Research Progress in microRNA-Based Therapy for Gastric Cancer

Abstract Gastric cancer (GC) is one of the leading causes of tumor-related mortality. In addition to surgery and endoscopic resection, systemic therapy remains the main treatment option for GC, especially for advanced-stage disease and for cases not suitable for surgical therapy. Hence, improving the efficacy of systemic therapy is still an urgent problem to overcome. In the past decade, the essential roles of microRNAs (miRNAs) in tumor treatment have been increasingly recognized. In particular, miRNAs were recently shown to reverse the resistance to chemotherapy drugs such as 5-fluorouracil, cisplatin, and doxorubicin. Synthesized nanoparticles loaded with mimics or inhibitors of miRNAs can directly target tumor cells to suppress their growth. Moreover, exosomes may serve as promising safe carriers for mimics or inhibitors of miRNAs to treat GC. Some miRNAs have also been shown to play roles in the mechanism of action of other anti-tumor drugs. Therefore, in this review, we highlight the research progress on microRNA-based therapy in GC and discuss the challenges and prospects associated with this strategy. We believe that microRNA-based therapy has the potential to offer a clinical benefit to GC patients, and this review would contribute to and motivate further research to promote this field toward this ultimate goal.


Introduction
Gastric cancer (GC) has an extremely high rate of mortality and is ranked as one of the leading causes of cancer-related death worldwide. 1,2 Besides adequate surgical resection and endoscopic resection, adjuvant and neoadjuvant therapies are generally used to improve the disease-free survival and overall survival of patients with GC. 3,4 However, it remains an important challenge to develop more effective systematic treatments for GC, especially for patients with inoperable disease at diagnosis or recurrent disease after resection. The development of resistance to standard chemotherapy drugs such as 5-fluorouracil (5-FU), cisplatin, and doxorubicin has been the main cause of chemotherapy failure. Moreover, first-line targeted therapies such as trastuzumab and ramucirumab are not suitable for the majority of patients with GC. 5,6 Hence, extensive research effort has focused on solving this current treatment challenge by gaining a better understanding of the underlying pathogenic mechanisms and conducting preclinical studies for ultimate clinical application.
In the last decade, exploration of the roles of miRNAs in carcinogenesis, treatment response, and as potential therapeutic targets has attracted widespread attention, with substantial progress made in better understanding mechanisms. 7 On the basis of mechanism-related studies, there has been increasing effort made to further explore the practical applications of these fundamental findings, which have revealed the essential roles of microRNAs in tumor therapy. 8 For example, with respect to drug resistance, over-expression of miR-939 was shown to enhance 5-FU-induced chemosensitivity by suppressing cellular growth and inducing apoptosis both in vitro and in vivo. 9 Moreover, caudally injected exosomes loaded with the inhibitor of miR-214 (exo-anti-214) could reverse the chemoresistance to cisplatin in a nude mouse model of GC. 10 In addition, synthetic carriers have been used to deliver the confirmed anti-tumor miRNAs to the precise tumor site so as to directly inhibit GC, such as "PPP", which was constructed by modifying phenylboronic acid onto the surface of a polyamidoamine dendrimer and poly (ethylene glycol)-poly(ε-caprolactone) copolymers (PEG-PCL) nanoparticles coated with trastuzumab. 11,12 Indeed, these studies have led to a major advance in the clinical application of miRNAs. Moreover, these findings suggested that in addition to their function as biomarkers, miRNAs can also play roles in the treatment of tumors. Nevertheless, despite these many important findings, there has been no study that summarizes these recent advances in microRNA-based therapy for GC. Therefore, in this review, we mainly focus on the advances in microRNA-based therapies for GC, including chemotherapy, targeted therapy, tumor immunotherapy, and others. In particular, we highlight the key miRNAs reported to affect the actions of commonly used chemotherapy drugs, which will facilitate further research on drug resistance. Moreover, the listed carriers that loaded mimics or inhibitors of miRNAs can emphasize the promising applications of anti-tumor miRNAs in targeted therapy and immunotherapy, which should promote their valuable consideration for clinical practice.

