Oncogenic MicroRNAs Characterization in Clear Cell Renal Cell Carcinoma

Antonio Luigi Pastore1, Giovanni Palleschi1, Domenico Autieri1, Yazan Al Salhi1, Samer Al Rawashdah1, Andrea Ripoli1, Antonino Leto1, Andrea Fuschi1, Gennaro Velotti1, Francesco Fazi2, Vincenzo Petrozza3, Antonio Carbone1
  • 1 "Sapienza" Università di Roma, Facoltà di Medicina e Farmacia, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, U.O. Urologia (Latina)
  • 2 "Sapienza" Università di Roma, Facoltà di Medicina e Farmacia, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, U.O. Patologia (Rome)
  • 3 "Sapienza" Università di Roma, Facoltà di Medicina e Farmacia, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, U.O. Patologia (Latina)

Objective

A key challenge for the improvement of ccRCC management could derive from a deeper molecular characterization of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. In several tumors, miRNAs expression profile is emerging as a relevant marker for diagnosis, prognosis and treatment of cancer . miRNAs are 22 nucleotides-long double strand small RNAs, typically excised from 60 to 110 nucleotide RNA precursor structures, which modulate gene expression generally at post-trascriptional level [6]. In fact, miRNAs show a developmental stage- and tissue-specific expression pattern and are present in complex regulatory circuits to regulate stem cells function, tissue differentiation and maintenance of cell identity during embryogenesis and adult life [7]. Notably, miRNA activity has also been correlated to the pathogenesis of cancer, since miRNAs have also been recently identified as a new class of genes with tumor-suppressor and oncogenic functions. To date, a molecular characterization of ccRCC is under investigation and several high-throughput analyses have been recently performed in order to identify miRNAs putatively involved in ccRCC tumorigenesis and progression. By using a retrospective cohort of 20 formalin-fixed paraffin-embedded (FFPE) tissue samples, we evaluated the levels of specific miRNAs differentially expressed in ccRCC vs. matched normal tissues. We evidenced miR-21-5p and miR-210-3p as the most significantly up-regulated in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC carcinogenesis.

Materials and Methods

This study was conducted on a retrospective cohort of ccRCC formalin-fixed paraffin-embedded (FFPE) tissue samples from 20 patients who underwent surgery between October 2011 and November 2013. For all the patients, FFPE-matched normal peritumoral kidney tissues were also considered. The patients were not treated with any neo-adjuvant therapy before surgery. Five patients were female (25%) and 15 patients were male (75%) with a mean age of 68.9 years old and a mean Body Mass Index (BMI) of 27.4 kg/m2. All the cases presented a clear cell histotype of RCC at the histological examination. The surgery procedures performed as treatments for these patients were: (i) open radical nephrectomy in 4 cases (20%); (ii) laparoscopic radical nephrectomy in 12 cases (60%); (iii) laparoscopic partial nephrectomy in 4 cases (20%). According to the tumor, node, and metastasis (TNM) classification, 10 patients have been identified as Stage I (50%), 5 patients have been identified as Stage II (25%), and 5 patients as Stage III (25%). Fuhrman’s grade has also been evaluated with 15% of cases belonging to the G1 grade (3 patients), 50% of cases belonging to G2 (10 patients) and 30% of cases to the G3 grade (6 patients). Only 1 patient actually showed a G2/3 grade. PCR quantification analysis of the SCARNA17 and miRNAs miR-21-5p, miR-210-3p, miR-185-5p, miR-221-3p and miR-145-5p, was performed using the miScript SYBR Green PCR kit (Qiagen, Chatsworth) with the miScript Primer Assay Hs-SCARNA17 (#MS00014014), Hs-miR-21-5p (#MS00009079), Hs-miR-210-3p (#MS00003801), Hs-miR-185-5p (#MS00003647), Hs-miR-221-3p (#MS00003857), Hs-miR-145-5p (#MS00003528) (Qiagen, Chatsworth, CA, USA).
The expression analyses of RNU19 and RNU66 were performed by TaqMan MicroRNA RT assay and TaqMan MiRNA® Assays according to the manufacturer’s protocol.
The p value was calculated by using a non-parametric Wilcoxon test with paired data and miRNAs whose differential expression was statistically significant (< 0.01) was indicated.

Results

Among the miRNAs deregulated in several human cancers, we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) to evaluate their expression in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues obtained from 20 ccRCC patients undergoing surgical nephrectomy resection. The characteristics of ccRCC patients and tumor specimens are reported in the Patients and Methods section and summarized in Table 1. A total of 20 matched ccRCC and adjacent normal tissue samples were collected. Interestingly, miR-21-5p and miR-210-3p resulted significantly up-regulated in ccRCC vs. normal tissues, with a p value of 0.0083 and 0.0010, respectively (Figure 1). miR-185-5p and miR-221-3p, although did not show any statistically significant modulation between tumor and normal tissues, show a trend of expression similar to miR-21-5p and miR-210-3p (Figure 1). Moreover, we analyzed miR-145-5p expression that usually results particularly down-regulated in several tumor samples compared to normal tissues. We evidenced that miR-145-5p did not show any statistically significant modulation between tumor and normal tissues.

Discussions

In this study we observed that specific miRNAs, previously reported as up-regulated in ccRCC vs. autologous normal tissues, also show increased expression levels in our series of 20 FFPE tumor samples relatively to their matched normal counterparts. Specifically, among the up-regulated miRNAs, we confirmed increased levels of miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p. miR-21-5p and miR-210-3p resulted significantly up-regulated in this patient cohort highlighting these onco-miRNAs as relevant players involved in ccRCC tumorigenesis.
Interestingly, the increased expression of miR-21, miR-210, miR-185, miR-221 was previously reported in ccRCC patients and their contribution to ccRCC tumorigenesis is currently under investigation. miR-221 was significantly increased in ccRCC tissues and cell lines, while its knock-down inhibited cell proliferation, migration and invasion of renal cancer cells [25]. miR-210 was significantly overexpressed in ccRCC relatively to normal kidney and patients with high levels of miR-210 show a statistically higher incidence of disease recurrence . Moreover, the down-regulation of miR-210 also reduced the migratory and invasive potential of metastatic RCC cells. Using ccRCC and matched normal kidney samples, it was also evidenced that the increased levels of miR-185 and miR-21 in tumors correlate with the loss of function of specific tumor suppressors such as PTPN13, SLC12A1 and TCF21. Noteworthy is that miR-21 not only shows up-regulated expression in tumor tissues but also its serum levels resulted to be significantly correlated with the clinical staging of ccRCC patients.

Conclusion

The selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression were evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in clear cell Renal Cancer Cell tumori – genesis. Concluding, this study confirms the deregulation of specific oncogenic miRNAs in renal tumor, reporting the identification of specific oncogenic miRNAs that are altered in ccRCC tissues, and further supports the potential clinical usefulness of these miRNAs in ccRCC management.

References

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