Aberrant expression of miRNAs may cause oncogenesis of several cancers, including AML (25). In this study, the expression levels of miR-100 and miR-101 involved in AML and their effects on genes of the mTOR/AKT/PI3K pathway in AML patients, which perform an essential function in AML pathogenesis, were investigated, were investigated. In addition, the correlation between these miRNAs and the aforementioned genes was considered. This study revealed the following remarkable results: The expression of miR-100 was increased in AML patients, whereas the expression of miR-101 was reduced. Moreover, mTOR and PI3K genes had a reduced expression compared to the control group, while our result showed no meaningful change in the expression of AKT1. Ultimately, we observed the association between miR-100 and miR-101 with the mTOR gene, which is an essential downstream effector of the PI3K/AKT pathway and plays an important role in controlling cancer cell growth.
The miR-100 is involved in various types of cancers; it serves as both tumor suppressor and oncomiR and shows bivariate expression changes in several malignancies. Thus, it is overexpressed in certain cancers such as renal cell carcinoma (26), small cell lung cancer (27), gastric cancer (28), and pediatric AML (29), whereas it is under-expressed in other cancers such as acute lymphoblastic leukemia (ALL) (30), chondrosarcoma (31) and adrenocortical tumors (32). Several studies have investigated the expression of miR-100 in the AML. For example, Sun et al. (33), who investigated the expression level of miR-100 in pediatric AML, indicated that the expression of miR-100 was increased in this group. Also was confirmed by Zhang et al.(1), who studied the expression of this miR in the AML. Furthermore, they indicated that the expression level of miR-100 increased in all subtypes of AML patients, and this enhanced expression was different among subtypes (1). Thus, the results of these studies suggest that miR-100 plays an essential part in the growth and pathogenesis of AML, whereas the molecular status and downstream targets of these miRs in AML patients need further studies. Therefore, our study sought to examine the correlation between miR-100 and the mTOR/AKT/PI3K pathway, which is downstream of these miRs and involved in the spread of different cancers, such as AML. The mTOR/AKT/PI3K cascade is an important signaling system that is dysregulated in numerous human cancers (34). This signaling cascade is activated by various extracellular signals, which in turn regulate protein translation, differentiation, proliferation, invasion, cell cycling, and apoptosis (35). More importantly, this signaling pathway has been thoroughly studied and explored as a therapeutic target in myeloid and lymphocytic leukemia. Several studies have investigated the inhibition of AKT in AML and APL and mTOR in CML, CLL, and ALL as therapeutic targets (36, 37). The mTOR/AKT/PI3K pathway has been studied as an essential signaling pathway in hepatocellular carcinoma development controlled by several miRs, including miR-99a, miR-100, and miR-149, which inhibit mTOR, and miR-9, miR-30, miR-125, miR-149 and miR-379 which inhibit the AKT (38). Similarly, the results of the current work in AML patients demonstrated a correlation between increase in miR-100 expression with a decrease in mTOR gene expression, which was statistically significant (P value: 0.041, r: -0.387); however, no significant correlation was observed with AKT and PI3K. It appears that overexpression of miR-100 controls the mTOR signaling pathway by targeting the direct 3'UTR region of mTOR, that attribute to the regulation of cell-cycle progression, particularly at the G1 to S-transition, this may occur by blockade the downstream messenger, p70 S6 kinase (p70S6K), and controlling the translation of some critical mRNAs needed for cell-cycle progression (39). In a research revealed by Xu and colleagues (15), using miR-100 inhibitors and miR-100 mimics, the link between miR-100 and mTOR in bladder cancer patients was confirmed as miR-100 directly targets mTOR. In another study by X-J Li and colleagues (30), in patients with acute lymphoblastic leukemia, miR-99a and miR-100 were shown to repress expansion and increase apoptosis by directly targeting FKBP51 and IGF1R / mTOR. Our results also demonstrated similar results findings.
