The identification of signaling pathways leading to the activation of epithelial to mesenchymal transition (EMT) during tumor invasiveness and other diseases provides insights into the plasticity of cellular phenotypes [
18]. EMT and the reverse process mesenchymal to epithelial transition (MET) play a vital role in different steps of metastasis formation, where at distant sites, a more epithelial phenotype is favorable, as cells have to adhere to their surroundings. Therefore, epithelial plasticity is essential for successful metastasis formation, as EMT can provide benign epithelial cancer cells with the required traits to travel to metastatic sites initiating secondary tumor growth. Reactivation of EMT during cancer progression enhances the metastatic phenotype [
19]. During EMT, epithelial cells lose their polarity, disassemble cell junctions, and gain migratory characteristics, acquiring a more mesenchymal phenotype. It is important to point out that during EMT there is no change in cell adhesion but rather a fundamental reorganization of cell topology [
20]. A number of genes involved in cell adhesion, migration, invasion, and mesenchymal differentiation are transcriptionally altered during the execution of EMT program. Amongst the EMT-inducing transcriptional factors associated with metastasis and tumor invasion are Snail, Slug, dEF1, SIP1, Twist1, FOXC2, and Goosecoid [
21]. It is hypothesized, since EMT causes the loss of epithelial characteristics, that it contributes directly to the variation of epithelial cell adhesion molecules (EpCam) and cytokeratin expression seen in circulating tumors. EMT-like phenotypes have been shown to bestow chemoresistance [
22]. The functional loss of E-cadherin gene promoter is a hallmark of EMT, which is detected in distant metastases, and in humans the E-cadherin promoter; the E-box elements are responsible for its transcriptional repression in non-E-cadherin-expressing mesenchymal cells. Several signaling molecules are implicated in the EMT process and carcinomas progress, including WNT, TGFβ, and Notch ligands [
23]. Other oncogenic pathways include STAT3, PIK3/Akt, ZEB, oncogenic miRNAs, and long non-coding RNAs (lncRNAs) [
24]. Carcinoma cells undergoing EMT establish mechanisms for initiating invasive and metastatic behavior for cells of epithelial cancers, generating life threatening manifestations of cancer progression. Additionally, they exhibit heightened resistance to various forms of existing anti-cancer therapies. Hallmarks of the initiation and early growth of primary epithelial cancers encompass uncontrolled epithelial cell proliferation and angiogenesis. During EMT, polarized immotile epithelial cells undergo multiple biochemical changes, enabling them to assume a motile mesenchymal phenotype. Cancer cells expressing the motile mesenchymal phenotype subsequently enter the invasive-metastasis cascade: intravasation, translocating through the bloodstream to microvessels of distant tissue, extravasation, formation of micrometastases, and finally colonization [
5], hence enhancing migratory capacity, invasiveness, resistance to apoptosis and production of extracellular matrix (ECM) components.
Amongst the several molecular processes involved in initiating and enabling EMT completion are the changes in the expression of specific miRNAs, which could be used as a biomarker to demonstrate the cell passage during EMT, possibly providing a lead for intervention [
18]. Chaffer et al., 2016. suggested that since cancer cells can reside in various phenotypic states along the EMT spectrum, and the fact that they can transition dynamically between the epithelial and mesenchymal phenotypes manifests their ability to survive and seed metastatic deposits, providing cancer cells with tumor initiating potential and the ability to maintain phenotypic plasticity [
23]. This thus promotes successful completion of the metastatic cascade outlined in . The ability of cancer cells to perform the metastatic cascade corresponds to the transition between the epithelial, hybrid E/M, and mesenchymal states. EMT promotes tumor aggressiveness by providing plasticity. Lately, there is evidence that carcinoma cells residing in a partially epithelial and partially mesenchymal state, i.e., a hybrid favor tumor progression and metastasis. That is because cancer cells at the metastatic cascade can reverse their phenotype by MET and form secondary tumors. A hybrid E/M state with both features increases the possibility of cells to acquire stem-like properties [
25]. Such cells have enhanced tumor-initiation potential and can integrate the properties of cell–cell adhesion and motility facilitating collective cell migration, forming clusters of circulating tumor cells (CTCs) accelerating the metastatic cascade even further. Transcription factor NRF2 can stabilize a hybrid E/M phenotype and even prevent a complete EMT. Its knockdown in RT4 BLCA cell line downregulates E-cadherin and ZEB-1, while its overexpression upregulates E-cadherin and ZEB-1, promoting a more hybrid E/M phenotype. Therefore, NRF2 is a hallmark of the hybrid E/M with maximal propensity to generate metastases, but NRF2 signaling could have different effects depending on the position of the cells in question in the EMT spectrum [
26].
