Polyphenols as Lung Cancer Chemopreventive Agents by Targeting microRNAs

Lung cancer is the second leading cause of cancer-related death worldwide. In recent decades, investigators have found that microRNAs, a group of non-coding RNAs, are abnormally expressed in lung cancer, and play important roles in the initiation and progression of lung cancer. These microRNAs have been used as biomarkers and potential therapeutic targets of lung cancer. Polyphenols are natural and bioactive chemicals that are synthesized by plants, and have promising anticancer effects against several kinds of cancer, including lung cancer. Recent studies identified that polyphenols exert their anticancer effects by regulating the expression levels of microRNAs in lung cancer. Targeting microRNAs using polyphenols may provide a novel strategy for the prevention and treatment of lung cancer. In this review, we reviewed the effects of polyphenols on oncogenic and tumor-suppressive microRNAs in lung cancer. We also reviewed and discussed the potential clinical application of polyphenol-regulated microRNAs in lung cancer treatment.


Introduction
Lung cancer originates from the bronchial mucosa or glands of the lung. Lung cancer can be mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Among all lung cancers, non-small cell lung cancer accounts for about 85-88%, while small cell lung cancer accounts for about 12-15% [1]. According to the reports of the World Health Organization, the incidence rate of lung cancer in 2020 was 22.4 cases per 100,000 people, ranking second in terms of cancers; while the mortality rate of lung cancer is as high as 18 cases per 100,000 people (https://www.wcrf.org/cancer-trends/ lung-cancer-statistics/ (accessed on 23 March 2022)). The existing treatment methods of lung cancer are mainly surgery, chemotherapy, and radiotherapy. These treatment methods have serious side effects and easily cause discomfort. Herb and plant derived-chemicals have the characteristics of less toxicity and side effects, showing better therapeutic effects, and can improve the quality of life of patients and weaken the deficiencies of existing therapeutic drugs [2]. expression of some signal pathways and oncogenes, lung cancer is also associated with an imbalance in microRNA expression [26].
The expression of miRNAs is complex. Some miRNAs are upregulated in tumors and play a role similar to oncogenes, while some miRNAs are downregulated in tumors and play the role of tumor suppressor genes. An abnormal miRNA molecule can affect the expression of hundreds of miRNAs. When an miRNA regulates key genes, it will have a great impact on the cell function [27]. In the pathogenesis and progression of SCLC and NSCLC, some miRNAs have been speculated as oncogenes, tumor suppressor genes, and cancer progression (Table 2 and Figure 1). Table 2. Oncogenic and tumor-suppressive miRNAs in lung cancer.

The Role of Polyphenols in Lung Cancer by Targeting microRNAs
More and more data show that miRNA is involved in the progression of lung cancer. It provides a new way to find more effective drugs for the treatment of lung cancer. Recent studies have shown that polyphenols play a pharmacological role in lung cancer by regulating miRNAs (Table 3 and Figure 2).

