MicroRNA-Based Combinatorial Cancer Therapy: Effects of MicroRNAs on the Efficacy of Anti-Cancer Therapies

The susceptibility of cancer cells to different types of treatments can be restricted by intrinsic and acquired therapeutic resistance, leading to the failure of cancer regression and remission. To overcome this problem, a combination therapy has been proposed as a fundamental strategy to improve therapeutic responses; however, resistance is still unavoidable. MicroRNA (miRNAs) are associated with cancer therapeutic resistance. The modulation of dysregulated miRNA levels through miRNA-based therapy comprising a replacement or inhibition approach has been proposed to sensitize cancer cells to other anti-cancer therapies. The combination of miRNA-based therapy with other anti-cancer therapies (miRNA-based combinatorial cancer therapy) is attractive, due to the ability of miRNAs to target multiple genes associated with the signaling pathways controlling therapeutic resistance. In this article, we present an overview of recent findings on the role of therapeutic resistance-related miRNAs in different types of cancer. We review the feasibility of utilizing dysregulated miRNAs in cancer cells and extracellular vesicles as potential candidates for miRNA-based combinatorial cancer therapy. We also discuss innate properties of miRNAs that need to be considered for more effective combinatorial cancer therapy.


Introduction
Although cancer cells may initially respond to treatment, not all cells are eliminated. This limited efficacy of cancer therapies can be due to several resistance mechanisms, ultimately leading to the recurrence of cancer and associated death. Biological factors underlying therapeutic resistance include the expression levels of drug transporters, which limit the cytoplasmic concentrations of therapeutic agents [1]. The efficient repair of damaged DNA in cancer cells also contributes to therapeutic resistance, especially for treatments aimed at damaging DNA. Besides, autophagy can act as a pro-survival mechanism by interrupting apoptosis induction in cancer cells, thereby restricting the efficacy of cancer treatments [2,3].
There are other factors responsible for cancer therapeutic resistance. Cancer stem cells (CSCs) are known to be resistant to cancer treatments due to several features, such as self-renewal potential, activation of the DNA damage response, and high levels of drug transporter [4]. Autophagy is also known to support the properties of CSCs [5,6]. Additionally, epithelial-mesenchymal transition (EMT) has been revealed 2.3.1. ABCB1 There is a possibility that the expression of transporters is transcriptionally regulated in an miRNA-dependent way. Wnt/β-catenin is one of the signaling pathways regulating the transcription of ABCB1 and causes multidrug resistance in cancers [32]. Frizzled 7 (FZD7), a receptor for Wnt ligands, has been identified as an miR-27-3p target. Ectopic introduction of miR-27-3p downregulates ABCB1 and augments 5-fluorouracil-induced cell death [33]. Recent studies also confirmed that miR-122-5p and miR-506-3p could elevate drug-induced cell death in hepatocellular carcinoma and colorectal cancer cells, respectively, by negatively regulating β-catenin and ABCB1 levels [34,35]. There is another possibility that miR-506-3p regulates ABCB1 expression by targeting the enhancer of zeste homolog 2 (EZH2), which is a possible transcription regulator of ABCB1 [36,37]. In addition, a decrease in ABCB1 expression can be mediated by miR-199a-3p and miR-218-5p via regulating mTOR and protein kinase C epsilon (PRKCE, also known as PKCε), respectively [38,39]. Since both mTOR and PKCε activate the signal transducer and activator of transcription 3 (STAT3), a transcription factor of ABCB1 [40][41][42], it is feasible that miR-199a-3p and miR-218-5p could downregulate ABCB1 levels through a common mediator. As noted above, miR-491-3p directly controls ABCB1 expression. Interestingly, miR-491-3p also transcriptionally represses ABCB1 levels by regulating Sp3, which is known as a transcription factor of ABCB1 [19]. In addition, it was intriguingly noted that miR-508-5p, which directly regulates ABCB1, also targets zinc ribbon domaincontaining 1 (ZNRD1), thereby negatively regulates the transcription of ABCB1 [21,43] (Figure 1 and Table 1).   Table 1. Drug transporter-related miRNAs and their effects on the susceptibility of cancer cells to anti-cancer treatments.

MiRNAs
Target Gene(s) Cancer Type Effect of MiRNAs Ref.

