Modulation of all-trans retinoic acid-induced MiRNA expression in neoplastic cell lines: a systematic review

A systematic search of the published literature form January 1, 2007 to November 12, 2018 was undertaken using the US National Library of Medicine MEDLINE/PubMed (www.​ncbi.​nlm.​nih.​gov/​pubmed) bibliographic search engine. Multiple PubMed searches were conducted using the following keyword combinations “(miRNA OR microRNA) AND (“retinoic acid” OR “vitamin A” OR retinol) AND (cancer OR neoplasm OR tumor).” The studies were then compiled into a single database and duplicates removed. An initial screening was performed by assessing the title and abstract. After reading the selected studies, the references section of each text was analyzed for additional relevant studies.

For this review, we selected studies written in the English language that evaluated changes in the expression profile of miRNAs in neoplastic human cell lines after treatment with ATRA. The exclusion criteria were other systematic reviews on the subject, bioinformatics analyses, short communications, and supporting information.

Data extraction was conducted by one reviewer (LL) and verified by the other authors. Extracted data included cancer type, cell type analyzed, conditions of ATRA treatment (dose and duration), miRNAs evaluated, and significant findings. For studies that determined ATRA-induced miRNA expression at multiple time points, the values obtained on the last day were considered. All studies are grouped according to the type of cancer.

NB originates from the aberrant development of primordial neural crest cells and is the most common extracranial solid tumor during childhood and the most common tumor in infants [35]. Several lines of evidence support a role for miRNAs in NB pathogenesis as well as the utility of miRNA profiling in NB diagnostics, classification, and prognosis [35]. Furthermore, the involvement of miRNAs in ATRA-induced differentiation of NB cells was recently reported [35]. In particular, cell lines derived from NB such as SK-N-BE, SH-SY5Y, and LAN-5 can be induced by ATRA treatment to undergo neural cell differentiation and are thus often used as model systems for studying biochemical pathways involved in differentiation [39].

To test the effects of ATRA on NB differentiation and the development of embryonic cells, Chen and Stallings [31] evaluated the expression of 34 human miRNAs in SK-N-BE cells treated with 5 μmol/L ATRA by replacing the culture medium every 24 h for 5 days. The expression profiles of 21 miRNAs were found altered when compared with that of untreated cells (Table 1). Of these miRNAs, 17 were upregulated in ATRA-treated cells and 4 were downregulated. In particular, miR-184 showed the most significant change, as its expression increased 9-fold following ATRA treatment. The authors also observed that the effects of ATRA treatment on miRNA expression were sustained for at least a short period of time after release; for example, after SK-N-BE cells were treated with ATRA for 5 days and released for 3 days, miR-184 levels were 25-fold higher than that of untreated cells, indicating sustained effects of ATRA on miRNA expression. In addition, their results suggested that treatment with ATRA is associated with apoptosis rather than differentiation induction in this cell line.

Laneve et al. [20] analyzed the expression pattern of 70 miRNAs in SK-N-BE cells treated with 10 μmol/L ATRA for 3, 6 and 10 days. They found that 14 miRNAs were upregulated (Table 1), 33 did not exhibit any changes in expression, and 23 could not be detected. Expression levels of the upregulated miRNAs were mostly induced after 3 days upon ATRA treatment and progressively increased after terminal differentiation (10 days). Moreover, the authors observed that the expression levels of miR-9, miR-125b, and miR-125a increased 1.7, 2.2, and 2.6-fold, respectively, compared with that of control cells, and that this increase led to a marked decrease in NB cell proliferation in vitro.

Evangelisti et al. [21] used the same ATRA concentration mentioned above and measured miR-128 expression in SH-SY5Y cell lines after ATRA treatment. The cells were fed every 48 h with ATRA and then treatment was stopped after 6 days. miR-128 expression was found upregulated by approximately 3-fold in treated cells compared with untreated ones.

