Non-coding RNAs in Regulating Tumor Angiogenesis

Non-coding RNAs (ncRNAs) are RNAs that do not encode proteins, but perform biological functions in various physiological and pathological processes, including cancer formation, inflammation, and neurological diseases. Tumor blood vessels are a key target for cancer management. A number of factors regulate the angiogenesis of malignant tumors. NcRNAs participate in the regulation of tumor angiogenesis. Abnormal expression of ncRNAs act as tumor suppressors or oncogenes to affect the development of tumors. In this review we summarized the biological functions of ncRNAs, and discussed its regulatory mechanisms in tumor angiogenesis. This article will provide new insights for the research of ncRNAs in tumor angiogenesis.


INTRODUCTION
It is well-acknowledged that cancer is one of the most severe diseases which leads to death and cripples life expectancy worldwide. Cancer therapy targeting solely tumor cells has been identified as the most extensive and efficient approaches in the past, but the clinical limitations still exist, such as toxic side effects (Montgomery et al., 2019). Since the rapid proliferation of tumor cells need new vascular systems to supply nourishment, angiogenesis plays a critical role in tumor maintenance, metabolic disorder, and tumor tissues dissemination/metastasis (Folkman, 2002). Therefore, antitumor angiogenesis treatment has emerged as an appealing solution in recent years.
Angiogenesis has been defined as a progression that new blood vessels are regenerated from the existing capillary network. An "angiogenic switch" is always activated in tumors, thus causing continuous new vessels generation. Tumor-associated neovasculature is a complex physiological incident, which is governed by a variety of pro-or anti-angiogenic cytokines and multiple signaling pathways, such as vascular endothelial growth factor (VEGF; Apte et al., 2019), angiopoietin (Carbone et al., 2018), etc.
As a class of significant RNA, non-coding RNAs (ncRNAs) are capable of performing biological functions at the RNA level. In high-grade organisms, up to half of DNA is transcribed into RNA, most of which are ncRNAs. It can be indicated that ncRNAs exert a key role in organismal development. Numerous studies have shown that ncRNAs participate in the occurrence and processing of tumors by functioning as oncogenes or tumor suppressor genes (Anastasiadou et al., 2018) thus these RNAs can be used as diagnostic and prognostic markers for cancer patients. New information has proved that a large number of ncRNAs involve in the modulation of tumor angiogenesis. NcRNAs [microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), small interfering RNA (siRNA), etc.] can interact with various angiogenic factors (VEGF, MMP2, etc.) and regulate signal pathways, such as Akt pathway and ERK1/2 pathway, in tumors (Safa et al., 2020;Wu et al., 2020). This article reviews and summarizes the major types of ncRNAs and their mechanisms in regulating tumor angiogenesis.

MicroRNAs
MicroRNAs are a type of eukaryotic endogenous small RNAs with a length of 18-25 nucleotides. Its main function is to regulate gene expression by binding to targeted RNAs (Beermann et al., 2016). The formation of miRNAs can be divided into two processes. First, the miRNA gene is transcribed into the initial transcription product (pri-miRNA), the pri-miRNA is recognized by the microprocessor and cut by the RNase III Drosha to form a hairpin structure called precursor miRNA (pre-miRNA), which is transferred from the nucleus to the cytoplasm under the mediation of Exportin-5 and RAN-GTP. Second, the pre-miRNA is cleaved into double-stranded miRNA under the action of RNase III Dicer. It then interacts with Argonaute to assemble the miRNA-induced silencing complex. The mature miRNA remains in the complex while the other strand is degraded (Ha and Kim, 2014). The mature miRNAs binds to targeted mRNA to perform post-transcriptional gene silencing role, thereby reducing the stability or inhibiting the translation of the target gene .

