MicroRNAs and Their Influence on the ZEB Family: Mechanistic Aspects and Therapeutic Applications in Cancer Therapy

Molecular signaling pathways involved in cancer have been intensively studied due to their crucial role in cancer cell growth and dissemination. Among them, zinc finger E-box binding homeobox-1 (ZEB1) and -2 (ZEB2) are molecules that play vital roles in signaling pathways to ensure the survival of tumor cells, particularly through enhancing cell proliferation, promoting cell migration and invasion, and triggering drug resistance. Importantly, ZEB proteins are regulated by microRNAs (miRs). In this review, we demonstrate the impact that miRs have on cancer therapy, through their targeting of ZEB proteins. MiRs are able to act as onco-suppressor factors and inhibit the malignancy of tumor cells through ZEB1/2 down-regulation. This can lead to an inhibition of epithelial-mesenchymal transition (EMT) mechanism, therefore reducing metastasis. Additionally, miRs are able to inhibit ZEB1/2-mediated drug resistance and immunosuppression. Additionally, we explore the upstream modulators of miRs such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), as these regulators can influence the inhibitory effect of miRs on ZEB proteins and cancer progression.


-untranslated region
. It has been demonstrated that the level of miRs has a negative relationship with the expression of their down-stream targets [62][63][64]. Moreover, one miR is able to target more than one messenger RNA (mRNA), again highlighting their widespread influence in many cellular processes [65]. In the synthesis of miR, a primary miR (pri-miR) is first produced by the action of RNA polymerase. The pri-miR is long with more than 500 nucleotides. It is then processed by Drosha/Pasha and DICER1 proteins, which cleave the pri-miR to generate a mature miR. Next, the mature miR is incorporated in a complex to form miR-RNA-induced silencing complex assembly [66][67][68][69][70].

MicroRNAs in Cancer Metastasis
When focusing on the cancer context, miRs can have oncogenic, or tumor suppressing properties. Onco-suppressor miRs that inhibit invasion of cancer cells undergo down-regulation during cancer development. Enhancing the expression of such miRs can aid in the down-regulation of factors involved in migration of cancer cells such as PRMT5 [71]. Additionally, MiR-506-3p up-regulation considerably reduces the viability and proliferation of ovarian cancer cells and stimulates apoptotic cell death. Investigation of underlying molecular pathways shows that miR-506-3p inhibits Akt/Forkhead box O3 (FOXO3a) by inhibition of sirtuin 1 (SIRT1) [72]. Elevating the expression of miR-506-3p is a potential strategy in ovarian cancer treatment. In the gastric cancer model, it was also observed that a reverse relationship between miR-612 and nin one binding protein (NOB1) helped reduce the migration and invasion of cervical cancer cells [73]. Similarly, in pancreatic cancer, it was also found that overexpression miRs could lead to better prognosis. Enhancing the expression of miR-519 appears to sensitize pancreatic cancer cells to apoptosis and inhibits their proliferation and migration. This miR prevents the activation of programmed death ligand 1 (PD-L1), under hypoxic conditions to suppress tumorigenesis [74]. In all, various studies have shown that miRs are efficient upstream mediators that target various molecular pathways. Enhancing the expression of tumor suppressor miRs may prove to be an advantageous strategy and extensive research is currently being performed to exploit this strategy [74][75][76][77][78]. Conversely, oncogenic miRs are able to elevate the malignancy and proliferation of cancer cells and are associated with poor prognosis. Their downregulation is of interest in cancer therapy [79,80]. For instance, miR-424-5p is able to induce anoikis resistance to promote migratory ability of cancer cells [81]. The targeting of miRs may therefore be considered a promising candidate in cancer therapy. Interestingly, EMT-TFs are considered as potential down-stream targets of miRs in cancer metastasis. MiR-582-3p and miR-582-5p suppress migration of cancer cells via down-regulation of TGF-β in cancer cells [82]. This concurs that miRs can play a significant role in the regulation of metastasis via targeting different pathways and mechanisms. In the following sections, we focus on the regulation of ZEB proteins by miRs and their association with cancer metastasis and chemoresistance.

MicroRNA, ncRNA, and ZEB: Role in EMT and Cancer Metastasis
This section specifically demonstrates the impact that miRs have on cell migration and invasion, through their targeting of ZEB proteins. Upstream modulators of miRs such as lncRNAs and circRNAs are also extensively discussed. As mentioned, miRs are able to act as both onco-suppressor as well as promoter of cancer dissemination. Particularly, they are able to exert these effects through their modulation of ZEB proteins, to result in changes in the EMT mechanism. For instance, it appears that miR-200c plays a dual role in cancer therapy. Some studies have demonstrated that miR-200c elevates the viability and proliferation of tumor cells, while another study showed that miR-200c sensitizes cancer cells into chemotherapy by targeting neurophilin 1 and reducing cancer malignancy [83][84][85][86]. It is believed that miR-200c exerts an inhibitory impact on TGF-β-mediated EMT through down-regulation of both ZEB1 and ZEB2 proteins [87]. expression of ZEB1 by suppressing Wnt and nuclear translocation of β-catenin. Conversely, lncRNA LINC00702 can activate Wnt/β-catenin signaling pathway by sponging miR-4652-3p to induce ZEB1 and promote the metastasis and invasion of malignant meningioma [120]. These studies highlight the fact that firstly, Wnt can promote metastasis of cancer cells via ZEB1 up-regulation; secondly, the Wnt/ZEB1 axis can be inhibited by onco-suppressor miRs; thirdly, miRs affect both expression of Wnt and nuclear translocation of β-catenin; and finally, lncRNAs can regulate miR/Wnt/ZEB1 axis. The mediation of the miR/ZEB1 axis by lncRNAs will be more extensively discussed in the next section.

