $P<$ 0.05 was considered significant (in bold). PSA, prostate-specific antigen.

Figure 3.

Association between circ-ITCH expression and survivals in prostate cancer patients. Circ-ITCH high expression was correlated with longer DFS (A) and OS (B) in prostate cancer patients. The difference of survival between patients with circ-ITCH low expression and high expression was illuminated with Kaplan-Meier curve and determined by log-rank test. $P<$ 0.05 was considered significant. DFS, disease-free survival; OS, overall survival; circ-ITCH, circular RNA Itchy E3 ubiquitin protein ligase.

Figure 4.

Difference in DFS between circ-ITCH high expression and low expression patients in subgroup analysis. Circ-ITCH high expression patients presented longer DFS compared to circ-ITCH low expression patients in subgroups including: age $>$ 60 years (B), PSA $\leqslant$ 10 ng/ml (C), PSA $>$ 10 ng/ml (D), Gleason score $\leqslant$ 7 (E), pT2 (G), pT3 (H), non-lymph node metastasis (I), surgical margin-negative (K) and surgical margin-positive (L). No difference in DFS between circ-ITCH low expression patients and circ-ITCH high expression patients was observed in subgroups including: age $\leqslant$ 60 years (A), Gleason score $>$ 7 (F) and lymph node metastasis (J). The difference in survival between patients with circ-ITCH low expression and high expression was illuminated with Kaplan-Meier curve and determined by log-rank test. $P<$ 0.05 was considered significant. DFS, disease-free survival; circ-ITCH, circular RNA Itchy E3 ubiquitin protein ligase; PSA, prostate-specific antigen.

3.4 Correlation of circ-ITCH expression with patients’ characteristics

Patients were divided into circ-ITCH low expression ( $N=$ 162) and high expression ( $N=$ 162) groups according to the median value of circ-ITCH expression (0.764) in tumor tissues (Table 2). Circ-ITCH low expression was associated with advanced pathologic T stage ( $P=$ 0.002) as well as higher risk of lymph node metastasis in prostate cancer patients ( $P=$ 0.047). No correlation of circ-ITCH expression with patients’ age, PSA level, Gleason score or surgical margin status was observed (All $P>$ 0.05).

3.5 Correlation of circ-ITCH expression with patients’ survival

Patients’ DFS and OS was evaluated by Kaplan-Meier curve analysis and comparison of survivals between circ-ITCH high expression and low expression groups were performed using log-rank test (Fig. 3). Circ-ITCH high expression was correlated with longer DFS ( $P<$ 0.001) (Fig. 3A) as well as better OS in prostate cancer patients underwent radical prostatectomy ( $P<$ 0.001) (Fig. 3B).

3.6 Comparison of DFS between circ-ITCH high expression and low expression patients in subgroup analysis

Patients were divided into subgroups according to their baseline characteristics, and in each subgroup, comparison of DFS between circ-ITCH high expression and low expression patients was carried out (Fig. 4). Compared to circ-ITCH low expression patients, circ-ITCH high expression patients presented longer DFS in subgroups including: age $>$ 60 years ( $P<$ 0.001) (Fig. 4B), PSA $\leqslant$ 10 ng/ml ( $P=$ 0.017) (Fig. 4C), PSA $>$ 10 ng/ml ( $P<$ 0.001) (Fig. 4D), Gleason score $\leqslant$ 7 ( $P<$ 0.001) (Fig. 4E), pT2 ( $P=$ 0.022) (Fig. 4G), pT3 ( $P=$ 0.001) (Fig. 4H), non-lymph node metastasis ( $P<$ 0.001) (Fig. 4I), surgical margin-negative ( $P=$ 0.002) (Fig. 4K) and surgical margin-positive ( $P<$ 0.001) (Fig. 4L). No difference in DFS between circ-ITCH low expression patients and circ-ITCH high expression patients was observed in subgroups including: age $\leqslant$ 60 years ( $P=$ 0.244) (Fig. 4A), Gleason score $>$ 7 ( $P=$ 0.261) (Fig. 4F) or lymph node metastasis ( $P=$ 0.452) (Fig. 4J).

