The miR-181b, miR-155 and miR-454 Expression in ctRNA in the Peripheral Blood Circulation of the Patients with Uveal Malignant Melanoma

doi:10.21203/rs.3.rs-3438754/v1

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The miR-181b, miR-155 and miR-454 Expression in ctRNA in the Peripheral Blood Circulation of the Patients with Uveal Malignant Melanoma

https://doi.org/10.21203/rs.3.rs-3438754/v1

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The Discovery of new non-invasive biomarkers in malignant melanoma is essential for the early diagnosis and follow-up of the disease. The aim of the study is to identify the expression levels of miR-181b and miR-155 and miR-454 in the ctRNA of serum/plasma samples of patients, and compare the differences with the healthy controls.

The expression levels of miR-181b, miR-155, and miR-454 were examined and compared with the healthy controls on the cell-free RNA (ctRNA) extracted from the peripheral blood serum/plasma samples of the 72 uveal malignant melanoma patients, and 72 healthy individuals.

The analysis showed that the expression level of the miR-181b has increased 9.25 fold, and expression level of miR-155 has increased 6.67 fold, and miR-454 expression level has increased 4.14 fold in the patient group compared with the levels in the control group. We found that the expression levels of the three miRNAs were statistically significant in patients compared with the expression levels in the healthy control group. The statistical evaluations between miRNA expression levels and clinical data showed that miR-155 had significant association with radiation therapy, and miR-454 showed a significant association with smoking and alcohol use.

The significantly higher expression levels of miR-181b, miR-155 and miR-454 in plasma/serum RNAs in the peripheral bloodstream of uveal melanoma patients compared with the levels in the control group suggest that these molecules can be evaluated both as the drug targets for the early diagnosis and follow-up of the disease.

Uveal Melanoma

miR-181b

miR-155

miR-454

ctRNA

Serum/Plasma

Uveal malignant melanoma (UMM) is a type of intraocular cancer of adults and seriously impairs patients' eyesight and the quality of life (Krantz et al. 2017). The tumor with a poor prognosis metastasizes in the early stages of the disease and shortens the survival time of patients (Shields CL et al. 2013; Shields JA and Shields CL 2015). With the current treatment possibilities, 50% of patients die due to metastases (Eskelin et al. 2000). Approximately 2000 new uveal malignant melanoma cases are diagnosed annually in the United States of America (Materin et al. 2011). Uveal malignant melanoma has a high potential for liver metastasis however, rarely spreads to the lymph nodes in the region (Dithmar et al. 2000). Uveal melanoma spreads in the iris, ciliary bodies, and highly in the choroid region in the eye. Some studies indicated that there are some very rare melanoma tumors that are located in the conjunctiva region (Koc and Kiratli 2020; Wong et al. 2014). Such type of melanoma is classified as ocular melanoma, or as cutaneous melanoma in different studies (Seregard 1998; Brownstein 2004). 40–50% of melanoma patients die within several months due to metastasis owing to the way it spreads and its metastatic properties (Kujala et al. 2003).

Although the diagnosis of uveal melanoma is possible during a clinical examination (Sun et al. 2015), the tumor is overlooked in many patients and the tumor may be found at an advanced stage (McLaughlin et al. 2005). Apart from its anatomical location, the higher mobility ability of the cilliary bodies and the excess number of vessels in this area not only complicate the early diagnosis, but also leads to the formation of an extra-vascular matrix pattern with a poor prognosis, and accordingly to the development of a highly metastatic potential (Chang et al. 1998; Johansson et al. 2010). Approximately 50% of patients with uveal melanoma develop metastasis (Yan et al. 2009). This cancer with high tendency to metastasize, makes distant liver metastasis by 90–95% through hematogenous spread (Chen et al. 2011). In other words, tumor cells have already spread throughout the body during tumor diagnosis, usually in 50% of patients (Singh et al. 2001). Therefore, there is a need for new noninvasive or highly sensitive biological biomarkers than can provide data with minimal invasive methods in uveal melanoma for the development of the treatment protocols, and for the early diagnosis and monitoring of the cancer.