ABCB4 and ABCG2
In addition to ABCB1, transcriptional regulations of ABCB4 (also called MDR3) and ABCG2 are associated with miRNAs. In breast cancer cells, doxorubicin resistance is regulated by spindlin1 (SPIN1), which is an upstream regulator of ABCB4 [45]. This study provided evidence that SPIN1 is targeted by the miR-148/152 family (miR-148a-3p, miR-148b-3p, and miR-152-3p) and that the miR-148/152 family thereby effectively improves the efficacy of doxorubicin-mediated cytotoxicity [45]. Although further research is necessary to unravel the precise pathways of the SPIN1-ABCB4 axis, it might be associated with peroxisome proliferator-activated receptor alpha (PPARα) owing to the fact that PPARα is a transcription factor of ABCB4 [47] and that SPIN1-mediated activation of Akt could inactivate glycogen synthase kinase 3 beta (GSK3β), which is a negative regulator of PPARα [48,49]. Moreover, cisplatin resistance could be reversed through miR-495-3p-mediated ABCG2 suppression [46]. In this study, it was uncovered that miR-495-3p directly modulates the expression of ubiquitin-conjugating enzyme E2 C (UBE2C), a transcription factor of ABCG2 [46] (Figure 1 and Table 1).

A MiRNA Regulating Degradation of a Drug Transporter
A recent study has shown that miR-20a-5p is involved in the post-translational regulation of ABCB1. In miR-20a-5p over-expressing cells, the levels of mitogen-activated protein kinase 1 (MAPK1, also known as ERK2) are down-regulated, leading to the alleviation of therapeutic resistance, together with the inactivation of ribosomal protein S6 kinase (RSK) [44]. RSK is known to destabilize ubiquitin-conjugating enzyme E2 R1 (UBE2R1), thereby protecting ABCB1 from the ubiquitin-proteasomal degradation pathways [50,51] (Table 1).

DNA Damage Repair in Cancer
Effective DNA damage repair followed by cancer therapies contributes to the limited efficacy of treatments and the appearance of therapeutic resistance [2]. Since the inhibition of DNA repair pathways could make cancer cells more vulnerable to anti-cancer therapies, the elucidation of precise mechanisms of DNA damage repair could lead to the development of a strategy for more successful cancer therapy [52]. For example, temozolomide induces DNA double-strand breaks that could be repaired by the homologous recombination pathway promoted by cyclin dependent kinase 1 (CDK1) and CDK2. Indeed, pharmacological inhibition of CDK1/2 sensitizes glioblastoma cells to temozolomide [53]. A recent study demonstrated that zinc finger protein 830 (ZNF830) plays a role in the repair of DNA double-strand breaks and that knockdown of ZNF830 sensitizes lung cancer cells to olaparib, a DNA-damaging agent [54]. In addition, the importance of miRNAs in the regulation of DNA repair mechanisms has been underscored. Thus, the modulation of miRNA levels has been suggested as a therapeutic strategy to advance the efficacy of cancer treatments.

MiRNAs Negatively Regulating DNA Repair Mechanisms
Several miRNAs have been identified to suppress cellular factors associated with DNA repair pathways, implying a possibility that the over-expression of an miRNA acting as the repressor of DNA repair could have a therapeutic benefit in cancer.

MiR-7-5p
Poly ADP-ribose polymerase 1 (PARP1) is known to recruit DNA repair factors to the site of DNA double-strand breaks. In lung cancer cells, the cytotoxicity of doxorubicin could be enhanced by miR-7-5p. PARP1 is down-regulated by miR-7-5p, leading to impaired DNA damage repair. Notably, miR-7-5p levels show a negative correlation with the status of doxorubicin resistance [55] (Table 2).

MiR-30-5p
It was demonstrated that doxorubicin resistance is related to p53 mutation in breast cancer. The expression of miR-30-5p could be transcriptionally regulated by wild-type p53, and this miRNA directly targets two DNA repair elements, Fanconi anemia complementation group F protein (FANCF) and REV1 DNA directed polymerase (REV1). In p53-mutated cells, both FANCF and REV1 are abundantly expressed due to the abolishment of miR-30-5p induction, thus advancing doxorubicin resistance [56] (Table 2).

MiR-138-5p
The excision repair cross-complementation group (ERCC) is known to participate in the nucleotide excision repair (NER) pathway [57]. Recent evidence suggested that miR-138-5p reverses drug resistance in gastric cancer cells as a result of targeting ERCC1 and ERCC4 genes [58] (Table 2). Additionally, several studies demonstrated that miR-138-5p is a tumor suppressor and suppresses the growth and metastasis of cancer cells [59,60]. However, this miRNA functions as an oncogenic miRNA in glioma stem cells via the regulation of several tumor-suppressive genes, including caspase-3 [61]. These findings indicate the complex role of miR-138-5p and suggest that further investigation into the effects of this miRNA on cellular signaling is necessary.