3.7 Comparison of OS between circ-ITCH high expression and low expression patients in subgroup analysis

For OS in subgroup analysis, better OS was observed in circ-ITCH high expression patients compared with low expression patients in subgroups including: age $>$ 60 years ( $P<$ 0.001) (Fig. 5B), PSA $\leqslant$ 10 ng/ml ( $P=$ 0.039) (Fig. 5C), PSA $>$ 10 ng/ml ( $P=$ 0.003) (Fig. 5D), Gleason score $\leqslant$ 7 ( $P<$ 0.001) (Fig. 5E), pT3 ( $P=$ 0.002) (Fig. 5H), non-lymph node metastasis ( $P=$ 0.006) (Fig. 5I), lymph node metastasis ( $P=$ 0.028) (Fig. 5J) and surgical margin-positive ( $P<$ 0.001) (Fig. 5L). No difference in OS between circ-ITCH low expression patients and circ-ITCH high expression patients was observed in subgroups including: age $\leqslant$ 60 years ( $P=$ 0.266) (Fig. 5A), Gleason score $>$ 7 ( $P=$ 0.226) (Fig. 5F), pT2 ( $P=$ 0.077) (Fig. 5G) or surgical margin negative ( $P=$ 0.059) (Fig. 5K).

Figure 5.

Difference in OS between circ-ITCH high expression and low expression patients in subgroup analysis. Better OS was observed in circ-ITCH high expression patients compared with low expression patients in subgroups including: age $>$ 60 years (B), PSA $\leqslant$ 10 ng/ml (C), PSA $>$ 10 ng/ml (D), Gleason score $\leqslant$ 7 (E), pT3 (H), non-lymph node metastasis (I), lymph node metastasis (J) and surgical margin-positive (L). No difference in OS between circ-ITCH low expression patients and circ-ITCH high expression patients was discovered in subgroups including: age $\leqslant$ 60 years (A), Gleason score $>$ 7 (F), pT2 (G) and surgical margin-negative (K). The difference in survival between patients with circ-ITCH low expression and high expression was illuminated with Kaplan-Meier curve and determined by log-rank test. $P<$ 0.05 was considered significant. OS, overall survival; circ-ITCH, circular RNA Itchy E3 ubiquitin protein ligase; PSA, prostate-specific antigen.

3.8 Factors affecting DFS in prostate cancer patients

From univariate Cox’s regression analysis, circ-ITCH (high vs. low) (HR $=$ 0.462, $P<$ 0.001) was correlated with longer DFS, whereas Gleason score ( $>$ 7 vs. $\leqslant$ 7) (HR $=$ 2.615, $P<$ 0.001), pathologic T stage (pT3 vs. pT2) (HR $=$ 2.293, $P<$ 0.001) and surgical margin status (positive vs. negative) (HR $=$ 1.907, $P=$ 0.001) were associated with shorter DFS (Table 3). Further multivariate Cox’s regression analysis revealed that circ-ITCH (high vs. low) (HR $=$ 0.476, $P<$ 0.001) independently predicted better DFS, while Gleason score ( $>$ 7 vs. $\leqslant$ 7) (HR $=$ 2.654, $P<$ 0.001), pathologic T stage (pT3 vs. pT2) (HR $=$ 2.277, $P<$ 0.001) and surgical margin status (positive vs. negative) (HR $=$ 2.022, $P<$ 0.001) were independent risk factors for worse DFS.

Table 3
Factors affecting DFS by Cox’s proportional hazards regression analysis

Items	Univariate Cox’s regression				Multivariate Cox’s regression
	$P$ value	HR	95% CI		$P$ value	HR	95% CI
			Lower	Higher			Lower	Higher
Circ-ITCH (high vs low)	$<$ 0.001	0.462	0.321	0.663	$<$ 0.001	0.476	0.329	0.690
Age ( $>$ 60 years vs $\leqslant$ 60 years)	0.160	0.781	0.554	1.102	0.057	0.713	0.503	1.011
PSA ( $>$ 10 ng/ml vs $\leqslant$ 10 ng/ml)	0.929	0.984	0.683	1.417	0.751	1.062	0.734	1.535
Gleason score ( $>$ 7 vs $\leqslant$ 7)	$<$ 0.001	2.615	1.760	3.886	$<$ 0.001	2.654	1.763	3.994
Pathologic T stage (pT3 vs pT2)	$<$ 0.001	2.293	1.620	3.246	$<$ 0.001	2.277	1.600	3.239
Lymph node metastasis (yes vs no)	0.162	1.329	0.892	1.981	0.206	1.302	0.865	1.960
Surgical margin status (positive vs negative)	0.001	1.907	1.322	2.752	$<$ 0.001	2.022	1.390	2.942

Factors affecting DFS were determined by univariate and multivariate Cox’s proportional hazards regression analyses. $P$ value $<$ 0.05 was considered significant (in bold). DFS, disease free survival; HR, hazard ratio; CI, confidence interval; PSA, prostate-specific antigen.