In this study, the expression of miR-101, which is recognized to be a tumor suppressor (by inhibiting proliferation, apoptosis, and metastasis) in various malignancies, was examined. Gonzales-Aloy et al.(14), revealed that miR-101 was decreased in newly diagnosed AML patients compared to the control group, and they also demonstrated the tumor suppressive role of miR-101 in MLL-rearranged AMLs. Down-regulation of miR-101 has also been showed in other malignancy, such as endometrial cancer (40), breast cancer (41), and T-cell acute lymphoblastic leukemia (T- ALL) (42). In a study conducted by Lei et al. (43), in patients with liver fibrosis, miR-101 was also found to cause down-regulation by targeting the mTOR/AKT/PI3K genes regulatory pathways and was proposed as an anti-fibrotic mechanism in medical fibrosis. Following, we demonstrated that there was a relationship between miR-101 and mTOR in patients with AML, and this correlation was statistically significant (P value: 0.029, r: 0.41), and the increase of miR-101 increased mTOR expression, while there was no significant relationship between the expression of miR-101 and AKT, PI3K genes in the present study. Lin et al.(20), pointed out that the expression of miR-101 notably decreased in osteosarcomas and was an inverse correlation with mTOR expression, confirming mTOR as a direct target and showing that miR-101 strongly decreased the expression of mTOR at mRNA and protein levels. miR-101 affects autophagy and AML function through mTOR signaling pathway, a significant serine-threonine protein kinase downstream of PI3K/Akt; Previous research also showed that miR-101 restricts autophagy to prevent tumor growth (40) (44). Li and colleagues (45) showed that octreotide could increase miR-101 expression and also impede autophagy by inactivating AMP-activated protein kinase (AMPK) and activating the mTOR cascade, Consequently reducing the incidence of intestinal mucosal inflammation after cancer therapy.
Based on a study of human cytomegalovirus (CMV) by Wang et al. (46), miR-100 has an expected target in the 3' UTR of Raptor, a partner of mTOR (mTORC1), and miR-101 has two expected targets in the 3' UTR of another partner of mTOR, Rictor (mTORC2). Blockade of mTOR is a promising strategy for the treatment of leukemia; factors that selectively target TORC1 (rapalogs) have restricted clinical action and negligible impact on AML therapy. Developing selective ATP catalytic blockers capable of blocking TORC1 functions and TORC2 has led to a new dynamic in research on mTOR targeting in AML. To overcome rapalog's limitations in treating leukemia, dual TORC1/2 or pan-PI3K-TORC1/2 blockers can be used. Previous studies have shown that dual blockers of TORC1/2 improve the effect of cytarabine on primary leukemia precursors of AML patients and enhance the antileukemic effect. These researches suggest a potential role for miR-100 and miR-101 in treating AML patients. Using dual TORC1/2 inhibitors and chemotherapeutic factors may make a promising strategy to target leukemia-initiating stem cells and improve the chances of curing AML patients (47).
Although most prior investigations have shown increased expression of mTOR/AKT/PI3K genes in patients with acute myeloid leukemia (48, 49), in the present study we found down-regulation in the genes of this pathway. Nevertheless, Nepstad et al.(50) showed that the expression of mTOR/AKT/PI3K depends on a part of the complex phenotype, such as cell communication, cell signaling and transcriptional regulation, which may be variable. In our study, this decrease in expression was probably influenced by the increase in expression of miR100 and miR101.
The present results showed there has no correlation between the expression of miR-100, miR-101, and mTOR, AKT, PI3K with gender, age, blast percentage, sample source, and subtypes of AML. However, Zheng et al. (1), noted that miR-100 depends on myeloid cell maturation and is differentially expressed in AML subtypes. In the current study, the samples were divided into two groups, AML-M3 and AML NON -M3; there was no considerable difference in the expression level of miR-100 and miR-101, which might be due to the limited sample size and the ethnicity of the patients. Further researches are required to investigate the relationship between miR-100, miR-101 and their downstream targets to clarify the regulators of this pathway. In addition, there is a significant association between the expression of mTOR genes and AKT and PI3K; this has also been shown by previous studies (51), according to these findings, it can be assumed that inhibitors of the mTOR gene, such as miR-100 and miR101, can affect other genes of this pathway.
The present study had some limitations that can be mentioned such as samples size and bone marrow sampling and storage. Despite these limitations, the results indicate that additional research is required to confirm the function of miR-100 and miR-101 and their direct targets in AML to control disease progression and prevent disease recurrence.