Exosomal miR-6803-5p as potential diagnostic and prognostic marker in colorectal cancer
Abstract
Accumulating data have suggested exosome-delivered microRNAs (miRNAs) play critical role in carcinogenesis and cancer progression. However, little is known about the influence of exosomal miR-6803-5p on the development and prognosis of colorectal cancer (CRC). Levels of serum exosomal miR-6803-5p were determined by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). Outcomes of overall survival (OS) and disease-free survival (DFS) of CRC patients were estimated by Kaplan-Meier analysis. We used cox regression analysis to investigate the association between exosomes-encapsulated miR-6803-5p and the clinicopathological factors of CRC patients. The exosomal miR-6803-5p was significantly increased in serum samples from patients with CRC in contrast to healthy controls. Significantly higher levels of serum exosomal miR-6803-5p were observed in CRC patients at later TNM stage or with lymph node metastasis as well as liver metastasis. Patients with elevated levels of serum exosomal miR-6803-5p had much poorer OS and DFS. Cox regression analysis revealed high levels of exosomal miR-6803-5p was associated with poor prognosis in CRC independent of other confounding factors. Thus, exosomal miR-6803-5p is a potential diagnostic and prognostic biomarker for patients with CRC.
1 INTRODUCTION
Colorectal cancer (CRC) is the second most common cancer in females and the third most common cancer in males, with high incidence of cancer-related deaths across the world.1 It is a multifactorial disease involving factors of genetics, environmental, and lifestyle. Accumulated evidence has documented several risk factors associated with the development of CRC, including physical inactivity, alcohol, smoking, and red and processed meat consumption.2, 3 Additionally, some kinds of diseases have been demonstrated to be related to increased risk of CRC, such as type 2 diabetes mellitus, obesity, and inflammatory bowel diseases.4-6 Increasing number of studies have implicated that non-coding RNAs play critical roles in the initiation and progression of CRC, many of which may even affect the prognosis of patients.7-9
MicroRNAs (miRNAs) are common non-coding RNAs with 19-25 nucleotides in length. They are small stable endogenous RNAs possessing the nature of inhibiting mRNA translation. Although miRNAs do not encode any proteins, they participate in the process of gene modulation and critical biological processes, including cell proliferation, differentiation, apoptosis as well as cancer initiation, and progression.10 Aberrantly expressed miRNAs can function as either oncogenes or tumor suppressors depending on the tumor environment.10 Accumulating studies have suggested that a number of miRNAs are abnormally expressed and stably exist in the blood or its components, making which as ideal circulating biomarkers for early detection in different cancers including CRC.11, 12 Recent evidence have implicated that exosomes and their encapsulated functional miRNAs have been recognized as potential biomarkers in cancers, including CRC.13, 14 Certain exosome-delivered miRNAs in human peripheral blood may influence the prognosis of CRC patients.15 We have previously found that several exosomes-encapsulated miRNAs in serum were identified as potential circulating biomarkers for CRC.16 Exosomal miR-6803-5p is one of the most significantly aberrantly expressed miRNAs in serum as assayed by microarray analysis. However, little is known about the modifying effect of serum exosomal miR-6803-5p on CRC. The aim of this study is to investigate the association between exosomes-encapsulated miR-6803-5p and the development and prognosis of CRC.
2 MATERIALS AND METHODS
2.1 Patients
A retrospective cohort study of 168 CRC patients including 100 males and 68 females were performed. MiRNAs microarray analysis was carried out at 5 years of follow-up. Table 1 presented characteristics of all patients histologically diagnosed with CRC. The mean age at diagnosis of CRC was 55.2 ± 10.8 years. No statistical significance was observed between miR-6803-5phigh group and miR-6803-5plow group regarding age, sex, TNM stage, tumor grade, vascular filtration, and status of metastasis. Twenty age and sex matched healthy controls receiving physical examinations were randomly selected from the same hospital. All participants in this study had signed the informed consent before study. The research was approved by the ethical committee of our hospital. Serum samples were collected from patients before surgery.