MiR-182-5p and MiR-4429
BRCA1 DNA repair associated (BRCA1) functions in the repair of DNA double-stranded breaks by enhancing the recombinase activity of RAD51 recombinase (RAD51) [62]. A recent study demonstrated that miR-182-5p regulates chemosensitivity via modulation of the expression of DNA repair genes. BRCA1 and RAD51 are targeted by miR-182-5p [63,64]. In particular, the treatment of panobinostat, a histone deacetylase inhibitor, could impede DNA repair following irradiation or 2 -C-cyano-2 -deoxy-1-β-d-arabino-pentofuranosyl-cytosine (CNDAC) treatments by inducing miR-182-5p [63]. Furthermore, another study provided evidence that there is a negative correlation between miR-4429 levels and radioresistance in cervical cancer cells. Indeed, the up-regulation of miR-4429 improves the efficacy of irradiation by repressing RAD51, suggesting the possibility of using miR-4429 mimics to defeat radioresistance, one of the major causes of cancer recurrence [65] (Table 2).

MiR-205-5p and MiR-211-5p
Zinc finger E-Box binding homeobox 1 (ZEB1) has been proven to be critical for the regulation of checkpoint kinase 1, which coordinates DNA damage response signaling [66]. Also, PKCε could activate the DNA-dependent protein kinase by modulating the nuclear accumulation of the epidermal growth factor receptor [67]. A recent study showed that miR-205-5p could impair DNA repair pathways by targeting ZEB1 and PKCε, leading to an elevated response to radiotherapy in prostate cancer cells [68]. Furthermore, the augmentation of carboplatin sensitivity can be achieved by miR-211-5p, owing to its ability to target multiple genes involved in DNA damage response, namely DNA polymerase eta (POLH), tyrosyl-DNA phosphodiesterase 1 (TDP1), ATRX chromatin remodeler (ATRX), mitochondrial ribosomal protein S11 (MRPS11), and ERCC excision repair 6 like 2 (ERCC6L2) [69] (Table 2).

MiRNAs Positively Regulating DNA Repair Mechanisms
By contrast, miRNAs are capable of promoting DNA damage repair and stabilizing the DNA replication fork, resulting in the aggravation of therapeutic resistance. This indicates that the knockdown of a resistance-associated miRNA could have a therapeutic benefit in cancer.

MiR-488-3p
Eukaryotic translation initiation factor 3 subunit A (EIF3A) has been demonstrated to down-regulate NER activity by regulating the levels of NER factors. Therefore, the knockdown of EIF3A interrupts DNA damage-induced cell death [71,72]. A recent study revealed that miR-488-3p levels are higher in cisplatin-resistant lung cancer cells than in parental cells, and NER is activated by miR-488-3p, which targets EIF3A [73] (Table 2).

MiR-493-5p
Stabilization of the DNA replication fork is one of the causes of therapeutic resistance. For example, the depletion of a chromatin-remodeling factor, such as chromodomain helicase DNA binding protein 4 (CHD4), confers cisplatin resistance in BRCA2-mutated cancer cells [74]. A similar conclusion was reached by investigating the role of miR-493-5p. Resistance to cisplatin and olaparib is mediated by miR-493-5p over-expression in BRCA2-mutated ovarian cancer cells because of the ability of miR-493-5p to target multiple genes (e.g., CHD4) involved in regulating single-strand annealing DNA repair and genomic stability [75] (Table 2). Down-regulation of miR-493-5p levels leads to enhanced responsiveness to cisplatin and olaparib [75] miR-520g-3p, miR-520h APEX1 Multiple myeloma Over-expression of both miR-520g-3p and miR-520h hampers the growth of bortezomib resistant multiple myeloma cells [69] miR-4429 RAD51 Cervical cancer Over-expression of miR-4429 enhances radiosensitivity [65] Cells 2020, 9, 29 9 of 32

General Mechanisms of Autophagy
Autophagy is a highly conserved cellular process by which cytoplasmic materials are isolated into double-membrane autophagosomes that fuse with lysosomes. The autophagic degradation activity (also known as autophagic flux) is known to maintain cellular homeostasis and protein/organelle quality control [76]. Several components are involved in the machinery of autophagy. Generally, mTOR complex 1 (mTORC1) represses unc-51-like autophagy-activating kinase (ULK), thus leading to the inhibition of autophagy induction. After being released from the inhibitory actions of mTORC1, the ULK complex, including the FAK family-interacting protein of 200 kDa (FIP200) and autophagy-related 13 (ATG13), is activated. Ultimately, the activated ULK complex induces autophagy by phosphorylating BECLIN1 and its binding partners, VPS34 (also known as phosphatidylinositol 3-kinase catalytic subunit type 3, PIK3C3) and ATG14 [77,78]. ATG12 is conjugated to ATG5 via ATG7, and then the ATG12-ATG5 conjugate forms a complex with ATG16L1. The ATG5-ATG12-ATG16L1 complex facilitates the lipidation of LC3I to LC3II, which is required for autophagosome formation [79]. Since multiple factors are necessary to accomplish autophagic processes, autophagy can be inhibited at several points.

