Modulation of AKT Pathway-Targeting miRNAs for Cancer Cell Treatment with Natural Products

Many miRNAs are known to target the AKT serine-threonine kinase (AKT) pathway, which is critical for the regulation of several cell functions in cancer cell development. Many natural products exhibiting anticancer effects have been reported, but their connections to the AKT pathway (AKT and its effectors) and miRNAs have rarely been investigated. This review aimed to demarcate the relationship between miRNAs and the AKT pathway during the regulation of cancer cell functions by natural products. Identifying the connections between miRNAs and the AKT pathway and between miRNAs and natural products made it possible to establish an miRNA/AKT/natural product axis to facilitate a better understanding of their anticancer mechanisms. Moreover, the miRNA database (miRDB) was used to retrieve more AKT pathway-related target candidates for miRNAs. By evaluating the reported facts, the cell functions of these database-generated candidates were connected to natural products. Therefore, this review provides a comprehensive overview of the natural product/miRNA/AKT pathway in the modulation of cancer cell development.

Apoptosis is a form of programmed cell death involving a series of activations for caspase signaling. Different miRNAs may exhibit functions opposite to apoptosis. Oncogenic In the following (Sections 2.1 to 2.8), AKT is connected to miRNA-mediated cell functions (Table 1). In the following (Sections 2.1-2.8), AKT is connected to miRNA-mediated cell functions (Table 1).
Several miRNAs reduce drug resistance and induce apoptosis in cancer cells (Table 1). Paclitaxel is a first-line drug for ovarian cancer therapy, but its resistance problem limits its treatment effects [66]. miR-181c binds to the 3 -UTR of glucose-regulated protein 78 (GRP78) and downregulates its expression, suppressing the resistance of ovarian cancer (SKOV3-PTX) cells toward paclitaxel by inducing apoptosis [54]. miR-181c is downregulated in paclitaxel-resistant ovarian cancer cells. In contrast, upregulating miR-181c alleviates this paclitaxel resistance by inactivating AKT [54]. Therefore, miR-181c reduces the paclitaxel resistance involving AKT.
Other apoptosis-inhibitory miRNAs, such as miR-14669, enhance drug resistance (Table 1). Drug resistance in breast cancer can be increased due to the activation of the PI3K/AKT/mTOR axis [67]. miRNA-mediated apoptosis in colon cancer cells and PI3K/AKT signaling may partly contribute to drug resistance [55]. Vincristine, which inhibits miR-14669, induces apoptosis in colon cancer cells and alleviates drug resistance by inactivating PI3K/AKT signaling, which is reversed by the upregulation of miRNA-14669 [55]. As mentioned above, some miRNAs regulate apoptosis with the involvement of AKT.

miRNAs Targeting AKT Regulate ER Stress in Cancer Cells
Several miRNAs show ER stress-modulating effects in cancer cells (Table 1). miR-495-3p mimics suppress the expression of ER chaperone GRP78 and inhibit the proliferation and migration of breast cancer (MDA-MB-231) cells, reducing pirarubicin resistance by inactivating AKT expression, which is reversed by miR-495-3p inhibition [56]. Although studies on the targeting of ER stress-associated AKT by miRNAs are rare (Table 1), the literature on ER stress-associated AKT effectors targeted by various miRNAs is discussed later (Section 3.3).

miRNAs Targeting AKT Regulate Ferroptosis in Cancer Cells
Several miRNAs show ferroptosis-modulating effects in cancer cells (Table 1). Clinically relevant radioresistant oral cancer cells [68] exhibit miR-7-5p (miR-7) overexpression, which is reversed by miR-7-5p inhibition [57]. miR-7-5p silencing enhances ROS and intracellular Fe 2+ content and upregulates ferroptosis gene (arachidonate 12-lipoxygenase, 12S type; ALOX12) expression and lipid peroxidation [57]. Although the reported study did not assess the involvement of AKT, miR-7-5p was predicted to target AKT3 according to the miRDB database (retrieval date: 12 October 2022) [69]. This warrants a detailed evaluation of the role of miR-7-5p in regulating ferroptosis through the targeting of AKT in the future. Although studies on the targeting of ferroptosis-associated AKT by miRNA are rare (Table 1), the literature on ferroptosis-associated AKT effectors targeted by various miRNAs is discussed later (Section 3.4).

miRNAs Targeting AKT Regulate DDR in Cancer Cells
Several miRNAs show DDR-modulating effects, such as DNA damage in cancer cells (Table 1). Bleomycin causes DNA damage in colon cancer (HCT116 and HT29) cells accompanied by downregulation of AKT1 protein expression, which is reversed by p53 and miR-374b knockdown [42]. Overexpressing p53 promotes expression of the bleomycininduced AKT1 regulator miR-374b, which is reversed by p53 knockdown. Hence, the p53/miR-374b/AKT1 axis regulates bleomycin-induced DNA damage in colon cancer cells.
In contrast, some miRNAs function as oncogenic miRNAs that inhibit cancer cell migration (Table 1). miR-136 is overexpressed in gastric cancer tissues and cells [44]. Downregulating miR-136 promotes antiproliferative effects and suppresses invasion in gastric cancer (MGC-803 and SGC-7901) cells. PTEN, a target of miR-136 and an inhibitor of AKT, shows low expression in gastric cancer tissues. miR-136 inhibition downregulates AKT phosphorylation [44]. Accordingly, some miRNAs regulate migration with the involvement of AKT.