Table 4

Factors affecting OS by Cox’s proportional hazards regression analysis

Items	Univariate Cox’s regression				Multivariate Cox’s regression
	$P$ value	HR	95% CI		$P$ value	HR	95% CI
			Lower	Higher			Lower	Higher
Circ-ITCH (high vs low)	0.001	0.399	0.237	0.672	0.001	0.415	0.245	0.703
Age ( $>$ 60 years vs $\leqslant$ 60 years)	0.324	1.279	0.784	2.087	0.622	1.132	0.691	1.855
PSA ( $>$ 10 ng/ml vs $\leqslant$ 10 ng/ml)	0.993	1.002	0.605	1.660	0.557	1.164	0.701	1.934
Gleason score ( $>$ 7 vs $\leqslant$ 7)	$<$ 0.001	3.222	1.866	5.564	$<$ 0.001	3.146	1.782	5.553
Pathologic T stage (pT3 vs pT2)	0.057	1.594	0.986	2.578	0.074	1.562	0.958	2.548
Lymph node metastasis (yes vs no)	0.132	1.525	0.881	2.640	0.101	1.608	0.912	2.835
Surgical margin status (positive vs negative)	0.001	2.283	1.396	3.735	0.001	2.280	1.374	3.785

Factors affecting OS were determined by univariate and multivariate Cox’s proportional hazards regression analyses. $P$ value $<$ 0.05 was considered significant (in bold). OS, overall survival; HR, hazard ratio; CI, confidence interval; PSA, prostate-specific antigen.

3.9 Factors affecting OS in prostate cancer patients

Circ-ITCH (high vs. low) (HR $=$ 0.399, $P=$ 0.001) was associated with better OS, whereas Gleason score ( $>$ 7 vs. $\leqslant$ 7) (HR $=$ 3.222, $P<$ 0.001) and surgical margin status (positive vs. negative) (HR $=$ 2.283, $P=$ 0.001) were correlated with worse OS (Table 4). Multivariate Cox’s regression analysis further illustrated that circ-ITCH (high vs. low) (HR $=$ 0.415, $P=$ 0.001) independently predicted longer OS, whereas Gleason score ( $>$ 7 vs. $\leqslant$ 7) (HR $=$ 3.146, $P<$ 0.001) and surgical margin status (positive vs. negative) (HR $=$ 2.280, $P=$ 0.001) independently predicted shorter OS.

4. Discussion

In the present study, we observed that: (1) Circ-ITCH was downregulated in tumor tissues, and its high expression was correlated with lower pathological T stage as well as reduced lymph node metastasis risk. (2) Circ-ITCH expression was positively associated with survivals in prostate cancer patients underwent radical prostatectomy.

CircRNAs are non-coding RNAs arising from exons and introns of the genome, and they are evolutionary conserved, relatively stable as well as abundantly expressed in mammals [16, 15]. Since the discovery, circRNAs are believed to be versatile in post-transcriptional regulation of gene expression via RNA-RNA interaction, and several endogenous circ-RNAs has been shown to serve as a sign of malignant degeneration [17, 16]. Circ-ITCH is a typical circRNA that works as microRNA sponge to increase ubiquitin E3 ligase ITCH level, and it has been reported to suppress cell proliferation of various cancers via inhibiting the Wnt/ $\beta$ -Catenin pathway whose activation contributes to development and progression of many cancers [12, 15, 18]. For instance, circ-ITCH expression is lowered in esophageal squamous cell carcinoma tissues and it cooperates with Dvl2 to inhibit Wnt/beta-Catenin pathway, thereby arrests tumor progression [15]. In lung cancer, circ-ITCH suppresses cell proliferation by enhancing ITCH and inhibiting the subsequent Wnt/beta-Catenin pathway [12]. These evidences illuminate anti-oncogenic function of circ-ITCH in different human cancers through regulating cells activities and signaling pathways.