Dual Roles of Autophagy in Cancer
In the context of cancer, autophagy plays a pivotal role in the regulation of cellular events, such as cell death, cancer stemness, and therapeutic resistance. Regarding cancer therapy, strategies of both stimulation and inhibition of autophagy have been considered. For example, the silencing of ATG5 and ATG12 could inhibit autophagy, consequently attenuating CSC properties [6,80]. The blockage of autophagic flux leads to cell death by augmenting stress-activated proteins, such as JNK and p38 [81]. It was also reported that treatment with paclitaxel can lead to the activation of autophagy, which, in turn, confers resistance to paclitaxel. Therefore, the inhibition of BECLIN1 enhances the anti-cancer activity of paclitaxel via the attenuation of cytoprotective autophagy in ovarian cancer cells [3]. Likewise, cell death induced by an epigenetic agent could be stimulated by suppressing BECLIN1 expression in drug-resistant leukemia stem cells [82].
By contrast, autophagy triggered by caloric restriction mimetics was proposed to deplete regulatory T cells, reinforce cancer immunosurveillance, and improve the efficacy of mitoxantrone as well as oxaliplatin [83]. Such discrepancy could be due to the fact that cellular factors involved in the process of autophagy regulate other cellular responses, such as cytokinesis, endocytosis, cell growth, and cell death, in an autophagy-independent manner, and that the effects of autophagy on the fate of cancer cells are dependent on p53 status [84][85][86]. In this review, we focus on the inhibitory roles of miRNAs in cytoprotective autophagy along with their effects on chemotherapeutic agents.

MiRNAs Regulating mTOR and mTORC1
As mentioned above, autophagy is negatively regulated by mTORC1. An association between mTORC1-dependent autophagy and miRNAs has been investigated in conjunction with the anti-cancer activity of therapeutic agents. Nuclear factor erythroid 2 like-2 (NRF2) is a mediator of therapeutic resistance to Trichostatin A. A mechanism underlying NRF2-mediated resistance suggested that NRF2 could stimulate autophagy induction following Trichostatin A treatment via transcriptionally up-regulating the levels of miR-129-3p, which targets mTOR [87] (Table 3). Actually, two functional miRNAs (miR-5p and miR-3p) can be derived from the same miRNA precursor [88]. Since both miR-129-3p and miR-129-5p (see Table 1) are derived from the same precursor, and both mature miRNA strands are functionally different in cells, it is feasible that over-expression of the miR-129 precursor in cancer cells using vector systems triggers cytoprotective autophagy, but inhibits ABCB1 levels. miR-410-3p HMGB1 Pancreatic cancer Over-expression of miR-410-3p improves gemcitabine-induced cell death and growth inhibition in drug-resistant cells [102] miR-520-3p ATG7 Hepatocellular carcinoma Replacement of miR-520-3p increases the sensitivity of drug-resistant cells to doxorubicin by enhancing cell death and growth inhibition [103] miR-874-3p ATG16L1 Gastric cancer Restoration of miR-874-3p sensitizes cells to 5-fluorouracil and cisplatin [104] Another recent study showed that RAB12 member RAS oncogene family (RAB12) is an endogenous mTORC1 inhibitory factor and protects cancer cells from drug-induced cell death by activating cytoprotective autophagy. RAB12 is targeted by miR-148-3p; therefore, the introduction of miR-148-3p in cancer cells can reverse therapeutic resistance [89] (Table 3). Since miR-148-3p negatively controls both cytoprotective autophagy and ABCB4 expression (see Table 1), this implies the possibility of the potential therapeutic application of miR-148-3p to suppress CSC properties.

MiRNAs Regulating HMGBs
The possibility of miRNA-mediated chemosensitization was also associated with the function of high mobility group box 1 (HMGB1) and HMGB2. In terms of autophagy, HMGB1 is known to promote autophagy by preventing the interaction between Bcl-2 and BECLIN1 in the cytoplasm [105]. A reduced form of HMGB1 in the extracellular space also facilitates BECLIN1-dependent autophagy [106]. Direct inhibition of HMGB1 by miR-34-5p and miR-410-3p impedes autophagy induction, reversing resistance to several chemotherapeutic agents [92,102]. Likewise, HMGB2 is presumed to promote autophagy by the same mechanisms as HMGB1. Negative regulation of HMGB2 levels in miR-23-3p over-expressing cells is one of the causes of autophagy inhibition [90] (Table 3).