It is important to investigate the miRNAs that will guide from a diagnostic and prognostic point of view in serum and plasma samples of patients for determining new biological markers in order to decrease the false negative and false positive clinical results in the diagnosis of uveal melanoma. The investigation of the circulating miRNAs is important for the early diagnosis as well as for monitoring each stage of the disease. Other than having non-invasive and easily accessible circulating biomarkers, their availability for investigation in any requested time interval with high precision and originality make these molecules worth examination (Chen et al. 2008; Gilad et al. 2008; Mitchell et al. 2008). MicroRNAs (miRNAs) are small, non-protein coding molecules with an average length of 22–24 nucleotides that regulate the suppression of gene expression, regulate the post-transcriptional activity of target mRNAs, and are also involved in the development of cancer (Bignotti et al. 2016; Bartel 2004). As with many cancers, the development of uveal malignant melanoma involves the exposure of oncogenes and tumor suppressor genes to multi-stage genetic and epigenetic changes. Some studies reported that the small noncoding RNAs have a role in the development of uveal melanoma (Li et al. 2020). The expression levels of different miRNAs were found to have increased compared with the normal cells/normal tissues in malignant melanoma cell lines and tumor tissues in studies which used different methods such as microarray and NGS (Sun et al. 2015; Zhang et al. 2018; Peng et al. 2017; Li et al. 2015). Currently, miRNA molecules are known to be used as the target molecule in cancer treatment in various cancer types, and as a biological biomarker in cancer diagnosis and monitoring of the treatment, and/or are known as the candidates for these procedures.

The miRNAs named miR-181b, miR-155 and miR-454 were reported to have high level of expression in uveal malignant melanoma cell lines (Sun et al. 2015; Zhang et al. 2018; Peng et al. 2017). The molecules belonging to the miR-181 family are suggested to negatively control the cell cycle and therefore they may be the drug targets (Zhang et al. 2018). miR-155 has been shown to be overexpressed in malignant cell line strains in the uveal malignant melanoma cell line studies investigating the miR-155, and suggested that miR-155 could have a role in proliferation, invasion and metastasis, and could be a therapeutic target (Peng et al. 2017). The miR-454 molecule was shown to have the direct target of PTEN gene in uveal malignant melanoma tumor tissues in the studies investigating the expression of miR-454. The study demonstrating that PTEN had overexpression in the same tissue, reported that miR-454 was effective in cell proliferation and invasion on the PTEN gene (Sun et al. 2015).

The aforementioned studies showed that the miR-181b, and miR-155 had overexpression only in uveal melanoma cell strains, however miR-454 was investigated in a very limited number of human tumor tissue and normal tissues in addition to uveal malignant melanoma cell strains (Sun et al. 2015; Zhang et al. 2018; Peng et al. 2017). There is no study in the literature which had identified the prognostic importance of miRNAs which were investigated in the plasma or serum samples in the peripheral blood circulation of uveal malignant melanoma patients. In the present study, the expression levels of miR-181b, miR-155 and miR-454 were compared in serum/plasma samples of 72 uveal malignant melanoma patients, and in an equal number of healthy people, and the diagnostic and prognostic significance of these miRNAs was evaluated.

Study Populations: The present study was initiated after the approval of the Clinical Research Ethics Board (Dated 20.09.2019/ No: 16) of Istanbul University, Istanbul Faculty of Medicine. The study was performed on the serum/plasma samples which were separated from the peripheral blood samples of uveal melanoma patients who presented to Istanbul University Oncology Institute, Department of Basic Oncology, and to the Department of Eye Diseases in Istanbul Faculty of Medicine. The experimental group consisted of 72 uveal malignant melanoma patients who presented to the Eye Diseases polyclinic in Istanbul University, Istanbul Faculty of Medicine in 2019, and the control group consisted of 72 healthy individuals who were matched for age, sex, and ethnicity with the patient group and had no history of cancer in the family in the last three generations. 3 out of 72 patients in the patient group were newly diagnosed to have the disease, the remaining patients were found in different stages of different treatment and follow up. All individuals included in the study were informed about the study before the peripheral blood samples were drawn, and their informed consent forms were received. The mean age of the patients was 54.68 years (± 13.69); the mean age of the healthy control group was 54.25 years (± 13.24), and the mean age of the patients at diagnosis was 48.19 years (± 12.97). There is no statistical difference between the mean age both in the control group consisting of healthy people and the group of patients with uveal malignant melanoma, and the groups showed similar distribution for age. The demographic, and clinical characteristics of the patients and the control group are given in Table 1 (Table 1).