LncRNAs as Modulators of miR/ZEB1 Axis
Long non-coding RNAs (lncRNAs) belong to a category of ncRNAs with regulatory effect on biological events [121,122]. They consist of at least 200 nucleotides and they are able to function as upstream mediators of miRs [111]. LncRNAs suppress the expression of miRs via acting as competitive endogenous RNA (ceRNA) [123]. The effect of lncRNAs on miR/ZEB1 axis has been investigated in cancer cells. For instance, miR-429 was found to inhibit EMT through ZEB1 inhibition and its expression is typically down regulated in pancreatic cancer cells. MiR-429 can be regulated by lncRNA XIST, which is up-regulated in pancreatic cancer cells to reduce miR-429 levels. This in turn increases ZEB1 expression and promotes EMT. Additionally, through targeting the miR-429/ZEB1 axis, XIST also affects morphology of cancer cells, such that silencing XIST results in a change in cell morphology, from the original spindle shape to a rounded one [124]. In another instance, LncRNA IUR is a onco-suppressor factor that has shown a great capability in suppressing tumorigenesis [125]. LncRNA IUR can inhibit the migration and metastasis of prostate cancer cells via enhancing the expression of miR-200, which in turn inhibits ZEB1 [126].
Conversely, lncRNA TDRG1 is an oncogenic factor that is able to regulate miRs in cancer cells [127,128]. In lung cancer cells, TDRG1 enhances the migration, metastasis, and malignancy of cancer cells by promoting ZEB1 expression through miR-873-5p down-regulation [129]. LncRNA TTN-AS1 is also considered an oncogenic factor that induces ZEB1 through miR-4677-3p down-regulation, leading to the enhanced migration and metastasis of NSCLC cells [130]. LncRNA (Nuclear Enriched Abundant Transcript 1) NEAT1 contributes to enhancing the malignancy of cancer cells [131]. It has been demonstrated that NEAT1 can target miRs to regulate cancer proliferation and migration [132]. In breast cancer cells, NEAT1 reduces the expression of miR-448 to elevate the metastasis and invasion of cancer cells through ZEB1 up-regulation [133]. LncRNA TP73-AS1 reduces the expression of miR-200a to up-regulate ZEB1, leading to the enhanced progression and malignancy of tumor cells. There appears to be a feedback loop, wherein TP73-AS1-activated ZEB1 has a stimulatory effect on the expression of TP73-AS1 to enhance its inhibitory activity on miR-200a, leading to increased induction of ZEB1 [90].
In renal cell carcinoma (RCC), miR-429 typically reduces the expression of ZEB1 to suppress RCC progression. However, miR-429 can be inhibited by SCAMP1, a lncRNA that is activated by oxidative stress [134]. This highlights that stimulation of oxidative stress negatively impacts cancer therapy. Generally, it is believed that enhancing level of oxidative stress can lead to a reduction in the viability of cancer cells by predisposing them into apoptosis [135,136]. However, as mentioned, increasing levels of oxidative stress may also activate lncRNAs involved in cancer metastasis. Therefore, careful considerations are warranted before using oxidative stress in cancer therapy, keeping in mind the possible adverse effects of this treatment method.
MiR-139-5 was also found to suppress ZEB1 levels. However, lncRNA human leukocyte antigen (HLA) complex 5 (HCP5) is able to induce ZEB1 and EMT by suppressing miR-139-5 [137]. LncRNA MAGI2-AS3 has also been explored in cancer and it appears that MAGI2-AS3 is able to modulate molecular pathways such as Fas/FasL to suppress breast cancer, bladder cancer, and hepatocellular carcinoma [138,139]. Particularly, in gastric cancer cells, miR-141/200a diminishes the invasion and migration of tumor cells via suppressing ZEB1. LncRNA MAGI2-AS3 down-regulates the expression of miR-141/200a to induce ZEB1, leading to the stimulation of EMT and enhanced invasion of tumor cells [125].
LncRNA LINC00511 is located on chromosome 17q24.3 and has been associated with increased malignancy in cancer [140]. In glioblastoma (GBM) cells, miR-524-5p inhibits ZEB1 to suppress GBM invasion and migration. LINC00511 has been found to decrease the expression of miR-524-5p to up-regulate YB1 [141]. YB1 is a transcription factor that can enhance the expression of ZEB1 in cancer [128]. The inhibition of miR-524-5p by LINC00511 promotes ZEB1 expression through YB1 up-regulation, leading to enhanced EMT and malignancy of GBM cells [141]. These studies again demonstrate that lncRNAs can disrupt inhibitory effects of miRs on ZEB1 to promote metastasis of cancer cells. In glioma cells, miR-205-3p inhibits TGF-β, while lncRNA linc00645 functions as an upstream mediator and activates TGF-β via suppressing miR-205-3p, leading to an increase in ZEB1 levels and subsequent EMT activation [137].
LncRNA MALAT1 located on the chromosome 11q13, is also suggested to be involved in elevating the malignancy of cancer cells. A variety of factors act as down-stream mediators for lncRNA MALAT1 and it appears that MALAT1 is capable of targeting miRs in cancer cells [105,142,143]. MALAT1 was found to enhance the expression of ZEB1 through miR-143-3p down-regulation, resulting in elevated migration and metastasis of tumor cells [144]. Another downstream target of MALAT1 is miR-429, which is considered as a potential biomarker for diagnosis of different cancers [145]. MALAT1 was found to inhibit miR-429 to accelerate the malignancy and invasion of cervical cancer cells [146]. Notably, miR-429 can inhibit the metastasis of cancer cells and stimulate apoptotic cell death through ZEB1 down-regulation [110]. Interestingly, it has been demonstrated that fine particulate matter (PM 2.5 , aerodynamic diameter, 2.5 µm) is able to induce oxidative stress, inflammation, genetic mutations, and DNA damage [147,148]. It has been found that miR-204 can reduce the expression of ZEB1 to suppress EMT. PM 2.5 activates MALAT1 via stimulation of NF-κB, as an inflammatory pathway. MALAT1 in turn induces ZEB1 through miR-204 down-regulation to enhance the malignancy and invasion of tumor cells via EMT induction [149]. These two studies demonstrate that lncRNAs can affect more than one downstream miR to mediate ZEB1 levels, and that other molecular pathways such as NF-κB can act as upstream mediator of lncRNA/miR/ZEB1 axis.
HOXA distal transcript antisense RNA (HOTTIP) is located at the distal end of HOXA gene cluster [150]. This lncRNA undergoes abnormal expressions in different cancers and it has been shown that HOTTIP is related to the increased proliferation and progression of cancer cells [120]. It is held that lncRNA HOTTIP down-regulates the expression of miR-101 to elevate ZEB1 levels, leading to an increase in EMT [151]. A study has also shown that miR-205 down-regulates the expression of ZEB proteins and HOXD9 to suppress the malignancy and invasion of cancer cells through EMT inhibition [152]. Finally, it has been demonstrated that lncRNA HOXC-AS2 induces ZEB1 by sponging miR-876-5p, leading to the stimulation of EMT and enhanced migration and invasion of tumor cells [153]. Taken together, the relationship between lncRNAs and miRs in the regulation of ZEB1 in cancer cells are dynamic and complicated, and understanding these pathways is an essential part of effective cancer therapy.

