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Abstract

BACKGROUND:

MicroRNAs (miRNAs) are emerging as promising molecules in the diagnosis, prognosis and treatment of urological tumours. Recently, our group performed two independent studies highlighting that miR-210-3p may be a useful biomarker not only for diagnosis but also for post-surgery clear cell Renal Cell Carcinoma (ccRCC) management.

OBJECTIVE:

The aim of this study is to further explore the effectiveness of miRNA as non-invasive biomarker for clinical outcomes and ccRCC response to the treatment.

METHODS:

We analyzed miR-210-3p levels in neoplastic and healthy tissue and in urine specimens collected at surgery and during follow-up of 21 ccRCC patients by RTqPCR.

RESULTS:

Firstly, we confirmed that the expression of miR-210-3p was upregulated in tumor tissues and in urine samples of analyzed cohort. Of note is that miR-210-3p expression was significantly reduced in urine samples from disease-free patients during follow-up (from 3 to 12 months) compared to the baseline levels observed at the time of surgery. In a small subgroup of patients presenting metastatic progression (such as bone, intestinal or lung metastasis), the urine levels of miR-210-3p correlated with responsiveness to the therapy.

CONCLUSIONS:

This pilot study highlights the relevance of secreted miR-210-3p as powerful non-invasive prognostic and predictive biomarker for the evaluation of clinical outcomes and treatment response during ccRCC follow up.

Keywords

microRNA miRNA ccRCC Cancer biomarkers Liquid biopsy metastasis m miR-210

1. Background

Renal cell carcinoma (RCC) represents the tenth most common cancer worldwide [1, 2]. Probability of occurrence is particularly high in men with excessive body weight. Apart from that smoking, hypertension, environmental pollutants, as well as genetic predisposition (e.g. mutation in VHL gene in von-Hippel-Lindau disease), are described as the main risk factors associated to this type of cancer. According to the 2016 WHO classification, RCC can be divided into three major clinical subtypes: the chromophobe RCC (chRCC), the papillary RCC (pRCC) and clear cell RCC (ccRCC). The latest represents the most frequent and aggressive phenotype, with a 30% of cases that developes distant metastases. ccRCC is also the most common cause of death among all kidney cancer patiens [1, 2]. To date, no useful markers are available in clinical practice not only for early diagnosis but also for better stratification and management of high-risk patients [3]. Identifying novel low-expensive and non-invasive tools for a more accurate definition of prognosis and response to therapy would improve patient clinical outcomes and quality of life therefore this is one of the major challenges in oncology research.

Among these novel tools, microRNAs (miRNAs) are emerging as one of the most relevant. MiRNAs are a class of small non-coding RNA of which expression is tissue-specific and highly regulated according to the cell line and its developmental stage [4]. Studies indicate that miRNAs are heterogenously expressed in cancer cells, in which they form unique expression patterns or signatures [5, 6]. Of note, specific miRNA-expression signatures are identified in correlation with tumor classification and are also emerging as predictive markers of metastasis [7, 8]. These small non-coding RNAs, to exercise their function, are assembled in the functional RNA induced silencing complexes (RISCs), where a member of Argonaute (Ago) family of proteins, Ago1-4, provides an unique platform for target recognition and gene silencing [9, 10, 11]. The miRNAs loaded in the RISC complex usually function as negative regulators of gene expression, mainly by targeting the 3’ untranslated region (UTR) of their target mRNAs [12].

MiRNAs and their protein complexes modulate a variety of developmental and physio-pathological processes by fine-tuning translation of specific target mRNAs [13].

MiRNAs are secreted by cells as free molecules and are bounded in a ribonucleoprotein complexes or in vesicles. Discovery of circulating miRNAs in biological fluids as blood plasma, serum and urine suggests the possibility of using this class of non-coding RNAs as non-invasive biomarkers for an early cancer diagnosis. Potentially, they could also serve as prognostic factors and predictors of response to the therapy [14, 15].

These miRNA secretion pathways are dysregulated in cancer, making miRNAs attractive candidate mole-cules for liquid biopsies in several types of cancer [16] and in particular in urologic tumors [17, 18]. Of note, several studies demonstrated that circulating miRNAs can be introduced to the cell by endocytosis or membrane fusion and affect recipient cell gene expression [19].