Table 1

The demographic, and clinical characteristics of the patients
Patients	Characteristics	Number of Patients and Percentage, n (%)
Age(years)	≤ 60 > 60	26 (36.11%) 46 (63.89%)
Histological Diagnosis	Iris Melanoma Ciliary Bodies Melanoma Choroidal Melanoma Conjunctival Melanoma Iris + Ciliary Bodies Melanoma Choroidal + Ciliary Bodies Melanoma	7 (9.72%) 1 (1.39%) 56 (77.78%) 3 (4.17%) 1 (1.38%) 4 (5.56%)
Stage	Early Stage (I, II) Advanced Stage (III, IV)	38 (52.77%) 34 (47.23%)
Metastasis (Liver, Kidney, Adrenal Gland)	Yes No	4 (5.56%) 68 (94.44%)
Risky Occupation	Yes No	31 (43.06%) 41 (56.94%)
Secondary Primary Tumors (Brain, Breast, Colon)	Yes No	4 (5.56%) 68 (94.44%)
Ciliary Bodies Involvement	Yes No	6 (8.33%) 66 (91.67%)
Smoking and Alcohol intake	Yes No	36 (50.00%) 36 (50.00%)

Cell Free RNA (ctRNA) Isolation from Plasma/Serum

First, the plasma/serum was separated from the peripheral blood sample taken from the patient and healthy control groups. Serum/plasma samples were stored in a liquid nitrogen tank for long-term storage. The serum/plasma samples were removed from the nitrogen tank after adequate number of samples were obtained. The ctRNA extraction from serum/plasma samples was performed using the Quick-cfRNA Serum and Plasma Kit (ZYMO RESEARCH) in accordance with the kit protocol. 1mL Digestion Buffer and 50 µL Proteinase K were added onto 1mL serum/plasma extracted from the nitrogen tank and were incubated at 37°C for 2 hours. After incubation, 400 µL of Quick cfRNA binding buffer was added. 100% Propanol approximately 1.5 fold of the total volume in the tube was added, and loaded into the Spin-away column. The Spin-Away column was connected to the vacuum device, liquid contents were transferred to the column. Then, 600 µL of RNA Prep Buffer was added to the Spin-away column and washed, respectively. Then, 200µL RNA Recovery Buffer was added to the column and the bottom liquid was discarded. The column was washed with 300 µL of 100% ethanol, 200 µL of RNA Prep Buffer, 200 µL and 400 µL RNA Wash Buffer twice. 15 µL DNase/RNase free dHO₂ was added and centrifuged for 2 minutes at 12.000g. The ctRNA was collected and immediately stored at -70°C.

Measurement of the RNA concentrations

The presence of the isolated total RNA was determined using the agarose gel electrophoresis. 5 µL sample of the obtained ctRNAs was mixed with1 µL of 10X BPB (bromine phenol blue) and were loaded onto the gel and conducted at 150 Volts and imaged under UV. The concentration measurements of ctRNA were performed on a NanoDrop 2000 Spectrophotometer (THERMO SCIENTIFIC) device. The purity and concentration of the isolated RNA were determined by the absorbance at wavelengths of A260/A280 nm. The ctRNAs with an absorbance range of 1.5-2.0 OD were considered as pure/suitable ctRNA samples.

cDNA Synthesis and RealTime PCR: The cDNA synthesis from ctRNA using miRNA-specific Reverse Transcriptase enzyme with the help of ID3EAL cDNA Synthesis System (MIRXES) kit for Real-Time PCR reaction was carried out on the BioRad PCR device. Then, the obtained cDNAs were diluted using distilled water in a ratio of 1:10. The Real-Time PCR Reaction was performed on the MIC-Real Time PCR Device (MIC qPCR Cycler) using the diluted cDNAs belonging to the miR-U6 reference gene and with the target miRNAs of MiR-181b, miR-155 and miR-454. The expression level of each sample was calculated using the Ct (Threshold Cycle) value. The gene expressions were evaluated using the formula 2-ΔΔCT. The triplicate results for each sample were determined according to the average of the Ct values with this method. In accordance with the Formula 2 − ΔΔCT, miR-U6 gene expression was taken as the reference, and the rate of increase or decrease of the expression levels of miR-181b, miR-155 and miR-454 was identified by comparing the rates for the patient group as in the control group.

Statistical Analysis

The assumption of normality between the groups was evaluated using the Kolmogorov-Smirnov, and Shapiro Wilk tests in accordance with the statistical analyses using the Statistical Package for the Social Sciences (SPSS) v21.0 program. The p value was found as p < 0.05 in accordance with the normality assumption results, therefore the groups were suggested to have no normal distribution, and the use of the nonparametric Mann-Whitney U test was found applicable in the statistical analysis of all data.