Long Non-coding RNAs
Long ncRNAs, with a length of more than 200 nucleotides, is transcribed by RNA polymerase II and involved in a variety of biological processes (Wong et al., 2018). LncRNAs exists both in the nucleus and cytoplasm, perform different functions according to its subcellular location (Kopp and Mendell, 2018;Miao et al., 2019;Li S. et al., 2020). The upstream promoter region encoding the protein interfered with the expression of downstream genes, and inhibits RNA polymerase II or recruits mediator proteins and chromatin remodeling enzymes to affect downstream gene transcription (Cho et al., 2018). LncRNA can be used as a scaffold to recruit RNA-binding proteins to form a nucleic acid-protein complex, and participate in chromatin remodeling and transcriptional regulation (Ban et al., 2020). In addition, it can also form a complementary double-strand with mRNA to interfere with the shearing of mRNAs (Romero-Barrios et al., 2018). Furthermore, lncRNAs interact with the protein bound to the 3 untranslated region (UTR) of mRNA in the cytoplasm to regulate the stability of mRNA . Antisense lncRNAs regulate the stability of mRNA by forming a double strand with mRNA (Liu et al., 2014). When lncRNAs is used as competing endogenous RNAs (ceRNAs), it can bind to miRNAs and prevent itself from inhibiting its targeted mRNAs . Moreover, it can positively or negatively regulate protein translation, encoding micropeptides with regulatory functions, and can also regulate signaling pathways in the cytoplasm, and bind to specific proteins to change the cellular localization of proteins (Hosen et al., 2018).

Circular RNAs
Circular RNAs have a closed-loop structure and is more stable than linear RNAs (Hanahan and Weinberg, 2011;Lee et al., 2020). In recent years, many circRNAs with important functions are coming to light and researchers began to focused on the properties and functions of circRNAs (Iurca et al., 2020). CircRNAs is divided into three types: exonic circular RNA (ecRNA), circular intronic RNA (ciRNA), and exon-intron circular (EIciRNA; Guo et al., 2014). Among them, ecRNA is the most common one, which is mainly locate in the cytoplasm, while ciRNA and EIciRNA are abundant in the nucleus (Liang and Wilusz, 2014). CircRNAs have been demonstrated as endogenous competitive RNAs that bind to miRNAs to inhibit targeted mRNA expression (Su et al., 2019).

Small Interfering RNAs
Small interfering RNAs are a kind of small double stranded RNA (dsRNA) with a length of 20-25 nucleotides, that are made from fully complementary long double-stranded RNA through dicer shearing (Leung et al., 2016). Exogenous dsRNA is cleaved by Dicer enzyme and TAR-RNA binding protein to form siRNA, then the siRNA loaded onto the Argonaute protein (AGO2) to form an RNA-induced silencing complex (RISC). RISC and targeted mRNA are partially or completely complementary paired, turning double-stranded siRNA into single-stranded siRNA. After combining with single-stranded siRNA, RISC become an active RISC. Targeting mRNA can be degraded through combining with activated RISC (Singh et al., 2018).