microRNA-Based Chemotherapy Fluorouracil Resistance
According to the National Comprehensive Cancer Network clinical practice guidelines in GC, 5-FU is recommended as the first-line anti-tumor drug. Several miRNAs have been identified to participate in the anti-tumor mechanism of 5-FU by decreasing the expression levels of various target genes. For example, overexpression of miR-939, miR-623, miR-429, miR-204, miR-124 or miR-31 was shown to enhance 5-FUinduced chemosensitivity by compromising tumor cell growth or inducing apoptosis. 9,13-19 Among the above-mentioned miRNAs, miR-939 and miR-204 were the only two miRNAs with confirmed ability of reversing drug resistance both in vitro and in vivo, providing more convening evidence than others. 9,15 A role of miR-31 in chemosensitivity was reported by two separate research groups and may have more obvious effects. [17][18][19] Moreover, reduced miR-6785-5p, miR-193-3p, or miR-147 expression could also increase the chemosensitivity of GC to 5-FU. [20][21][22] Interestingly, miR-193-3p and miR-147 both target phosphatase and tensin homolog (PTEN) to exert their functions in GC. 21,22 In addition to miR-193-3p and miR-147, PTEN was identified as the target gene of numerous other miRNAs and to play a role in other drug resistance and treatment mechanisms of GC, which will be discussed in further detail below. The full list of miRNAs reported to be involved in the 5-FU resistance in GC to date is provided in Table 1.

Cisplatin Resistance
Cisplatin (DDP) is another first-line chemotherapy drug, and the mechanism of cisplatin resistance has been the most frequently studied according to our review of the literature. As shown in Table 2, the majority of reported miRNAs (34 at the time of writing this review) can reverse DDP resistance, whereas some miRNAs (13 included in this review) could induce DDP resistance. Overexpression of miR-421, miR-320a, miR-192-5p, miR-181a, miR-148a-3p, miR-145 and miR-let-7b, and reduced expression of miR-135b-5p and miR-21-5p could enhance the chemosensitivity to DDP, which was observed in both GC cell lines and in animal tumor models. [23][24][25][26][27][28][29][30] These miRNAs could become more promising targets for further clinical research after resolving some key technical problems, such as selecting suitable carrier for mimics and inhibitors of miRNAs. Moreover, although miR-218, miR-200c, miR-198, miR34a, and miR-30a were only investigated in cell lines, each of these microRNAs was reported to be involved in DDP resistance in more than one separate study, [32][33][34][35][36][37][38][39][40] suggesting that they warrant further attention.

Other Single-Drug Resistance
Some studies have also indicated roles of miRNAs in the resistance of other clinically used chemotherapy drugs, including doxorubicin (DOX; also named adriamycin, ADR), paclitaxel (PTX), oxaliplatin (OXA), vincristine (VCR), taxol, and docetaxel (Table 3). DOX, as an anthracycline-based anti-tumor agent, is widely used for the treatment of various solid tumors such as GC. Increased expression levels of miR-494, miR-422a, miR-140, miR-103/107, and miR-16-1, and reduced levels of miR-501-5p and miR-21-5p promoted the chemosensitivity to DOX (for more details, refer to Table 3). 57-64 Among these, miR-103 and miR-107 were the only miRNAs demonstrated to have effects on resistance both in vitro and in vivo. 60 Another important miRNA that is taking the spotlight in chemotherapy resistance research is miR-21. miR-21-5p targets PTEN to induce both DOX and DDP resistance. 30,48,64 Other studies showed that miR-21 targets P-gp to induce PTX resistance. 65 Notably, PTEN is not only a target gene of miR-21-5p but is also a target of miR-193-3p, miR-147, and miR-106a. 21,22,46 Through targeting PTEN, miRNAs regulate 5-FU, DDP, and DOX resistance in GC. 21,22,30,46,48,64 Hence, we speculated that miR-21-5p and its target gene PTEN may play essential and broad roles in regulating drug resistance in GC.
As shown in Table 3, limited studies have been conducted to investigate the roles of miRNAs in the resistance mechanisms to other chemotherapy drugs. MiR-155-5p, miR-34c-5p, and miR-21 were found to be related to PTX resistance; 65-67 miR-361-3p, miR-135a, and miR-27a affect OXA resistance; 68-70 miR-1284 and miR-647 regulated VCR resistance; 71,72 and miR-200a inhibited taxol resistance, while miR-361-5p suppressed docetaxel  [19] Notes: *Targets genes were confirmed by Western blotting, RT-qPCR or dual-luciferase. **Upregulation (↑) or downregulation (↓) of miRNAs enhance chemosensitivity. ***The drug resistance properties of microRNAs were investigated in cell lines or in vivo.   [127] (Continued) resistance. 73,74 Among the above-mentioned miRNAs, only miR-135a, miR-1284, and miR-647 were studied both in vitro and in vivo. 69,71,72 Interestingly, miR-361-3p and miR-361-5p were reported to regulate the resistance of different drugs. 68,74 This indicates that to best exploit the potential of microRNA-based tumor therapies, we need to pay attention to both the 3p-and 5p-arms of the same miRNA in addition to considering the functions of different miRNAs.