The cell free RNAs extracted from serum/plasma samples separated from the peripheral blood of 72 healthy individuals who were matched with the patient group in terms of age, sex and ethnicity were used as the control group with the samples of 72 patients with uveal malignant melanoma in our study. The results were evaluated among 72 patients with uveal malignant melanoma, and 72 healthy controls.

63.89% of the patient group was aged over 60 years, and 36.11% were aged below 60 years. The mean age of the patients was 54.68 years (± 13.69), and the mean age of the healthy control group was 54.25 years (± 13.24). The mean age at diagnosis in the patient group was calculated as 48.19 years (± 12.97). 77.7% of the patients were histologically classified as choroidal melanoma. 52.77% were in Stage I-II, and 47.23% were in Stage III-IV. Metastasis was present in 5.56% (4/72) of the patients at the time of diagnosis. 50% (2/4) of the patients with metastases had only liver metastases, 25% (1/4) had liver + kidney metastases, and 25% (1/4) had liver + kidney + adrenal gland metastases. In addition to uveal malignant melanoma, 5.56% (4/72) of the patients were diagnosed with secondary primary tumors, including breast cancer in 2, colon carcinoma in 1, and brain tumor in 1 individual. The ciliary body involvement was found in 97.97% of the patients.

The graphs indicating the increase or decrease in the expression levels (2-∆∆C t) in uveal malignant melanoma patients compared to the levels in the healthy group are given in Fig. 1 (Fig. 1). Considering the Fold & Change (|FC|≥2) value in the comparison of the gene expression levels of the uveal malignant melanoma patients with the results of the healthy control group showed that miR-181b increased 9.25 fold, miR-155 by 7.67 fold and miR-454 increased 4.14 fold.

We found that the expression levels of miR-181b, miR-155, and miRNA-454 showed statistical difference (p < 0.05) in the patient group compared with the levels in the control group in the calculations based on the 2-∆∆Ct values between the patient and control groups in accordance with the evaluations with the Mann-Whitney U test. The expression levels of miR-181b, miR-155 and miRNA-454 in the uveal malignant melanoma patients were found statistically highly significant compared with the levels in the healthy controls (p = 0.005).

The association of the expression levels of miR-181b, miR-155 and miR-454 in patients with uveal malignant melanoma with the variables as the age, age at diagnosis, clinical stage, tumor size, metastasis status, treatment options as surgery, radiotherapy or chemotherapy, the risky occupational group status, and smoking and alcohol use were analysed and evaluated using the Mann-Whitney U test. No significant association was detected with other variables except radiotherapy and smoking and alcohol use, with the expression levels of miR-181b, miR-155 and miR-454, however, miR-155 expression level was found higher in patients receiving radiotherapy compared with the nonreceiving patients, and compared with the healthy group, and this association was found statistically significant (p = 0.040). Furthermore, a statistical significance was found between the miR-454 expression level with smoking and alcohol use as P = 0.009, and P = 0.026, respectively (Tables 2, 3 and 4).

Table 2

The relationship between miR-155 expression levels and radiotherapy.
miRNA-155 Expression Levels
Expression Levels Decreased Increased n (%) n (%)	Total n(%)
Radyotherapy Not received 1 (1.4%) 36 (50.0%) Received 6 (8.3%) 29 (40.3%)	37 (51.4%) 35 (48.6%)
Total n(%) 83 (9.7%) 44 (90.3%)	72 (100%)	P-value = 0.040

Table 3

The relationship between miR-454 expression levels and smoking.
miRNA-454 Expression Levels
Expression Levels Decreased Increased n (%) n (%)	Total n(%)
Smoking Yes 2 (2.8%) 33 (45.8%) No 11 (15.3%) 26 (36.1%)	35 (48.6%) 37 (51.4%)
Total n(%) 13 (18.1%) 59 (81.9%)	72 (100%)	P-value = 0.009

Table 4

The relationship between miR-454 expression levels and alcohol intake.
miRNA-454 Expression Levels
Expression Levels Decreased Increased n (%) n (%)	Total n(%)
Alcohol intake Yes 11 (15.3%) 58 (80.6%) No 2 (2.8%) 1 (1.4%)	69 (95.8%) 3 (4.2%)
Total n(%) 13 (18.1%) 59 (81.9%)	72 (100%)	P-value = 0.026

It was striking to find out that the expression levels of miR-181b, miR-155, and MiR-454 in patients with metastases at the time of diagnosis and in patients with secondary primary tumors were at higher extremes than the mean levels in the healthy control group. In addition, the highest expression level of these miRNAs was observed in 3 patients who had just been diagnosed and had not yet undergone any treatment or surgical procedure. We suggest that these extremely higher levels of miR-181b, miR-155 and miR-454 expression are formed by the effect of the metastatic cells in the circulation in addition to the role of the active uveal melanoma tumor cells, and confirms that miRNAs have diagnostic power in each stage of the disease.