| Factors | miR-6803-5phigh (n = 101) | miR-6803-5plow (n = 67) |
|---|---|---|
| Age, n (%) | ||
| <59 years | 34 (33.7) | 36 (53.7) |
| ≥59 years | 67 (66.3) | 31 (46.3) |
| Sex, n (%) | ||
| Females | 40 (39.6) | 28 (41.8) |
| Males | 61 (60.4) | 39 (58.2) |
| TNM, n (%) | ||
| I | 16 (15.9) | 5 (7.5) |
| II | 29 (28.7) | 19 (28.3) |
| III | 38 (37.6) | 30 (44.8) |
| IV | 18 (17.8) | 13 (19.4) |
| Tumor grade, n (%) | ||
| High | 36 (35.6) | 16 (23.9) |
| Moderate | 46 (45.6) | 17 (25.4) |
| Low | 19 (18.8) | 34 (50.7) |
| Vascular infiltration, n (%) | ||
| Yes | 23 (22.8) | 18 (26.9) |
| No | 78 (77.2) | 49 (73.1) |
| Lymph node metastasis, n (%) | ||
| No | 28 (27.7) | 19 (28.4) |
| Yes | 73 (72.3) | 48 (71.6) |
| Liver metastasis, n (%) | ||
| Yes | 23 (22.8) | 12 (17.9) |
| No | 78 (77.2) | 55 (82.1) |
2.2 Purification of exosomes
Fresh peripheral blood samples were centrifugated at 1200g for 10 min at 4°C for the preparation of serum samples. All serum samples were stored at −20°C for subsequent experiments. Serum exosomes were isolated by Invitrogen™ Total Exosome Isolation Kit (Invitrogen, Carlsbad, CA) according to the product protocol. Briefly, Proteinase K was incubated with serum samples for 30 min at 4°C. Then, the precipitated exosomes were recovered by centrifugation at 10 000g for 5 min at room temperature, and resuspended in phosphate buffered solution (PBS).
2.3 Extraction of exosomal RNAs
Total RNAs were extracted from serum exosomes by the miRNeasy mini kit (Qiagen, Venlo, Netherlands) and stored at −80°C for subsequent experiments. Briefly, serum exosomes were lysed with 1 mL Lysis Reagent. Total RNAs including miRNAs were purified from the lysed products based on the manufacturer's protocol, and used as a template for quantitative real-time PCR.
2.4 Quantification of miRNA by real-time PCR
Expression of serum exosomes-encapsulated miR-6803-5p was determined by the real-time PCR. We used ABM miRNA EasyScript cDNA Synthesis kit (Cat No.G269) for cDNA synthesis. ABM miRNA primers were applied for PCR (miR-16 Primers: Cat.No. MPH01191, ABM, Peterborough, Canada; miR-6803-5p Primers: Cat.No. MPH03637, ABM, Canada). The relative expression of exosomal miR-6803-5p in serum samples from patients with CRC was estimated by 2−ΔΔCT. MiR-16 was used as an internal control.
2.5 Outcome estimation
The association between levels of serum exosomes-encapsulated miR-6803-5p and CRC outcomes of overall survival (OS) and disease-free survival (DFS) was estimated by the Kaplan-Meier survival curve analysis and log-rank tests.
2.6 Statistical analysis
All data were presented as mean ± SEM. Methods of independent-samples t-test or one-way ANOVA were selectively used for statistical analysis. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) were calculated by Cox regression analysis. The adjusting factors mainly included age, sex, TNM stage, tumor grade, vascular filtration, and status of metastasis. We used Graphpad (V5.0) and STATA (V12.0) softwares for statistical analysis.