Recently, our group performed a retrospective cohort study of tissue samples obtained from patients subjected to partial or radical nephrectomy for ccRCC and evaluated the expression of miR-21-5p, miR-210-3p, miR185-5p and miR-221-3p either in FFPE neoplastic tissue and in paired normal parenchyma [20]. miR-21-5p and miR-210-3p resulted significantly up-regulated in ccRCC vs matched normal tissues.

To further support the potential clinical usefulness of these miRNAs in ccRCC management we started a prospective study [21] where we evidenced that miR-21-5p, miR-210-3p and miR-221-3p were up-regulated in fresh frozen ccRCC tissues vs matched healty tissues. Moreover, by using two independent cohorts of patients we evidenced that miR-210-3p was significantly up-regulated in urine specimens collected from ccRCC patients at the time of surgery compared to samples from healthy donors [21].

In this study, to further assess the robustness of miR-210-3p as a diagnostic and prognostic biomarker, we analyzed its levels in tissues and urine specimens collected at surgery and during follow-up visits (from 3 to 12 months) from 21 ccRCC disease-free patients and in a small subgroup of patients presenting metastatic progression.

2. Methods

2.1 Patients and urine samples collection

This study includes a cohort of ccRCC patients (totally 21 patients) who underwent surgical resection between September 2016 and March 2017 (as described in Table 1) at Regina Elena National Cancer Institute of Rome. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Regina Elena National Cancer Institute (Project identification code: CI IRE 19.11.2012 and 431-13.6.2017).

Table 1
Clinical characteristics of patients with ccRCC

#	Gender/ age	Weight (kg) and height (cm)	Smoking history	Serum creatinin (mg/dl)	Histology	Tumor size (cm) and site	Tumor grade	Tumor stage	Tumor metast.	Follow-up 1-3/ 6/12 (months)
1	M/64	99/180	current	1,3	ccRCC	6 (R)	G3	T3a	M0	1-3/6/12
2	M/75	73/160	former	1,21	ccRCC	3 (R)	G3	T1a	M0	1-3/6
3	F/50	62/165	former	1,2	ccRCC	3,5 (R)	G2	T1a	M0	1-3/6
4	M/52	85/185	former	1,19	ccRCC	8 (L)	G3	T2a	M1 (Bone)	1-3/6
5	M/62	92/165	former	1,32	ccRCC	7 (R)	G3	T3b	M1 (Intes.)	1-3/24
6	M/43	n/a	current	1,36	ccRCC	7,5 (L)	G3	T3a	M0	6/12
7	M/75	84/183	never	1,1	ccRCC	5,4 (L)	G3	T3a	M0	1-3/6/12
8	F/78	78/165	never	1,1	ccRCC	6 (L)	G4	T1b	M0	1-3/6/12
9	M/46	80/175	never	1,26	ccRCC	11 (L)	G2	T3a	M1(Lung)	1-3/6/12
10	M/74	90/175	never	1,4	ccRCC	4 (L)	G3	T1a	M0	1-3
11	M/63	69/164	never	1,18	ccRCC	7,2 (R)	G2	T2a	M0	1-3/6
12	M/46	69/170	current	1	ccRCC	2,5 (R)	G2	T1a	M0	1-3/6/12
13	F/63	80/160	never	0,88	ccRCC	5,5 (R)	G2	T1b	M0	1-3/6/12
14	F/83	65/155	never	0,66	ccRCC	3,5 (R)	G2	T1a	M0	1-3/6/12
15	F/69	80/160	former	0,89	ccRCC	5 (L)	G2	T1b	M0	6/12
16	F/65	n/a	n/a	1	ccRCC	3 (L)	G2	T1a	M0	6
17	F/50	n/a	n/a	1,1	ccRCC	6 (L)	G2	T1b	M0	6
18	F/54	n/a	n/a	1,04	ccRCC	9 (R)	G3	T2a	M0	1-3/6
19	M/46	n/a	n/a	1,16	ccRCC	4,2 (L)	G2	T1b	M0	6
20	M/73	n/a	n/a	1	ccRCC	2,5 (R)	G3	T1a	M0	1-3/6/12
21	M/74	n/a	n/a	1,08	ccRCC	2,6 (R)	G3	T1a	M0	1-3/6/12

F: Female; M: Male; R: right; L: left: Bone: Bone matastasis; Intes: Intestinal metastasis; Lung: lung metastasis.

Neoplastic and healthy kidney tissues samples from 13 patients were included. Additional 8 neoplastic samples were considered. Urine specimens were collected at the time of surgery from all the 21 patients and during follow-up when available (from 3 to 12 months, as reported in Table 1). Urine samples were frozen within 30 minutes from collection and stored at $-$ 80 ${}^{\circ}$ C until RNA extraction.