2.3. Mechanism of Metastatic Inhibition or Induction in BLCA Via miRNA
There are many lines of evidence demonstrating the involvement of miRNAs in signaling pathways of proteins regulating metastasis in BLCA (). Onco-suppressor miRNAs are downregulated in BLCA cells and tissues to ensure proliferation and migration. In the previous sections, we discussed specific miRNAs dysregulation in regards to cell–cell and cell–matrix adhesions, here we will be covering alterations of some direct miRNA target genes and pathways that enhance metastasis in BLCA, as shown in .
Figure 2. Metastatic inhibition and induction by miRNA in BLCA.
Table 3. miRNAs involved in metastasis induction/inhibition in BLCA.
microRNA |
Samples/Cell Culture |
Targets/ Regulators |
Function |
Patient’s Prognosis |
References |
miR-124 |
Human bladder transitional cancer cell lines J82 and T24 |
UHRF1 |
miR-124 can impair the proliferation or metastasis of human BLCA cells by down-regulation of UHRF1 |
|
[56] |
miRNA-139-5p and miRNA-139-3p |
human BLCA cell lines: T24 and BOY |
MMP11 |
Downregulation of both miRNAs enhanced BLCA cell migration and invasion |
Higher expression of MMP11 predicted shorter survival of BLCA patients (p = 0.029) |
[55] |
pre-miR-145 and miR-145-5p |
Clinical tissue: 62 BLCA patients at Kagoshima University Hospital between 2003 and 2013 |
UHRF1 |
Overexpression of miR-124 in vitro, attenuated cellular proliferation, migration, invasion, and angiogenesis by downregulating UHRF1 |
|
[55] |
miR-328-3p |
Cell lines: Urinary epithelial SV-HUC-1 and 5637, T24, J82 BC cell lines Clinical tissue:28 pairs of BLCA tissues and adjacent normal samples were acquired from the Yinzhou Hospital |
ITGA5 |
miR-328-3p inhibited the development of BLCA by targeting ITGA5 and inactivating the PI3K/AKT pathway |
Downregulation of miRNA predicted poor prognosis in BLCA patients |
[57] |
miR-15 |
T24, BIU87, HT1376 BLCA cell lines, and normal uroepithelial cell lines SV-HUV-1 |
BMI1 |
Overexpression of miR-15 inhibited EMT and PI3K/AKT pathway |
|
[58] |
miR-24 |
Human BLCA cell lines T24, UMUC-3, J82, 5637 and normal transitional epithelial cell line SV-HUC-1 |
CARMA3 |
Upregulation of miR-24 inhibited proliferation, invasion, EMT, and induced apoptosis of T24 and UMUC-3 cells. Additionally, upregulation of miR-24 decreased the protein levels of cyclin D1, CDK4, CDK6, p-Rb, and Bcl-2 |
|
[59] |
miR-23b |
T24 and J82, and normal SV-HUC-1 |
3′UTR of Zeb1 |
Overexpression of miR-23b suppressed the oncogene ZEB1 suppressing cell proliferation, invasion apoptosis, and cell cycle arrest |
Patients with higher miR-23b expression had longer OS compared to patients with low miR-23b expression. Additionally, its expression distinguished malignant from normal tissues |
[60] |
miR-203 |
T24, RT4, normal urothelial cell line SV-HUC-1 |
Twist1 |
miR-203 mimic significantly reduced BLCA cell proliferation, migration, and invasion, and induced apoptosis by targeting Twist1 |
|
[61] |
miR-22 |
T24, UM-UC-3, as well as one normal bladder cell line SV-HUC-1 |
MAPK1 and Snail |
miR-22 was found to suppress cell proliferation/apoptosis by directly targeting MAPK1 and inhibiting cell motility by targeting both MAPK1 and Snail |
Low-expression of MAPK1 or Snail is an independent prognostic factor for better OS |
[62] |
miR-132 |