Similarly, Le et al. [32] treated SH-SY5Y cells with 10 μmol/L ATRA but over the course of 5 days. They analyzed the expression profiles of 175 human miRNAs and found that 12 miRNAs were significantly upregulated (miR-106, let-7b, miR-199a*, miR-124a, miR-143, miR-125b, miR-7, miR-189, miR-199a, miR-27a, miR-21, and miR-214) during treatment; however, after validation with Northern blot analysis, only six were found upregulated during differentiation (Table 1).

Beverigde et al. [33] treated SH-SY5Y cells with 10 μmol/L ATRA for an additional 1 day longer than Le et al. [32]. They performed microarrays and RT-qPCR to examine miRNA expression profiles. Microarray assays identified 44 miRNAs with altered expression after treatment, of which 12 were significantly upregulated and 32 downregulated (Table 1); however, only 10 miRNAs were confirmed altered by RT-qPCR, of which 3 were upregulated (miR-128a, miR-10a, and miR-124a) and 7 downregulated (miR-301, miR-20a, miR-106a, miR-19a, miR-29b, miR-134, and miR-15b). Interestingly, miR-9 exhibited a different expression pattern depending on the method of analysis. It was found upregulated via microarray analysis and downregulated via RT-qPCR. ATRA also induced the downregulation of the entire miR-17 cluster (microarray results). Moreover, five miRNAs that exhibited increased expression (miR-9, miR-124a, miR-128a, miR-208, miR-210, and miR-423) were previously demonstrated to be brain-specific or brain-enriched miRNAs and are considered to play important roles in brain development, neuronal maturation, and neuronal differentiation.

Ragusa et al. [34] analyzed the expression profiles of three miRNAs (miR-152, miR-200b, and miR-338) after ATRA treatment. SK-N-BE cells were treated with ATRA 10 μmol/L and observed on day 5 and 10 after treatment. After 10 days of treatment, all three miRNAs analyzed were upregulated, with the most significant increase observed for miR-200b.

In keeping with the above findings, Meseguer et al. [35] showed that ATRA treatment of SH-SY5Y cells resulted in profound changes in miRNA expression patterns. The authors treated SH-SY5Y cells with 1 μmol/L ATRA and assessed changes in miRNA expression at 0, 24, 48, and 96 h. The expression levels of 42 miRNAs were significantly changed [26 upregulated and 16 downregulated (Table 1)]; in particular, miR-10a and 10b showed the most prominent changes in expression. Furthermore, these changes induced by ATRA contributed to the regulation of SH-SY5Y NB cell differentiation and the associated changes in migratory and invasive activities.

Das et al. [36] treated SK-N-BE NB cells with 5 μmol/L ATRA by replacing the culture medium every 24 h for 7 days to determine changes in methylation patterns and gene expression. In addition, they investigated whether upregulated miRNAs are causally associated with the downregulation of a gene known to cause genome-wide demethylation events. They performed expression analysis of 368 miRNAs using low-density TaqMan arrays and found 17 upregulated miRNAs (≥ 2-fold increase) and 17 downregulated miRNAs (≥ 2-fold decrease; Table 1). Among the upregulated miRNAs possibly involved in controlling DNA methylation, ectopic overexpression of miR-152 significantly decreased cell invasiveness and anchorage independent growth, contributing in part to ATRA-induced differentiation. miR-152 expression was also analyzed in three other NB cell lines (SH-SY5Y, LAN-5 and SK-N-AS) that received the same treatment. As observed in SK-N-BE cells, ATRA treatment induced miR-152 upregulation in SH-SY5Y and LAN-5 cell lines; however, this miRNA was downregulated in SK-N-AS cells.

In a subsequent study with the same treatment regimen, Das et al. [37] observed a correlation between changes in miRNA expression profiles and methylation following ATRA treatment. In SK-N-BE cells, 20 miRNAs were found upregulated and 24 downregulated following ATRA treatment, whereas 13 miRNAs were upregulated in SH-SY5Y cells (the authors did not specify which miRNAs are altered). Only miR-340 was upregulated in both cell lines and showed the highest expression levels. In addition, overexpression of miR-340 was associated with decreased cell viability and limited colony-forming ability.

