Anti-Tumor Mechanisms Associated With Regulation of Non-Coding RNA by Active Ingredients of Chinese Medicine: A Review

Cancer has become the second leading cause of death worldwide; however, its complex pathogenesis remains largely unclear. Previous research has shown that cancer development and progression are closely associated with various non-coding RNAs, including long non-coding RNAs and microRNAs, which regulate gene expression. Target gene abnormalities are regulated and engaged in the complex mechanism underlying tumor formation, thereby controlling apoptosis, invasion, and migration of tumor cells and providing potentially effective targets for the treatment of malignant tumors. Chemotherapy is a commonly used therapeutic strategy for cancer; however, its effectiveness is limited by general toxicity and tumor cell drug resistance. Therefore, increasing attention has been paid to developing new cancer treatment modalities using traditional Chinese medicines, which exert regulatory effects on multiple components, targets, and pathways. Several active ingredients in Chinese medicine, including ginsenoside, baicalin, and matrine have been found to regulate ncRNA expression levels, thus, exerting anti-tumor effects. This review summarizes the scientific progress made regarding the anti-tumor mechanisms elicited by various active ingredients of Chinese medicine in regulating non-coding RNAs, to provide a theoretical foundation for treating tumors using traditional Chinese medicine.


INTRODUCTION
The incidence of cancer has tripled over the past three decades and is expected to increase five-fold by 2030 (1). Nevertheless, the precise mechanism associated with cancer pathogenesis remains largely unclear as it is highly complex and diverse. Since the 1970s, gene therapy has become an increasingly attractive strategy for the treatment of various diseases. Accordingly, more recently, the focus has shifted toward identifying specific genetic etiologies of cancer to design effective gene therapies to overcome the challenges associated with traditional cancer treatment modalities. As the human genome is gradually deciphered, RNA has been shown to play an auxiliary role as an intermediate vector of genetic information, and an increasing number of regulatory functions have been ascribed to this class of molecules. In particular, non-coding sequences, which account for 99% of the total human genome, have received greater attention (2). Most of the identified non-coding RNAs (ncRNAs), including long non-coding (lnc)RNAs, micro (mi) RNAs, small interfering (si)RNAs, Piwi-interacting (pi)RNAs, ribosomal (r)RNAs, transfer (t)RNAs, and small nuclear (sn) RNAs, participate in the translation, modification, and other cellular functions. Among these molecules, lncRNAs and miRNAs have been researched more intensively to decipher their fundamental roles in many diseases, including cancer (3). In fact, lncRNAs were initially believed to be the "noise" of genome transcription, a by-product of RNA polymerase II transcription, and were considered biologically non-functional (4). However, recent studies have shown that lncRNAs play an essential role in cancer signal transduction pathways by interacting with proteins and RNA (5) and are reportedly associated with various cancers, including those of the stomach (6)(7)(8), lung (9,10), breast (11,12), and prostate (13,14). Specifically, inhibition of lncRNA H19 and lncRNA PVT1 expression can effectively inhibit the cancerization, metastasis (15), and angiogenesis (7) of gastric cancer.
The emergence of ncRNA provides new alternatives for cancer treatment, but its effective application in clinical cancer treatment remains challenging. At present, chemotherapy is one of the most important means of treating malignant tumors. However, its efficacy is limited by systemic toxicity and tumor cell resistance (27). Traditional Chinese medicine has been practiced for thousands of years because of its safety and minimal side effects. To date, many cancer patients have used traditional Chinese medicine as an alternative therapy for cancer treatment (28). Moreover, some of the active ingredients of Chinese medicine have been shown to exert anti-cancer effects by regulating ncRNAs and acting on various signaling pathways and cancer-related molecular targets, thus, inhibiting tumor proliferation, metastasis, and invasion and inducing cancer cell apoptosis (29). For instance, the isoflavone calycosin inhibits nasopharyngeal carcinoma cell growth by regulating the lncRNA EWSAT1 and its downstream TRAF6 pathways (30). Additionally, curcumin upregulates miR-145 expression to inhibit cell proliferation and invasion in vitro, while inducing cell cycle arrest (31). These reports suggested that ncRNA have anti-tumor abilities that regulate cancer cell apoptosis, proliferation, resistance, metastasis, and invasion.