Human BLCA cell lines T24 and human normal urothelial cell line SV-HUC-1 Clinical tissue:32 patient samples |
SMAD2 |
miR-132 may play a suppressive role in the metastasis of BLCA cells via TGFβ1/Smad2 signaling pathway. Overexpression of miR-132 suppressed expression of mesenchymal cell markers (N-Cadherin, Zeb1, Snail, and Vimentin) |
|
[63] |
miR-484-5p |
Cell lines: Human bladder cancer cell lines SW780, T24, HT1376, and HT5637 and human bladder epithelial cell lines HU609 and HEK293 cells Clinical tissue:15 patient samples who had undergone surgery and primary therapy |
HMGA2 |
miR-485-5p exerts a suppressive effect, partly through the suppression of HMGA2 |
|
[64] |
miR-126 |
Cell lines: HUC, EJ138, MCF10A, TCCSUP, J82, and 293FT cells Clinical samples: TaLG (n = 3), TaHG (n = 3), CIS (n = 3), T1LG (n = 3), T1HG (n = 3), and T2HG (n = 6) |
ADAM9 |
miR-126 exerts its tumor suppressive role by targeting ADAM9 to inhibit cell invasion |
|
[65] |
miR-199a-5p |
T24 and SV-HUC-1 cell lines Clinical samples:40 BLCA tissue samples and adjacent normal tissue from patients who underwent transurethral bladder tumor resection or radical cystectomy |
3′UTR of CCR7 |
miR-199a-5p was confirmed to be able to target the 3′ UTR of CCR7 and regulate the expression of CCR7, MMP-9, and vimentin and E-cadherin |
|
[66] |
miR-493 |
SV-HUC-1, T24, J82, and TCCSUP cells Clinical samples: human bladder cancer tissue array from US Biomax |
RhoC and FZD4 |
miR-493 possibly a tumor suppressive, inhibiting cell invasion and migration by blocking FZD4 and RhoC, implicating the Wnt-PCP pathway in bladder carcinogenesis |
|
[67] |
miR-497 |
BOY and T24 cell lines Clinical samples: 5 BLCA patients and five normal epithelial samples of patients who underwent cystectomy or transurethral resection. |
BIRC5 and WNT7A |
Downregulation of miR-195/497 contributed to BLCA progression and metastasis |
|
[68] |
Induction |
miRNA-135a |
Normal human SVHUC-1 epithelial cells, EJ, T24, BIU87, ScaBER, and 5637165 Clinical samples: paired BLCA tissues and adjacent normal tissues were obtained from patients with BLCA who had undergone a bladder resection |
GSK3β |
miR-135a accelerates the EMT, invasion, and migration of BLCA cells by activating the Wnt/β-catenin signaling pathway through the downregulation of GSK3β expression. |
|
[37] |
miR-96 |
HT1376 |
FOXQ1 |
TGF-β1 could change the expression of FOXQ1 induced by miR-96, which revealed that TGF-β1 regulates miR-96/FOXQ1 signaling |
|
[69] |
miR-221 |
RT4 and T24 |
3′UTR of STMN1 |
miR-221 can facilitate the TGFβ1-induced EMT process in human BLCA cells by suppressing STMN1 |
|
[70] |
miR-301b |
J82, UM-UC-3, T24, 5637 BLCA cell line and normal SVHUC-1 cell line Clinical tissue: 31 paired BLCA and normal tissue obtained from patients who underwent radical cystectomy. |
EGR1 |
miR-301b promotes the proliferation, migration, and aggressiveness of human BLCA cells by inhibiting the expression of EGR1. |
|
[70] |
The expression of most of the below mentioned miRNAs was found to be downregulated in BLCA cells. In cancers, miR-124 is involved in epigenetic regulatory programs with DNA methylation and chromatin remodeling and is also responsible for transcription factor SOX9. In BLCA, UHRF1 is a direct target of miR-124 downregulation. Overexpression of miR-124 in T24 cell line resulted in reduced levels of VEGF, MMP-2, and MMP-9. Additionally, overexpression of miR-124 in vitro attenuated cellular proliferation, migration, invasion, and angiogenesis, while in vivo tumor growth downregulated UHRF1, showing an inverse relationship between miR-124 and UHRF1. In T24 and J82 cell lines, silencing UHRF1 suppresses migration and hinders invasion, thus having an oncogenic role in BLCA, with a role in angiogenesis [