Relationship between miRNA, AKT Effectors, and Cell Functions
In the following (Sections 3.1-3.8), AKT effectors are connected to miRNA-mediated cell functions (Table 2).

EBP1-Targeting miRNAs and Apoptosis
Several miRNAs inhibit apoptosis in cancer cells with the involvement of 4EBP1 (Table 2). miR-149-3p (miR-149*) is overexpressed in T-cell acute lymphoblastic leukemia (T-ALL) [115]. miR-149-3p mimics enhance T-ALL cell proliferation and suppress apoptosis in T-ALL cells by upregulating 4EBP1 and S6K (PRS6KB1), which is reversed by miR-149-3p inhibitors [115]. In contrast, several miRNAs promote apoptosis in cancer cells with the involvement of SREBP1. miR-101-3p (miR-101) is downregulated and mTOR and 4EBP1 are upregulated in endometrial cancer cells, promoting proliferation and invasion and suppressing apoptosis, which is reversed by the miR-101-3p mimic and/or si-mTOR [118]. Hence, several miRNAs modulate 4EBP1 expression in the regulation of apoptosis.

mTOR-, AKT1S1-, and DEPTOR-Targeting miRNAs and Autophagy
Several miRNAs induce the autophagy of cancer cells with the involvement of mTORC1 (Table 2). miR-126-3p shows a lower level in colon cancer tissues and cells than normal. miR-126-3p binds to 3 -UTR to downregulate mTOR expression. The overexpression of miR-126-3p inhibits proliferation and mTOR expression and induces autophagy and apoptosis in colon cancer cells, which is reversed by autophagy inhibitor bafilomycin A1, suggesting that miR-126-3p-induced apoptosis depends on autophagy through the modulation of mTOR expression [95].
Under hypoxia, miR-210 is overexpressed and induces autophagy and radioresistance in colon cancer cells by upregulating HIF1A (Table 2) [120]. Hence, miRNA may regulate autophagy with the involvement of HIF1A. This warrants further identification of HIF1Atargeting miRNAs in the regulation of autophagy.
Several studies have shown interactions between ER stress and miRNAs involving FOXO (Table 2). miR-494-3p is upregulated by ER stress inducers, such as tunicamycin and thapsigargin. miR-494-3p pretreatment suppresses tunicamycin-induced ER stress and promotes cell proliferation, which is reversed by miR-494-3p inhibition [80]. Hence, miR-494-3p shows reciprocal regulation with ER stress. Although this study did not assess the involvement of AKT effectors, miR-494-3p was predicted to target FOXO3 in accordance with the miRDB database (retrieval date: 12 October 2022) [69]. This warrants a detailed evaluation of miR-494-3p in the regulation of ferroptosis through the targeting of AKT effectors in the future.

c-Myc-Targeting miRNAs and ER Stress
Several miRNAs modulate ER stress in cancer cells with the involvement of c-Myc (Table 2). IRE1α, an ER stress sensor, is directly targeted by miR-1291 [85]. IRE1α enhances prostate tumor growth by activating c-Myc [131]. The inhibition of IRE1α downregulates c-Myc expression in natural killer cells. Accordingly, c-Myc is involved in miRNA-regulated ER stress.

mTOR-and DEPTOR-Targeting miRNAs and ER Stress
Several miRNAs regulate ER stress in cancer cells with the involvement of mTORC1 (Table 2). mTOR, one of the targets of miR-99b-5p and miR-100-5p (miR-100), can trigger amyloid β-induced apoptosis by causing ER stress [96]. DEPTOR silencing causes multiple myeloma death without activating the UPR [132]. The role of DEPTOR in regulating ER stress in conjunction with miRNAs needs further investigation.

c-Myc-Targeting miRNAs and DDR
Several miRNAs modulate DDR in cancer cells with the involvement of c-Myc (Table 2). c-Myc directly targets the miR-1245 promoter and enhances its expression, as well as causing downregulation of BRCA2 DNA repair-associated (BRCA2) gene expression and suppressing the ability for homologous recombination in breast cancer cells [88]. X-rays induce DNA damage, and they upregulate miR-449a but downregulate c-Myc in prostate cancer cells [92]. Accordingly, c-Myc is involved in regulating miRNA-mediated DDR.