Other Non-coding RNAs
Piwi-interacting RNAs are small single-stranded RNAs with a length of 24-32nt. They have strong sense and antisense strand specificity, and the first nucleotide at the 5 -end is uracil-prone, while the 3 -end is modified by 2 -O-methylation. This type of end modification can prevent the degradation of mature piRNA genes. PiRNA must interact with PIWI protein form a piRNA silencing complex to play its regulatory role. Current studies  have shown that piRNA and PIWI abnormally expressed in gastric cancer, breast cancer, kidney cancer, colon cancer and lung cancer, and are involved in the occurrence, development and metastasis of cancers. PiRNAs could be potential prognostic and diagnostic biomarkers, and cancer treatment targets. PiRNA clusters are mainly distributed around centromeres and subtelomeres. They are transcribed into precursor piRNAs by RNA PolII, and are transported to the cytoplasm through primary processing pathways to form primary piRNAs, the pre-processed antisense piRNAs are loaded on Aub (Aub belongs to the Piwi subfamily) in the cytoplasm, and target the sense reverse transcript, resulting in the production of sense piRNA. These sense piRNAs are loaded onto Argonaute 3 (AGO3), and process the precursor antisense piRNA into mature piRNA. This amplification cycle continues with the continuous expression of Aub and AGO3, thereby causing a large amount of piRNA in the cell to be amplified. This phenomenon is called the "Ping-Pong" cycle (Soleimani et al., 2020). PiRNA mainly binds with PIWI or AGO3 protein, a member of the PIWI subfamily, to maintain genome stability by silencing transposable elements and regulating coding mRNA (Ng et al., 2016).
Small nuclear RNAs are a class of 50-200 nucleotides small RNAs exist in the nucleus. They are the main components of eukaryotic RNA spliceosomes. SnRNA is rich in uracil and usually numbered U1-U7. All snRNAs (except U6) have a 2,2,7trimethylated 5 -guanosine cap (Bohnsack and Sloan, 2018). In addition to regulating the correct expression of histone mRNA and the production of rRNA, it is involved in the formation of snRNAs complexes with proteins to catalyze the splicing of precursor mRNA (Guiro and Murphy, 2017).
Small nucleolar RNAs, with a length of 60-300 nucleotides located in the nucleolus, are divided into box C/D snoRNAs and box H/ACA snoRNAs. Box C/D snoRNAs mainly mediate 2 -O-methylation at specific sites of rRNA, box H/ACA snoRNA mainly mediate the pseudo uridylation of rRNA specific sites. Both can combine with ribonucleoprotein to play a key role in rRNA processing, participate in the splicing process of rRNA and other small RNA genes (Gong et al., 2017).

TUMOR ANGIOGENESIS
Tumors need to rapidly develop new vascular networks to support the rapid proliferation of cancer cells. Angiogenesis, which is defined as regeneration of new blood vessels from the existing capillary network, participates in the entire process of tumor development (Liu et al., 2011). Relevant studies have shown that solid tumors cannot grow to more than 2-3 mm without inducing their own blood supply. This view explains the association between angiogenesis and tumor development   . Therefore, angiogenesis plays an important role in the occurrence and development of tumors. In order to develop new drugs for anti-tumor angiogenesis, it is necessary to better understand the cellular and molecular mechanisms involved in tumor angiogenesis. Tumor angiogenesis is a complex process, including degradation of basement membrane, proliferation and migration of endothelial cell, and other steps.
As the key pro-angiogenic factors, VEGF and its receptors play a vital role in the whole process of tumor angiogenesis. VEGF family members are able to mediate a series of intracellular signal transduction pathways activation by fully binding to three significant tyrosine kinase receptors (VEGFR1, VEGFR2, and VEGFR3) and act on endothelial cells, which results in cell mitosis and capillary formation (Holmes et al., 2007). After combining with VEGFR2, several VEGF family members can significantly stimulate the differentiation and proliferation of vascular endothelial cells, promote angiogenesis and enhance the permeability of capillaries (Chang et al., 2009). VEGFR2 regulates the expression of related genes through PLC-γ-MEK-MAPK pathway, which leads to EC proliferation (Downward, 2004). VEGFR-2 can modulate cell migration by activating PI3K pathway. The activation of PI3K/Akt pathway in tumor microenvironment can inhibit endothelial cell apoptosis, ensure ECs survival and contribute to angiogenesis. It has been verified that PI3K/Akt pathway regulate hypoxia inducible factor-1 (HIF-1) and VEGF expression by activating kinases p70S6K1 and HDM2 in tumor tissue (Skinner et al., 2004;Jiang and Liu, 2008). Moreover, PTEN can inhibit tumor angiogenesis by promoting PI3K/Akt/VEGF/eNOS signaling pathway (Ma et al., 2009).
Tumor angiogenesis is a complex process, depends on the synergistic effect of multiple regulatory factors (Sahraei et al., 2019). Tumor internal environment can induce HIF-1 activating cells, to release a large amount of vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-2 (VEGF-2), fibroblast growth factor-2, and stromal cell-derived factor-1α/β (SDF-1α/β). These factors stimulate blood vessel formation and remodeling . In addition, pre-stimulation-angiogenic cells secrete matrix metalloproteinases (MMPs) to digest the basement membrane and accelerate vascular remodeling. The stability of the vascular network is an important factor that influence the development of tumors. The platelet particles release PDGF-BB and transforming growth factor-α (TGF-α), angiopoietin-1 (ANG-1) to promote the stability and maturity of the complex vascular network. Signaling pathways of tumor angiogenesis as shown in Figure 2.

