Role of miR-221/222 in Tumor Development and the Underlying Mechanism

MicroRNA-221/222 (miRNA-221/222, miR-221/222) is a noncoding microRNA which is widely distributed in eukaryotic organisms and deeply involved in the posttranscriptional regulation of gene expressions. According to recent studies, abnormal expressions of miR-221/222 are closely related to the occurrence and development of various kinds of malignant tumors. The role of miR-221/222 in tumor development and their potential molecular mechanism in various cancers, including liver cancer, colorectal cancer, cervical cancer, ovarian cancer, and endometrial carcinoma, are summarized and reviewed in this paper. Moreover, the potential translational biomarker role of abnormal miR-221/222 level in tumor or blood circulation for tumor diagnosis is also discussed.


Introduction
MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs with a length of 18-25 nucleotides. In the human genome, several thousands of miRNAs are encoded, which regulate more than 30% of the genes, thereby participating in the regulation of almost all cellular functions, such as cell differentiation, proliferation, growth, migration, and apoptosis. As illustrated, microRNA-221/222 (miRNA-221/222, miR-221/222) is a noncoding microRNA which is widely distributed in eukaryotic organisms and deeply involved in the posttranscriptional regulation of gene expressions [1][2][3][4].
Hsa-miR-221 and hsa-miR-222 are encoded tandemly in chromosome Xp11.3, which are highly homologous miRNAs sharing the same "seed sequence." e present study investigated the role of miR-221/222, which plays an important regulatory role in the development and progression of tumors, both in promoting or suppressing cancers [5]. Since there are different subtypes like miR-221/ 222 3p or 5p, whose heterogeneity may perform different biological functions, their new progresses are also reviewed in this paper. e biogenesis of miR-221/222 is similar to other miRNAs' biogenesis, which is initially transcribed principally by either RNA polymerase II or RNA polymerase III as long primary transcriptions and is further processed by the nuclear RNase Drosha and cytoplasmic RNase Dicer to generate precursor miRNAs and mature miRNAs, respectively ( Figure 1) [6]. In the cytoplasm, miRNAs recognize and bind to partially complementary sites in the 3′ untranslated region (3′UTR) of target mRNAs, resulting in either translational repression or target degradation [7].
Recently, accumulated investigations showed that the abnormal expression of miR-221/222 involved in various tumor initiation and progressions. As noted, abnormal miR-221/222 expression promotes cancer cell proliferation, invasion and metastasis, stemness promotion, cell survival, chemoresistance, or the relapse of cancer cells [5,8]. In this review, the role and its research progress of potential mechanism of miR-221/222 in tumor development are summarized and discussed.