56]. By regulating UHRF1, the pre-miR-145 and guide strand miR-145-5p and passenger strand miR-145-3p act as anti-tumor miRNA in BLCA, thus indicating that both the guide and passenger strands of miRNA have a biological role through the regulation of several genes in BLCA. Their restoration significantly inhibited BLCA cell migration and invasion, therefore possibly downregulating genes that promote metastasis [
55].
miR-328-3p is found to be downregulated in BLCA predicting poor prognosis. It acted as an inhibitory miRNA directly targeting ITGA5, via inactivating protein kinase B signaling pathway (PI3K/Akt), consequently inhibiting EMT and tumorigenesis [
58]. Another miRNA that inhibits EMT and decreases BLCA invasion through PI3K/Akt signaling pathway is miR-15. B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a direct target of miR-15 and via regulating BMI1 through PI3K/Akt pathway, miR-15 inhibited in vivo BLCA cell progression and tumor growth. miR-15 enhanced N-cadherin and Vimentin expression and suppressed E-cadherin levels, thereupon regulating cell invasion and migration by EMT regulation as well as repressed the phosphorylation of AKT expression levels [
58]. In T24 and UMUC-3 BLCA, cell lines upregulation of miR-24 inhibited proliferation and induced G1 phase arrest and apoptosis. Its upregulation downregulated the mesenchymal markers including MMP9 amongst others at protein levels, indicating that it can inhibit invasion and EMT. Card-containing MAGUK3 (CARMA3), a tumor suppressor gene required for G protein-coupled receptors (GPCR), shares a link with NF-κB activation. That is of special interest, as downstream targets of NF-κB include cyclin D1, Bcl-2, and MMP9, and thus blocking its activation could inhibit metastasis and induce apoptosis. CARMA3 is negatively regulated and a direct binding target of miR-24 in BLCA with its suppression crucial for miR-24 inhibited proliferation, invasion, and EMT, potentially by downregulating the CARMA3/NF-κB pathway [
59].
Compared to normal bladder tissue, miR-23b was found to be downregulated in BLCA and non-malignant cell lines acting as a tumor suppressor. Ectopic expression of miR-23b in J82 and T24 cell lines decreased cell proliferation and colony formation, albeit its re-expression increased cells in the G0/G1 phase while decreasing cells in the S-phase of the cell cycle, triggering G0/G1 arrest and inducing apoptosis. ZEB1, a crucial regulator of EMT and in BLCA responsible for enhanced motility, is a direct target of miR-23b. In BLCA, miR-23b overexpression suppressed the oncogene ZEB1. Thus, miR-23b can mediate EMT, as it post-transcriptionally regulates ZEB1 via targeting its 3′UTR, downregulating its levels [
60]. miR-203 in T24 and RT4 cell lines induced cell apoptosis via inhibiting the expression of Bcl-2 and procaspase 3 proteins, while enhancing the expression of Bax and cleaved caspase 3. Twist1 is a direct target of miR-203, with miR-203 acting as a tumor suppressor by negatively targeting Twist1 [
61].