3 RESULTS
3.1 Expression of exosomal miR-6803-5p in the serum and its association with clinicopathological factors of CRC patients
In comparison with healthy controls, higher levels of exosomal miR-6803-5p were observed in the serum of patients with CRC (Figure 1A). Besides, more significantly elevated levels of serum exosomal miR-6803-5p were found in CRC patients at III/IV TNM stage (Figure 1B). However, no significant difference for the levels of exosomal miR-6803-5p in serum was identified between the two groups of CRC patients with and without vascular infiltration (Figure 1C). Moreover, there were much higher levels of exosomal miR-6803-5p in serum samples from CRC patients with lymph node metastasis and liver metastasis (Figures 1D and 1E). Taken together, the exosomal miR-6803-5p in serum was increased in patients with CRC, particularly those at later TNM stage or with lymph node metastasis as well as liver metastasis.
3.2 Association between serous exosomal miR-6803-5p and the OS of patients with CRC
Kaplan-Meier analysis showed the evidence that the overall survival (OS) was shorter in CRC patients with higher levels of exosomal miR-6803-5p in serum than those patients with lower levels of exosomal miR-6803-5p in serum (P < 0.001, Figure 2A). As shown in Table 2, the OS was much shorter in CRC patients at III/IV TNM stage and with liver metastasis. In addition, the cox regression analysis suggested that CRC patients with higher levels of serum exosomal miR-6803-5p was associated with much poorer prognosis adjusted by age, sex, TNM stage, lymph node metastasis as well as liver metastasis (Table 2).
| OS | DFS | |||
|---|---|---|---|---|
| HR (95%CI) | P | HR (95%CI) | P | |
| Age (≥59 years) | 1.26 (0.72-2.21) | 0.422 | 0.94 (0.54-1.62) | 0.822 |
| Sex (Males) | 1.10 (0.66-1.84) | 0.706 | 1.07 (0.64-1.80) | 0.793 |
| TNM stage (III/IV) | 2.96 (1.57-5.58) | 0.001 | 3.02 (1.63-5.58) | <0.001 |
| Vascular infiltration | 0.72 (0.30-1.73) | 0.461 | 0.70 (0.29-1.66) | 0.413 |
| Lymph node metastasis | 2.16 (0.93-5.01) | 0.072 | 1.58(0.70-3.56) | 0.272 |
| Liver metastasis | 4.53 (2.37-8.65) | <0.001 | 6.18 (3.23-11.81) | <0.001 |
| High levels of serum exosomal miR-6803-5p | 2.93 (1.35-6.37) | 0.007 | 3.26 (1.56-6.81) | 0.002 |
- OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval; TNM, tumor node metastasis.
3.3 Association between serum exosomal miR-6803-5p and the DFS of patients with CRC
Similarly, the disease-free survival (DFS) of CRC patients with increased levels of exosomal miR-6803-5p in serum was much lower (P < 0.001, Figure 2B). CRC patients at later (III/IV) TNM stage and with liver metastasis had much poorer DFS, as suggested by the cox regression analysis (Table 2). Furthermore, increased levels of exosomal miR-6803-5p in serum was related to lower DFS independent of other confounding factors, including age, sex, vascular infiltration, TNM stage and metastasis of lymph node, and liver (Table 2).
3.4 Serum exosomal miR-6803-5p was a good biomarker for the diagnosis of CRC
As shown in Figure 3, the area under curve (AUC) was equal to 0.7399, which suggested that exosomal miR-6803-5p in serum could be a good diagnostic marker for CRC.
4 DISCUSSION
Our study firstly shows strong evidence for the important influence of serum exosomal miR-6803-5p in the development and progression of CRC, suggesting a promising biomarker of diagnosis, and prognosis in this deadly disease. Compared with healthy controls, levels of serum exosomal miR-6803-5p in CRC patients was significantly elevated. Most interestingly, significantly increased levels of serum exosomes-delivered miR-6803-5p were found in CRC patients at later TNM stage or with lymph node metastasis and liver metastasis. In addition, Patients who had higher levels of exosomal miR-6803-5p in serum usually had poorer OS and DFS. Thus, the present study shed some light on the potential role of exosomal miR-6803-5p as a diagnostic and prognostic biomarker in CRC.