MiRNAs Regulating the Lipidation of LC3
In the case of miR-23-3p, another identified target is ATG12. This further provides a mechanism whereby miR-23-3p sensitizes cancer cells to chemotherapeutic agents [90]. It was also found that ATG12 is directly targeted by miR-214-3p [99]. This association between ATG12 and miR-214-3p explains the role of this miRNA in promoting radio-sensitivity, since the exposure of irradiation can induce ATG12-mediated autophagy, which, in turn, leads to radio-resistance [99] ( Table 3). However, miR-214-3p protects lung cancer cells from radiotherapy-induced cell death [107], indicating that the overall effects of miR-214-3p on cancer therapeutics could be distinct in a cellular context-dependent manner.
Additional studies have also shown the effects of autophagy-regulating miRNAs on therapeutic agents. ATG7, a mediator of ATG12-ATG5 conjugation, is targeted by miR-520-3p, and over-expression of miR-520-3p sensitizes drug-resistant cells to chemotherapy [103]. ATG5 can be targeted by multiple miRNAs, including miR-137-3p, miR-142-3p, and miR-224-3p. The ability of these miRNAs to restrain autophagy contributes to the sensitization of cancer cells to anti-cancer agents [95,97,100]. In addition to ATG5, the inhibitory effects of miR-142-3p on therapeutic resistance can be mediated by targeting ATG16L1 [97]. Moreover, a recent study also indicated that autophagy inhibition in miR-874-3p over-expressing cells abrogates therapeutic resistance [104] (Table 3).

Cancer Stem Cells
Cancer stem cells (CSCs), also known as cancer stem-like cells and tumor-initiating cells, have been identified in cancers, and they have notable characteristics of stemness, such as self-renewal and multilineage differentiation capabilities. Several signaling pathways, such as Wingless (Wnt)/β-catenin, Notch, Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), Hedgehog, nuclear factor kappa B (NF-κB), ERK, and PI3K/Akt, regulate the induction and maintenance of CSC properties [111,112]. In terms of cancer therapies, CSCs are known to be resistant to anti-cancer treatments, as discussed in the introduction. Therefore, targeted therapy against signaling pathways involved in CSCs has been suggested to improve the anti-cancer activity of therapeutic agents [4,113]. Indeed, accumulating evidence from recent studies evidently demonstrates the noticeable effects of miRNAs on CSC-associated signaling pathways ( Figure 2 and Table 4).  Table 4. Silencing of miR-93-3p and miR-105-5p enhances the sensitivity to cisplatin and chemoradiotherapy [116] miR-124-3p USP14 Lung cancer Over-expression of miR-124-3p increases the effects of gefitinib on apoptosis and growth inhibition [120] miR-136-5p NOTCH3 Ovarian cancer Over-expression of miR-136-5p escalates paclitaxel-induced cell death in drug-resistant cells [124] Ectopic expression of miR-139-5p enhances the sensitivity of CD44+/CD133+ cells to  Table 4.

Notch Signaling and CSCs
Since Notch signaling functions as one of the potential mediators of CSC maintenance, the targeted inhibition of Notch signaling has been considered as a strategy to eradicate CSC populations. For example, an application of pharmacological or endogenous inhibitors of Notch receptors down-regulates stemness factors (e.g., OCT4) and potentiates the efficacy of anti-cancer agents, including cisplatin and sorafenib, in renal cell carcinoma cells [121].

MiRNAs Regulating Notch Receptor 1 and 3
Notch signaling is also affected by miRNAs that post-transcriptionally regulate the levels of Notch receptors. Notch 1 is targeted by miR-34-5p and miR-139-5p in breast cancer and colorectal carcinoma cells, respectively [122,123]. In doxorubicin-resistant breast cancer cells, miR-34-5p is down-regulated compared to parental cells, implying the possibility that this miRNA is pertinent to therapeutic resistance. In addition, over-expression of miR-34-5p represses the self-renewal capacity of breast cancer stem cells [122]. In colorectal cancer cells, expression of miR-139-5p is lower in oxaliplatinand vincristine-resistant carcinoma cells than in non-resistant cells. These drug-resistant cells have more CD44-and CD133-positive populations than non-resistant cells. As expected, the over-expression of miR-139-5p renders CD44-and CD133-positive cells sensitive to several anti-cancer drugs [123]. Furthermore, Notch 3 is directly targeted by miR-136-5p; therefore, CSC spheroid formation is restricted by this miRNA [124] (Table 4).

A MiRNA-Regulating Notch Receptor 2 and its Downstream Signaling Factor
Recombination signal-binding protein for immunoglobulin kappa J region (RBPJ) is the transcription mediator of Notch signaling. Upon ligand binding, the Notch intracellular domain (NICD) is released from the plasma membrane and interacts with RBPJ to activate target genes [125]. It was demonstrated that miR-195-5p suppresses the levels of stemness factors (e.g., CD133 and SOX2) in colorectal CSCs by targeting Notch 2 and RBPJ [126] (Table 4).