CircRNAs as Modulators of miR/ZEB1 Axis
Circular RNAs (circRNAs) are endogenous, conserved ncRNAs that are sometimes employed as biomarkers for cancer diagnosis [154,155]. Similar to lncRNA, circRNAs are able to modulate the expression of their targets [156]. In lung cancer cells, hsa-circ-0023404 decreases the expression of miR-217 to enhance the expression of its target, ZEB1, leading to the increased migration and invasion of cancer cells [157]. The laryngeal carcinoma is considered as one of the common cancers among head and neck tumors and is mainly diagnosed in elder people [158]. In spite of the low incidence rate, this cancer results in high mortality worldwide [159]. It was discovered that miR-200c is capable of inhibiting ZEB1 to prevent the metastasis and invasion of laryngeal cancer cells. Hsa-circ-005748 up-regulates ZEB1 by sponging miR-200c, leading to the metastasis of these cancer cells [160]. Therefore, inhibition of hsa-circ-005748 may in turn increase miR-200c expression to suppress ZEB1 and cancer metastasis. Similarly, in lung adenocarcinoma (LUAD) cells, miR-665 is able to inhibit cancer metastasis via ZEB1 down-regulation. The circ-TSPAN4 enhances the expression of ZEB1 by miR-665 down-regulation to promote the metastasis of LUAD cells [161]. These studies concur that down-regulation of onco-suppressor miRs in cancer cells may also be mediated by upstream circRNAs. This in turn promotes up-regulation of ZEB1 and enhanced metastasis of cancer cells.

