A cluster of specified microRNAs in peripheral blood as biomarkers for metastatic non-small-cell lung cancer by stem-loop RT-PCR

Tumor metastasis is one of the most important prognostic factors for advanced NSCLC, which often results in treatment failure. So it seems urgent to detect metastasis in a convenient way at a relatively early stage.

It has been well established that miRNAs are present in human’s peripheral blood. And some researches have focused on the sources of circulatory miRNAs. In healthy individuals, by measuring miRNAs in the peripheral blood and blood cells, it was found that circulatory miRNAs came from blood cells. In cancer patients, many reports have clearly shown that cancer affect miRNA levels in the bloodstream, although it is still unclear how tumor miRNAs make their way into the bloodstream. So far, there has been no definitive conclusion of cancer-related miRNAs sources, but it is for certain that they have no relation with blood cells (Chen et al. 2008). However, there are mainly two conjectures about the sources of cancer-related miRNAs. They may originate from cancer cells in the circulation (Mitchell et al. 2008). They may be present as a result of tumor cells dying and getting lysed or tumor cells releasing miRNAs into the surrounding environment (Chin and Slack 2008). The other conjecture is that they may come from tumor-derived exosomes. Some researchers detected certain miRNA levels of cancer cells and circulating tumor exosomes from ovarian cancer patients by using a modified MACS procedure with anti-EpCAM, and it was proposed that cancer-related miRNAs in the circulation came from tumor-derived exosomes (Taylor and Gercel-Taylor 2010).

Quantification of miRNAs by stem-loop RT-PCR and high-flux sequencing technology were used to confirm that the vast majorities of the small RNAs were miRNAs in peripheral blood (Chen et al. 2008). Other qualitative, quantitative, or semiquantitative experiments also indicated that miRNAs exist in human’s peripheral blood (Lawrie et al. 2008; Resnick et al. 2009; Chim et al. 2008; Gilad et al. 2008). In fact, stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. This method enables fast, accurate, and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases (Chen et al. 2005). This present study applied the stem-loop RT-PCR method to show that certain miRNAs such as miR-125a-5p, miR-126, miR-183, miR-200, miR-221, and miR-222 were present in peripheral blood. At the same time, the serum samples can be obtained atraumatically and conveniently.

Besides, miRNAs are stable in peripheral blood. Given that serum has been reported to contain high levels of RNase activity, miRNAs exist in a form that is resistant to serum nuclease activity. Studies have shown that the levels of endogenous miRNAs remain stable in many other harsh conditions such as prolonged room temperature incubation, repeated freeze-thawing procedures, boiling, superacid, and peralkaline. The intrinsic stability of miRNAs may attribute to their small size and chemical structure, but not the extrinsic conditions (Mitchell et al. 2008; Resnick et al. 2009; Chim et al. 2008).

In addition, miRNA measurements are substantially identical in serum compared with in plasma, indicating that both serum and plasma samples will be suitable for the investigations of miRNAs as blood-based biomarkers. Studies also indicate that there is not any significant difference of miRNAs level between men and women (Chen et al. 2008; Mitchell et al. 2008). Our study demonstrated that the serum levels for miR-125a-5p, miR-126, miR-183, miR-200, miR-221, and miR-222 between the male and female controls were not significantly different, which kept identical with the previous studies.

Four major hallmarks—unlimited cell proliferation, autonomous growth, resistance to growth inhibitory signals, and escape from apoptosis—are central to the transformation of a normal cell to a cancer phenotype, the initial step of tumorigenesis. MiRNAs have recently been implicated as important modulators of these cell growth and survival processes, and their deregulation can initiate malignant transformation and unchecked cellular growth (Du and Pertsemlidis 2010). The regulatory mechanisms of miRNA expression includes various ways such as epigenetic changes (methylation for example),transcription factor misadjustment that controls the expression of miRNA coding gene, chromosome abnormality, genic mutation, single-nucleotide polymorphism, mechanism disorders of miRNA biosynthesis, and so on (Du and Pertsemlidis 2010). And as continuously being shown, miRNAs can act as both oncogenes and tumor suppressors (Esquela-Kerscher and Slack 2006). Through various techniques, numerous groups have shown that different cancer types including NSCLC have distinct miRNA profiles. This may explain why some cancer-related miRNAs are up-regulated and others are down-regulated.