Multidrug Resistance
Although chemotherapy resistance is a challenge for all cancer treatments, it is considered to be a particularly more complex problem in the clinical treatment of GC. In addition to singledrug resistance, multidrug resistance (MDR) may be even more common in clinical practice. Some studies have focused specifically on the roles of miRNAs in MDR (Table 4). Both in vitro and in vivo studies confirmed that miR-508-5p, miR-23b-3p, and miR-590-5p strongly regulate MDR. [75][76][77][78][79] Shang's group revealed that the restoration of miR-508-5p reversed the 5-FU, DDP, DOX, and VCR resistance of a xenograft in an animal model by targeting ATP binding cassette subfamily B member 1 (ABCB1) and zinc ribbon domain-containing 1 (ZNRD1). 75 The same group further identified that the miR-27b-CCNG1-P53-miR-508-5p axis regulated the MDR of GC. 76 They transplanted SGC7901/VCR cells into the right flanks of mice, which were then treated with an agomir for miR-27b or control oligonucleotides before 5-FU or DDP therapy, demonstrating that miR-27b overexpression reversed drug resistance. An et al 77 found that miR-23b-3p sensitized GC cells to 5-FU, DDP, and VCR by targeting autophagyrelated gene 12 (ATG12) and high-mobility group box 2 (HMGB2). Subsequently, another study showed that the long non-coding RNA MALAT1 regulates autophagy-associated chemoresistance via the miR-23b-3p/ATG12 pathway. 78 Shen et al 79 found that miR-590-5p reduces the sensitivity of GC cells to DDP and PTX. To further verify the effect of miR-590-5p, stable miR-590-5p-expressing SGC7901 cells were inoculated into nude mice and the tumor-bearing mice were treated with DDP, demonstrating that miR-590-5p reduced chemosensitivity. Compared with other reported miRNAs related to MDR (see Table 4 for the full list), there is more sufficient and reliable preclinical evidence for these three above-mentioned miRNAs, demonstrating their suitability for further research toward their potential clinical application. Figure 1 schematically illustrates the proposed relationship between chemotherapy drugs and relevant miRNAs based on the results of studies of single-drug resistance and MDR. Figure 2 shows the relationship between miRNAs that have an effect on drug resistance and their target genes. It is now clear that some miRNAs can regulate the resistance of different drugs simultaneously. Therefore, this review should be beneficial for the design of future drug resistance studies to select the most suitable miRNAs according to different clinical application purposes. Given the pace of this field along with continuous advances in sequencing technologies, we also believe that more miRNAs that participate in the drug resistance mechanisms will be discovered with further research.

microRNA-Based Targeted Therapy
Thus far, the majority of research focused on microRNAbased targeted therapy has focused on miRNAs that affect the resistance to targeted drugs (trastuzumab and lapatinib) and development of new types of transport carriers (synthetic nanoparticles/compounds and exosomes).