ACTIVE INGREDIENTS OF TRADITIONAL CHINESE MEDICINE REGULATE THE ANTI-TUMOR MECHANISM OF MIRNA
Over the past decades, miRNA has been shown to play a regulatory role in many cancer signaling pathways (32). The active ingredients in traditional Chinese medicine act through different mechanisms to upregulate the expression of tumorsuppressing miRNAs and downregulate the expression of oncogenic miRNAs (33). They also inhibit tumor occurrence and development by inducing cell apoptosis to inhibit tumor metastasis, enhancing cell cycle arrest to reduce drug resistance, and by downregulating other pathways (34) ( Table 1).

ACTIVE INGREDIENTS OF CHINESE MEDICINE TARGETING MIRNA INDUCE TUMOR APOPTOSIS
Apoptosis is a basic biological phenomenon, that involves the activation, expression, and regulation of a series of genes. The active ingredients of traditional Chinese medicine regulate target genes by influencing an abnormal expression of miRNAs and inducing tumor cell apoptosis ( Figure 1). miR-21 inhibits apoptosis in various tumor cells (71)(72)(73). Ginsenoside Rh2 is a natural monosome of ginseng total saponin, and its anti-cancer effects have been demonstrated in various tumors. A former report showed that ginsenoside Rh2 inhibited Bcl-2 by increasing miR-21 levels, which induced apoptosis and significantly decreased leukemia cell viability (35). PTEN is a classical anti-oncogene, the inhibition of PTEN is key for cell apoptosis, mainly relying on the phosphorylation and dephosphorylation of Akt. In FTC-133 human follicular thyroid cells, the alkaloid Chinese medicine active ingredient matrine is induced by upregulating the PTEN/ Akt signaling pathway via the downregulating miR-21 (39) it induces apoptosis of TPC-1 human thyroid cancer cells (40). Similarly, the reverse quantitative polymerase chain reaction of an extract of Magnolia officinalis revealed that magnolol could induce abnormal expression of miRNA in human osteosarcoma cells, with miR-21 showing a very strong ability to downregulate mRNAs. Recent evidence has suggested that Honokiol is able to suppress the PI3K/AKT signaling pathway; however, it was reactivated by miR-21 overexpression. Honokiol inhibits proliferation and induces apoptosis by regulating the miR-21/ PTEN/PI3K/AKT signaling pathway in human osteosarcoma cells (41).
A previous report described that Rh2 reduces the expression levels of MCL1 and Nrf2, suppresses colony formation, and induces HepG2 cell apoptosis by inhibiting miR-146a-5p in HepG2 cells (36). Berberine is a quaternary ammonium alkaloid isolated from Coptis chinensis. It inhibits the growth of non-smallcell lung cancer via the miR-19a/TF/MAPK axis and promotes apoptosis. A previous study of the mechanism of anti-tumor baicalin in human colon cancer showed that baicalin induced colon cancer cell apoptosis via the Wnt signaling pathway mediated by miR-217/DDK1, in which DDK1 was identified as a direct downstream target gene of miR-217 (33). A previous study showed that resveratrol regulated the apoptosis and cell cycle of breast cancer cells by regulating miRNAs such as miR-125b-5p, miR-200C-3p, miR-409-3p, miR-122-5p, and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key antiapoptotic and cell cycle proteins, including Bcl-2, X-linked inhibitor of apoptosis protein, and CDKs, which are critical for its activity. Among these, miR-542-3p and miR-122-5P play key roles in resveratrol-mediated apoptosis of MCF-7 and MDA-MB-231 breast cancer cells, respectively (44). Resveratrol significantly reduced miR-196b/miR-1290 expression in the T-ALL (T-cell acute lymphoblastic leukemia) TTL-104 and B-ALL (B-cell acute lymphoblastic leukemia) SUP-B15 cell lines and upregulated the expression of IGFBP3. As a miR-196b/miR-1290 inhibitor, resveratrol was further demonstrated to exert antitumor effects on ALL cells including antiproliferation, cell cycle arrest, apoptosis and inhibition of migration (45). Pyruvate kinase PKM2 is highly expressed in various tumors. The expression of miR-326 was Enhance chemosensitivity (70) increased after resveratrol treatment, and miR-326/PKM2mediated stress and mitochondrial dysfunction were involved in apoptosis induced by resveratrol (46). Camptothecin, a cytotoxic quinoline alkaloid, is an anti-cancer compound found in plants.
Deep sequencing analysis of miRNA expression profiles during the camptothecin-induced apoptosis showed that 79 miRNAs were downregulated post-treatment (74). A study of camptothecin verified that miR-125b was down-regulated in camptothecin induced apoptosis in cancer cells. Camptothecin induced apoptosis in cancer cells through miR-125b-mediated mitochondrial pathways by targeting the 3'-untranslated (UTR) regions of Bak1, Mcl1, and p53 (53). In glioblastoma stem cells, miR-143 expression was downregulated after shikonin administration, whereas the regulation factor BAG3 was upregulated. Notably, miR-143 overexpression reversed this phenomenon and enhanced the anti-tumor activity of shikonin in glioblastoma stem cells (58). Celastrol is found in the root of Celastrus orbiculatus, belonging to the family Celastraceae. It can regulate apoptosis-promoting signaling pathways (75). Celastrol suppresses cellular proliferation and induces apoptosis of ovarian cancer OVCAR3 cells by downregulating miR-21 to inhibit the PI3K/P-Akt-NF-kB signaling pathway, thereby revealing a potential therapeutic approach (63).