Beyond the capability as gene regulators, the correlation of circ-ITCH with tumor progression and severity in human cancers has been increasingly reported [19]. For example, circ-ITCH is downregulated in tumor tissues compared with normal adjacent tissues in papillary thyroid cancer, and overexpression of circ-ITCH impairs tumor growth, hence attenuates cancer severity in papillary thyroid cancer patients [20]. In addition, circ-ITCH is downregulated in colorectal cancer (CRC) and suppresses cancer progression in CRC patients [18]. These previous studies imply that circ-ITCH is downregulated in various cancers and suppress tumor progression, while information about circ-ITCH in prostate cancer development and progression is still limited. In the present study, we compared the expression of circ-ITCH between tumor tissue and paired adjacent tissue and we observed that circ-ITCH was downregulated in prostate tumor tissue with great value on distinguishing tumor tissues from paired adjacent tissues. Besides, circ-ITCH high expression was associated with lower pathological T stage as well as reduced lymph node metastasis risk in prostate cancer patients underwent radical prostatectomy. These might be due to that: (1) Circ-ITCH might work as anti-oncogene by sponging oncogenic microRNAs (e.g. miR-17, miR-224 and miR-145) to suppress oncogenes (e.g. P21 and PTEN) or elevate level of cancer-suppressor genes (e.g. ITCH and RASA1), therefore, circ-ITCH high expression was correlated with reduced pathological stage and lower risk of lymph node metastasis in prostate cancer patients [13, 18, 21]. (2) Circ-ITCH might inhibit cell proliferation, migration, invasion and metastasis through suppressing signaling pathways (e.g. Wnt/beta-Catenin signaling pathway) in prostate cancer, thereby attenuated tumor progression in prostate cancer patients.

In the meantime, the correlation of circ-ITCH expression with prognosis of cancer patients has been discovered. One previous study demonstrates that circ-ITCH exerts inhibitory effect on HCC and its high expression is associated with improved OS in hepatocellular carcinoma (HCC) patients [14]. Another study also discloses a positive association between circ-ITCH expression and OS in bladder cancer patients [13]. These studies present a positive correlation between circ-ITCH expression and survival profiles in cancer patients, whereas for prostate cancer, the prognostic value of circ-ITCH is still obscure. In accordance with the previous studies, our study revealed that circ-ITCH expression was independently correlated with better DFS and OS in prostate cancer patients underwent radical prostatectomy. The possible explanations were: (1) Circ-ITCH might suppress cancer cell proliferation, migration and invasion by inhibiting Wnt/beta-Catenin pathway, attenuating tumor progression and improving the prognosis in prostate cancer patients. (2) Circ-ITCH high expression was correlated with lower pathological stage and reduced lymph node metastasis as described above, therefore, it might predict better survival in prostate cancer patients. (3) Circ-ITCH might increase the sensitivity of cancer cells to the subsequent chemotherapies or radiotherapy after radical prostatectomy and result in better treatment responses, hence favoring the survival of prostate cancer patients. However, further validation was needed for this supposed explanation.

To our knowledge, this is the first study investigating the role of circ-ITCH in prostate cancer, whereas there still remained several limitations in our study. Firstly, tissue samples were used for detection of circ-ITCH whose collection was invasive, whereas for biomarker detection, it was critical that the sample was easy to access. Therefore, other sample sources, such as blood samples, that were non-invasive and easier to obtain could be evaluated in further studies. Secondly, the sample size was small with all patients originated from single hospital in China, resulting in weak statistical power and regional selection bias. Thirdly, GAPDH was the only one internal reference used in analysis of qPCR results, while more housekeeping genes could lead to more robust results. Finally, this single study identified a potential biomarker for prostate cancer, whereas we did not investigate the molecular mechanism of circ-ITCH in development of prostate cancer. Therefore, further experimental studies were needed for validation of our findings.

In conclusion, circ-ITCH is downregulated in prostate cancer tissues, and its low expression correlates with advanced pathologic T stage, high lymph mode metastasis risk and poor survivals in prostate cancer patients underwent radical prostatectomy.

Footnotes

Conflict of interest

None.

References

Bray

Ferlay

Soerjomataram

Siegel

R.L.

Torre

L.A.

and Jemal

, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J Clin 68 (2018), 394–424.

C. Global Burden of Disease Cancer Fitzmaurice

Akinyemiju

T.F.

Al Lami

F.H.

Alam

Alizadeh-Navaei

Allen

Alsharif

Alvis-Guzman

Amini

Anderson

B.O.

Aremu

Artaman

Asgedom

S.W.

Assadi

Atey

T.M.

Avila-Burgos

Awasthi

Ba Saleem

H.O.

Barac

Bennett

J.R.

Bensenor

I.M.