Chen et al. [38] observed significant changes in the expression profiles of 12 miRNAs, of which half were upregulated and half downregulated, when SH-SY5Y cells were treated with 10 μmol/L ATRA for 48 h (Table 1). Foley et al. [39] treated NB cell lines (SK-N-BE, SH-SY5Y, and LAN-5) with 5 μmol/L ATRA by exchanging the culture medium every 24 h for 7 days, and then analyzed the expression profiles of 364 miRNAs. In SK-N-BE cells, several miRNAs exhibited altered expression levels, of which 53 miRNAs showed significant changes—30 were positively regulated and 23 negatively regulated (Table 1). miRNAs with the highest changes in expression (410-fold increase by day 7) included miR-132, miR-10a, and miR-10b. Similarly, these miRNAs were significantly upregulated in response to ATRA in SK-N-BE, SHSY-5Y, and LAN-5 NB cells.

Das and Bhattacharyya [22] treated SH-SY5Y cells for 7 days with 10 μmol/L ATRA and investigated the expression levels of 96 miRNAs on day 3 and 7. They found 31 upregulated miRNAs, of which 14 exhibited high expression levels (miR-9, miR-9 *, miR-15b, miR-16, miR-34a, miR-100, miR-124, miR-125b, miR-126, miR-132, miR-134, miR-137, let-7a, and miR-432), and 33 downregulated miRNAs (Table 1). Interestingly, miR-185 presented with two different patterns of expression, downregulation on day 3 and upregulation on day 7.

These results support the notion that miRNA regulation plays a key role in the differentiation of NB cells induced by ATRA and in the phenotypic changes linked to the expression of genes associated with these miRNAs. In addition, multiple lines of evidence indicate that NB treated with ATRA exhibits lower migration and invasion abilities. Changes in miRNA expression profiles in NB cell lines after ATRA treatment are summarized in Table 1.

AML is characterized by abnormal differentiation and uncontrolled proliferation of immature hematopoietic cells [56]. In hematopoietic malignancies, several miRNAs have been reported to exhibit tumor-suppressive or oncogenic roles in leukemogenesis [44]. Accordingly, it has been suggested that miRNAs are important in the molecular pathogenesis of leukemia by interfering with essential pathways of hematopoietic differentiation [40].

Acute promyelocytic leukemia (APL), the main subtype of acute myeloid leukemia in which positive responses are observed after treatment with ATRA [56], is characterized by chromosomal translocations involving the RA receptor-α (RARA) gene that result in clonal expansion of hematopoietic precursors blocked at the promyelocytic stage of differentiation [40]. Several miRNAs upregulated upon ATRA treatment of APL cell lines have already been identified in different studies [40].

Garzon et al. [23] cultured APL NB4 cells with or without 10^− 1 μmol ATRA over 4 days and identified nine upregulated and one downregulated miRNA after comparing expression levels between both groups (Table 2). They also observed that treatment of NB4 cells with ATRA induced granulocytic differentiation, as evidenced by morphological changes and increased expression of the surface antigens CD11b and CD15.

Marchis et al. [40] treated NB4 APL cells with 1 μmol/L ATRA and confirmed that ATRA induced a marked and selective increase in miR-342 expression, starting 24 h after treatment and progressively increasing over 3 days. They also found increased expression of the miR-342 host gene Enah/Vasp-like (EVL) during ATRA treatment. These results indicated that EVL and miR-342 expression is co-regulated, with miR-342 contributing to the ATRA-mediated granulocytic differentiation program of promyelocytic precursors in ATRA-treated NB4 cells.