STRING Analysis

The miR-181b, miR-155 and miR-454 and their target genes investigated in the present study were determined using the “mirTarBase” and “TargetScan ” databases. The target genes of miR-181b were found as RALA, PTEN, KRAS, MAPK1, MAPK8, AKT3, and the target genes of miR-155 were FOS, ETS1, TP53INP1, MDM2, MYB, RAP1B, CBL and the target genes of miR-454 were MDM4, PTEN, WNT1, WNT2B, MAPK1, MET, and HGF. The STRING (Protein-protein interaction analysis) analysis was performed to determine the interaction between miR-181b, miR-155 and miR-454 and the targets of these genes. The STRING analysis results showed a strong interaction between the genes targeted by miR-181b and the associated proteins with the KRAS and MAPK1 proteins in the RAF/MAPK pathway (p = 0.000118). In addition, a strong interaction was found between MAPK1, and MAPK8 in the same analysis. Furthermore, the strong and significant associations of KRAS and RALA; KRAS with MAPK1; MAPK1 with MAPK8; MAPK8 with AKT3 proteins were also evaluated as remarkable in this analysis (p < 0.05) (Fig. 2).

The results of the STRING analysis performed for miR-155 showed that the molecules that interact most with miR-155 were JUND, FOS and ETS1 molecules. Although the FOS molecule intensively interacted with JUND, it also interacts with CBL, RAP1B, and LCP2 proteins via PLCG1 (p < 0.05). (Fig. 3). We found that the interaction of FOS with PLCG1 was also indirectly established via MDM2, which was remarkable.

The STRING analysis performed for the target proteins of miR-454 showed that the greatest interaction was between the MET and HGF; and there was a significant association between MET and CDH1; PTEN and MDM4; MDM4 and MDM2 molecules (p = 0.000129). The studies in the literature reported that there were interactions between the MDM2, FOXO and TP53 proteins in the PTEN and MDM4 signal transduction pathway. An intense association was detected between the MAPK1 and MAP2K2 molecules in the performed analysis. In addition, the analysis also showed that the WNT1 and WNT2B molecules, which have the closest sequence similarity, were also in close interaction (p < 0.05) (Fig. 4).

We examined the diagnostic performance of the miR-181b, miR-155 and miR-454 molecules as the candidate biomarkers -in other words, the differentiation power of the uveal malignant melanoma patients with the healthy control group using the Receiver Operator Characteristics (ROC) analysis. In Fig. 5, the ROC-AUC values determined for each miRNA, and 95% CI (confidence interval), and the diagnostic power of the molecules miR-181b, miR-155 and miR-454 in the diagnosis of patients with malignant uveal melanoma were found statistically highly significant (p = 0.000) (Fig. 5).