2.2 RNA extraction and microRNA expression analysis

Fresh Frozen samples were homogenized by gentle dissociator (Miltenyi Biotec) in 700 $\mu$ l of Qiazol (Qiagen, Chatsworth, CA, USA) and RNA was extracted following the manufacturer’s protocol. The concentration and purity of total RNA were assessed using a Nanodrop TM 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). A quantity of 150 ng of total RNA was reverse transcribed in 10 $\mu$ l using miScript II RT kit (Qiagen, Chatsworth) and 1 $\mu$ l of cDNA dilution (1:5) was used for quantitative Real time PCR (RT-qPCR) experiments. All reactions were performed in triplicates. PCR quantification analysis of the SNORD68 and miR-210-3p was performed using the miScript SYBR Green PCR kit (Qiagen, Chatsworth) with the miScript Primer Assay Hs-SNORD68 (#MS00033712) and Hs-miR-210-3p (#MS00003801) (Qiagen, Chatsworth, CA, USA).

As normalizer we chose the small nucleolar RNA SNORD68 (miScript Primer Assay Hs-SNORD68 # MS00033712) for its low variability expression between fresh tissue samples. To quantify miRNA expr-ession in RT-qPCR, we used a standard curve method; standard curve was performed with serial dilutions of a reference cDNA, obtained from RNA extracted from a tumor sample, to quantify miR-210-3p and SNORD68. Ratio of miR-210-3p over SNORD68 was then used to present the relative expression data.

Total RNA from 200 $\mu$ l of urine samples was extracted using miRNAeasy serum/plasma kit (Qiagen, Chatsworth, CA, USA) following the manufacturer’s instructions. The concentration purity and quality of total RNA were assessed using a Nanodrop TM 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). A quantity of 25 ng of total RNA was reverse transcribed in 10 $\mu$ l using miScript II RT kit (Qiagen, Chatsworth) and 1 $\mu$ l of cDNA dilution (1:5) was used for quantitative PCR (qPCR). qPCR for miR-210-3p was carried out by miScript Primer Assay (Qiagen, Chatsworth, CA, USA), using the miScript SYBR Green PCR kit (Qiagen, Chatsworth, CA, USA).

A Spike-in Control (#219610) (Qiagen, Chatsworth, CA, USA), a synthetic miRNA, C. elegans miR-39 miRNA mimic, to monitor RNA recovery and reverse transcription efficiency. 1.6 $\times$ 10 ${}^{8}$ copies/ul of Spike-in were added to each sample during extraction prior to phase separation by the addition of chloroform. Quantification of miR-210-3p levels was evaluated over the Spike-in Control through $\Delta\Delta$ CT method.

All reactions were performed in triplicates. microRNA expression was evaluated on urine samples from the ccRCC cohort and compared to urine samples from healthy donors.

2.3 Statistical analysis

All statistical analyses were performed using the GraphPad Prism 6.0 software (GraphPad Software, Inc., USA). The $P$ values for tissues and urine samples were calculated by using: i) Paired parametric T-test for matched samples; ii) Unpaired parametric T-test for unmatched samples.

3. Results

By using a cohort of fresh frozen renal tissues obtained from 21 ccRCC patients subjected to surgical resection we evaluated the expression level of miR-210-3p. The clinical and demographic details of ccRCC patients and tumor specimens included in this study are summarized in Table 1.

In particular, thirteen patients were males (62%) and 8 patients were females (38%) with a median age of 63 years old (range 43–83) and a median Serum Creatinin concentration of 1.1 mg/dl (range 0.66–1.36). All the cases presented a clear cell histotype of RCC at the histological examination and according to ISUP grade classification, 10 cases (47.6%) were G2 grade, 10 cases (47.6%) were G3 grade and only 1 case (4.7%) was G4 grade. Information about smoking habit, as a main risk factor, was available for 15 patients; among these, 3 patients (20%) were cigarette smokers, 5 patients (33.3%) have a history of smoking and 7 patients (46.6%) were non-smokers (Table 1). The surgery procedures performed as choice treatment for these patients were: Robotic Radical Right Nephrectomy in 1 cases (4.7%), Robotic Partial Right or Left Nephrectomy in 14 cases (66.6%), Open Radical Left Nephrectomy in only 1 case (4.7%) and Laparoscopic Right or Left Nephrectomy in 5 cases (23.8%). All the patients included in the study did not receive any neo-adjuvant therapy before surgery.