MiRs as Modulators of ZEB2
MiR-124 is suggested to be an onco-suppressor miR. Recently, an effort has been made to suppress the prostate cancer invasion. It is held that cationic polymer nanoparticles are able to deliver miR-124 in prostate cancer cells to inhibit their proliferation, motility, and colony formation [162]. In TNBC cells, miR-124 effectively decreases the malignancy and invasion of tumor cells by EMT inhibition through ZEB2 down-regulation [163]. Similarly, miR-145 has been widely established as a tumor suppressor. It negatively affects the invasion and migration of thyroid carcinoma cells by down-regulation of NF-κB signaling pathway [164]. Furthermore, lncRNA-ROR down-regulates the expression of miR-145 to remove its inhibitory impact and induce EMT in tumor cells [165]. Importantly, it was found that miR-145 decreases the expression of ZEB2 to inhibit EMT, and consequently, suppress the proliferation, progression, and migration of NSCLC cells [166]. These studies demonstrate that the downregulation of ZEB2 by onco-suppressor miRs can lead to a decrease in the metastasis of cancer cells.
Another onco-suppressor miR is miR-30a. In breast cancer cells, miR-30a suppresses the nuclear translocation of β-catenin to attenuate cancer proliferation and progression, and is associated with favorable prognosis of patients with breast cancer [167]. Furthermore, miR-30a appears to be beneficial in sensitizing cancer cells to chemotherapy via affecting Akt signaling pathway [168]. MiR-30a was found to inhibit ZEB2 to result in a reduction of triple negative breast cancer (TNBC) cells malignancy [169]. Finally, miR-3653 is an onco-suppressor that is down-regulated in hepatocellular carcinoma (HCC) cells [170]. MiR-3653 was found to bind to the 3 -UTR of ZEB2 to diminish its expression, leading to the reduced invasion and malignancy of colon cancer cells [171]. MiR-138-5p uses a same strategy in inhibition of lung adenocarcinoma cell malignancy, by suppressing EMT through ZEB2 inhibition to attenuate metastasis of tumor cells [172].
Osteosarcoma typically has a high recurrence rate and low survival rate [173,174]. Therefore, understanding the pathways involved in malignancy and cancer progression may pave the road for improved treatment of this type of cancer. Investigation of molecular pathways has shown that miR-101 up-regulation inhibits ZEB2 and affects proliferation and metastasis of osteosarcoma cells [175]. Unfortunately, miR-101 is down-regulated in osteosarcoma cells compared to the normal cells. Enhancing the expression of miR-101 may reduce malignancy and progression of osteosarcoma cells.

LncRNAs as Modulators of miR/ZEB2 Axis
In the previous section, we demonstrated that lncRNAs are able to function as ceRNA in affecting miR expression. Notably, increasing evidence has also demonstrated that lncRNAs can effectively target ZEB2 via affecting miRs. For instance, LncRNA HOTAIRM1, which has dual properties as it interacts with both onco-suppressor and oncogenic miRs. HOTAIRM1 is located on human HOXA gene cluster and suggested to be involved in myeloid cell development [176]. A newly published article has shown the anti-tumor activity of lncRNA HOTAIRM1 by up-regulation of ARHGAP24 through miR-106a-5p inhibition [177]. However, it has been found that HOTAIRM1 is related to the elevated migration and invasion of tumor cells [178]. It is held that lncRNA HOTAIRM1 diminishes the expression of miR-873-5p to induce ZEB2, resulting in an increase in cancer cell proliferation and suppressing apoptotic cell death [179].
MiR-505 is also considered an onco-suppressor miR that interacts with IGF-1 and HMGB1 to suppress the growth and malignancy of tumor cells [180][181][182]. Various studies have demonstrated that lncRNAs such as lncRNA CRAL, LEF-AS1, and DLX6-AS1 are able to target miR-505 in different cancers such as gastric cancer, CRC, and breast cancer [183][184][185]. In cervical cancer, lncRNA CTS was found to target miR-505. MiR-505 down-regulates ZEB2 levels to inhibit EMT and invasion of cervical cancer cells. LncRNA CTS, therefore, stimulates ZEB2-mediated EMT through miR-505 sponging, leading to the enhanced viability, proliferation, and malignancy of cervical cancer cells [186]. Taken together, stimulation of ZEB2 by lncRNAs not only enhances metastasis of cancer cells via EMT induction, but also promotes cell proliferation. This decrease in apoptosis by ZEB2 induction is of importance in chemotherapy, since cancer cells can attain chemoresistance via reducing their sensitivity into chemotherapy-mediated apoptosis. As such, targeting miR/ZEB2 axis may be a promising strategy in cancer therapy, as it increases the sensitivity of cancer cells toward chemotherapy.
In gastric cancer cells, miR-203 diminishes cancer metastasis through ZEB2 down-regulation. LncRNA UCA1 enhances the progression and metastasis of tumor cells through disrupting the miR-203/ZEB2 axis [187]. Particularly, lncRNAs can affect upstream transcription factors of ZEB2 in cancer metastasis. In NSCLC cells, slug was found to behave as an upstream mediator to induce EMT through increasing ZEB2 levels. MiR-218 was able to disrupt the Slug/ZEB2 axis to suppress NSCLC migration. Conversely, miR-218 undergoes down-regulation by lncRNA SNHG12 to stimulate Slug/ZEB2 and promote metastasis of NSCLC cells [188].
Glioma is an intracranial tumor that emanates from neuroglial stem or progenitor cells [189]. Again, this is an alarming cancer with high mortality and morbidity rate [190,191]. The migration and invasion of cancer cells into neighboring cells and tissues reduces the survival time of patients [192,193]. It has been demonstrated that lncRNA SNHG5 can inhibit miR-205-5p expression. Reduced miR-205-5p expression triggers the induction of ZEB2, which in turn enhances the migration ability of tumor cells [194]. Up-regulation of miR-205-5p may therefore be beneficial in reducing glioma malignancy.