Bhakta

Brenner

Cahuana-Hurtado

Castaneda-Orjuela

C.A.

Catala-Lopez

Choi

J.J.

Christopher

D.J.

Chung

S.C.

Curado

M.P.

Dandona

das Neves

Dey

Dharmaratne

S.D.

Doku

D.T.

Driscoll

T.R.

Dubey

Ebrahimi

Edessa

El-Khatib

Endries

A.Y.

Fischer

Force

L.M.

Foreman

K.J.

Gebrehiwot

S.W.

Gopalani

S.V.

Grosso

Gupta

Gyawali

Hamadeh

R.R.

Hamidi

Harvey

Hassen

H.Y.

Hay

R.J.

Hay

S.I.

Heibati

Hiluf

M.K.

Horita

Hosgood

H.D.

Ilesanmi

O.S.

Innos

Islami

Jakovljevic

M.B.

Johnson

S.C.

Jonas

J.B.

Kasaeian

Kassa

T.D.

Khader

Y.S.

Khan

E.A.

Khan

Khang

Y.H.

Khosravi

M.H.

Khubchandani

Kopec

J.A.

Kumar

G.A.

Kutz

Lad

D.P.

Lafranconi

Lan

Legesse

Leigh

Linn

Lunevicius

Majeed

Malekzadeh

Malta

D.C.

Mantovani

L.G.

McMahon

B.J.

Meier

Melaku

Y.A.

Melku

Memiah

Mendoza

Meretoja

T.J.

Mezgebe

H.B.

Miller

T.R.

Mohammed

Mokdad

A.H.

Moosazadeh

Moraga

Mousavi

S.M.

Nangia

Nguyen

C.T.

Nong

V.M.

Ogbo

F.A.

Olagunju

A.T.

Park

E.K.

Patel

Pereira

D.M.

Pishgar

Postma

M.J.

Pourmalek

Qorbani

Rafay

Rawaf

D.L.

Roshandel

Safiri

Salimzadeh

Sanabria

J.R.

Santric Milicevic

M.M.

Sartorius

Satpathy

Sepanlou

S.G.

Shackelford

K.A.

Shaikh

M.A.

Sharif-Alhoseini

She

Shin

M.J.

Shiue

Shrime

M.G.

Sinke

A.H.

Sisay

Sligar

Sufiyan

M.B.

Sykes

B.L.

Tabares-Seisdedos

Tessema

G.A.

Topor-Madry

Tran

T.T.

Tran

B.X.

Ukwaja

K.N.

Vlassov

V.V.

Vollset

S.E.

Weiderpass

Williams

H.C.

Yimer

N.B.

Yonemoto

Younis

M.Z.

Murray

C.J.L.

and Naghavi

, Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2016: A Systematic Analysis for the Global Burden of Disease Study, JAMA Oncol 4 (2018), 1553–1568.

Chistiakov

D.A.

Myasoedova

V.A.

Grechko

A.V.

Melnichenko

A.A.

and Orekhov

A.N.

, New biomarkers for diagnosis and prognosis of localized prostate cancer, Semin Cancer Biol 52 (2018), 9–16.

Wise

H.M.

Hermida

M.A.

and Leslie

N.R.

, Prostate cancer, PI3K, PTEN and prognosis, Clin Sci (Lond) 131 (2017), 197–210.

Sardana

Dowell

and Diamandis

E.P.

, Emerging biomarkers for the diagnosis and prognosis of prostate cancer, Clin Chem 54 (2008), 1951–1960.

S.M.

H.L.

Tang

Wang

S.G.

Z.Q.

and Hu

, The putative tumour suppressor miR-1-3p modulates prostate cancer cell aggressiveness by repressing E2F5 and PFTK1, J Exp Clin Cancer Res 37 (2018), 219.

Bond

D.R.

Naudin

Carroll

A.P.

Goldie

B.J.

Brzozowski

J.S.

Jankowski

H.M.

Cairns

M.J.

Ashman

L.K.

Scarlett

C.J.

and Weidenhofer

, miR-518f-5p decreases tetraspanin CD9 protein levels and differentially affects non-tumourigenic prostate and prostate cancer cell migration and adhesion, Oncotarget 9 (2018), 1980–1991.

Pellegrini

K.L.

Sanda

M.G.

and Moreno

C.S.

, RNA biomarkers to facilitate the identification of aggressive prostate cancer, Mol Aspects Med 45 (2015), 37–46.