MiR-21
MiR-21 is encoded by a gene containing miR-21 in the intron region of the TMEM49 gene (Ma et al., 2011). The primary transcript, pri-miR-21, is produced by RNA polymerase II transcription in the nucleus and processed into mature miR-21 in the cytoplasm . MiR-21 is involved in almost every aspect of tumor growth, such as promoting cell proliferation, invasion and metastasis, genome instability and mutation, inflammation, replication immortalization, metabolic abnormality, angiogenesis, evasion of apoptosis, immune destruction, and growth inhibition (Pfeffer et al., 2015). Studies have shown that miR-21 induces tumor angiogenesis by targeting PTEN and activates the AKT and ERK1/2 signaling pathways, thereby enhancing the expression of HIF-1 and VEGF . HIF-1 is a key target of miR-21 in regulating tumor angiogenesis. Liu et al. (2016) have shown that miR-21 leads to signal transducer and activator of transcription-3 (STAT3) activation and increases the level of VEGF in recipient cells, thereby promoting angiogenesis. Sahraei et al. (2019) suggested that the overexpression of miR-21 in tumor-associated macrophages (TAMs) led to the overall reorganization of the transcriptional regulatory network, which favored the formation of pro-inflammatory blood vessels, and promote tumor angiogenesis and tumor cell growth. In addition, miR-21-5p is highly enriched in endothelial progenitor cellsexosomes, and specifically inhibits the expression of angiogenesis inhibitor thrombospondin-1 (THBS1; Hu et al., 2019). Wu et al. (2019) found that under hypoxic conditions, the expression of miR-21-5p in the exosomes of papillary thyroid carcinoma BCPAP cells was significantly up-regulated. MiR-21-5p directly target and inhibit TGFBI and COL4A1, increasing endothelial cell proliferation, promoting tumor angiogenesis.

MiR-126
MiR-126 is encoded by a single gene located in the intron of the encoding protein 7 with an EGF-like domain (EGFL7) and is located on chromosome 9q34.3. MiR-126 is considered to be one of the most important miRNAs for maintaining the integrity of blood vessels, of which both miR-126-3p and miR-126-5p have biological activity (Casciaro et al., 2018). Jing et al. (2017) found that miR-126 inhibited tumor angiogenesis by downregulating the VEGF-A signaling pathway. In addition, miR-126 acts as a tumor suppressor in breast cancer cells, blocking tumor cell growth and metastasis by inhibiting tumor angiogenesis (Alhasan, 2019). Inhibition of miR-126 can induce the upregulation of the pro-angiogenic gene adrenomedullin to promote angiogenesis of cervical cancer (Huang and Chu, 2014).
In colorectal cancer , the oncogene YAP1 forms a complex with β-catenin/TCF4, which binds to the MALAT1 promoter and miR-126-5p, promotes the expression of VEGFA, SLUG and TWIST, and regulates the angiogenesis of colorectal cancer. Du et al. (2014) found that that miR-126-3p significantly inhibits HCC cell migration and invasion of extracellular matrix, and inhibits capillary formation of endothelial cells in vitro.
Overexpression of miR-126-3p significantly reduced tumor volume and microvessel density in vivo, and LRP6 and PIK3R2 were the targets. The level of miR-126-3p is negatively correlated with LRP6 and PIK3R2 in HCC tissues. In addition, rescue experiments showed that the function of angiogenesis of miR-126-3p is mediated by LRP6 and PIK3R2.