Urine specimens were collected at the time of surgery from all the 21 patients and subsequently during follow-up (from 3 to 12 months, as reported in Table 1). Urine samples were also collected from 16 healthy donors with characteristics comparable to the ccRCC patients included in the study (median age: 59.6; males: 50% and females: 50%).

Figure 1.

miR-210-3p levels in tissue and urine specimens collected at surgery and during follow-up of ccRCC patients. (A) Box plot showing the modulation of miR-210-3p in a cohort of 21 fresh frozen tissues from ccRCC patients. A total of 13 matched ccRCC tumor (T) and adjacent healthy tissue (N) samples, and additional 8 tumor samples were analyzed by RT-qPCR. The expression value of miR-210-3p was normalized over the SNORD68 expression. $P$ value is referred to the matched samples of the cohort and was calculated by Paired parametric T-test (as reported in the Supplementary Fig. 1A) and a value of $P\leqslant$ 0.05(*) was considered statistically significant. (B) Box plot showing the expression level of miR-210-3p in urine collected from 18 patients at the time of surgery (T), during follow-up (3, 6,12 months of time from surgery) and from 16 healthy donors (N), evaluated by RT-qPCR and normalized over a Spike-In Control. $P$ value was calculated by Unpaired parametric T-test for the N vs T samples or Paired parametric T-test for the follow-up matched samples (as reported in the Supplementary Fig. 1B and C), and a value of $P\leqslant$ 0.05(*) was considered statistically significant. (C) Box plot showing the expression level of miR-210-3p in urine collected from 3 metastatic patients at the time of surgery and during follow-up, as indicated (months from surgery). The arrows specify the time of therapy.

Three patients developed metastasis (to the lungs, bones or intestines). The patient with bilateral lungs metastasis was treated with Sunitinib (Sutent ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ ) at the time indicated by the arrows in the Fig. 1 and the urine samples were collected 15 days after each treatment. Between the first and the second treatment a pulmonary invasion and progression was proved. The patient with vertebral sacral bone metastasis did not receive any treatment and he was waiting for radiotherapy. The patient with intestinal metastasis in the peritoneal and lateroconal right band was treated with Sunitinib and Pazopanib (Votrient ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ ) at the time indicated by the arrows in the Fig. 1 and the urine samples were collected 15 days after treatments. After 4 months, the treatment was interrupted for severe side effects. A urine sample was also collected at 24 months from the surgery when an increase of the omental nodule was evidenced and a second-line of adjuvant therapy had not been administered yet.

In agreement with the previous studies [20, 21], we evidenced that miR-210-3p is up-regulated in ccRCC vs adjacent healthy tissue (Fig. 1A and Supplementary Fig. 1A).

To further evaluate the relevance of miR-210-3p as novel low-expensive and non-invasive clinical biomar-ker for ccRCC, we analyzed miR-210-3p expression in 21 urine specimens collected at the time of surgery and during follow-up (1–3, 6 and 12 months of time from surgery) from the same cohort of ccRCC patients (18 disease-free and 3 in metastatic progression); urine specimens from healthy donors with similar characteristics were analyzed as well.

Of note, miR-210-3p was significantly upregulated in urine specimens collected from ccRCC patients at the time of surgery, compared to healthy donor samples (Fig. 1B and and Supplementary Fig. 1B). Importantly, miR-210-3p urinary level was significantly reduced during follow-up in disease-free ccRCC patients of the same cohort, for which follow-up urine samples were available (Fig. 1B and and Supplementary Fig. 1C).

Of note, in a small subgroup of patients presenting metastatic progression to the lungs, bones or intestines, the urine levels of miR-210-3p were down regulated after the treatment (pts #9 and #5) or after surgical resection (pt #4) in comparison to their basaline levels (time of surgert). Patient #4 who was waiting for radiotherapy presented a recovery of high miR-210-3p urine levels (Fig. 1C). However, when a small increase of the omental nodule was evidenced after 24 months from surgery in patient #5 (with intestinal metastasis), the expression levels of miR-210-3p did not correlate with the relapse of the disease.

4. Discussion

In this study, we evidenced that at the time of diagnosis the miR-210-3p is up-regulated not only in ccRCC vs matched healthy tissues but also in urine specimens from ccRCC patients compared to healthy donors.