Increased ectopic expression of miR-22 increased E-cadherin expression, but decreased that of N-cadherin, vimentin, Snail, Slug, and GSK-3β phosphorylation, therefore suppressing EMT in BLCA acting as a tumor suppressor. MAPK and Snail were found to be direct target genes of miR-22. Overexpression of MAPK1 correlated with poor survival and induced the transcriptional activity of Slug, upregulating vimentin expression, whereas overexpression of miR-22 reversed MAPK or Snail-induced migration and invasion, thus playing an important role in EMT progression and the MAPK1/Slug/vimentin feedback loop. Additionally, knockdown of MAPK1 suppressed Slug and vimentin expression, indicating that Slug possibly acts as a scaffold in MAPK1-induced vimentin expression in BLCA cells. They also performed western blot (WB) assay to reveal the effect of silencing vimentin and found that it suppressed ERK2 phosphorylation, implying that vimentin can activate MAPK1, forming a MAPK1/Slug/vimentin feedback loop in BLCA cells [
62].
Another study performed in T24 BLCA cell line examined the expression of TGFβ1/Smad2 following transfection with miR-132 inhibitor. Their results revealed that migration and invasion capabilities, as well as EMT related markers and TGFβ1/Smad2 expression levels, were increased, with a negative expression of Smad2 and miR-132 in BLCA tissue. Additionally, they found miR-132 levels of expression reduced in BLCA tissue with lymph node metastasis. Furthermore, miR-132 suppressed the mesenchymal markers including N-cadherin, Zeb1, Snail, and Vimentin. Hence, miR-132 might suppress EMT via TGFβ1/Smad2 signaling pathway [
63]. Through the suppression of HMGA2 expression, miR-484-5p was also found to inhibit metastasis and EMT partly [
64]. As mentioned earlier, miRNAs have the unique ability to regulate several protein coding genes, and since BLCA is a heterogeneous disease, when it becomes invasive and metastatic, it substantiates poor prognosis. miR-126 impaired the invasive potential of BLCA by direct downregulation of ADAM9 [
66], miR-199a-5p by targeting CCR7 [
66], miR-493 by downregulating RhoC and FZD4 [
67], and miR-497 by targeting BIRC5 and WNT7A [
68]. These miRNAs could present potential therapeutic approaches in treating invasive BLCA.
We formerly stated that the mode of action of miRNAs depends on their target genes in cells or tissues, ergo they not only act as oncosuppressor, but also as oncogenes. One of those oncogenes is miRNA-135a. In colorectal cancer, miR-135a activates the downstream Wnt pathway by suppressing the tumor suppressor gene adenomatous polyposis coli (APC). In BLCA, when compared with adjacent normal tissue, the expression of miR-135a, β-catenin, cyclinD1, vimentin mRNA, and protein expression increased, while GSK-3β and E-cadherin mRNA and protein expression decreased. EMT was induced when miR-135a overexpression inhibited GSK-3β mRNA and protein expression following that activation of Wnt/β-catenin signaling pathway by promoting its related genes including GSK3β, since miR-135a binds specifically to it. Therefore, when miR-135a targets GSK3β, it accelerates BLCA proliferation, migration, and invasion and suppresses apoptosis [
37]. Other oncogenic miRNAs are capable of enhancing the expression of EMT transcription factors such as miR-96. TGF-β1 is essential for EMT and cancer progression, and miR-96 induced EMT driven by TGF-β1, which could also regulate the expression of miR-96 target, FOXQ1. miR-96 binds to the 3′untranslated region (UTR) of forkhead box O3 (FOXO3), which triggers apoptosis via regulating genes necessary for cell death, thus inhibiting its function [
70]. Furthermore, in RT4 and T24 cell lines, miR-221 and STMN1 were found to be involved in TGF-β1 induced EMT with the expression of miR-221 being upregulated. The microtubule-destabilizing protein, stathmin 1/oncoprotein 18 (STMN1), is an oncogenic protein enhancing invasion and metastasis and during mitosis influences cell cycle progression. STMN1 was downregulated by TGF-β1, and miR-221 suppressed STMN1 expression by targeting 3′UTR. In BLCA, miR-301b levels were overexpressed and induced EMT via early growth response gene 1(EGR1) downregulation [
70]. A bioinformatics study found that miR-21 could serve as a prognostic factor for OS and a good indicator of metastasis and tumor recurrence in BLCA with p53, Akt, and PTEN as its target genes [
11].