Trastuzumab and Lapatinib
Trastuzumab as a HER2-targeting antibody has been successfully used in combination with chemotherapy for the treatment of HER2-neu overexpressing GC. Trastuzumab resistance is considered to be a difficult problem in clinical application. Increased miR-223, miR-200c, and reduced miR-21 were reported to reverse trastuzumab resistance via different mechanisms. 80,81 MiR-223 could modulate apoptosis to enhance the sensitivity of a HER2-positive GC cell line to trastuzumab through targeting F-box and WD repeat domain-containing 7 (FBXW7), 80 whereas miR-200c inhibits TGF-β-induced epithelial-mesenchymal transition to restore  [28] Notes: *Targets genes were confirmed by Western blotting, RT-qPCR or dual-luciferase. **Upregulation (↑) or downregulation (↓) of miRNAs enhance chemosensitivity. ***The drug resistance properties of microRNAs were investigated in cell lines or in vivo.
trastuzumab sensitivity through inhibiting zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2, 81 and miR-21 significantly suppressed trastuzumab-induced apoptosis and decreased the sensitivity of GC cells to trastuzumab through regulating PTEN expression. 82 In addition to the above three miRNAs, Sun et al 83 reported a correlation between miR-125b and trastuzumab resistance according to the clinicopathologic characteristics of patients with GC. Nishida et al 84 reported that the inhibitory effect of miR-125a-5p on GC proliferation was enhanced in combination with trastuzumab.
As an alternative strategy, nanoparticles coated with trastuzumab were used as carriers to transfer miRNAs or   chemotherapy drugs to the target, which will be discussed further in the following section. Lapatinib, a type of tyrosine kinase inhibitor, is also commonly used in GC targeted treatment. The resistance to lapatinib can be reversed by miR-494. 85 Like trastuzumab, lapatinib can induce miR-16 upregulation in GC sensitive cells via inhibition of c-Myc and the PI3K/AKT and Erk1/2 pathways. 86

Synthetic Nanoparticles and Compounds
As mentioned above, some studies have explored the applications of nanoparticles loaded with mimics/inhibitors of miRNAs or some other tumor-targeting compounds. Song's team constructed a tumor-targeted gene carrier, PPP, through modification of phenylboronic acid onto the surface of a polyamidoamine dendrimer. 11 The carrier PPP showed favorable miRNAs binding and condensation ability, protected miRNAs against nuclease degradation, and mediated the cellular uptake of miRNAs. 11 Jang's team used nanovesicles containing poly (l-lysine-graft-imidazole) (PLI)/ miRNA complexes (NVs/miR) to systemically deliver miRNA to the target site. 87 Incorporation of hydrophilic PEG molecules on the nanoparticle surface could further prolong the blood circulation. Li's team loaded sorafenib (SRF) and all-trans retinoic acid (ATRA) in PEGylated solid lipid nanoparticles (SLNs) composed of Gelucire, and loaded miRNA onto the surface of the SLNs based on the electrostatic interaction. 88 Liu's team employed intelligent gelatinases-stimuli nanoparticles to co-deliver miRNA and docetaxel to inhibit cancer stem cells. 89 Hu's team and Wu's team each introduced PCL-PEG nanoparticles coated with trastuzumab (HER-PEG-PCL NPs) to control the delivery of the inhibitor of miRNA. 12,90 These studies, respectively, verified the inhibitory function of miR-34a, miR-21, miR-542-3p, and miR-200c on GC in vitro or in vivo. Importantly, these strategies involve the use of synthetic nanoparticles and compounds to attempt to solve the problem of improving microRNAs-targeted transport to tumors so as to promote the clinical application of microRNA-based targeted therapies.