ACTIVE INGREDIENTS OF CHINESE MEDICINE TARGET MIRNAS TO BLOCK TUMOR CELL CYCLE
The occurrence of most tumors is related to the disruption of cell cycle regulation, leading to uncontrolled cell growth. Many active ingredients of Chinese medicine play an anti-tumor role by blocking the tumor cell cycle and inhibiting cell proliferation ( Figure 1). For instance, in patients with acute lymphoblastic leukemia (ALL), a study demonstrated that the expression of IGFBP3 was decreased in ALL patients. The authors further identified that miR-196b and miR-1290 were overexpressed in T-ALL TALL-104 and B-ALL SUP-B15 cell lines, respectively. As an miR-196b/miR-1290 inhibitor, resveratrol was further demonstrated to exert antitumor effects on ALL cells including cell cycle arrest. Resveratrol blocks T-ALL T-ALL-104 cells during the G1 phase and the B-ALL SUP-B15 cells in the S phase by inhibiting miR-196b/miR-1290 (45). Baicalin upregulates miR-3127-5p, which increases p21/CDKN1A and P27/CDKN1B expression to inhibit cell proliferation arresting the cell cycle in S and G2/M phases in Bel-7402 cells (43). Celastrol caused G2/M cell cycle arrest that was accompanied by the down-regulation of miR-21 expression. Further study showed that celastrol inhibited p27 protein degradation by inhibiting the miR-21 and mTOR signaling pathways in BGC-823 and MGC-803 cells. The effect of celastrol on cell cycle arrest of gastric cancer cells was due to an increase in the p27 protein level via inhibition of the miR-21-mTOR signaling pathway (66).