2.4. Mechanism of Angiogenesis Inhibition Via miRNA in BLCA
miRNAs have a vital role in angiogenesis by regulating proliferation, differentiation, apoptosis, migration, and tube formation of angiogenesis related cells (). An imbalance between miRNA regulation and angiogenesis could result in the occurrence and development of cancer and vascular diseases. In BLCA, vascular endothelial growth factor-C (VEGF-C) has an anti-apoptotic and proliferative role, as shown in . It is an angiogenic factor that may affect cancer growth, is associated with lymphangiogenesis and regional lymph node metastasis, and can directly act on cancer cell receptors. VEGF is responsible for the growth and permeability of vascular endothelial cells, vasculature, and angiogenesis by inhibiting the apoptosis of endothelial cell lining newly formed vessels. The activation of VEGFR-2 signals through the PI3K/AKT pathway produces the most angiogenic effects accredited to VEGF, as the PI3K/AKT is vital for regulating cellular functions. VEGF-C is a direct target and negatively regulated by miR-128 in BLCA, with it being upregulated while miR-128 expression is downregulated. miR-128 is tissue/cell specific, behaving differently according to its location, but mostly acts as a tumor inhibitor. Its expression suppresses migration and invasion [
71]. miR-128 expression in UBC associates with pelvic lymph node metastasis and poor prognosis [
72].
Figure 3. Inhibition of angiogenesis by miRNA in BLCA.
Table 4. miRNAs involved in angiogenesis inhibition in BLCA.
microRNA |
Samples/Cell Culture |
Targets/Regulators |
Function |
References |
miR-128 |
Human bladder epithelial cell line SV-HUC-1, and the BLCA cell lines T24, 5637, 3-UM-UC-3, and RT4 |
VEGF-C |
Overexpression of miR-128 inhibited growth rate, proliferation, migration, and invasion capacities. |
[71,72] |
miR-122 |
BLCA cells BIU-87, T24, SW780, HT1376, 5637, RT4, and normal bladder epithelial cell line SV-HUC-1 |
3′-UTR of VEGF-C |
miR-122 regulated cell proliferation through the VEGFC/AKT/mTOR signaling pathway. |
[12] |
miR-27a |
Clinical samples: Urothelial carcinoma and normal urothelial tissue samples were collected from 59 patients undergoing surgery |
AGGF1 |
Down-regulation of AGGF1 expression by hypoxia-induced miR-27a expression represents a signaling network for development of high-grade UBC. |
[7] |
miR-214 |
138 patients with primary urothelial carcinoma of the urinary bladder and 144 healthy controls |
|
The urinary levels of cell-free miR-214 were significantly higher in the NMIBC patients than in the controls. Thus, they could be used as a prognostic marker for NMIBC. |
[73] |
miR-34s |
Cell lines: 5637, T24, HT-1376, J82, SCABER, and EJ Clinical samples: BLCA tissue and adjacent normal tissue specimens |
CD44 |
miR-34a overexpression can inhibit bladder cell migration, invasion, tube formation in vitro, and metastasis and angiogenesis in vivo. Additionally, CD44-mediated functions can be reversed by miR-34a in bladder cells. |
[52] |
miR-124 |
Hek293, human normal cell SV-HUC-1 and BLCA T24, 5637, J82, and UM-UC-3 Clinical tissues: 83 bladder tissues and their adjacent non-tumor tissues |
CDK4 |
Overexpression of miR-124 induced by mimic transfection was observed to inhibit the cells viability, angiogenesis, and proliferation. |
[74] |
miR-200c |
Human bladder epithelial cell line, SV-HUC-1, BLCA cell lines 5637 and T24 |
Akt2 |