Wang

Gong

Yang

Zhang

Xiong

Xiang

Zhou

Liao

Zhang

Zeng

and Xiong

, Circular RNAs in human cancer, Mol Cancer 16 (2017), 25.

10.

Zhong

Yang

Fan

Xiong

Ren

Wang

Wei

Guo

Zeng

and Xiong

, Circular RNAs function as ceRNAs to regulate and control human cancer progression, Mol Cancer 17 (2018), 79.

11.

Kong

Wan

Zhang

Huang

and Li

, Androgen-responsive circular RNA circSMARCA5 is up-regulated and promotes cell proliferation in prostate cancer, Biochem Biophys Res Commun 493 (2017), 1217–1223.

12.

Wan

Zhang

Fan

Cheng

Z.X.

Sun

Q.C.

and Wang

J.J.

, Circular RNA-ITCH suppresses lung cancer proliferation via inhibiting the Wnt/beta-catenin pathway, Biomed Res Int 2016 (2016), 1579490.

13.

Yang

Yuan

Yang

Wang

Han

Tao

Yang

and Zhang

, Circular RNA circ-ITCH inhibits bladder cancer progression by sponging miR-17/miR-224 and regulating p21, PTEN expression, Mol Cancer 17 (2018), 19.

14.

Guo

Zhang

Cao

Zhang

Wang

Wen

Yang

Shi

Pan

and Ye

, Polymorphisms and expression pattern of circular RNA circ-ITCH contributes to the carcinogenesis of hepatocellular carcinoma, Oncotarget 8 (2017), 48169–48177.

15.

Zhang

Deng

Zheng

and Zhou

, Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/beta-catenin pathway, Oncotarget 6 (2015), 6001–6013.

16.

Memczak

Jens

Elefsinioti

Torti

Krueger

Rybak

Maier

Mackowiak

S.D.

Gregersen

L.H.

Munschauer

Loewer

Ziebold

Landthaler

Kocks

le Noble

and Rajewsky

, Circular RNAs are a large class of animal RNAs with regulatory potency, Nature 495 (2013), 333–338.

17.

Zhao

Z.J.

and Shen

, Circular RNA participates in the carcinogenesis and the malignant behavior of cancer, RNA Biol 14 (2017), 514–521.

18.

Huang

Zhu

Shi

Cai

and Chen

, cir-ITCH plays an inhibitory role in colorectal cancer by regulating the Wnt/beta-catenin pathway, PLoS One 10 (2015), e0131225.

19.

Hansen

T.B.

Jensen

T.I.

Clausen

B.H.

Bramsen

J.B.

Finsen

Damgaard

C.K.

and Kjems

, Natural RNA circles function as efficient microRNA sponges, Nature 495 (2013), 384–388.

20.

Wang

Chen

and Cong

, CircRNA circ-ITCH suppresses papillary thyroid cancer progression through miR-22-3p/CBL/beta-catenin pathway, Biochem Biophys Res Commun 504 (2018), 283–288.

21.

Wang

Chen

Gao

Zhao

Huang

Jiang

Zhou

and Chen

, The circular RNA circ-ITCH suppresses ovarian carcinoma progression through targeting miR-145/RASA1 signaling, Biochem Biophys Res Commun 505 (2018), 222–228.

Downregulated circular RNA itchy E3 ubiquitin protein ligase correlates with advanced pathologic T stage,high lymph node metastasis risk and poor survivals in prostate cancer patients

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Methods

2.1 Patients

2.2 Data collection

2.3 Detection of circ-ITCH in tumor and paired adjacent tissue

2.4 Statistical analysis

3.1 Study flow

Table 1 Baseline characteristics of patients with prostate cancer

3.3 Circ-ITCH expression in tumor tissue and paired adjacent tissue

Table 2 Correlation of circ-ITCH relative expression with patients’ characteristics

3.5 Correlation of circ-ITCH expression with patients’ survival

3.6 Comparison of DFS between circ-ITCH high expression and low expression patients in subgroup analysis

3.7 Comparison of OS between circ-ITCH high expression and low expression patients in subgroup analysis

Table 3 Factors affecting DFS by Cox’s proportional hazards regression analysis

4. Discussion

Footnotes

Conflict of interest

References

Table 1
Baseline characteristics of patients with prostate cancer

Table 2
Correlation of circ-ITCH relative expression with patients’ characteristics

Table 3
Factors affecting DFS by Cox’s proportional hazards regression analysis