ACTIVE INGREDIENTS OF CHINESE MEDICINE TARGETING MIRNA INHIBIT TUMOR CELL PROLIFERATION
Many traditional Chinese medicines regulate cell proliferation through miRNAs (Figure 2). Paeoniflorin is a monoterpene glycoside with various anti-cancer activities and is derived   (77), lungs (78), breast (79), and pancreas (80). miR-124 levels are significantly increased in paeoniflorin-treated MGC-803 cells, which inhibits PI3K/Akt and p-STAT3 expression. A PI3K agonist or STAT3 overexpression can reverse effects of paeoniflorin on MGC-803 cell proliferation (55). Shikonin, a natural naphthoquinone isolated from traditional Chinese herbs. miR-106b is one of the most significantly downregulated miRNAs among the several miRNAs dysregulated by shikonin. miR-106b targets the tumor suppressor gene phosphatase and tensin homolog (PTEN), thereby modulating AKT/mTOR signaling pathway and ultimately inhibiting the proliferation of endometrial cancer cells (59). An earlier report investigated changes in the proliferation of the retinoblastoma cell lines Y-79 and Weri-RB-1 after shikonin administration. The results revealed that shikonin upregulated miR-34a and miR-202 expression and directly targeted the oncogene MYCN to degrade its mRNA while inhibiting the proliferation of retinoblastoma cells (62).
Several studies have shown that celastrol can inhibit tumor cell proliferation in several types of cancers. For example, in colon cancer cells, the overexpression of miR-21 enhanced cell viability, inhibited apoptosis, increase Bcl-2 expression, and decreased Bax levels; these effects were reversed by celastrol. As enzymes are involved in cell survival, the PI3K/AKT/GSK-3b pathway provides important signals for tumor cell proliferation Clastrol may inhibit colon cancer cell proliferation by negatively regulating the PI3K/AKT/GSK-3 pathways via miR-21 (64). Similar results were reported in lung adenocarcinoma, in which celastrol inhibited cell proliferation and induced apoptosis by regulating the expression levels of miR-24 and miR-181b (69).
Another study showed that ginsenoside Rh2 inhibited the proliferation of prostate cancer cells in a dose-dependent manner, and no CDKN1A cell cycle inhibitor was observed in the increased protein of ginsenoside Rh2. Screening all candidate miRNAs for binding to the 3′-untranslated region of CDKN1A showed that miR-4295 was dose-dependently inhibited by ginsenoside Rh2. Therefore, comprehensive experimental investigations revealed that ginsenoside Rh2 inhibits prostate cancer cell growth by inhibiting miRNA-4295, which activates CDKN1A (37).

ACTIVE INGREDIENTS OF CHINESE MEDICINE TARGETING MIRNA INHIBIT TUMOR CELL METASTASIS AND INVASION
The metastasis and invasion are essential feature characterizing the biological behavior of malignant tumors. The active   (82). In NCI-N87 cells, shikonin inhibited the proliferation, migration, and invasion by regulating miR-195 to inhibit the PI3K/AKT signaling pathway (60). Matrix metalloproteinases (MMPs) play important roles in mediating angiogenesis, metastasis, and invasion. MMP-2/9 expression is related to the progression of many tumors, such as colon cancer (83), neuroblastoma (84), and bladder cancer (85). Paeoniflorin regulated miR-16/29b, which targeted MMP-9/2 (56) to inhibit the growth and invasion of multiple myeloma cells (57). Celastrol can downregulate miR-21 and MMP9 and regulate the expression of the cell migration protein vimentin; this reduces the migration and invasion ability of MKN45 gastric cancer cells (65). Resveratrol significantly inhibited cell migration in T-cell ALL T-ALL-104 and B-cell ALL SUP-B15 cells by inhibiting miR-196b/miR-1290 (45).

ACTIVE INGREDIENTS OF CHINESE MEDICINE TARGETING MIRNA REVERSE TUMOR CELL RESISTANCE
Increasing evidence has revealed that dysfunctional miRNAs significantly affect chemotherapy resistance. Active ingredients of Chinese medicine play an important role in reducing the toxic and side effects of chemotherapy and improving resistance to chemotherapy (Figure 3). A previous study showed that lowinvasive breast cancer cells were resistant to amycin and this resistance was reversed by resveratrol, which also targeted the regulatory inhibitor miR-122-5p to influence the cell cycle and apoptosis (49). Increased autophagy during chemotherapy can promote tumor apoptosis or mediate autophagy-related apoptosis. miR-15a and miR-16 effectively induce autophagy,  enhancing the therapeutic effect of camptothecin (54). Celastrol reduced mTOR, P-P70S6K, and p-4EBP1 expression by increased miR33a-5p to inhibited tumor growth (70).