Role of miR-221/222 in Tumorigenesis and Progression
2.1. Upstream Regulation of miR-221/222. In recent years, studies have found that in miRNA biogenesis (Figure 1), the pri-miRNA hairpins by Drosha and Dicer could result in a change of the length at the 5′-or 3′-end of miRNA [9]. us, the isoform of miRNA is longer or shorter than the consensus miRNA length [10]. e heterogeneity at the 5′-end could result in "seed shifts," thereby changing the expected pool of target genes [11], while the occurrence of 5′ variation is relatively rare. In contrast, the 3′ heterogeneity is discovered as more common situations [12][13][14]. As an essential endonuclease to produce mature miRNAs, more researchers suggested Dicer is related to the regulation of miR-222. Ueda et al. reported [15] that Dicer-disrupted cells exhibited upregulated intercellular cell adhesion molecule-1 (ICAM-1) expression and enhanced susceptibility to CTL-mediated cytolysis and directly demonstrated that miR-222 functionally targeted the 3′ UTR of ICAM-1 mRNA according with luciferase reporter assays. After transfected with Dicer siRNA, or inhibitors of miR-222, the results of flow cytometric analyses and immunoblotting suggested the inhibition of Dicer or miR-222 led to an increase in ICAM-1 expression in human malignant glioma cells and promoted their susceptibility to cytotoxic T-lymphocytes (CTLs  [18]. Cytokines play key regulatory roles in the expression of miR-221/222 subtypes ( Figure 2). Human polynucleotide phosphorylase (hPNPaseold-35), a highly evolutionarily conserved gene that catalyzes 3′-to-5′ phosphorolysis as well as 5′-to-3′ polymerization of RNA, acts as a trimer in the expression of miR-221/222 subtypes. Das et al. [19] reported that the overexpression of hPNPaseold-35 by type-I IFN treatment resulted in robust and preferentially targeted downregulation of miRNA-221/222 with consequent upregulation of its suppressed target cyclin-dependent kinase inhibitor p27 Kip1 . Nejad et al. discovered that type-I IFN stimulation selectively decreases the levels of longer miR-221-3p and miR-222-3p isoforms, while sparing the shorter ones [20]. Yu et al. [21] analyzed the endogenous length variation across breast cancer tumor samples and observed the frequent occurrence of templated 3′ isomiRs of miRNA-221/222. In breast cancer cell line MCF10A, the longer miR-222 (extending 2-4 nucleotides at the 3′ end) reduced cell fusion and promotes cell apoptosis by downregulating PI3K-AKT, and the longer miR-222 displayed an increased proclivity for nuclear localization, which indicates that different subtypes of miRNA may have different functions. And when these subtypes are regulated, cellular activity is also affected. Panneerselvam et al. [22] found that IL-24 could decrease the levels of miR-222-3p and -5p, thus increasing the target PPP2R2A level of miR-222, inhibiting the activation of protein kinase B (PKB, AKT), and inhibiting the expression of AKT in lung cancer cells. Lodge reported [23] that the cytokine TNF-α produced by macrophages could enhance the expression of miR-221/222 and that miR-221/222 binds to CD4 mRNA. It could also be found that cytokines not only regulate the expression of miR-221/222 but also selectively reduce the level of allogeneic cells in order to realize the regulation of cell activity.

Regulation of Competing Endogenous RNA on miR-221/
222. e proposition of competing endogenous RNA (ceRNA) was based on a series studies on the "talk" between RNAs [24]. ey present a hypothesis about ceRNA as that the reversed RNAs influence each other's levels by competing for a limited pool of miRNAs, which is essentially referred to "RNA ⟶ miRNA" and also a supplement to the traditional theory of "miRNA⟶RNA." In the context of cancer, pseudogenes and long noncoding RNAs (IncRNAs) could act as potential tumor suppressors and oncogenes through their ceRNA function by binding miRNA response elements (MREs). Lu et al. used molecular network-based identification of ceRNA (MNIceRNA) to construct miRNA-mRNA-IncRNA networks in thyroid carcinoma, while the hsa-miR-221/222 acted as key driver RNAs during the process [25]. Liu et al. verified that the expression of IncRNA GAS5 was significantly lower in the HCT116 and SW480 cell lines compared with that in the normal NCM460 cell line, whereas the expression of miR-222-3p was significantly higher [26]. e dual-luciferase reporter assays, RNA immunoprecipitations (IP), and RNA pull-down assays revealed that miR-222-3p could specifically bind to PTEN and IncRNA GAS5 and suggested that IncRNA GAS5 could regulate PTEN through competitively binding to miR-222-3p.

miR-221/222's Targets and Molecular Modulation
Pathways. PTEN, TIMP3 (tissue inhibitor of metalloproteinases 3), p27, and p57 are well-known tumor suppressor genes, but their expressions are often dysregulated in human cancer [27]. e research showed that both miR-221 and miR-222 directly targeted the 3′UTR of p27 and p57 mRNA and decreased the protein level of p27 and p57, thus activating the AKT pathway [28]. On the other hand, miR-221 and miR-222 also targeted PTEN and to induce TRAIL tolerance and enhance cell migration [29]. PUMA is a member of Bcl-2 protein family and has a strong proapoptotic effect [30], while miR-221/222 could bind to 3′UTR of PUMA mRNA to inhibit the expression of PUMA and then resist normal apoptosis [31] (Table 1).