The up-regulation of miR-210-3p in ccRCC tissue was also evidenced by several studies described in the literature [22, 23, 24, 25]. Our study supports and confirms the relevance of this miRNA as molecular diagnostic biomarker for ccRCC evaluation and classification. Moreover, different studies evidenced also the correlation of miR-210-3p expression and different clinical features. In particular, a significant inverse correlation between miR-210 expression and survival in ccRCC tumours was found by Neal et al. [26]. Interestingly, Samaan et al. evidenced that patients with a higher level of miR-210 expression have significantly shorter overall survival than patients with lower miR-210 expression and that miR-210 expression is independent from pathologic stage and tumor size [27]. However, surprisingly, McCormick et al. [22] evidenced the association of miR-210 with good clinico-pathological parameters in ccRCC in terms of a better prognosis and lower grade/stage of tumours.

Our study suggests that the urine level of miR-210-3p was significantly reduced during follow-up samples highlighting this onco-miRNA as a potential biomarker useful not only for diagnosis but also to assess the complete resection and disease-free survival in ccRCC patients. Moreover, in metastatic patients miR-210-3p might represent a useful tool for the evaluation of the treatment response during ccRCC follow-up. Interestingly, these results further support the relevance of miRNAs as potential prognostic tool for urological tumours [15, 17] and the detection of the urine levels of miR-210-3p as biofluid marker for ccRCC management [18, 21].

Dysregulation of miR-210 expression have great relevance in ccRCC pathogenesis. Indeed, this miRNA is not only an hypoximiR, but is also involved in multiple molecular pathways related to different metabolic processes, including autophagy and mitochondrial respiration, linking hypoxia to metabolic reprograming [28]. In this scenario, it is emerging that ccRCC is characterized by reprogramming of metabolism. In particular, the metabolic flux through glycolysis is partitioned [29], and mitochondrial bioenergetics and oxidative phosphorylation are impaired [30]. To date, a several number of metabolic pathways are under investigation, to identify potential therapeutic targets and relevant tools for diagnostic and prognostic biomarkers of ccRCC, including for example glutathione metabolism [31]. Moreover, ccRCC is a tumor highly vascularized and the pathways of VHL/hypoxia – VEGFR in this type of neoplasms are well studied and targeted by actual molecular treatments with monoclonal antibodies such as bevacizumab, or tyrosine kinase inhibitors such as sunitinib or sorafenib [32].

In this context, the characterization of miRNAs contribution to ccRCC establishment and progression could be relevant for the improvement of the clinical management of this tumor.

5. Conclusions

In conclusion, this study highlights miR-210-3p, which is altered in ccRCC tissues and urine specimens, as non-invasive biomarker for ccRCC management but also suggests this miRNA as an emerging biomarker in the monitoring of tumor progression. Further investigation including larger cohorts of patients will allow to establish the associations existing between miR-210-3p levels and insurgence of metastasis in ccRCC patients.

Footnotes

Acknowledgments

Contribution of Sapienza University of Rome was greatly appreciated.

List of abbreviations

Supplementary data

Supplementary Fig. 1.

(A) Box plot showing the modulation of miR-210-3p in fresh frozen tissues from 13 matched samples of the cohort reported in Fig. 1A. $P$ value was calculated by Paired parametric T-test and a value of $P\leqslant$ 0.05(*) was considered statistically significant. (B) Box plot showing the expression level of miR-210-3p in urine collected from 18 patients at the time of surgery (T) and from 16 healthy donor samples (N) of the cohort reported in Fig. 1B (left panel). $P$ value was calculated by Unpaired parametric T-test and a value of $P\leqslant$ 0.05(*) was considered statistically significant. (C) Box plot showing the expression level of miR-210-3p in matched urine samples from patients of the cohort reported in Fig. 1B at the time of surgery (T) and during follow-up (3, 6, 12 months of time from surgery). $P$ value was calculated by Paired parametric T-test and a value of $P\leqslant$ 0.05(*) was considered statistically significant.

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Emerging role of secreted miR-210-3p as potential biomarker for clear cell Renal Cell Carcinoma metastasis

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Background

2. Methods

2.1 Patients and urine samples collection

Table 1 Clinical characteristics of patients with ccRCC

2.3 Statistical analysis

3. Results

5. Conclusions

Footnotes

Acknowledgments

List of abbreviations

Supplementary data

References

Table 1
Clinical characteristics of patients with ccRCC