THE ACTIVE INGREDIENTS OF TRADITIONAL CHINESE MEDICINE REGULATE THE ANTI-TUMOR MECHANISM OF LNCRNAS
Generally, lncRNAs are defined as molecules comprising more than 200 nucleotides lacking protein-coding capacity. However, more recently, lncRNAs have been reported to regulate gene expression (86). Meanwhile, various active ingredients in Chinese medicines, such as curcumin and resveratrol, modulate tumor development via lncRNA expression regulation in vitro and in vivo ( Table 2). In gemcitabine-resistant pancreatic ductal adenocarcinoma cell, a phenolic compound extracted from turmeric, curcumin, desensitizes chemotherapy-resistant pancreatic ductal adenocarcinoma via inhibiting the PRC2-PVT1c-Myc axis. Hence, emerging evidence suggested that curcumin may be an effective sensitizing agent for chemotherapeutic drugs (96). Moreover, curcumin induces the expression of the lncRNA PINT to inhibit acute lymphoblastic leukemia cell growth (88).
Resveratrol is a non-flavonoid polyphenol compound with a wide pharmacological spectrum of anti-cancer, anti-inflammatory, anti-microbial, and antioxidant activity (97). One study on lung cancer reported the upregulation of 21 lncRNAs and downregulation of 19 lncRNAs resveratrol treated A549 cells. Among these, decreased levels of the lncRNA AK001796 weakened the inhibitory effect of resveratrol on cell proliferation (91). Resveratrol inhibits cell proliferation, migration, and invasion by downregulating AK001796 and NEAT1 in lung cancer and multiple myeloma (91,92). Similarly, triptolide and isoflavone calycosin inhibit tumor cell growth by inhibiting specific lncRNAs in nasopharyngeal cancer. Triptonide inhibits human nasopharyngeal carcinoma cell growth via disrupting lncRNA THOR-IGF2BP1 signaling. Conversely, ectopic lncRNA THOR overexpression inhibits Triptonide-induced cytotoxicity in NPC cells (95).
One of the representative lncRNAs, H19, is recognized as a cancer biomarker and is associated with the occurrence of esophageal cancer (98), colorectal cancer (99), liver cancer (100), breast cancer (101), bladder cancer (102), and stomach cancer. Furthermore, reduced expression of H19 can inhibit cancer development (103,104). Specifically, curcumin inhibited cell proliferation via c-Myc/H19 pathway, which reduced the expression of H19 in stomach cancer cells. This indicated that curcumin is a potential drug for gastric cancer (87). Moreover, microarray data identified H19 as a potential target of Huaier (a fungal parasite on locust trees), the extract from which reduced the expression of H19, while also reducing the viability of breast cancer cells by inducing apoptosis via regulation of the H19-miR-675-5p-CBL axis (105). Besides, certain lncRNAs and miRNAs mutually restrict and regulate target genes to achieve tumor inhibition. For example, in bladder cancer, H19 can directly bind miR-29b-3p to derepress the target DNMT3B. Further, upregulating H19 antagonizes miR-29b-3p-mediated proliferation, migration, and epithelial-mesenchymal transition (EMT) suppression in bladder cells. This evidence demonstrated, for the first time, that H19 may function as a competing endogenous RNA (ceRNA) for miR-29b-3p and relieve the suppression of DNMT3B, leading to EMT and metastasis of bladder cancer (106). In 2011, a new theory was proposed that ceRNAs and miRNA response elements could mediate the interactions between mRNA pseudogenes and some ncRNAs to form a large-scale regulatory network in the transcriptome and serve as a "new language" for "mutual conversation" (107) (Figure 4). These networks are characterized by sponge activity, in which ncRNA interacts with the target gene to competitively bind or inhibit. In addition, ceRNAs have been identified as key regulatory factors in cancer (108,109). PVT1, located downstream of the proto-oncogene Myc in chromosome 8q24, was used as a ceRNA of miR-216b and miR-152 in non-smallcell lung cancer and osteosarcoma to promote the tumor resistance to anti-cancer drugs (110,111). In prostate cancer cells, lncRNA-ROR and the stem cell marker Oct4 mRNA contain binding regions for miR-145 and directly compete with this microRNA. Curcumin reduced the expression of endogenous lncRNA-ROR and effectively increased the available concentration of miR-145 in human prostate cancer stem cells, where miR-145 prevented cell proliferation by decreasing Oct4 expression (31). The ceRNA hypothesis has revealed new mechanisms of RNA interactions, which incentivized the analysis of ncRNA role in cancer development.