miR-200c could suppress HIF-1α/VEGF expression in BLCA cells and inhibit angiogenesis, and these regulations were achieved by targeting Akt2/mTOR. |
[75] |
miR-153 |
T24, UMUC3, 5637, and J82 cell lines and an immortalized human normal bladder epithelial cell line SV-HUC-1 Clinical tissue: normal and cancerous tissue specimens from 45 BLCA patients |
IDO1 |
IDO1 mediated miR-153 anti-tumor activity in BLCA via inactivating the IL6/STAT3/VEGF pathway. |
[76] |
The effect of miR-122 on angiogenesis was tested by using chorioallantoic membrane (CAM) system in HT1376 cells. Overexpression of miR-122 decreased HT1376 xenograft tumor growth without significant toxicity and reduced the amount of microvessels in a CAM model. Several targets of miR-122 have been identified including ADAM10 and VEGF-C. miR-122 was found to downregulate VEGF-C expression, inhibiting BLCA growth and angiogenesis via targeting 3′UTR of VEGF-C mRNA. miR-122 inhibited angiogenesis by inhibiting VEGF-C expression, thus blocking Akt/mTOR signaling pathway. Additionally, miR-122 increased BLCA chemo-sensitivity to cisplatin treatment in a VEGFC-dependent manner. Therefore, miR-122 was found to have an inhibitory role in in vivo and in vitro BLCA angiogenesis and growth [
12]. AGGF1 is a critical vasculogenesis and angiogenesis factor. In hypoxic conditions, miR-27a mimics significantly decreased the expression level of AGGF1 with up-regulation of miR-27a in UBC. Thus, down-regulation of AGGF1 expression by hypoxia-induced miR-27a expression could possibly represent a pathway for the development of high grade UBC [
7]. Additionally, miR-200c has been considered as a regulator of tumor angiogenesis, while AKT2/mTOR is considered a regulator of VEGF and HIF1α. miR-200c was able to negatively regulate the expression of angiogenesis related proteins HIF1α/VEGF expression in BLCA via targeting AKT2/mTOR as did miR-27a, inhibiting angiogenesis and affecting the process of tubule formation [
75].
As mentioned earlier, CD44 helps promote invasion and angiogenesis of tumor cells. Following stable transfection of miR-34s precursor, which plays a role in BLCA progression, VEGF and CD44 levels were reduced. In vitro overexpression of miR-34a suppressed angiogenesis by targeting CD44 and reduced tube formation. Thus, miR-34a reduced angiogenesis in BLCA both in vitro and in vivo [
52]. CDK4 was a direct target of miR-124, and its overexpression in T24 and 5637 cells increased VEGF expression and impaired the suppressive functions of miR-124 on BLCA angiogenesis, cell viability, and proliferation [
76]. miR-153 inhibits in vitro and in vivo T24 and UMUC3 BLCA growth by promoting tumor cell apoptosis, cell migration, invasion, and EMT. In vitro and in vivo assessment of miR-153 expression on BLCA induced HUVEC angiogenesis, and showed that miR-153 suppressed IDO1, a rate limiting enzyme in tryptophan metabolism, which plays a role in tumor cell escape. Ergo, miR-153 suppressed tryptophan metabolism and angiogenesis. Last but not least, STAT3 expression enhances tumor angiogenesis. In BLCA, miR-153 anti-tumor activity was mediated by targeting IDO1, further inactivating IL6/STAT3/VEGF signaling pathway [
76].
In NMIBC, urinary miR-214 served as a non-invasive prognostic biomarker of BLCA, distinguishing NMIBC patients from noncancerous controls, and the authors suggested that miR-214 levels could relate to the inhibition of angiogenesis, cell proliferation, and tumor recurrence [
73].