Exosomes, discoid vesicles of 40-100 nm in diameter, are firstly found in reticulocytes of sheep, which can be secreted by a number of cells in normal, and pathological conditions.17 Exosomes are naturally found in common body fluids, including blood, saliva, urine, and breast milk. Accumulated data have implicated that exosomes are involved in intercellular communications through transporting protein, nucleic acid, and other constituents with biological activities.18, 19 Given the crucial role of exosomes in the regulation of biological processes, relevant research becomes hot, particularly in cancer research. Exosomes generated by tumor cells can mediate angiogenesis and the proliferation and immune escape of cancer cells, while dendritic cells-derived exosomes can induce effective anti-tumor immune response. Those exosomes-specific proteins, nucleic acids, lipids, and even some important signaling molecules are expected to be targets in the early diagnosis and treatment of certain diseases.20, 21 Currently available studies have suggested that exosomes are expected to be novel way of drug administration and gene therapy vectors in cancer, in that exosomes can pass through biological barriers and deliver exosomes-encapsulated proteins, miRNAs, and so on, between cells.22, 23 It has been well established that miRNAs can serve as diagnostic and prognostic biomarkers due to their important modifying effects in the initiation and progression of cancer, including CRC.24 MiRNAs are a family of small, non-coding RNA molecules, which are highly conserved across different species, and can regulate gene expression.24-26 A recent study have implicated that miRNA 375, a potential therapeutic target for CRC, could regulate the proliferation and migration of CRC cells through suppressing the CTGF-EGFR signaling pathway.27 Apart from the regulation of proliferation and migration of cancer cells, certain miRNAs may influence the survival of CRC patients, suggesting prognostic value of miRNAs and promising drug targets for CRC.28 Increasing studies show the evidence that exosomes-delivered miRNAs may serve as circulating biomarkers for CRC.16, 29 In this study, we have found that miR-6803-5p is up-regulated in serum exosomes of CRC patients compared with healthy controls. Most importantly, high levels of exosomal miR-6803-5p in serum were associated with decreased survival of patients with CRC, suggesting miR-6803-5p might serve as a prognostic biomarker for this disease.
Previously, we have identified the specific miRNAs profile in serum exosomes in patients with CRC, some of the aberrantly expressed miRNAs may serve as disease biomarkers and novel therapeutic targets for CRC.15 The exosomal miR-6803-5p was firstly found to be significantly increased in serum samples from CRC patients. Additionally, its expression was associated with the TNM stage, lymph node metastasis, and liver metastasis status of patients with CRC. Moreover, CRC patients with elevated levels of serum exosomal miR-6803-5p had poor prognosis. Taken together, the exosomal miR-6803-5p in serum may not only serve as a diagnostic biomarker but a prognosis-related biomarker. Nevertheless, little is known of the underlying molecular mechanisms of miR-6803-5p in cancer initiation and progression. More functional and mechanical studies are warranted to elucidate the biological effects of exosomes-encapsulated miR-6803-5p in CRC.
In summary, the present study, for the first time, provides strong evidence that serum exosomes-derived miR-6803-5p is a novel promising biomarker for the diagnosis and prognosis estimation in CRC. Although the association between serum exosomal miR-6803-5p and the survival of CRC patients is well documented, the underlying molecular mechanisms involved in colorectal carcinogenesis and progression remain largely unknown. Future studies with high quality are warranted for further investigation.
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science Foundation for Young Scholars (81601408, 31570941, and 31270993), Natural Science Foundation of Shandong Province (Project Number: ZR2016HQ12), the Medical and Health Science and Technology Development Program of Shandong Province (2016WS0681 and 2016WS0686), and Weifang Science and Technology Development Program (2016YX016 and 2017YX019).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