MiR-93
MiR-93 is involved in angiogenesis and tumor growth. Experimental studies have shown that miR-93 promotes tumor angiogenesis by reducing the expression of EPLIN (Ashrafizadeh et al., 2020). Upregulation of miR-93-5p can increase the angiogenic ability of HUVECs, thereby improving blood vessel density, increasing proliferation and migration of cancer cells (Liang et al., 2017). Fang et al. (2011) showed that overexpression of miR-93 can promote the proliferation, growth, migration and tube formation of endothelial cells, induce blood vessel formation, and extend blood vessels to tumor tissues at high density. The expression of miR-93 enhances the survival and invasive ability of cells, promotes tumor angiogenesis and metastasis by inhibiting the expression of LATS2. The formed tumor is rich in blood vessels (Fang et al., 2012). Fabbri et al. (2015) found that in the glioma cell lines U251 and T98G, proand antago-miR-93 can reversely regulate the expression of VEGF and IL-8 genes and protein release, which is associated with angiogenesis in glioma.

Long Non-coding RNAs Regulate Tumor Angiogenesis
Abnormal expression of lncRNAs has been observed in many human cancers, and their role as tumor suppressors or oncogenes is associated with the staging and grading of tumors (Bhan et al., 2017). Different lncRNAs play different roles in tumor regulation. LncRNAs such as HOTAIR (Li et al., 2014;Yu and Li, 2015;Fu et al., 2016;Zhou et al., 2020), MALAT1, ANRIL and SRA are up-regulated in tumors and play the role of oncogenes, while MEG3, GASS and LncRNA-p21 are down-regulated in tumors and play a role of tumor suppressors. H19 (Conigliaro et al., 2015;Jia et al., 2016;Jiang et al., 2016;Lv et al., 2017;Yuan et al., 2019;Liu Z.Z. et al., 2020) plays both oncogene and suppressor roles in tumors. LncRNA H19, HOTAIR, and MVIH (Yuan et al., 2012;Lei et al., 2016;Wang Y. et al., 2020) mainly regulate the proliferation, migration, invasion and angiogenesis of tumor cells by regulating VEGF, VASH2, and miR138/HIF1α axis. LncRNAs that regulate tumor angiogenesis are shown in Table 2.

H19
The H19 gene is mainly expressed in endoderm and mesodermderived tissues and locates on human chromosome 11p15.5. Its expression is down-regulated after birth (Yang et al., 2021). Studies have found that upregulation of H19 is related to angiogenesis (Rolla et al., 2021). Conigliaro et al. (2015) found that H19, which is highly expressed in CD90 + Huh cells, enters endothelial cells through exosomes to up-regulate the production and release of VEGF, which ultimately promotes angiogenesis and affects its tumor microenvironment. Jia et al. (2016) found that the upregulation of H19 in glioma tissues and glioma-associated endothelial cell (GEC) microvessels can promote the proliferation, migration and tubular formation of vascular endothelial cells in gliomas. H19 gene targets the 3 -UTR region of angiostatin 2 (VASH2) by inhibiting the expression of miR-29a. H19 is significantly overexpressed in glioblastoma tissues and promotes the angiogenesis of cells in vitro (Jiang et al., 2016;Lv et al., 2017). Yuan et al. (2019) found that H19 can interact with histone methyltransferase enhancer 2 (EZH2) to promote angiogenesis. Liu Z.Z. et al. (2020) found that the up-regulated H19 in glioma cells can promote tumor cell proliferation, migration, invasion, and angiogenesis through the miR138/HIF1α axis.