In our present study, all of the six specified miRNAs have been reported to be involved in lung cancer metastases in vitro; however, the serum level for some of them was of no significant difference between the healthy controls and the patients. Only hsa-miR-126, hsa-miR-183, and hsa-miR-222 showed significant difference between the serum of the Stage IV NSCLC patients and the controls. And the expression levels for these three miRNAs were lower in the patients than in the controls. It seems difficult to understand the relation and function of tissue and serum miRNAs levels.

As described above, there is a hypothesis that tumor cells use exosomes/microvesicles to transport genetic information, including miRNAs, to surrounding cells and thereby support tumor growth and progression (Skog et al. 2008). Moreover, circulating miRNAs might modulate immune responses (Valenti et al. 2007; Baj-Krzyworzeka et al. 2007): For example, micro vesicles derived from human melanoma and colon cancer can promote tumor growth and immune escape by mediating the differentiation of monocytes toward TGFβ-secreting myeloid suppressive cells (Valenti et al. 2007). However, it has not been investigated if these effects are mediated by the miRNAs contained in the microvesicles.

There are also contradictive assumptions about the question. Wang et al. (2009b) concluded from their study that specific miRNAs are released into the blood stream after drug induced liver injury leading to a down-regulation in the tissue. In contrast to that, Tanaka et al. 2009 speculated that tumor cells rely on the specific intake of miRNAs from circulating micro vesicles. These different viewpoints clearly demonstrate a need for additional studies to elucidate the relation of tissue and serum miRNAs expression levels.

In fact, many researches have indicated that miRNAs play essential roles in tissue differentiation during normal development and tumorigenesis (Mascaux et al. 2009). Certain miRNAs are differentially expressed in lung squamous cell carcinomas and adenocarcinomas. A group of these miRNAs shows considerable tissue specificity (Lu et al. 2005). For example, miR-205 is believed to be a marker of stratified squamous epithelium (Ryan et al. 2006), and its expression is retained in squamous cell cancers of the upper and lower respiratory tracts (Yanaihara et al. 2006; Jiang et al. 2005). Actually, the differential expression of miR-205 in lung squamous cell cancers but not in adenocarcinomas provides a rationale for the classification of NSCLCs based on miRNA expression profiling. Quantification of miR-205 level has recently been optimized for formalin-fixed and paraffin-embedded samples, so that levels of miR-205 can now be used to classify NSCLCs with a high degree of sensitivity and specificity (Lebanony et al. 2009). It was found that it had 100% sensitivity and 100% specificity in differentiating squamous cell cancers and adenocarcinomas for the lung cancer resections by applying quantitative reverse transcription-PCR to analyze the hsa-miR-205 and hsa-miR-21 expression together (Bishop et al. 2010). However, so far there have been no data about the serum level of miR-205 in NSCLCs. Our study demonstrated that there was no significant difference in the serum levels for certain miRNAs including miR-125a-5p, miR-126, miR-183, miR-200, miR-221, and miR-222 between the lung squamous cell cancer patients and adenocarcinoma patients, indicating that the serum levels of these miRNAs may have no relation to the pathological types of NSCLCs.