To uncover the regulation of miR-15a and miR-16-1 induced by ATRA, Gao et al. [41] employed two models, NB4, HL60, and U937 cell lines and fresh leukemic cells extracted from 10 AML patients; both experimental underwent 1 μmol/L ATRA treatment for 72 h. The authors found that treatment with ATRA increased miR-15a and miR-16-1 expression in NB4 cells, which in turn culminated in the inhibition of leukemic cell differentiation. They also observed that miR-15a and miR-16-1 were upregulated in 8 of 10 patient samples. These findings indicated that upregulation of miR-15a and miR-16-1 may be associated with the differentiation induced by ATRA. In addition, they evaluated the time (0 to 72 h) and concentration-dependent (10^− 2 to 10 μmol/L ATRA) expression of miR-15a and miR-16-1 in response to ATRA treatment, which induced differentiation in NB4 cells. They found that an increase in time and ATRA concentrations between 10^− 1 to 10 μmol/L ATRA were associated with elevated miR-15a and miR-16-1 expression; however, 10^− 2 μmol/L ATRA did not induce differentiation and failed to modulate the expression of miR-15a and miR-16-1.

In another study, Lin et al. [42] treated NB4, HL60, and K562 cells with 1 μmol/L ATRA over 72 h to monitor the in vitro expression levels of CDX2 and miR-125b. In NB4 cells, miR-125b expression progressively decreased to 30% of baseline after 72 h following differentiation induction with ATRA. The researchers also demonstrated decreased CDX2 mRNA expression levels that were positively correlated with miR-125b levels after ATRA treatment, suggesting a potential function of CDX2 and miR-125b in inhibiting cell differentiation in AML cells and promoting leukemogenesis.

Morris et al. [43] further examined AML cell lines (NB4, HL60, PL21, and THP-1) that can be induced to differentiate along the granulocytic or monocytic lineage after exposure to ATRA (10^− 3 to 1 μmol/L) for 2 (THP-1), 3 (NB4) or 4 (HL60 and PL21) days. The authors found that these AML cells lines express very low levels of endogenous miR-150 in contrast to normal CD34+ progenitors cell lines after exposure to ATRA. However, in AML cell lines, differentiation of miR-150-expressing cells occurs independently of RARA signaling.

Zhuang et al. [44] revealed the downregulation of miR-181a in NB4 cells during treatment with 1 μmol/L ATRA over 36 h. They also observed that ATRA induced NB4 cell differentiation. Their study strongly supported the therapeutic role of ATRA on APL. Furthermore, Lin et al. [45] observed significant upregulation of miR-638 during ATRA-induced myeloid differentiation of HL60 and NB4 cell lines compared with that of untreated control. HL60 cells were treated with 3 μmol/L ATRA for 72 h while NB4 cells were treated with 2 μmol/L ATRA for 4 days. In HL60 cells, upregulation of miR-638 coincided with increased expression of the myeloid-specific surface markers CD14 and CD11b. Moreover, CDK2 overexpression eliminated the inhibitory effect of miR-638 in HL60 cells, which was more significant upon ATRA treatment.

To evaluate changes in miRNA expression mediated by ATRA, Bräuer-Hartmann et al. [46] cultured NB4 cell lines in the presence of 10^− 1 μmol/L ATRA and analyzed the expression profiles of miR-181 family members (miR-181a–d) 24 h after treatment. In this study, a significant downregulation of all miR-181 family members was observed. In parallel, they also investigated granulocyte differentiation associated with changes in the expression patterns of the miR-181 family in U937, HL60, and NB4 cells treated with 1 μmol/L ATRA. They found significant downregulation of miR-181a/b in APL cell lines (NB4); however, no significant changes were found in the non-APL cell lines (U937 and HL60). Moreover, inhibition of the miR-181a/b cluster by ATRA treatment effectively repressed cell proliferation and induced apoptosis in APL cells (NB4).

After treatment of AML HL60 cells with 0.1 or 1 μmol/L ATRA for 72 h, Yan et al. [47] found a significant reduction in the expression levels of the miR-17–92 cluster upon ATRA treatment, which decreased by almost 50% in 3 days. In addition, they found that HL60 cell proliferation was reduced after exposure to ATRA, which activated their terminal differentiation into granulocytes.