CircRNAs as Modulators of miR/ZEB2 Axis
Increasing evidence highlights the role of miR-377 as an onco-suppressor in cancer cells. MiR-377 can target Akt signaling to suppress the proliferation and invasion of tumor cells, and induce cell cycle arrest [195]. Normally, miR-377 reduces the expression of ZEB2. In bladder cancer cells, the expression of miR-377 undergoes down-regulation by circZFR to promote cancer metastasis through ZEB2 stimulation [196]. MiR-653 also appears to be an onco-suppressor miR in bladder cancer cells. CircRNA ciRs-6 reduces miR-653 expression to induce March1, leading to the increased proliferation of tumor cells [110]. MiR-653 is similarly suppressed in breast cancer cells, by another circRNA hsa-circ-0004771. Knockdown of hsa-circ-0004771 sensitizes cancer cells to apoptosis and inhibits their progression through miR-653 up-regulation and subsequent inhibition of ZEB2 [197]. Evidently, ZEB2 induction dually enhances proliferation and metastasis of cancer cells. Hence, targeting the circRNA/miR/ZEB2 axis can pave the way into effective inhibition of proliferation and migration of cancer cells.
In renal cancer, patients typically have poorer survival rates and treatment strategies can be improved [198][199][200]. MiR-153 was found to exert inhibitory impact on ZEB2 expression to suppress renal cancer, while circPCNXL2 stimulates ZEB2 expression via miR-153 sponging to elevate the invasion and proliferation of renal cancer cells [201]. Therefore, decreasing the expression of circPCNXL2 may yield an up-regulation of miR-153 and suppresses ZEB2 expression to eliminate renal cancer.
In all, these studies highlight the extensive influence that miRs have on ZEB proteins (Figures 1  and 2). Across a wide range of cancers, different miRs work to inhibit ZEB and halt cancer progression. Additionally, lncRNAs and circRNAs are able to act as upstream mediators of miRs to affect ZEB2 expression. Through the revealing of these molecular pathways, we may better understand these promising candidates in cancer therapy.

MicroRNAs, ZEB, and Their Role in Tumor Resistance
Multidrug resistance (MDR) is a complicated and challenging phenomenon accounting for cross-resistance towards structurally unrelated drugs [202,203]. It is estimated that approximately 70% of solid and hematological tumors demonstrate MDR. This percentage elevates after chemotherapy, since cancer cells are able to switch among molecular pathways to obtain chemoresistance, and frequent application of chemotherapeutic agents speeds up MDR [204,205].

MicroRNAs, ZEB, and Their Role in Tumor Resistance
Multidrug resistance (MDR) is a complicated and challenging phenomenon accounting for cross-resistance towards structurally unrelated drugs [202,203]. It is estimated that approximately 70% of solid and hematological tumors demonstrate MDR. This percentage elevates after chemotherapy, since cancer cells are able to switch among molecular pathways to obtain chemoresistance, and frequent application of chemotherapeutic agents speeds up MDR [204,205].

MicroRNAs, ZEB, and Their Role in Tumor Resistance
Multidrug resistance (MDR) is a complicated and challenging phenomenon accounting for cross-resistance towards structurally unrelated drugs [202,203]. It is estimated that approximately 70% of solid and hematological tumors demonstrate MDR. This percentage elevates after chemotherapy, since cancer cells are able to switch among molecular pathways to obtain chemoresistance, and frequent application of chemotherapeutic agents speeds up MDR [204,205]. Therefore, when trying to understand the role of miR and ZEB proteins in cancer, it also important to explore how miR's modulation on ZEB can affect tumor resistance. Particularly, miR is implicated in tumor resistance. For instance, cisplatin is a potential chemotherapeutic agent with the ability of inhibiting the proliferation and viability of various cancers [206]. In ovarian cancer, it has been reported that miR-137 reduces the expression of MCL1 to sensitize tumor cells into cisplatin-induced apoptosis [207]. In this section, we seek to understand how miR modulation on ZEB can contribute to tumor resistance.

Paclitaxel Resistance
Paclitaxel (PTX) is a chemotherapeutic agent that is frequently employed in cancer therapy to prevent cell proliferation due to its anti-mitotic capabilities [208]. Unfortunately, PTX resistance is an important obstacle, which has reduced the feasibility of this agent [209][210][211][212]. Notably, ZEB1 can promote cancer cells resistance towards PTX, and down-regulation of ZEB1 may be a key toward re-sensitizing cancer cells to PTX chemotherapy [213]. MiR-124-3p suppresses ZEB1 to sensitize gastric cancer cells into PTX therapy. Circular RNA Circ-PVT1 reverses this axis by sponging miR-124-3p and elevating the expression of ZEB1 to induce PTX resistance in gastric cancer cells [214]. LncRNA NEAT1 was also found to mediate PTX resistance in ovarian cancer cells. Normally, miR-194 undergoes up-regulation to inhibit ZEB1 and subsequently, reduce the malignancy and invasion of cancer cells. LncRNA NEAT1 suppresses the inhibitory effect of miR-194 on ZEB1 to induce the resistance of ovarian cancer cells into PTX chemotherapy [215].