REGULATION OF THE ANTI-TUMOR MECHANISM BY CIRCRNA AND OTHER NCRNAS
As potential targets for the active ingredients of Chinese medicine, circRNA, siRNA, rRNA, and other non-coding RNAs are involved in tumor development. Some circRNAs affect cancer biogenesis in diverse manners, such as by functioning as miRNA sponges, combining with RNA-binding proteins, acting as transcription factors, and affecting protein translation (112). Matrine decreased circRNA-104075 and Bcl-9 expression significantly via inhibition of PI3K/AKT and Wnt-bcatenin pathways,it suppressed cell viability while induceing apoptosis and autophagy in glioma cell line U251 (113). Another study shows that matrine down-regulated the levels of circ_0027345 and HOXD3, and up-regulated miR-345-5p expression. Meanwhile, matrine restrained tumor growth, invasion and promoted autophagy of HCC by regulating the circ_0027345/miR-345-5p/HOXD3 axis in vivo (114). Curcumin has antioxidant and anti-cancer properties, and it has also been used as a radiosensitizer. A study compared the differences in circRNA levels in NPC cell lines after radiotherapy and after treatment with curcumin, using a high-throughput microarray. Finally, it was demonstrated by reverse transcriptionquantitative polymerase chain reaction assay and wound healing assay that curcumin could enhance radiosensitization of NPC cell lines via mediating regulation of tumor stem-like cells by the "hsa_circRNA_102115"-"hsa-miR-335-3p"-"MAPK1" interaction network (115). At present, although circRNA has shown significant activity in the treatment of cancer, there are few reports on the regulation of circRNA by Active Ingredients of Chinese Medicine, which is also an important direction for future researchers to concern and research. circRNA has been expected to become a new molecular biomarker for the clinical diagnosis, treatment and prognosis, and the potential target for targeted therapy. siRNAs are double-stranded RNAs of 20-25 nucleotides and are involved in RNA interference; they regulate gene expression in a specific manner. Multiple synthetic siRNAs can achieve long-term silencing of target genes without interfering with endogenous microRNA pathways. Ginsenoside Rh2 downregulated P-STAT3/STAT3 and intracellular oxidative stress by upregulating PPAR. In response to siRNA-mediated knockdown of PPAR, STAT3 and intracellular oxidative stress were increased (116). rRNA is the most abundant type of RNA in cells. In lung cancer cells, triptolide interrupts rRNA synthesis by inhibiting transcriptional activation of RNA Pol I and UBF, thereby activating the apoptosis regulators caspase 9 and caspase 3 to inhibit BCL2 and induce apoptosis and cell cycle arrest (117).

DISCUSSION
During cancer development, abnormal ncRNAs modulate cell proliferation, migration, and invasion by regulating the expression of proto-oncogenes and tumor suppressor genes. Many active ingredients of traditional Chinese medicine, such as resveratrol, matrine, and berberine, have been evaluated in vivo and in vitro to target specific ncRNA and shown to play anticancer roles. This review summarized the finding regarding 16 active ingredients of traditional Chinese medicine that can target miRNAs, lncRNAs, and other ncRNAs, thereby playing an effective role in suppressing cancer growth. Among the ncRNAs regulated by Chinese medicine active ingredients, miR-21 is the most reported ncRNA and is extensively studied in various cancers. It is involved in most of the cancer-related processes, such as cell apoptosis, proliferation, migration, and cell cycle. These findings indicate that miR21 is one of the promising ncRNAs to develop targeted therapeutic agents for many types of cancer. Some active ingredients of traditional Chinese medicine, such as Ginsenoside Rh2 and Resveratrol, promote apoptosis by regulating ncRNAs to target common apoptosis-related target genes, such as BCL2 and Caspase3.  In conclusion, traditional Chinese medicine's active ingredients significantly ameliorate malignant neoplasms via ncRNA regulation, suggesting that active ingredients of traditional Chinese medicine may become alternative therapeutic agents for cancer in the future. At present, most studies have reported that the active ingredients of traditional Chinese medicine mainly target one kind of ncRNA for cancer treatment. However, ceRNA mechanism suggests that several kinds of ncRNA have complex interactions in cancer treatment. Therefore, we need to further explore the detailed anti-cancer mechanism and clinical safety of each of the active ingredients of traditional Chinese medicine. We hope that this review on the regulation of ncRNA by active ingredients of traditional Chinese medicine on tumor will be helpful for future research studies on anti-cancer of traditional Chinese medicine and provide a reference for their clinical application.

AUTHOR CONTRIBUTIONS
TL and SH participated in writing, editing, and making figures. ZQ and DL read and approved the final manuscript. All authors contributed to the article and approved the submitted version.