The expression data of human genes in the current studies show that miRNAs have an important role in the occurrence and progression of cancer (Zhang et al. 2007; Aughton et al. 2020; Catalanotto et al. 2016; He and Hannon 2004). Various studies have shown that the miRNAs are the effective molecules that can be used in the diagnosis and follow-up of cancer and in the treatment of cancer as a therapeutic agent or therapeutic target (Erson and Petty 2009; Lagana et al. 2010; Lamy et al. 2006). To date, the miR-181b and miR-155 in many cancer types were shown to have high expression in the serum samples of high-risk breast cancer patients before surgery and/or treatment, and different studies have shown various times that the expression levels of these miRNAs were decreased after treatment (Sochor et al. 2014; Liu et al. 2013). Researchers in another study reported that the expression level of miR-155 in the circulating serum samples of patients with esophageal cancer increased compared with the levels in healthy individuals, and might be used as a noninvasive biologic biomarker (Liu et al. 2012). The studies associated with miR-454 expression reported that the miR-454 expression level showed differences in the cell lines of liver carcinoma (HCC) (Li et al. 2019), cervical cancer (Song et al. 2019), breast cancer (Ren et al. 2019), glioma (Shao et al. 2015) and uveal melanoma in the literature. The abnormal miRNA expression levels shown in the uveal melanoma cell lines and tumor tissue were reported as closely associated with cell proliferation, invasion, and metastasis in different studies (Zhang et al. 2016; Peng et al. 2017; Eedunuri et al. 2015; Li et al. 2014; Sun et al. a 2015; Venza et al. 2016; Wang et al. 2018; Zhou et al. 2016; Ling et al. 2017; Li et al. 2019; Wang YC et al. 2018; Wang Y et al. 2018). The bioinformatics and functional analysis results in a study which investigated the miR-181b in uveal melanoma showed that miR-181b precipitated the cell cycle by targeting the CTDSPL (small phosphatase-like molecule) of the carboxy-terminal region and created an oncogenic effect (Zhang et al. 2016). Researchers reported that the molecules of the miR-181 family, which are known to negatively control the cell cycle, may be the drug targets (Zhang et al. 2016). Similarly, miR-155 has been shown to create an oncogenic effect by promoting cell proliferation in uveal melanoma (Peng et al. 2017). Researchers in the studies investigating the miR-155 in uveal malignant melanoma cell lines suggested that miR-155 was overexpressed in tumor tissue and may be a therapeutic target (Peng et al. 2017). In addition, the miR-454 was found to have overexpression in the tumor tissue, and the direct target of the miR-454 molecule was the PTEN gene in the study investigating the miR-454 (Sun et al. b 2015).

Researchers in many studies investigating the uveal malignant melanoma reported in the literature that different miRNAs act as tumor suppressors and/or oncogenes in uveal malignant melanoma. The miR-367, miR-21, miR-21a, miR-155, miR-181b, miR-92a-3p were shown to have oncogenic effect in uveal malignant melanoma cell lines and tumor tissue, however, miR-137, miR-144, miR145, miR-296-3p and miR-23A were shown to have tumor suppressor role (Li et al. 2020). Most studies summarized so far have been conducted on uveal malignant melanoma cell lines (Zhang et al. 2016; Peng et al. 2017; Eedunuri et al. 2015; Li et al. 2014; Sun et al. a 2015; Venza et al. 2016; Wang et al. 2018; Zhou et al. 2016; Ling et al. 2017; Li et al. 2019; Wang YC et al. 2018; Wang Y et al. 2018), there was no study investigating the expression levels of miRNAS on ctRNA in serum/plasma samples of patients in the literature.

We investigated the expression levels of miR-181b, miR-155 and miR-454 on the ctRNAs differentiated from the serum samples of the uveal malignant patients, and of the healthy controls who were matched with the patient group for age, sex, and ethnicity with no history of cancer in the past three generations in the family. We found that the miR-181b was increased 9.25 fold, miR155 increased 7.67 fold, and the miR-454 was increased 4.14 fold in the comparison between the patient and healthy control groups who had no statistical difference between the mean age. The higher levels of miRNA expression, which were found statistically significant, suggest that miRNAs have oncogenic properties considering the activities of the targeted genes.

We found no statistical significance between the expression levels of miR-181b, miR-155 and miR-454 with having a risky occupation, tumor size, clinical stage, tumor location and metastasis status, however, there was a statistical significance between high miR-454 expression level with smoking and alcohol use, and between high miR-155 expression level with radiotherapy in our study. Demonstration of the higher expression levels of miR-181b, miR-155 and miR-454 molecules on ctRNA, in all stages, and on the histological subtypes of uveal malignant melanoma suggest that these molecules have a diagnostic value in all stages, and histological subtypes. This result was confirmed by the Receiver Operator Characteristics (ROC) analysis performed in our study. The ROC analysis showed that the ability of the miR-181b, miR-155 and miR-454 molecules to differentiate between the patient diagnosed with uveal melanoma and healthy control group was statistically significant (P = 0.000). All these results suggest that the higher expression levels of miR-181b, miR-155 and miR-454 detected in serum/plasma can be used as non-invasive biological biomarkers both in the diagnosis and follow-up of the disease. It will be meaningful to evaluate the power of these molecules, whose diagnostic power has been proven in our study, to be a prognostic and therapeutic target/agent by large-scale future studies.