HOX Transcript Antisense Intergenic RNA
HOX transcript antisense intergenic RNA (HOTAIR) contains more than 2,100 nucleotides and locates in the 12q13.13 region of chromosome. It has a trans-acting (Cai et al., 2014) and promoting effect on the proliferation, metastasis, angiogenesis and metabolism of cancer cells (Yu and Li, 2015). In cervical cancer patients, elevated HOTAIR levels are significantly Silencing STAT3 and HIF-1α leads to an increase in the concentration of macrophages (CD11b + cells) and M1 macrophages that infiltrate the tumor microenvironment (CD169 + cells). It also leads to the reversal of the tumor-promoting function of TAMs-mainly angiogenesis and tumor cell activation. Shobaki et al., 2020 Frontiers in Cell and Developmental Biology | www.frontiersin.org associated with poor prognosis. Fu et al. (2016) found that HOTAIR promotes tumor cell growth and angiogenesis by directly activating VEGFA and Ang2 expression in nasopharyngeal carcinoma cells. Li et al. (2014) found that in metastatic breast cancer, HOTAIR promotes the angiogenesis of breast epithelial cells through transcriptional activation of VEGF-C, thereby promoting tumor metastasis.

MVIH
MVIH locates in the intron region of the ribosomal protein S24 gene (RPS24) and overlaps the exons of RPS24 . MVIH is associated with microvascular invasion of liver cancer. Yuan et al. (2012) found that in liver cancer, MVIH promotes tumor growth and intrahepatic metastasis by activating angiogenesis. Wang Y. et al. (2020) found that MVIH inhibits the secretion of PGK1 to activate colorectal cancer angiogenesis through interacting with RPS24 (ribosomal protein S24), which is highly expressed in colorectal cancer.

CricRNAs Regulate Tumor Angiogenesis
CircRNAs act as signaling molecules in regulating tumor growth, angiogenesis, invasion, metastasis, and chemotherapeutic sensitivity. In addition, circulating exosome circRNAs can affect tumor progression and malignant characteristics. CircRNA has great value in tumor diagnosis and prognosis, and is a promising non-invasive biomarker (Wang M. et al., 2020). Circ-ATXN1 , circ-SHKBP1 (Xie et al., 2020), and circ-001971  mainly regulate VEGF through sponging miRNAs and activate PI3K/AKT signaling pathway to promote tumor cell proliferation, migration and angiogenesis. Common circRNAs that regulate tumor angiogenesis are shown in Table 3.
Previous studies have shown that  angiogenesis plays an important role in the occurrence and development in gliomas. The expression of circ-ATXN1 significantly enhances the cell viability, migration and tube formation in GECs. Circ-ATXN1 functionally targets miR-526b-3p in the RISC and affects the angiogenesis of vascular endothelial cells by negatively regulating the expression of MMP2/VEGFA. Xie et al. (2020) found that circ-SHKBP1 is expressed in gastric cancer tissues and serum of patients. As a sponge molecule, circ-SHKBP1 can adsorb miR-582-3p to increase the expression of HUR, enhance the stability of VEGF mRNA, and promote angiogenesis of gastric cancer cells. Circ-001971 acts as a ceRNA by relieving miR-29c-3p-induced VEGFA inhibition, thereby aggravating the proliferation, invasion and angiogenesis of colorectal cancer . Zheng et al. (2020) found that the expression of circ-0056618 increased in colorectal cancer tissues and colorectal cancer cell lines. Circ-0056618 acts as a sponge molecule to adsorb miR-206 and eliminate the inhibitory effect of miR-206, thereby upregulating CXCR4 and VEGF-A in colorectal cancer. Circ-PRRC2A acts as a sponge molecule to adsorb miR-514a-5p and miR-6776-5p to prevent the degradation of the mRNA of tissue-specific oncogene TRPM3, promoting angiogenesis and tumor metastasis (Li W. et al., 2020). CircRNA-MYLK can directly bind to miR-29a and reduce the inhibition of VEGFA, thereby activating the VEGFA/VEGFR2 signaling pathway, ectopic expression of circRNA-MYLK promotes the proliferation, migration, tubular formation and cytoskeleton rearrangement of HUVEC . Hsa-circ-0000515 is up-regulated in breast cancer tissues. Hsa-circ-0000515 binds to miR-296-5p to prevent it from inhibiting CXCL10 expression, promotes cell cycle progression, cell proliferation and invasion of breast cancer cells, and increases the potential of cancer cells to promote angiogenesis (Cai et al., 2021). He et al. (2019) showed that circ-DICER1 acts as a molecular sponge to adsorb miR-103a-3p and miR-382-5p, and weaken its negative regulatory effect on ZIC4 in GECs. ZIC4 upregulates the expression of its downstream target Hsp90β, and Hsp90 activates the PI3K/AKT signaling pathway and promotes cell viability, migration and tubular cell formation.