JAK/STAT3 Signaling and CSCs
JAK/STAT3 signaling contributes to CSC maintenance, as well as therapeutic resistance. For instance, the inhibition of JAK/STAT3 signaling with ruxolitinib results in a reduction in CSC hallmarks, such as spheroid formation capacity [127]. JAK/STAT3 signaling also induces carnitine palmitoyltransferase 1B (CPT1B) and activates the fatty acid beta oxidation (FAO) pathway, which is a critical regulator of CSC self-renewal. Indeed, an inhibition of JAK/STAT3 and FAO pathways sensitizes cells to chemotherapies [128].

MiRNAs Regulating Negative Regulators of JAK/STAT3 Signaling
JAK/STAT3 pathways could be modulated by miRNAs, eventually affecting the therapeutic efficacy of anti-cancer agents. For instance, miR-196-5p is highly expressed in colorectal cancer tissues compared to non-cancerous tissues. Both the stemness and therapeutic resistance of colorectal cancer cells are promoted by miR-196-5p. It was identified that miR-196-5p directly targets the suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3), which are negative regulators of JAK/STAT3 signaling [129].
The transcriptional activity of STAT3 is hampered by protein tyrosine phosphatase non-receptor types (PTPNs) [130]. A recent study demonstrated that multiple negative regulators of JAK/STAT3 signaling, including SOCS2, SOCS5, PTPN1, and PTPN11, are known to be directly suppressed by miR-589-5p. Therefore, CSC properties can be reinforced by this miRNA in hepatocellular carcinoma cells [131] (Table 4).

A MiRNA and Hedgehog Signaling
Activation of Hedgehog (Hh) signaling is triggered by the binding of Hh ligands to patched (PTCH) receptors. Interactions of Hh ligands with PTCH receptors stimulate smoothened (SMO) and glioma-associated oncogene (GLI) transcription factors [132,133]. The inhibition of SMO and GLI decreases the number of aldehyde dehydrogenase (ALDH)-positive CSCs in melanoma [134]. This indicates the significance of Hh signaling in CSC maintenance. A recent finding indicated that miR-324-5p could directly control SMO and GLI1 expression, and subsequently inhibit the colony formation of multiple myeloma cells in a stem cell medium [135] (Table 4).

MiRNAs Regulating NF-κB Signaling and PD-L1
NF-κB signaling regulates multiple cellular processes, including metastasis and CSC properties [136]. A recent study demonstrated that miR-423-5p targets inhibitor of growth 4 (ING4), a negative regulator of NF-κB. Therefore, this miRNA contributes to the augmented expression of glioma stem cell factors, including CD133 and SOX2 [137] (Table 4). Recently, miR-423-5p was also reported to promote the metastasis of lung adenocarcinoma and gastric cancer cells [138,139], implying that knockdown of miR-423-5p may have a therapeutic benefit in several cancer types.

Indirect Regulation of Stemness Factors
Cancer stemness is also maintained by other factors, such as Golgi phosphoprotein 3 (GOLPH3), yin and yang 1 (YY1), and neurofilament light (NEFL). These factors are capable of modulating the expression levels of various stemness factors in a positive manner.

GOLPH3
In bladder cancer cells, the over-expression or knockdown of GOLPH3 increases or decreases the expression of CSC factors (CD44, KLF4, ALDH1, and SOX2), respectively. The levels of miR-34-5p are down-regulated in gemcitabine-and cisplatin-resistant bladder cancer cells, contributing to enriched CSC populations [147] (Figure 2 and Table 4). Furthermore, the ability of miR-34-5p to target multiple genes (see Tables 3 and 4) can explain why this miRNA functions effectively with several chemotherapeutics.

EMT and Cancer Stemness
EMT is known to confer CSC properties on cancer cells. For example, transforming growth factor-beta (TGF-β) and hepatocyte growth factor (HGF) signaling often induces EMT by activating EMT transcription factors and enhances the stemness of cancer cells [7,151,152]. This indicates that inhibition of the mechanisms underlying EMT can improve therapeutic responses by blocking the transformation of cancer cells to the CSC state.

MiRNAs Regulating TGF-β Signaling
Owing to the effort to screen EMT-regulating miRNAs, miR-509-5p was identified to repress the EMT process by targeting vimentin (VIM) and HMGA2, both of which are positive regulators of TGF-β signaling [153]. In this study, miR-1243 was also demonstrated to inhibit the EMT process by targeting SMAD family member 2 (SMAD2) and SMAD4. Certainly, over-expression of either miR-509-5p or miR-1243 augments gemcitabine efficacy [153]. Recently, it was demonstrated that treatment of 5-fluorouracil reduces the expression of miR-204-5p, which was identified to target TGF-β receptor 2 (TGFBR2) [154] (Table 5).
Integrins are known to induce the transmembrane signaling pathways to modulate the EMT process. For instance, it was noted that the translational activation of integrin subunit β 3 (ITGB3) supports TGF-β pathways and advances malignant phenotypes, such as EMT. Thus, targeting integrins with antibodies has been attempted for cancer therapy [155,156]. A recent study of miR-483-3p indicated that this miRNA enhances the effectiveness of gefitinib by targeting ITGB3 [154] (Table 5). Although multiple signaling pathways coordinately regulate cancer stemness, the dysregulation of miR-483-3p may play a partial role in the ITGB3-mediated regulation of stemness in cancer, since ITGB3 was reported to drive cancer stemness [157].