Gemcitabine Resistance
Gemcitabine is a chemotherapeutic agent isolated from deoxycytidine, which is frequently applied in the treatment of breast cancer [216]. Gemcitabine triggers cell cycle arrest by binding into DNA or suppressing ribonucleotide reductase [217,218]. It appears that ZEB1 contributes to the gemcitabine resistance in TNBC cells. This study found that ZEB1 associates with Yes associated protein (YAP) to enhance cancer progression and proliferation and induces chemoresistance. Importantly, ZEB1 was found to be a target of miR-873, and that increasing miR-873 expression down-regulates the expression of YAP and ZEB1, and sensitizes tumor cells into gemcitabine therapy [219].

Cisplatin Resistance
Another important factor to consider when exploring acquired tumor resistance is lncRNA, which can regulate miR, to in turn affect ZEB levels. Prostate cancer-associated transcription 1 (PCAT1) undergoes up-regulation in cancer cells to suppress cell death [220]. In gastric cancer cells, PCAT-1 induces the resistance of cancer cells into cisplatin therapy by stimulation of ZEB1 through miR-128 inhibition, leading to the enhanced progression and malignancy of gastric cancer cells [221]. Therefore, targeting the miR/ZEB1 axis may alleviate cisplatin resistance.

5-Fluorouracil
The most common chemotherapeutic agent in treatment of cancer is 5-FU [222]. Different molecular pathways are involved in resistance into 5-FU, and miRs are key players [223,224]. LncRNA NEAT1 have been found to possess oncogenic activity and enhance the progression and malignancy of cancer cells via targeting miRs such as miR-144-3p and miR-410 [225,226]. In CRC cells, NEAT1 is involved in 5-FU resistance through miR-34a regulation [227] (Figure 3).

ZEB2
In osteosarcoma, miR-200b diminishes the progression and motility of tumor cells by inhibition of PI3K/Akt and AMPK signaling pathways, leading to the downregulation of vascular endothelial growth factor (VEGF). LncRNA CCAT2 reverses this axis by induction of VEGF through miR-200b inhibition [228]. It is worth mentioning that enhancing the expression of miR-200b is beneficial in sensitizing cancer cells into chemotherapy, so that arrestin domain containing 3 (ARRDC3) elevates the efficacy of chemotherapy in TNBC cells via miR-200b up-regulation [229]. It appears that upregulation of miR-200b enhances apoptosis in lung cancer cells and remarkably increases the efficacy of chemotherapy [230]. Another member of miR-200 family, known as miR-200c, sensitizes gastric cancer cells to cisplatin and enhances chemotherapeutic efficacy by suppressing ZEB2 expression [215].

MicroRNAs Target ZEB Family in Immune Cells
Other than ZEB's prevalent role in EMT, ZEB's involvement with the tumor microenvironment and immune system is also crucial in its mediation of cancer dissemination and development. Tumor cells use immunosuppressive cells such as CD4+ T cells to escape from the anti-cancer activity of CD8+ T cells [231][232][233]. Notably, it has been demonstrated that cytotoxic CD8+ tumor infiltrating lymphocytes (CD8+ TILs) are able to eliminate cancer cells [234], while sustained exposure of tumor cells into CD8+ TILs reduces their anti-tumor activity [235]. It is worth mentioning that PD-1/PD-L1 axis may be involved in driving CD8+ T cell exhaustion and therapies targeting PD-L1 have been explored [236][237][238][239][240]. PD-L1 binds to PD-1 to induce apoptotic cell death in CD8+ T cells and ensure the survival of cancer cells [241][242][243][244].
In diffuse large B cell lymphoma (DLBCL) cells, miR-8890-3p is capable of suppressing ZEB1, while lncRNA SNHG14 conversely reduces the expression of miR-8890-3p to activate ZEB1. Consequently, ZEB1 stimulates PD-L1 to protect cancer cells against the cytotoxic effects of immune cells, resulting in promoting the survival and migration of DLBCL cells [245].
In another instance, it has been reported that ZEB1 is an efficient factor in elevating the malignancy of tumor cells, through the induction of PD-L1 expression to enhance the levels of CD8+ T-cell immunosuppression and cancer metastasis. Enhancing the expression of miR-200 disrupts ZEB1 expression to suppress PD-L1 and immunosuppression, resulting in decreased metastasis and invasion of cancer cells [246]. ZEB1 can induce EMT in breast cancer cells via activation of PD-L1. It

ZEB2
In osteosarcoma, miR-200b diminishes the progression and motility of tumor cells by inhibition of PI3K/Akt and AMPK signaling pathways, leading to the downregulation of vascular endothelial growth factor (VEGF). LncRNA CCAT2 reverses this axis by induction of VEGF through miR-200b inhibition [228]. It is worth mentioning that enhancing the expression of miR-200b is beneficial in sensitizing cancer cells into chemotherapy, so that arrestin domain containing 3 (ARRDC3) elevates the efficacy of chemotherapy in TNBC cells via miR-200b up-regulation [229]. It appears that up-regulation of miR-200b enhances apoptosis in lung cancer cells and remarkably increases the efficacy of chemotherapy [230]. Another member of miR-200 family, known as miR-200c, sensitizes gastric cancer cells to cisplatin and enhances chemotherapeutic efficacy by suppressing ZEB2 expression [215].