Small Interfering RNAs Regulate Tumor Angiogenesis
Small Interfering RNAs silences targeted genes to inhibit angiogenesis of cancer cells and tumor growth. In vivo, siRNAs can significantly influence tumor angiogenesis by regulating related genes and pathways. Common siRNAs that regulate tumor angiogenesis are shown in Table 4. Yang F. et al. (2020) used polymer micelles as a carrier to deliver triptolide and siRNA to retinoblastoma (RB) cells. The micelle carrier loaded with triptorelin and HIF-1 siRNA showed effective cell internalization, inhibited the expression of HIF-1α and VEGF in RB cells, leading to inhibition of the HIF-1α/VEGF/VEGFR signaling pathway, and the proliferation, migration, and invasion of vascular endothelial cells. Bastaki et al. (2021) generated trimethyl chitosan and thiolated chitosan nanoparticles (NPs) conjugated with HIV-1-derived TAT peptide and HA (hyaluronic acid). These NPs exhibited prominent physicochemical characteristics, notable siRNA encapsulation, serum stability, non-toxicity, controlled siRNA release, and extensive cellular uptake by cancer cells. The siRNAs silenced targeted genes, immune checkpoint molecule programmed cell death ligand 1 and oncogene transcription factor STAT3, which significantly inhibits the proliferation, migration and angiogenesis of cancer cells, inhibits tumor growth in the body. Shan et al. (2020) found that chitosan magnetic nanoparticles (CMNPs) carrying Ang-2 small interfering RNA plasmids have inhibitory effects on malignant melanoma, and can significantly inhibit the growth of melanoma. In vivo, Ang2-CMNP significantly inhibits tumor angiogenesis and promotes cell apoptosis by adjusting the ratio of Bax/Bcl-2 and increasing the expression of caspase-3. Shobaki et al. (2020) using optimized load CL4H6-LNP (CL4H6 is a novel, pHsensitive cationic lipid, LNP is a lipid nanoparticle) siRNA targeting TAMs, and anti-tumor response is obtained in the same tumor model. The anti-tumor therapeutic response was obtained through the silencing of STAT3 and HIF-1α, which resulted in an increase in the level of infiltrated macrophage (CD11b + cells) into the tumor microenvironment as well as a tendency to increase the concentration of M1 macrophages (CD169 + cells). The treatment also resulted in reversing the protumorous functions of TAMs -mainly angiogenesis and tumor cell activation.

Other Non-coding RNAs Affect the Occurrence and Development of Tumor
Although the association of other ncRNAs such as piRNAs, snRNAs and snoRNAs with tumor angiogenesis has not been reported, they also play important roles in the development of tumors. Common other ncRNAs affect the occurrence and development of tumor are shown in Table 5.
Both PIWI protein and piRNA are mainly expressed in germline cells and abnormally expressed in a variety of cancer cells. Weng et al. (2018) found that piR-1245 is overexpressed in colorectal cancer, and the overall survival of patients with piR-1245 overexpression is significantly shortened. PiR-1245 acts as an oncogene and promotes tumor development by targeting tumor suppressor genes such as ATF3, BTG1, DUSP1, FAS, NFKBIA, UPP1, SESN2, TP53INP1, and MDX1.
U1 snRNA, as one of the most abundant ncRNAs in human cells, has a high recurring A > C somatic mutation at the third base (Shuai et al., 2019). This mutation changes the splicing pattern of multiple genes, including known cancer driver factors, accounting for the mechanism of abnormal snRNA in cancers. Siprashvili et al. (2016) compared 5,473 pairs of tumornormal genome pairs. They found that in 12 common cancers, 10-40% of snoRNA loci were deleted, the deletion of snoRNAs promoted tumorigenesis, but the mechanism is still unknown.
Piwi-interacting RNAs, snRNAs, and snoRNAs are abnormally expressed in a variety of cancer cells, and play important roles in the occurrence and development of cancers. However, their relationship with tumor angiogenesis has not yet been reported. Because tumor angiogenesis plays an indispensable role in the development of tumors, the relationship between these ncRNAs and tumor angiogenesis warrants further explored.