MiRNAs Directly Regulating EMT-Related Transcription Factors and Markers
The chemosensitization effects of miR-204-5p are also mediated by targeting ZEB1 in addition to TGFBR2 [165]. Besides, it has been shown that the expression of EMT-promoting transcription factors is directly modulated by other miRNAs. In ovarian cancer cells, miR-363-3p targets snail family transcriptional repressor 1 (SNAI1). As a consequence, the over-expression of miR-363-3p in cancer cells reverses EMT-mediated drug resistance [166]. In addition, the regulation of ZEB1 expression is mediated by miR-574-3p and miR-708-3p in gastric and breast cancer cells, respectively. Both miRNAs negatively regulate the process of EMT and render cancer cells susceptible to chemotherapy agents [133,167]. In the case of miR-708-3p, it also regulates EMT markers, cadherin 2 (CDH2, also known as N-cadherin) and VIM, by directly binding to their 3 UTRs [133]. In addition, miR-128-3p and miR-873-5p were identified to make cancer cells more responsive to anti-cancer agents by suppressing ZEB1 expression [162,169] ( Table 5). ZEB1 can transcriptionally induce expression of PD-L1 [170]. Therefore, miR-873-5p has a great possibility of potentially regulating ZEB-1/PD-L1 axis, since this miRNA also targets PD-L1 (see Table 4).

MiRNAs Indirectly Regulating EMT-Related Transcription Factors
Death-effector domain-containing DNA-binding protein (DEDD) inversely controls the process of EMT by attenuating the expression of EMT-promoting elements, such as SNAI1 and Twist family BHLH transcription factors (Twist) [171]. In gastric cancer cells, a reduction in miR-17-5p interrupts the EMT process by up-regulating its target, DEDD, thereby affecting the therapeutic resistance of gastric cancer cells [161] (Table 5).

Extracellular Vesicles (EVs)
There is mounting evidence that EVs (exosomes and microvesicles) play a critical role in the intercellular communication by transferring cargo molecules, such as cytosolic proteins, lipids, and RNA [172]. Exosomes are released into extracellular space by fusion of multivesicular endosomes with the cell membrane, whereas microvesicles are developed directly from the cell membrane [172]. It has been demonstrated that EVs from neighboring cells can affect various biological properties of cancer cells, such as proliferation, metastasis, hypoxia tolerance, and therapeutic resistance [173,174]. For example, mitochondrial DNA in EVs derived from cancer-associated fibroblasts (CAFs) contributes to the development of resistance to hormone therapy in breast cancer [175]. Also, cancer cells exposed to anti-cancer agents can deliver EVs harboring drug efflux and pro-survival proteins into circumjacent cancer cells, thereby playing a part in enhancing cell survival and therapeutic resistance [176,177].

EVs from CAAs, TAMs, and CAFs
In addition to DNA and proteins in EVs, miRNAs are also enriched in EVs derived from cancer-associated cells, hence affecting the expression of cellular factors involved in therapeutic resistance.

EVs from Drug-Resistant Cancer Cells and CSCs
It has been underscored that drug-resistant cancer cells also secrete EVs to transfer miRNAs into adjacent cancer cells and that transferred miRNAs contribute to the development of therapeutic resistance in non-resistant cells.
The enriched expression of miR-32-5p, which targets PTEN, was identified in exosomes secreted from 5-fluorouracil-resistant hepatocellular carcinoma cells. The treatment of non-resistant cancer cells with these exosomes confers therapeutic resistance to several agents, such as 5-fluorouracil, oxaliplatin, gemcitabine, and sorafenib, owing to the downregulation of PTEN and the activation of PI3K/Akt pathways [180] (Figure 3).

MiR-155-5p
Exosomes harboring miR-155-5p are secreted by paclitaxel-or doxorubicin-resistant gastric and breast cancer cells, as well as breast CSCs. Delivery of these exosomes can convert the phenotype of drug-sensitive cells to drug-resistant cells through miR-155-5p-mediated alteration of the levels of negative regulators of EMT, including GATA binding protein 3 (GATA3), tumor protein p53-inducible nuclear protein 1 (TP53INP1), CCAAT enhancer-binding protein beta (CEBPB), and forkhead box O3A (FOXO3A) [181,182] (Figure 3). However, miR-155-5p acts as a tumor-suppressive miRNA and sensitizes multiple myeloma cells to bortezomib [9], indicating that either the replacement or knockdown of this miRNA can be applied for cancer therapy with other anti-cancer agents, depending on the type of cancer.