In summary, these studies demonstrated that several miRNAs are highly expressed in specific hematopoietic cell lineages and that modulation of their expression induced by ATRA treatment may be correlated with changes in cellular properties or differentiation and may thus represent an effective treatment strategy. miRNAs induced in AML cell lines after ATRA treatment are summarized in Table 2.

Breast cancer is the most common malignancy in women [57] and represents a heterogeneous group of tumors with varying responses to therapeutic agents, including retinoids [48]. Therefore, only a few reports have assessed the effects of retinoids on miRNAs in this carcinoma.

For example, Terao et al. [48] determined the differential profiles of miRNA expression in MCF-7 and MDA-MB-231 cells cultured with or without 1 μmol/L ATRA over 72 h. Although ATRA did not affect the miRNA profiles of MDA-MB-231 cells—as they are estrogen receptor-negative (ERα-)—the retinoid significantly increased expression levels of a single miRNA, miR-21, in MCF-7 cells (estrogen receptor-positive; ERα+) after 2 h, which leveled off at 24 h.

To investigate the potential modulation of miR-10a expression in breast cancer cells, Khan et al. [49] analyzed SKBR3 cells exposed to higher ATRA concentrations (1 and 5 μmol/L)—albeit for shorter duration (24 h). For both concentrations, an increase in miR-10a expression was observed, with higher expression levels induced upon exposure to 5 μmol/L ATRA. The authors also analyzed T47D cells exposed to 1 or 5 μmol/L ATRA for 24 h and found that miR-10a expression was similarly upregulated upon stimulation under both ATRA concentrations.

Fisher et al. [50] treated SKBR3 cells with 10^− 1 μmol/L ATRA for up to 36 h and then observed the expression profiles of 57 miRNAs, of which 37 were upregulated and 20 downregulated. The authors chose to only analyze 30 of these miRNAs in detail (Table 3), because they are strongly associated with positive or negative regulation of more than 500 mRNAs involved in different stages of tumor progression (e.g., apoptosis, motility, and cell invasion). This indicates a possible role for ATRA in the control of the metastatic invasiveness of breast cancer. In addition, the authors treated MCF-7, MDA-MB-453, MDA-MB-231, and MDA-MB-157 cells with 1 μmol/L ATRA for up to 36 h. In MCF-7 cells, ATRA negatively regulated 12 miRNAs, which were also downregulated in SKBR3 cells. They also observed a significant ATRA-dependent downregulation of miR-125a-5p and miR-210-3p in MDA-MB-157 cells. On the other hand, none of the miRNAs were found modulated in response to ATRA in MDA-MB-231 and MDA-MB-453 cells. miRNA expression profiles in breast carcinoma cell lines after ATRA treatment are summarized in Table 3.

Lung cancer is responsible for the largest number of deaths among all cancer types in both men and women [3]. Recently, it has been demonstrated that miRNAs are directly involved in lung cancer development and progression [3].

Zhu et al. [3] treated non-small cell lung cancer A549 cells with 10 or 100 μmol/L ATRA for 12 or 24 h and H1299 cells with 100 μmol/L ATRA for 48 or 72 h. The authors observed changes in the expression profiles of 13 miRNAs in A549 cell lines (Table 4); however, they focused on miR-512-3p because it exhibited a significant response to ATRA treatment and pro-apoptotic tumor suppressor abilities. In A549 cells, miR-512-3p expression increased by approximately 100% at 12 h and reached a peak at 24 h upon 100 μmol/L ATRA treatment, whereas expression in H1299 cells increased by only 7-fold after 72-h treatment with 100 μmol/L ATRA and showed no change at 48 h. In addition, they demonstrated that miR-512-3p overexpression inhibited adhesion, migration, and invasion of A549 and H1299 lung cancer cells.