Role of miR-221/222 in Various Kinds of Cancers
miR-221/222 is not only acting as a biomarker in patients with HCC but also directly targets on key targets in liver cancer (Figure 3). Fornari et al. [43] demonstrated that caspase-3 is a direct target of miR-221 in HCC, further suggesting miR-221 inhibition as a therapeutic approach aimed to contrast resistance to sorafenib. Moreover, the roles of miRNAs are cell context-dependent. For example, Han et al. [50] confirmed that α2δ1 (encoded by the gene CACNA2D1) is a surface marker that marks HCC tumorinitiating cells (TICs), and they identified miR-222 as a tumor suppressor in HCC by controlling the stemness and tumorigenicity of α2δ1 + TICs by directly targeting on pre-Bcell leukemia homeobox 3 (PBX3), which activated the expression of CACNA2D1.

Role of miR-221/222 in Colorectal Cancer.
Colorectal cancer (CRC) is one of the most common cancers and the fifth most common cause of cancer-related death world widely [97]. Several reports revealed miR-221/222 levels are elevated in human CRCs [58]. Early effective detection is critical for disease prevention; however, CRC is asymptomatic in the early stage and difficult to diagnose until advanced stages. us, there is a compelling need to identify molecular biomarkers for mass screening and early diagnosis of CRC. A report showed that miR-221 (p < 0.0001) was significantly higher in peripheral blood of 71 patients with colorectal cancer in comparison with 80 matched healthy control individuals [107]. Yau and colleagues evaluated the plasma miR-221 level [108] and the stool miR-221 level [109] and found that the plasma miR-221 had a high sensitivity (86%) but poor specificity (41%) and an AUC of only 0.61. In contrast, the stool miR-221 had an AUC of 0.73, a sensitivity rate of 62%, and a specificity rate of 74%; hence, the detection of miR-221 in stool is a better way.
ere are some reports that studied the changes of miR-221/ 222 in circulating tumor cells (CTCs) and miRNAs from CTCs during CRC treatment and found the changes in counts reflected the disease progression and/or response to chemotherapy; however, the miRNA levels (including miR-221/222) from CTCs showed transient expression and did not correlate with CTC counts [110]. e oncogene KRAS induces increased expression of miR-221/222 in human CRC cell lines [111]. It has been demonstrated that p27/CDKN1B, p57/CDKN1C, and PTEN have been identified as targets of miR-221/222 in CRC cells [ (Figure 4) [112,113]. Qin and Luo [114] found miR-221 modulates CRC cell migration and invasion in vitro and in vivo, and they demonstrated that miR-221 could directly bind to reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) 3′UTR and promotes metastasis in CRC. More reports demonstrated that mammalian STE20-like protein kinase 3 (MST3) may be the miR-222 target, and therefore, overexpression of miR-222 plays a critical role in regulating CRC cell migration and invasion [56]. Gao et al. [55] demonstrated that miR-222 enhances the migration and invasion in CRC cells by specifically targeting on the 3′UTR of melanoma inhibitory activity member 3 (MIA3). However, miR-222 may be a tumor suppressor miRNA in CRC [92]. Scientists investigated the role of ADAM-17 (a disintegrin and metalloprotease 17) as a novel multidrug resistance (MDR) mechanism in multidrug-resistant CRC and found that miR-222 was directly targeting on ADAM-17, which was downregulated in multidrug-resistant CRC cells and increased cancer cells' apoptosis.