Conclusions
Resistance to anti-cancer treatments is intricate, and divergent mechanisms are involved and act together. In light of this, the simultaneous inhibition of signaling components has been considered to improve the current condition of cancer treatment. For instance, a recent study demonstrated that combined targeting of the Hh pathway and autophagy efficiently inhibits cell proliferation and induces cell death in drug-resistant cells [188], providing evidence that combination therapy is a reasonable approach to enhance therapeutic efficacy in cancers. However, the occurrence of therapeutic resistance continues to be a significant cause of the failure of current combination therapy. It has been demonstrated that the acquisition of resistance can occur following combination treatment and that the use of a third agent can address the emergence of resistance by inhibiting a factor involved in the resistance [10,11,189]. This implies that the multiple targeting of resistance-mediating factors is better than the current approach to increase the cellular response to cancer therapy and that the development of new combination strategies is still required to improve treatment outcomes.
A growing number of investigations have successfully demonstrated that single miRNA can control the effectiveness of anti-cancer treatments by regulating the expression of resistance-related factors, as highlighted in this article. Most of the investigations have identified a single target gene for an miRNA, implying that the identified target of an miRNA at least partly accounts for the regulation of efficacy of cancer therapeutics. Since an miRNA can regulate diverse biological pathways owing to its multiple targets, it is probable that there are other identified/unidentified targets, which modulate multiple resistance-related signaling pathways. Further research is required to address the extensive miRNA-target gene interactions. Additionally, there are some considerations relating to the characteristic features of miRNAs when designing miRNAs for therapeutic purposes.
Some miRNAs have dual roles, depending on cancer type, since they can target both oncogenes and tumor suppressors. Therefore, the appropriate selection of miRNAs that are particularly relevant to the specific cancer type is necessary to achieve a better response to treatments through miRNA-based combinatorial cancer therapy. For this purpose, further investigations are required to comprehensively identify the dysregulated miRNAs in therapy-resistant cancer cells and tissues. In addition, miRNAs that influence the efficacy of anti-cancer therapies could also influence the physiological processes of non-cancerous cells. For example, miR-138-5p was proposed to negatively control the osteogenic differentiation of human mesenchymal stem cells [190]. In the case of miR-155-5p, knockdown of this miRNA induces the levels of inflammatory cytokines [191]. This emphasizes the need to consider cancer-specific modulation of miRNA levels to avoid unintended side effects of miRNA-based combinatorial cancer therapy. Indeed, the targeted delivery of miRNA modulators to cancer cells has been developed to minimize feasible side effects [192,193].
A recent study evidently demonstrated that vector-based miRNA expression could generate both miR-202-3p and miR-202-5p [194]. In this study, miR-202-3p inhibits the proliferation of colorectal cancer cells, but miR-202-5p has no effect on cell proliferation. Although miR-202-5p is not associated with the growth regulation of colorectal cancer cells, another study demonstrated that miR-202-5p regulates the sensitivity of doxorubicin in breast cancer cells [195]. As discussed in Section 4.3, miR-129-3p and miR-129-5p also have opposite functions in the regulation of drug efficacy. Therefore, functional differences between miR-3p and miR-5p are an additional concern that should be considered when evaluating the effects of miRNAs on other anti-cancer therapies. In addition, the modulation of single miRNA expression could only partially regulate signaling pathways that regulate the cellular response to cancer therapeutics since (1) over 2000 mature miRNAs have been described in miRBase [196], (2) multiple miRNAs function cooperatively with other miRNAs [197], (3) the same gene is undoubtedly regulated by multiple miRNAs, and (4) alternative pathways can be induced by the modulation of miRNA expression owing to the direct and/or indirect effects of a miRNA on transcription factors [198]. Therefore, it appears essential to investigate whether simultaneous replacement or knockdown of functionally relevant miRNAs is more potent than single miRNAs for miRNA-based combinatorial cancer therapy.
A considerable number of studies have been conducted and demonstrated the benefit of miRNA-based combinatorial cancer therapy in combating cancer. However, it is essential to carefully consider several characteristic features of miRNAs, including their dual roles, side effects, and the functional differences between miR-3p and miR-5p, when designing and evaluating the efficacy of miRNA-based combinatorial cancer therapy. Further investigation of miRNA's target genes is also necessary to comprehensively elucidate the functions of miRNAs and to analyze miRNA-signaling pathway networks in cells. Advanced knowledge of the multifactorial nature of miRNAs will enable miRNA-based combinatorial cancer therapy to move toward clinical application in the future.