CONCLUSION
Neovascularization is of indispensability for tumor development and metastasis, with multiple formation patterns and complicated regulatory mechanisms. The main physiological processing of tumor angiogenesis is as follows: One of the critical outcomes of rapid tumor growth is oxygen and nutrients absence in tumor microenvironment. Hypoxia can immediately trigger the secrete of various angiogenic factors in tumor tissue. After binding to the surface receptors of ECs, these cytokines involve in promoting the proliferation and directional migration of ECs. New sprouts are shaped from the degradation of subendothelial basal membrane afterward. Upon the stimulation of several growth factors, ECs could sharply proliferate, cross bloody sprouts and move forward to neoplasm location. Next, the newly-formed ECs are able to produce massive adhesion molecules, which specifically connect with the original ECs and bridge the stretch of vascular sprouts. Consequently, a complete vessel network has established as a result of interaction between the newly-formed ECs, vascular extracellular matrix and stromal cells. In summary, the proliferation, migration and invasion of ECs and microtubule formation induced by pro-angiogenic factors are significant for tumor angiogenesis process.
As described above, a variety of ncRNAs exert multi-roles in the secretion of angiogenic cytokines, the proliferation, migration, invasion of ECs and the establishment of vascular system. Common ncRNAs that regulate the critical steps in tumor angiogenesis are shown in Figure 3. For example, it has been demonstrated that miR-21, miR-93, H19, HOTAIR, MVIH, circ-ATXN1, circ-SHKBP1, circ-001971, circ-0056618, and circRNA-MYLK are able to effectively modulate the expression of VEGF. In addition, miR-21, H19, circ-PRRC2A, and circ-DICER1 are proved to involve in the proliferation of ECs. Similarly, miR-93, H19, Hsa-circ-0000515 and circ-0056618 can affect the course of ECs migration and invasion. Moreover, regarding the establishment of vascular system, miR-21, miR-93, H19, and circ-ATXN1 play an important role in this process. For the multi-functions of ncRNAs in tumor angiogenesis, miR-21, miR-93, circ-ATXN1, and circ-0056618 participate in regulating several key aspects of this course and H19 involve in the whole processing. Of note, miR-126 exerts an adverse effect on tumor angiogenesis: it inhibits the production of VEGF, the migration and invasion of ECs and eventually abates the vascular system.

FUTURE PERSPECTIVES
Although the biological functions and mechanisms of ncRNA in regulating tumor angiogenesis still need to be further investigated, novel advances in past several years have been achevied in exploring the regulatory role of ncRNAs in tumor angiogenesis. For example, ncRNAs accumulation specificly in some tumor cells can be exploited to develop new medical surveillance technologies, potentially allowing faster and more accurate detection of tumor initiation and progression. In addition, RNA sponges, specific interfering molecules targeting ncRNAs that function as proto oncogenes and ncRNAs that serve as tumor suppressors have been synthesized for anti-tumor angiogenesis therapy. Therefore, the emerging relationship between ncRNAs and tumor angiogenesis opens up new horizons for its diagnosis and treatment. Notably, although researchers have investigated multiple methods to transform ncRNAs to applicable biomarkers or the targeted drugs, several problems still need to overcome in present, such as the instability of RNA itself, the indetermination of temporal and spatial expression of ncRNAs, and the unknown other side effects. In short, ncRNAs regulate tumor angiogenesis and could be targets of novel drug development for cancer treatment. One can hope that in the near future, the relationship between ncRNAs and angiogenesis will be better understood, with their value provided original and potential strategies for cancer management.