As for the serum levels for the six cited miRNAs, our study showed significant difference for hsa-miR-126, hsa-miR-183, and hsa-miR-222 between the Stage IV NSCLC patients and the controls. Further study showed that there was no statistical difference for hsa-miR-126, hsa-miR-183, and hsa-miR-222 between the controls and Stage I/II patients, which demonstrated that the expression of hsa-miR-126, hsa-miR-183, and hsa-miR-222 kept stable at the early stage of NSCLC. Furthermore, it showed statistical difference for hsa-miR-126 and hsa-miR-183 between the Stage I/II patients and Stage IV patients, but not for hsa-miR-222, which indicated that the serum levels for hsa-miR-126 and hsa-miR-183 changed, but hsa-miR-222 kept at a stable level as the metastases developed. So we concluded that the levels of hsa-miR-126 and hsa-miR-183 were closely related to lung cancer metastases and these two miRNAs had the potential to serve as serum biomarkers for diagnosing NSCLC metastases. Interestingly, our study demonstrated that the three miRNAs—hsa-miR-126, hsa-miR-183, and hsa-miR-222— were all down-regulated in the patients’ serum, which indicated that they could inhibit cancer progression, while hsa-miR-126 and hsa-miR-183 could play an important role in inhibiting lung cancer metastases.

The data of this study showed that the serum level for miR-126 was 2.5-fold decrease in the Stage IV NSCLC patients versus the controls. By analyzing a miRNA profile of SCC samples of Chinese lung cancer patients, Yang et al. (2010) identified that miR-126 was one of the miRNAs that were down-regulated in the tumors compared with non-cancerous lung tissues. The over-expression of miR-126 results in a decrease in Crk protein, which is a member of a family of adaptor proteins involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. As increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness, so miR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion, and the effects on invasion may be partially mediated through Crk regulation (Crawford et al. 2008).

With regard to hsa-miR-183, our data showed that the level was up to tenfold decrease in the Stage IV NSCLC patients versus the controls. It was identified that several potential downstream effectors including ezrin and more than 30 genes were involved in metastases in pulmonary giant cell cancer (Wang et al. 2008). Further study showed that ezrin and parathyroid hormone-related protein were up-regulated at sites of bone metastasis as detected by RT-PCR and immunohistochemistry in HARA lung squamous carcinoma cells, which offers new mechanistic insights into the metastasis of lung cancer and provides potential targets for the prevention and treatment of lung cancer metastasis (Deng et al. 2007).

In our study, the serum level for miR-222 was nearly twofold decrease in the Stage IV NSCLC patients versus the controls with significant difference. However, the serum level for miR-221 between the two groups showed no significant difference. Some previous studies proved that miR-221 & 222 function in the same way. MiR-221 & 222 induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases by targeting PTEN and TIMP3 tumor suppressors. And the MET oncogene is involved in miR-222 and miR-221 activation through the c-Jun transcription factor (Garofalo et al. 2009). Our study showed that miR-222 was at a relatively higher level, indicating that examination in the serum may be less sensitive or perhaps only a part of microRNAs in the tumor cells can be released to the serum. In this way, it is easy to understand that the serum levels for other three miRNAs miR-125a-5p, miR-200, and miR-221 were of no significant difference between the patients and the controls.

For patients with suspected metastatic lung cancer, histopathological diagnosis is the gold standard; however, sometimes it is difficult to obtain tissue for diagnosis. Computed tomography (CT) even combined with positron emission tomography (PET) is usually used to detect the metastasis or stage the disease. Even though it is a widely used tool for diagnosing and managing suspected lung cancer, it has limited sensitivity and specificity, besides PET is very expensive. In contrast, detecting the serum level of certain miRNAs by stem-loop RT-PCR is relatively convenient and economical. Additionally, it may be applied to improve the accuracy for diagnosing metastatic lung cancer combined with other currently widely used tools.

Although currently there are some problems with using miRNAs as cancer biomarkers such as the unclearly cut reference range and the lack of large-sample data, our study extends the potential of hsa-miR-126 and hsa-miR-183 to serve as molecular markers for human primary lung cancer. The serum levels for these miRNAs could be identified as an indicator for metastatic NSCLCs. And they may be applied clinically combining with other lung cancer-specific miRNAs such as miR-21, miR-210, and miR-486-5p in the future (Shen et al. 2011). Since there have been some meaningful findings, we are planning to further study these miRNAs in NSCLCs.