Role of miR-221/222 in Cervical Cancer.
Although a great deal of scientific research has been made in recent years, the mortality rates of gynecological cancers continue to rise [97,115]. Early diagnosis and limited options of treatment for advanced gynecological cancers are the factors to their high mortality. However, the expression level of miRNAs in gynecological cancers is closely related to its disease diagnosis, clinic pathological features, and prognostic markers, while it has been found that miR-221/222, as a common ectopic miRNA, is almost at the abnormal expression level in gynecological cancer [116]. Sun et al. [67] observed that the expression of miR-222 in cervical cancer tissue was 2.48 times higher than that in normal tissues adjacent to cancer according to the samples of 18 patients. e upregulation of miR-222 was correlated with the deterioration of cervical cancer, and the analysis showed that p27/Kip1 and PTEN were negatively correlated with miR-222, which affected the proliferation and migration of Hela cells and SiHa cells, suggesting that miR-222 might be a new target for cervical cancer therapy. Wilting et al. [95] analyzed different expression profiles of miRNAs in the development of cervical cancer, and they found that the high expression of miR-221 was a specific marker of HPV positive cervical cancer. Cervical squamous cell carcinoma (CSCC), the most common histological subtype of cervical cancer, spreads principally by migrating into the lymphatics or by invading adjacent soft tissue. Wei et al. [116] demonstrated that there was a higher level of miR-221-3p, in human primary cervical cancer tissues derived from cervical cancer patients with or without lymph node metastasis, and it enhanced the malignancy of cervical cancer cells. e calculation and experiment showed that twist homolog 2 (TWIST2) targeted on the promoter region of miR-221-3p.
As a consequence of the metastasis of cervical cancer lymphocytes, the transcription and expression of miR-221-3p were stimulated, and miR-221-3p targeted on the 3′UTR of twist homolog 2, thus accelerating the metastasis of cervical cancer lymphocytes. Some latest reports confirms that the miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro and facilitate lymph angiogenesis and lymph node (LN) metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, they identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through  bioinformatics target prediction and luciferase reporter assays [64]. High-mobility group AT-hook1 (HMGA1, formerly HMG-I/Y), an architectural transcription factor, participates in a number of tumor biological processes. Fu et al. [68] have shown that HMGA1 is highly expressed in CSCC tissue, which promotes the migration and invasion of cervical cancer cells and showed that the mechanism of HMGA1 cancer promotion is targeting the promoter region of miR-221/222 to enhance the expression of miR-221/222. Among them, miR-221/222 targeted on the 3′UTR of tissue inhibitor of metalloproteinases 3 (TIMP3), while TIMP3 downregulated and MMP2/MMP9 upregulated, and miR-221/222-TIMP3-MMP2/MMP9 axis participates in the migration and invasion process ( Figure 5).

Role of miR-221/222 in Ovarian Cancer.
Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, accounting for 90% of the total ovarian cancer, and the 5-year survival rate is relatively low, which is the main cause of death in gynecological malignant tumors [117,118]. erefore, early detection and chemosensitivity are essential to control disease development and reduce mortality. Some research found that the expression of miR-221 and miR-222 in human ovarian cancer tissues and cell lines reaches the highest levels of miRNA relative to immortalized ovarian surface epithelial cultures [119,120]. Studies have shown that miR-222 is overexpressed in epithelial ovarian cancer cases and promotes cell proliferation through downregulation of p27 Kip1 [71]. Shang et al. indicted that patients with positive expressions of human epidermal growth factor receptor 2 (HER2), signal transducer, and activator of transcription 3 (STAT3) and p-STAT3 or with negative expressions of suppressors of cytokine signaling 3 (SOCS3) had shorter survival time [72]. Moreover, Ying et al. indicate miR-222-3p was enriched in exosomes released from EOC cells and it could be transferred to macrophages. Overexpression of miR-222-3p in macrophages induced polarization of the M2 phenotype. TargetScould prediction and the luciferase assay proved that the 3′ UTR of SOCS3 was targeted by miR-222-3p in these studies. Downregulation of SOCS3 correlated with an increased expression of STAT3 activation and induced activation of JAK/STAT pathway, which increased M2 macrophages and promoted angiogenesis and lymphangiogenesis in tumor microenvironment, which accelerated progression of EOC [121].
More studies showed that the miR-221 was upregulated in 63 samples of ovarian cancer tissues, and miR-221 level was upregulated with larger tumor size, deeper tumor invasion, and higher FIGO stage. ere was a negative correlation between the expression of apoptosis protease activator 1 (APAF1) protein and miR-221 in 5 of 63 ovarian  Journal of Oncology cancer tissues and 6 cell lines, including A2780, OVCAR3, SKOV3, and 3AO5. e APAF1 gene was confirmed to be a direct target of miR-221, which induced the proliferation of ovarian cancer cells and hindered the apoptosis of ovarian cancer cells in vitro [69]. MiR-221 also targets on BCL-2 modifying factor (BMF) promoting cell proliferation in ovarian cancer cell line SKOV3 [70]. Amini-Farsani et al. demonstrated that, in human epithelial ovarian cancer cell lines A2780 S and A2780/CP, miR-221/222 was expressed at a higher level in A2780/CP cells. An in vitro cell viability assay showed that downregulation of miR-221/222 sensitized A2780/CP cells to cisplatin-induced cytotoxicity. Bioinformatics analysis and luciferase reporter assays proved that miR-221/222 was found to directly target on PTEN, and miR-221/222 induced cisplatin resistance by targeting PTEN-mediated PI3K/Akt pathway [73].
Like the abovementioned cancers, other studies have also found that the high expression of miR-222-3p and miR-221-3p has an anticancer effect. For example, high expression of miR-222-3p could inhibit the deterioration of cancer cells, and the patients with high expression seem owning higher survival rate and longer survival time. Fu et al. analyzed miR-222-3p expression with qRT-PCR in 74 EOC patients, and the results suggest the higher the mean expression level of miR-222-3p, the longer the median overall survival time of EOC. e expression of miR-222-3p in six ovarian cancer cell lines (Tara R182, SKOV3, SKOV3DDP, SKOV3IP, HO8910, and HO8910-PM) was detected with qRT-PCR analysis, and then, low level of miR-222-3p was found in SKOV3/DDP and Tara R182 cells with high cell growth rate. SKOV3 and HO8910-PM cells with low cell growth rate expressed high level of miR-222-3p. e overexpression of miR-222-3p in the SKOV3/DDP cell line demonstrated that the migration of the tumor cells was blocked. When miR-222-3p was overexpressed in six cell lines, it was found that miR-222-3p targets on AKTphosphorylation protein regulator G protein alpha inhibiting activity polypeptide 2 (GNAI2), which inhibited AKT phosphorylation to reduce the proliferation of ovarian cancer cells [91]. Recently, there are also some investigations showing that higher expression of miR-221-3p is associated with better overall survival in EOC patients. Wu et al. examined the expression level of miR-221-3p in three EOC cell lines (SKOV3, SKOV3-IP, and SKOV3-R), which showed lower expression level in SKOV3-IP and SKOV3-R but showed more proliferations and migrations. In vitro experiments indicated that miR-221-3p inhibited EOC cell proliferation and migration. By performing subsequent systematic molecular biological, bioinformatics analyses and luciferase reporter assay, scientists found and confirmed ADP-ribosylation factor (ARF) 4 is miR-221-3p′s targeting genes [90]. Interestingly, Wurz et al. [66] found that the expression of CDKN1C (p57) protein was negatively correlated with miR-221/222 in EOC, while the expression of CDKN1B (p27) protein was not correlated with miR-221/222 in EOC ( Figure 6).