To explore the effects of RA on the expression profile of miR-512-p, Chu et al. [4] also treated H1299 and A549 cells with 10 or 100 μmol/L ATRA for 24, 48, and 72 h. miRNA expression levels in A549 cells increased 3-fold when exposed to 10 μmol/L ATRA across all time points compared with that of control. When ATRA concentration increased to 100 μmol/L, miR-512-5p expression increased by 5, 17, and 3-fold at 24, 48, and 72 h, respectively. In H1299 cells, miR-512-5p expression increased by approximately 4-fold and 10-fold when treated with 10 μmol/L and 100 μmol/L ATRA, respectively, at all three time points compared with that of control. They also found that apoptosis was stimulated in both lung cancer cell lines. Overexpression of miR-512-5p in A549 and H1299 cells led to a 40 and 46% increase, respectively, in apoptotic cells compared with that of control cells. In addition, ATRA treatment and the consequent increase in miR-512-p expression led to a sharp decrease in glucose uptake by tumor cells and attenuated cell migration capacity by 39% compared to that of control. miRNA expression profiles of lung cancer cell lines after ATRA treatment are summarized in Table 4.

Weiss et al. [51] investigated the importance of miR-10a in pancreatic cancer metastasis and the possible interactions between miR-10a expression and RA. The authors employed the pancreatic tumor cell lines PaTu8988-S and PaTu8988-T and administered 1 μmol/L ATRA or a selective RARα antagonist for 72 h. They found that miR-10a expression in nonmetastatic pancreatic cells was positively regulated by ATRA stimulation, whereas miR-10a expression significantly decreased in cells treated with a selective RARα antagonist.

In another study, Xia et al. [52] treated U343 and U251 glioma cell lines with 1 μmol/L ATRA for 48 h and found that ATRA treatment negatively regulates miR-125b expression in both cell lines, although U343 cells were more sensitive to ATRA than were U251 cells. Furthermore, ATRA-mediated miR-125b overexpression led to reduced cell proliferation and increased apoptosis in both cell lines.

Human glioblastoma cells (U87 MG) were administered different doses of ATRA (10, 20, 40, and 60 μmol/L) and analyzed after 24, 48 and 72 h in the study by Chen et al. [53]. ATRA was found to positively regulate different members of the miR-302 cluster (Table 5), especially miR-302b, in a dose-dependent manner. Treatment with 40 μmol/L ATRA more than doubled miR-302b expression 12 h after exposure, and this overexpression was positively associated with cell death, suggesting that an increase in miR-302b expression is associated with suppression of tumorigenesis. Moreover, cell death associated with ATRA treatment may be regulated via miR-320b expression.

In another study by Chen et al. [54], pluripotent human embryonal carcinoma cells (NT2/D1) were treated with 10 μmol/L ATRA for 21 days. A decrease in the expression levels of the transcription factor FOXM1 was observed, which was mediated by the ATRA-induced overexpression of miR-134. Moreover, miR-134 overexpression resulted in decreased NT2/D1 cell pluripotency. ATRA was found to function by binding to the ligand-inducible transcription factors that activate or repress the transcription of downstream target genes, controlling cell growth and differentiation in both embryonic and adult cells.

Liu et al. [55] further investigated the association between miR-3666 expression and the effects of ATRA treatment on human colorectal cancer (CRC) cells. After HCT116 cells were treated with increasing doses of ATRA (10, 20, 40, and 60 μmol/L) for 24 h, ATRA concentrations between 20 and 60 μmol/L were found to increase miR-3666 expression in a dose-dependent manner. Moreover, ATRA-induced positive modulation of miR-3666 in HCT116 cells was associated with regulation of cancer cell viability, apoptosis, migration, and invasiveness. The authors also reported that 40 μmol/L ATRA treatment can decrease CRC cell viability by 50%, which was mediated by miR-3666 expression. Thus, ATRA may be a potential preventative agent against CRC development by regulating miR-3666 expression.