We also identified the target genes and gene pathways of miR-181b, miR-155 and miR-454 molecules which were found to have higher expression in ctRNAs of the serum/plasma samples of uveal malignant melanoma using the STRING analysis. The KRAS and MAPK1 protein interactions were found strong between the genes targeted by the miR-181b and between their associated proteins (p < 0.05). These two proteins are involved in the RAF/MAPK pathway (Chakraborty et al. 2016) and are among the most deteriorated pathways in cancer. We also found that there is a strong connection between the KRAS and RALA proteins in addition to MAPK1 and MAPK8 proteins of miR-181b. The STRING analysis performed for miR-155 showed that the molecules with the most interaction of the miR-155 were CBL and RAP1B proteins along with FOS and ETS1 (p < 0.05). These proteins, which are effective in the MAPK and PI3K pathways, coordinate the normal functioning of that gene pathway. The increase in certain proteins due to the overexpression of miR155 is known to be effective in the development of cancer by working in the direction of proliferation of the gene pathway (Basuyaux et al. 1997). Our study results are totally compatible with this data. The STRING analysis performed for miR-454 showed that the MET and HGF molecules were the target proteins that mostly interact with miR-454. Researchers in previous studies reported that MET and HGF molecules regulate various physiologic process including proliferation, morphogenesis, invasion, and survival with the receptor tyrosine kinase activity which transfer the signals from extracellular matrix to the cytoplasm through HGFR-HGF interaction (Danilkovitch-Miagkova and Zbar 2002; Gentile et al. 2008). The activation of PI3K-AKT-mTOR signaling pathways are known to be performed by the MAPK pathway with the autophosphorylation of the intracellular MET (Liu et al. 2020). These activated pathways play a role in the development of cancer by leading to cell survival by cell proliferation (Liu et al. 2020). The detection of higher level of miR-454 molecule in the peripheral blood of patients with uveal malignant melanoma supports that malignant formation occurs by this mechanism. The STRING analysis also showed that miR-454 interacts with MDM2, FOXO and TP53 proteins in the PTEN and MDM4 signaling pathways, as well as molecules in different pathways such as WNT1 and WNT2B. These results suggest that miR-454 is an extremely effective molecule that affects different gene pathways in carcinogenesis. All these protein-protein interactions should be individually investigated in future studies.

The weaknesses of our study were that the miR-181b, miR-155 and miR-454 molecules which were proven to be used for diagnostic purposes were not investigated in benign and other malignant eye tumors, and the peripheral blood samples taken from patients in specific periods were not investigated for identifying their prognostic value. However, the aim is to examine and evaluate these missing points in future studies.

In conclusion, our study is the first to investigate the expression levels of miR-181b, miR-155 and miR-454 in the ctRNA in the peripheral bloodstream of patients with uveal malignant melanoma. This study showed that the miR-181b, miR-155 and miR-454 molecules can distinguish uveal malignant melanoma patients from healthy people and are diagnostically valuable. The detectability of these molecules in serum/plasma suggests that they can be used as the non-invasive biomarkers. The prognostic and therapeutic target/drug potential of these molecules, whose diagnostic power has been proven by our study, will be evaluated in future studies.

ACKNOWLEDGEMENTS

Thanks to the technicians who were Arzu BurnUva and Turkan Şen Ferhadoğlu for assistance and laboratory organizations.

FUNDING

This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University (Grant number: TDK-2019-35336).

AVAILABILITY OF DATA AND MATERIALS

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

AUTHORS’ CONTRIBUTIONS

ST recruited and referred patients to the study. MH performed the experiments and help writing the manuscript. MH and DAÖ performed statistical and STRING analysis. All experiments were required in the laboratory of HY. All the chemicals and KITs required for the experimental processes of the study was provided by HY and MH. MH, TD and HY also wrote the manuscript, contributed towards the interpretation of data, writing of the manuscript and increased the scientific value of writing adding intellectual information. MH recruited healthy cases to the study and also collected the data and drawing pedigrees and fill up demographic information forms and prepared all documents for the samples. HY criticized the manuscript according to literature intellectually.

COMPETING INTERESTS OF AUTHORS

The authors declare that they have no competing interests.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was provided by the parents/guardians of the participants included in the study. The study was approved by the Ethics Committee of Istanbul Medical Faculty in Istanbul University (Approval document: Dated 20.09.2019/ No: 16).

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The miR-181b, miR-155 and miR-454 Expression in ctRNA in the Peripheral Blood Circulation of the Patients with Uveal Malignant Melanoma

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