Exosomes
Exosomes are nanosized extracellular membrane-derived vesicles that are secreted by various cells. In 2007, miRNAs were first identified to be transferred in exosomes. 91 In a study  on the DDP resistance mechanism, miR-21 was identified within the exosomes secreted by tumor-associated macrophages and could be directly transferred from macrophages to GC cells to promote DDP resistance. 30 Similarly, the exosomal transfer of miR-501-5p from DOX-resistant GC SGC7901/ADR cells to SGC7901 cells enhanced recipient cell growth, metastasis, and chemoresistance. 62 Moreover, the exosomal delivery of miR-155-5p was suggested to induce chemoresistant phenotypes from paclitaxel-resistant GC cells to sensitive cells. 66 Given the potential of genetic exchange between cells via exosomes, they have been considered as an alternative promising tool for therapeutic purposes, including in microRNA-based treatment. To support this possibility, one study transfected inhibitors of miR-214 and a negative control into HEK293T cells, and exosomes extracted from the cell medium after 48 h were verified to contain the inhibitor of miR-214, which reversed the resistance to DDP both in GC cells and in vivo. 10 In applications for reversing drug resistance or directly suppressing tumors, exosomes containing mimics or inhibitors of miRNAs are likely safer than synthetic nanoparticles and compounds. However, more research is needed to solve exosome-related technical problems, such as the lack of methods to interfere with the packaging of cargo or with vesicle release, before microRNA-based targeted therapies can become a reality. 92

microRNA-Based Tumor Immunotherapy
In the past decade, tumor immunotherapy has made great progress in both scientific research and clinical application. Novel drugs target immune checkpoint molecules, such as programmed cell death protein 1 (PD-1), PD-1

microRNA-Based Other Therapies
The upstream regulatory mechanism of miRNAs, such as hypermethylation in the promoter region and transcription factor dysregulation, affects the expression of miRNAs. Some miRNAs play key roles in GC tumorigenesis, proliferation, metastasis, and recurrence. In theory, many genes and miRNAs in these processes could become potential therapeutic targets. However, there are too many possible miRNAs and related mechanisms involved in GC to give a detailed    description of them in just one paper. Some miRNAs, regarded as potential therapeutic targets, have also had been reviewed elsewhere. [94][95][96] Our review focused more on discussing miRNAs that are more promising for clinical use. We believe that miRNAs that have an effect on GC treatment (including chemotherapy, targeted therapy, immunotherapy, and others) could also become core targets in microRNA-based therapies. It is reported that some drugs with anti-tumor effects may play roles by altering the expression of miRNAs and their related pathways. For example, cinobufacini, which is widely used in the treatment of advanced cancers, was reported to suppress cell proliferation and induce apoptosis in GC by regulating the miR-494-BAG-1 (BCL2 associated athanogene 1) axis. 97,98   Cell lines (AGS) [157,158] miR-17-5p Shenqifuzheng Notes: *The drug resistance properties of microRNAs were investigated in cell lines or in vivo.
exploration of mechanisms but are now actively trying to realize the goal of clinical application and translation of achievements from the laboratory to clinical practice. Hence, microRNAs-based therapy is expected to become a promising research direction and ultimately benefit patients with GC. The goal of the present review was not only to summarize the relevant literature and advances on the roles of miRNAs in GC treatment, but to further attempt at answering several existing questions. First, we wondered which aspects of GC treatment will microRNA-based therapies be best suited for. As reviewed herein, miRNAs can function in reversing drug resistance, directly inhibiting tumors, or enhancing the anti-tumor effects of drugs. Here, we have mainly focused on the potential for curbing drug resistance and the enhancing the anti-tumor effects of drugs. In terms of directly inhibiting tumors, we have highlighted only some key representative studies, as this field is extremely rich and therefore a comprehensive review is outside of the overall scope. Second, we have tried to assess which miRNAs are most suitable for therapeutic applications. We believe that the miRNAs verified in animal experiments and in more than one study are more reliable for further investigation. Of course, we also acknowledge that this perspective is limited by the extent and scope of current research. Therefore, further verification and screening are needed in future studies. Third, we want to determine the best method to realize the utilization of these miRNAs. Synthesized oligonucleotides of miRNA mimics or inhibitors have been used in an animal tumor model for several years with good results. However, before clinical application, more safety, targeting, and stability issues of this approach need to be considered. Recently, the application of synthetic nanoparticles/compounds and exosomes provides more promising possibilities; however, these techniques also require the combined efforts of doctors, pharmacists, and scientists of other disciplines.
Overall, we believe that this field is currently at an explosive stage of discovery, and hope that this review will promote further applications of this research to realize the ultimate goal of improving the quality of life and outcome of patients with GC.