MicroRNAs Target ZEB Family in Immune Cells
Other than ZEB's prevalent role in EMT, ZEB's involvement with the tumor microenvironment and immune system is also crucial in its mediation of cancer dissemination and development. Tumor cells use immunosuppressive cells such as CD4+ T cells to escape from the anti-cancer activity of CD8+ T cells [231][232][233]. Notably, it has been demonstrated that cytotoxic CD8+ tumor infiltrating lymphocytes (CD8+ TILs) are able to eliminate cancer cells [234], while sustained exposure of tumor cells into CD8+ TILs reduces their anti-tumor activity [235]. It is worth mentioning that PD-1/PD-L1 axis may be involved in driving CD8+ T cell exhaustion and therapies targeting PD-L1 have been explored [236][237][238][239][240]. PD-L1 binds to PD-1 to induce apoptotic cell death in CD8+ T cells and ensure the survival of cancer cells [241][242][243][244].
In diffuse large B cell lymphoma (DLBCL) cells, miR-8890-3p is capable of suppressing ZEB1, while lncRNA SNHG14 conversely reduces the expression of miR-8890-3p to activate ZEB1. Consequently, ZEB1 stimulates PD-L1 to protect cancer cells against the cytotoxic effects of immune cells, resulting in promoting the survival and migration of DLBCL cells [245].
In another instance, it has been reported that ZEB1 is an efficient factor in elevating the malignancy of tumor cells, through the induction of PD-L1 expression to enhance the levels of CD8+ T-cell immunosuppression and cancer metastasis. Enhancing the expression of miR-200 disrupts ZEB1 expression to suppress PD-L1 and immunosuppression, resulting in decreased metastasis and invasion of cancer cells [246]. ZEB1 can induce EMT in breast cancer cells via activation of PD-L1. It has been reported that miR-200 overexpression reduces the levels of ZEB1 to inhibit EMT through interfering with PD-L1 activation, as an immunosuppressive factor [247]. Unfortunately, there are currently no reports about the relationship between miRs and ZEB2 in cancer immunotherapy, and further studies can focus on revealing relationship between miR/ZEB2 axis and cancer immunotherapy. Tables 1-6 demonstrate the regulation of ZEB1 and ZEB2 by various miRs proteins in mediating cancer metastasis. Upstream mediators of miR such as lncRNAs and circRNAs are also highlighted in Table 1 through  Table 6. Figure 4 further summarizes the effect of miR/ZEB axis on immune system. has been reported that miR-200 overexpression reduces the levels of ZEB1 to inhibit EMT through interfering with PD-L1 activation, as an immunosuppressive factor [247]. Unfortunately, there are currently no reports about the relationship between miRs and ZEB2 in cancer immunotherapy, and further studies can focus on revealing relationship between miR/ZEB2 axis and cancer immunotherapy. Tables 1-6 demonstrate the regulation of ZEB1 and ZEB2 by various miRs proteins in mediating cancer metastasis. Upstream mediators of miR such as lncRNAs and circRNAs are also highlighted in Table 1 through Table 6. Figure 4 further summarizes the effect of miR/ZEB axis on immune system.    The lncRNA SNHG16 functions as an oncogenic factor and neutralizes the inhibitory effect of miR-140-5p on ZEB1 to induce EMT and enhance the migration and invasion of cancer cells [312] MiR-205 ZEB1 Osteosarcoma SNHG16 reduces the expression of miR-205 to elevate the expression of ZEB1, resulting in an increase in the viability, proliferation, and migration of cancer cells [313] LncRNA HOTAIR MiR-217 ZEB1 Osteosarcoma By reducing the expression of miR-217, lncRNA HOTAIR enhances the expression of ZEB1 and improves their malignancy [314] MiR-23b-3p ZEB1 Hepatocellular carcinoma The miR-23b-3p inhibits ZEB1 and lncRNA HOTAIR prevents the inhibitory effect of miR-23b-3p on ZEB1 to induce EMT [315] lncRNA UCA1 Has-miR-145 ZEB1/2-FSCN1 Bladder cancer There is a reverse relationship between lncRNA UCA1 and has-miR-145. Decreased expression of has-miR-145 enhances the expression of ZEB1/2 and FSCN1 to elevate the migration and invasion of cancer cells [316] MiR-204-5p ZEB1 Glioma cells By sponging miR-204-5p, lncRNA UCA1 stimulates ZEB1 and activates EMT mechanism [317]  The reverse relationship between ZNF469-3 and miR-574-5p paves the road for up-regulation of ZEB1 and subsequent activation of EMT, leading to the cancer progression and malignancy The expression of miR-3666 reduces to neutralize its inhibitory impact of ZEB1, and consequently, elevate the metastasis and progression of cancer cells [289]