FOXO-and c-Myc-Targeting miRNAs and Migration
Several miRNAs modulate migration with the involvement of FOXO (Table 2). FOXO1 is the direct target of miR-135a [79], which is overexpressed in liver cancer tissues and cells. Overexpression of miR-135a promotes the migration and invasion of liver cancer cells by upregulating MMP2 and downregulating FOXO3a, which is reversed by miR-135a inhibition [79]. Moreover, osteosarcoma tissues and cells exhibit low miR-33b-5p expression. miR-33b-5p inhibits osteosarcoma cell migration and invasion by downregulating c-Myc expression [90]. The CD44 and c-Myc genes are the targets of miR-34a-5p. Overexpression of miR-34a-5p suppresses the invasion of bladder cancer (UMUC3) cells by downregulating CD44 and c-Myc [93]. Accordingly, FOXO and c-Myc are involved in regulating miRNAmediated cancer cell migration.
HIF1A can modulate several hypoxia-induced miRNAs ( Table 2). miR-200c inhibits HIF1A expression and blocks the migration of lung cancer cells [126]. Therefore, overexpression of miR-200c may exhibit anticancer effects in tumors associated with hypoxia. Accordingly, SREBP1, 4EBP1, and HIF1A regulate the miRNA-mediated migration of cancer cells.

Modulation of miRNAs by Natural Products
Several natural products have been reported to modulate oncogenic and tumorsuppressor miRNAs and to control their targeting genes in the regulation of cancer cell proliferation [144][145][146]. Up-to-date, detailed information on the miRNAs, cancer cells, and functions related to these natural products is summarized in Table 3.
All chemical structures mentioned in Table 3 are provided in Figure 2.  Table 3.

Modulation of AKT-and AKT Effector-Targeting miRNAs by Natural Products
Many natural products show AKT-and AKT effector-modulating functions [197] with anticancer effects. However, a systemic understanding of the potential roles of miRNAs in regulating cell functions in coordination with natural products that modulate AKT and AKT effectors has not yet been achieved.

Modulation of AKT-and AKT Effector-Targeting miRNAs by Natural Products
Many natural products show AKT-and AKT effector-modulating functions [197] with anticancer effects. However, a systemic understanding of the potential roles of miRNAs in regulating cell functions in coordination with natural products that modulate AKT and AKT effectors has not yet been achieved.
Notably, most studies listed in Table 3 did not investigate the involvement of AKT and its effectors. Utilizing bioinformatics (the miRDB database [69]), the miRNAs listed in Table 3 were input into the miRDB database to check the predicted target genes related to AKT and AKT effectors. The retrieval results for the natural product-modulating miRNAs from Table 3 are summarized in Table 4. For example, miR-7-1-3p was predicted to target AKT1, while miR-103a-3p, miR-107, miR-124-3p, miR-148a-3p, miR-29b-3p, and miR-29c-3p were predicted to target AKT2 (Table 4). Many miRNAs were predicted to target AKT3, as shown in Table 4. The above natural product-affected miRNAs were derived from Table 3. Some AKT effectors are not listed because they could not be retrieved using the miRDB database (date: 11 November 2022).
Moreover, DEPTOR and HIF1A were also predicted to be targeted by several natural product-modulating miRNAs that were not reported to be associated with AKT and AKT effectors. Therefore, several miRNAs connected to natural product studies have bioinfor-matic predictions related to AKT and AKT effectors that should be verified in experiments. The contributions of AKT and AKT effectors to the modulation of miRNAs by natural products can thus be explored.

Conclusions
Many miRNAs regulate various cancer cell functions by targeting several genes. AKT and AKT effectors are highly expressed in various cancer cells and accompanied by the regulation of cell functions (apoptosis, autophagy, ER stress, ferroptosis, necroptosis, DDR, senescence, and migration). However, the connection between miRNAs and AKT and AKT effectors in controlling cell function remains unclear. Although several natural products may modulate miRNAs and the AKT pathway, their relationships lack systematic organization.
This review provided comprehensive information concerning the relationships between miRNAs and cancer cell functions (Figure 3). The roles of AKT and AKT effectors in miRNA-regulated cancer cell functions were clarified. Moreover, there is abundant information on the modulation of many miRNAs by natural products, but the involvement of AKT and AKT effectors has rarely been reported. Utilizing bioinformatics, the miRDB database was chosen for the prediction of AKT and AKT effectors related to miRNAs regulated by natural products. Consequently, the gaps between AKT, AKT effectors, miRNAs, and natural products were filled ( Figure 3).  However, the predictions of the targets for miRNAs obtained by searching miRDB must be carefully examined via detailed experiments because these predictions may be derived from specific types of cancer cells that may have different responses to other types of cancer cells. This review sheds light on the connections between natural products, miRNAs, and AKT pathways and can provide a future direction for exploring the regulation of cancer cell functions by natural products.  However, the predictions of the targets for miRNAs obtained by searching miRDB must be carefully examined via detailed experiments because these predictions may be derived from specific types of cancer cells that may have different responses to other types of cancer cells. This review sheds light on the connections between natural products, miRNAs, and AKT pathways and can provide a future direction for exploring the regulation of cancer cell functions by natural products.