CLL is the most prevalent leukemia in adults which remains a public health problem in the world (24). This cancer has poor prognosis and diagnosis at different stages (25). In the past, it was difficult to diagnose B-CLL patients, but today it is partially detectable using symptoms and analysis of prognostic laboratory biomarkers (26). Therefore, the identification of new biomarkers with high sensitivity and specificity is crucial for early diagnosis of B-CLL. Several reports have shown that changes in miRNAs expression are related to the development and progression of different cancers in human (27). Additionally, miRNAs are stable in serum and plasma, which might be due to their protection by exosomes, microvesicles and Argonaute 2 (28). Therefore, these molecules as non-invasive biomarkers can be useful for detection of different cancers including B-CLL (29, 30).
On the other hand, the APRIL is known as an oncogene that is rarely expressed in normal tissues but its high levels are detectable in tumor cells in vivo and in vitro and cancers such as thyroid and colon (31). Additionally, APRIL is a soluble molecule secreted from the Golgi apparatus (20). Some studies have demonstrated that APRIL, as an oncogene, upregulated in non-canonical NF-kB signaling pathway and that dysregulation of this signaling pathway contributes to development and evolution of B-CLL (32). Therefore, considering the role of APRIL in the pathogenesis of B-CLL, this molecule seems to be a suitable choice for miRNAs targeting studies. In the present study, we predicted miRNAs targeting APRIL gene using bioinformatics software, and determined the expression levels of these miRNAs and the APRIL gene using RT-qPCR in the plasma samples of B-CLL patients and healthy individuals. ROC curve analyses were also performed to investigate the possibility of using plasma levels of predicted miRNAs as new and potential biomarkers for detecting B-CLL.
The bioinformatics predictions presented here indicated that four miRNAs including miR-145-5p, miR-185-5p, miR-6132 and miR-4644 could target the transcript of the APRIL gene. However, miR-145-5p and miR-185-5p had higher scores and were chosen for experimental study.
The results of RT-qPCR in the current study showed that the plasma levels of these two miRNAs were significantly lower in the B-CLL patients than in the healthy individuals. The expression of miR-145-5p and miR-185-5p were downregulated by -14.28 fold (P = 0.001) and − 3.44 fold (P = 0.001), respectively. On the other hand, the plasma levels of APRIL were upregulated by 102 fold (P = 0.001) in the B-CLL patients than in the healthy individuals. The decreased expression of miR-185 has been reported in the several solid tumors (33). Zanette et al., showed that five miRNAs including miR-135b, miR199s, miR-142-5p, miR-181c and miR-185 had the lowest expression levels in CLL (29). The role of miR-185 has been investigated in cancers including lung cancer (34), osteosarcoma (35) and breast cancer (36) by targeting SOX9, HK2, DNMT1 and E2F6, respectively. On the other hand, miR-145 was reported as a tumor suppressor miRNA because its expression is reduced in various tumor, including colon, breast, ovarian, CLL and burkitt lymphoma (37, 38). These finding suggest that miR-145-5p and miR-185-5p act as a tumor suppressor in the B-CLL by direct-targeting of APRIL mRNA. Therefore, B-CLL progression may be prevented by the expression of miR-145-5p and miR-185-5p in cancer cells through decreases in the expression of APRIL gene. The accuracy of miR-145-5p and miR-185-5p levels to discriminate between patients with B-CLL and healthy individuals using ROC curve analyses revealed that these two miRNAs are specific and sensitive for the detection of B-CLL but miR-145-5p presented the greatest AUC, sensitivity, and specificity as 90%, 95%, and 0.952, respectively, at a cutoff of 0.034. Therefore, differences in the expression of miR-145-5p could distinguish healthy individuals from patients with B-CLL. Akao et al., also confirmed that miR-145-5p can be used as biomarker to differentiate normal cells from malignant B cells (38). Therefore, according to the findings of present study, it can be concluded that serum miR-145-5p and miR-185-5p levels could be applied as potential and efficient biomarkers in B-CLL.