Flavonoids
Epigalocatechin gallate (EGCG), the main component of green tea polyphenols, is a catechin monomer isolated from tea [54]. Studies showed that the expression levels of miR-212 were decreased and the expression of miR-155 were increased in EGCG-treated A549 by regulating the MAPK signaling pathway, which in turn inhibited cancer cell proliferation and migration [55]. Wang et al. found that EGCG, through the upregulation of HIF-1α and the expression of mir-210, inhibited the growth of lung cancer cells [56]. At the same time, EGCG can enhance the expression of has-miR-4855p, significantly inhibit the growth of NSCLC cells, and induce apoptosis [57]. Another study showed that EGCG inhibited cancer stem cell-like properties by upregulating the expression of miR-485 and reducing the expression of CD44 [58]. Meanwhile, some studies have found that EGCG can inhibit the expression of hsa-miR-98-5p and upregulate the expression of p53, thereby enhancing the efficacy effects of cisplatin on A549 cells [59]. Skullcapflavone I is a natural product found in Scutellaria baicalensis, Andrographis paniculata, and other organisms [60]. Skullcapflavone I can downregulate the expression levels of miR-21, enhance the expression levels of PP2A in A549 cells, and inhibit the proliferation of human lung cancer cells [61]. Quercetin is a widely distributed flavonoid alcohol compound with a variety of biological activities in plants [62]. Studies found that the expression level of miR-16 was upregulated with quercetin treatment, in turn mediating the decrease in Claudin-2 expression and inhibiting the invasion and migration of lung adenocarcinoma cells [55,63]. Genistein is a soybean isoflavone and phytoestrogen with antitumor activity [64]. Genistein-treated A459 cells showed decreased expression of miR-27a and increased expression of MET, which in turn promoted the apoptosis of A459 cells [55].
Kaempferol is an organic compound with the chemical formula c15h10o6 and is a flavonoid. After kaempferol treatment, the expression of mir-340 increased, the expression of the target gene cyclin D1 decreased, and the expression of PTEN increased, which inhibited proliferation and promoted the apoptosis of A549 cells [55]. Similarly, Han et al.
found that the expression of mir-340 was upregulated, the level of PTEN increased, the phosphorylated levels of PI3K and AKT were decreased, the proliferation of A549 cells was inhibited, and the apoptosis and autophagy of A549 cells were increased after kaempferol treatment compared with the control group [65]. Baicalin is a flavonoid extracted and isolated from the dried roots of Scutellaria baicalensis Georgi, a dicotyledonous Labiatae plant [66]. Recent studies found that the expression levels of miR-340-5p and the target gene NET1 were increased after baicalin treatment, in turn inhibiting the proliferation and invasiveness of A549 and H1299 cells [67]. Meanwhile, Baicalein inhibited cell growth and increased the sensitivity of A549 and H460 cells to cisplatin through the miR-424-3p-targeted PTEN/PI3K/AKT pathway [68]. The Radix Tetrastigma hemsleyani flavone (RTHF) is extracted from a traditional Chinese medicinal herb T. hemsleyani [69]. The increase in has-miR-410-3p in A549 cells caused by RTHF may play a role in the inhibition of A549 cells via downregulating the expression of MMP14 and MMP16 [69]. Moreover, the downregulation of miR-1303 by RTHF may occur through targeting CLDN18, GSK3β, and SFRP1, thereby inhibiting the proliferation, migration, and invasion of A549 cells [70]. Apigenin mainly exists in Daphneceae, Verbenaceae, and Selaginellaceae plants, especially in celery [71]. It was found that apigenin may induce apoptosis by upregulating miR-34a-5p in A549 cells and downregulating SNAI1 [72].
The soy isoflavone genistein is usually present in genistein and daidzein. It is a bioflavonoid in soybean products and other plants [73]. In NSCLC cells treated with the soy isoflavone genistein, miR-873-5p inhibited cell proliferation, migration, and invasion and increased apoptosis by regulating FOXM1 [74]. Licochalcone A (Lico A) is a post chalcone isolated from the root of Glycyrrhiza uralensis, a plant from Xinjiang Province in China [75]. It is reported that LiCo A can significantly promote the expression of miR-144-3p, downregulate the expression levels of Nrf 2, and finally induce apoptosis in lung cancer cells [76]. Chen et al. also found that Lico A can activate the unfolded protein response (UPR) and induce autophagy in H292 cells, thereby inducing apoptosis [77].
Puerarin is a C-glycosyl compound and a hydroxyisoflavone [78]. Purerin inhibits the expression of CCND1 by upregulating miR-342; inhibits cell viability, migration, invasion, and the cell cycle process; and enhances the apoptosis of NSCLC cells [79]. Nobiletin is a natural product found in Ageratum conyzoides and Viburnum tinus [80]. Sp et al. found that nobiletin inhibited the expression of PD-L1 through the EGFR/JAK2/STAT3 signaling pathway, while the expression levels of STAT3 and PD-L1 were regulated by miR-197, thereby enhancing the antitumor immunity [81]. Recent studies have shown that the downregulation of miR-106b by grape seed procyanidin (GSE) induced the expression levels of tumor inhibition cycle-independent kinase inhibitor 1A (CDKN1A) and p21, which further promotes the antitumor effect of GSE [82]. Another study found that grape seed procyanidin significantly downregulated the expression of miR-19a and-19b in tumor cells, increased the mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homologue (PTEN), and significantly inhibited tumor growth [83].
Hesperidin is a flavanone glycoside, which is found in citrus fruits [84]. Hesperidin can promote the apoptosis of lung cancer cells by increasing the expression of miR-132 and reducing the expression of ZEB2, so as to inhibit the proliferation of lung cancer cells [85]. Breviscapine is found in Indian wood, perilla, and other organisms [86]. Zeng et al. found that breviscapine enhanced the expression of miR-7, upregulated Bax/Bcl-2, and promoted apoptosis [87]. It was found that Nepeta cataria L. extract can regulate the expression of miR-126 and regulate the PI3K-Akt signaling pathway to perform the anticancer effect [88]. Luteolin is a natural product found in Cryptomeria japonica and Epimedium [89]. Luteolin upregulates the expression of miR-34a-5p by targeting MDM4, inhibits tumorigenesis, and induces the apoptosis of NSCLC cells [90]. Orientin is a C-glycosyl compound, and it is believed that orientin regulates the expression of COX-2/PGE-2 in the A549 cell line through miR-26b and miR-146a and reduces the proliferation, migration, and invasion of A549 cells [91]. Rhamnetin is a natural product found in Liriodendron tulipifera and Albizia julibrissin [92]. Cirsiliol is a natural product found in Salvia lineata and Teucrium chamaedrys. Rhamnetin and Cirsiliol can inhibit the EMT of lung cancer cells through the miR-34a-mediated downregulation of Notch-1 expression [93]. Icaritin exists in Epimedium bicolor, Epimedium aculeatum, and Epimedium wushanese [94]. Icaritin inhibits NSCLC cell proliferation by downregulating miR-10a, which could regulate the expression of PTEN [95].