MiR-132 ZEB2
Colorectal cancer Reducing the invasion and metastasis of cancer cells through ZEB2 down-regulation [340] Lung cancer Diminishing the migration and invasion of cancer cells through ZEB2 inhibition [341] MiR-138 ZEB2 Bladder cancer The miR-138 binds to the 3 -UTR of ZEB2 to inhibit the metastasis and invasion of cancer cells [289] MiR-141 ZEB2

Hepatocellular carcinoma
The miR-141 decreases the expression of ZEB2 to induce apoptosis and diminish viability and proliferation of cancer cells [342] Renal cancer The administration of honokiol is associated with miR-141 induction and subsequent downregulation of ZEB2 to inhibit the malignancy of cancer cells [343] MiR-145 ZEB2 Non-small cell lung cancer MiR-145 acts as an onco-suppressor miR that negatively affects the expression of ZEB2 to inhibit the progression and malignancy of cancer cells [166] Prostate cancer There is a negative feedback loop between miR-145 and ZEB2, so that overexpression of miR-145 down-regulates the expression of ZEB2 to ensure the reduced viability and proliferation of cancer cells [344]

MiR-545
Wnt-β-catenin/ZEB2 Non-small cell lung cancer The miR-545 reduces the expression of Wnt/β−catenin to down-regulate the expression of ZEB2, leading to the decreased migration and invasion of cancer cells [368] MiR-598 ZEB2 Non-small cell lung cancer The in vitro experiment demonstrated that miR-598 decreases the expression of ZEB2 to inhibit the migration and metastasis of cancer cells [369] MiR-622 ZEB2 Glioma The increased expression of miR-622 is related to the desirable prognosis via ZEB2 down-regulation [370] MiR-769-3p Wnt-β-catenin/ZEB2 Glioma The miR-769-3p down-regulates the expression of Wnt and inhibits nuclear translocation of β−catenin to suppress ZEB2, leading to the decreased viability, proliferation and invasion of cancer cells [371] MiR-940 ZEB2 Glioma Inhibition of cancer progression and EMT through ZEB2 down-regulation [372] MiR-1179 ZEB2 Hepatocellular carcinoma The miR-1179 reduces the expression of ZEB2 to inhibit cancer progression and malignancy [373] MiR-3653 ZEB2 Colon cancer Suppressing metastasis and EMT by inhibition of ZEB2 [171]

LncRNA HOTAIRM1
MiR-873-5p ZEB2 Glioma LncRNA HOTAIRM1 decreases the expression of miR-873-5p by sponging to up-regulate the expression of ZEB2, leading to an increase in progression of glioma cells and a decrease in apoptotic cell death [179]  MiR-653 reduces the expression of ZEB2 and is associated with desirable prognosis. Hsa-circ-0004771 diminishes miR-653 expression to induce ZEB2, leading to the inhibition of apoptosis and enhanced migration and invasion of cancer cells [197]

Conclusions
In this article, we provided a comprehensive review about the relationship between miRs and ZEB family in cancer cells and how this relationship affects the progression and metastasis of tumor cells. After miRs discovery, an exponential amount of research has been performed to understand their role in different biological processes such as cell differentiation, apoptosis, and migration. We typically observe aberrant miR expression in cancer cells and restoring the normal expression of miRs may be crucial in cancer therapy. It is also vital to explore the relevance of ZEB1 and ZEB2 proteins in cancer therapy. It has been reported that ZEB proteins are able to enhance the proliferation and malignancy of tumor cells. One of the most important pathways affected by ZEB proteins is the EMT mechanism. It appears that induction of EMT by ZEB proteins not only enhances the progression and metastasis of cancer cells, but also stimulates drug resistance. Therefore, revealing the underlying molecular pathways involved in ZEB regulation can be beneficial for further studies in the field of cancer therapy and elevating the efficacy of chemotherapy. In this review, we also detailed how and which miRs affect ZEB proteins in various cancers. We consolidated the factors that may function as upstream modulators to negatively affect miRs, leading to the induction of ZEB expression. As it is shown in Tables 1-6, lncRNAs and circRNAs can act as oncogenic factors. These upstream mediators induce and enhance the expression of ZEB1 and -2 through sponging their target miRs, resulting in an increase in malignancy and invasion of tumor cells. Identification of these factors and further targeting of them can significantly diminish the malignancy of tumor cells and pave the road for the effective cancer therapy. Finally, we highlighted ZEB1's role in immunosuppression. Through it, we identified a crucial knowledge gap wherein the relationship between miRs, ZEB2, and immune cells in the cancer context is still a mystery. In all, we dissected the different effects of miR on ZEB proteins, which may in turn help us develop better treatment strategies in attenuating metastasis of cancer cells.

Conflicts of Interest:
The authors declare no conflict of interest.