Phenolic Acids
Caffeic acid phenethyl ester (CAPE) is a natural product found in Euonymus alatus and Alibertia macrophylla, and is the phenethyl alcohol ester of caffeic acid and a bioactive component of honeybee hive propolis, with antineoplastic, cytoprotective, and immunomodulating activities [96]. Mo et al. found that CAPE treatment downregulated the expression of YAP1 and C-MYC, thereby inducing H446 cell apoptosis. Moreover, they found that miR-3960 upregulated the expression of C-MYC and participated in CAPEinduced SCLC cell apoptosis [97]. Cucurbitacin B is a cucurbitacin derived from the hydrides of lanosterol [98]. Cucurbitacin B inhibits the proliferation and promotes the apoptosis of lung cancer cells through the lncRNA XIST/miR-let-7c axis [99].

Stilbenes
Resveratrol, a non-flavonoid polyphenol organic compound, is an antitoxin produced by many plants when stimulated [100]. In lung cancer cells treated with resveratrol, cell proliferation was inhibited via the miR-622/k-Ras axis [101]. Moreover, resveratrol can also inhibit the expression of FOXC2 and tumor activity by regulating the miR-520h-mediated signal cascade [102]. Lu et al. found that resveratrol inhibited NSCLC cell proliferation via miR-345-and miR-498-regulated MAPK/CFO and Akt/BCL2 signaling pathways by directly targeting MAPK1 and PIK3R1, respectively, which increased the sensitivity of NSCLC cells to gefitinib and induced apoptosis [103].

Lignans
Honokiol is found in Cryptomeria fortunei, star anise, and other organisms [104]. Honokiol inhibited the proliferation and migration of NSCLC cells and induced the apoptosis of NSCLC cells through miR-148a-5p and miR-148a-3p, probably by targeting ERBB3 and itga5 through the PI3K/Akt signaling pathway [105]. Treatment with Phyllanthus emblica L (PEL) extract could effectively prevent precancerous lesions of lung cancer by regulating the IL-1β/miR-101/LIN28B signaling pathway [106]. Ailanthone comes from Ailanthus altissima, and can inhibit the proliferation of lung cancer cells and promote the apoptosis and autophagy of lung cancer cells [107]. Hou et al. found that Ailanthone induced the apoptosis and autophagy of lung cancer cells by upregulating the expression of miR-195 [108].

Clinical Trials Using Polyphenols for Lung Cancer Treatment
To date, there have been 12 clinical trials of polyphenols in lung cancer (http:// clinicaltrials.gov/ (accessed on 8 August 2022), listed in Table 4). Among these clinical trials, flavonoids are the major ones used for treatment. Zhao et al. studied the side effects and optimal dose of EGCG in patients with non-small cell lung cancer. The initial dose of EGCG was 400 mg administered twice a day. The second incremental dose was 800 mg, the third incremental dose was 1200 mg, the fourth incremental dose was 1600 mg, and the fifth incremental dose was 2000 mg (NCT01317953). The results showed that oral EGCG is feasible, safe, and effective, and the recommended concentration of EGCG in patients with non-small cell lung cancer in the second stage of treatment is 440 µM [124]. Scott et al. determined that the maximum tolerated dose of green tea extract in patients with advanced lung cancer was 3 g/m 2 /day. At this dose, the green tea extract was well tolerated and the toxicity was no more than grade 3 or 4 [125]. Siegenthaler et al. found that flavor aesthetic acid (NSC.347512, LM975) had slight antitumor activity against NSCLC [126]. However, the results of most clinical trials have not been published. Therefore, whether polyphenols mediate antitumor effects through miRNAs in clinical trials has not been clarified.

Conclusions and Future Perspectives
In the past two decades, miRNAs have been proven to play a major role in the pathogenesis of lung cancer and have become candidate therapeutic targets. Preclinical studies have shown that polyphenols can downregulate pro-tumor-associated microRNAs or upregulate tumor-associated microRNAs, thereby exerting their antitumor function in lung cancer. However, the therapeutic effects of using miRNAs for lung cancer treatment need to be demonstrated in clinical trials. Thus, further studies are needed to explore this promising field. Therefore, in future clinical trials, we could study the effects of polyphenols on miRNAs in lung cancer patients in vivo by using new technologies such as metabolomics and single-cell sequencing. Special attention should be paid to the cancer-promoting or cancer-suppressing miRNAs that were found to be affected by polyphenols in preclinical experiments. We could screen for different polyphenols targeting specific types of miRNAs associated with cancer through the application of polyphenols in clinical settings.
Author Contributions: J.L., X.Z., C.P. and Z.X. wrote the first draft of the manuscript, Y.Z. and J.S. performed the first revision. All authors revised the manuscript for the second time before submission. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare that they have no conflict of interest.