Role of miR-221/222 in Endometrial Carcinoma.
Endometrial carcinoma (EC) is the fourth most common malignant tumor in women in developed countries and the most common cancer in female genital tract; however, the incidence of endometrial carcinoma has been moving to the young population due to obesity and lifestyle factors in China [122]. Estrogen is a classic etiological factor for endometrial tumorigenesis [123]. In endometrial carcinoma, deregulated ERα caused by genomic or epigenetic aberrations was a prevalent phenomenon, which reduced the expression of ERα and associated with extensive invasion and high-stage and poor prognosis [124,125].
Liu et al. found miR-222-3p expression was much lower in ERα-positive than in ERα-negative endometrial carcinoma tissue samples, and the level of miR-222-3p expression was lower in tumors of lower grades and earlier stage. TargetScould and luciferase reporter assays revealed that ERα was a target of miR-222-3p. erefore, miR-222-3p was overexpressed in ERα-negative endometrial carcinoma tumors and was associated with high grade, late stage, and nodal metastasis, and high level of miR-222-3p was a mechanism for raloxifene resistance in endometrial carcinoma therapy. Moreover, a significant increase in serum miR-222 levels of endometrial carcinoma patients was found by qRT-PCR miRNA analysis in 46 EC patients and 28 women without cancer history. It may be used as a proper marker for the diagnosis of endometrial carcinoma in future [126].

Conclusion
As summarized above, microRNA-221/222 (miR-221/222) is a noncoding microRNA which is widely distributed in eukaryotic organisms and deeply involved in the posttranscriptional regulation of gene expressions. It is important that the miR-221/222 expression level is closely related to tumor stage and prognosis. It may be used as a biomarker for the diagnosis of premalignant tumors [104] and provide a new target for tumor therapy [8-33, 35, 38, 51, 94], as a therapeutic tool for drug resistance or sensitivity to anticancer drugs [127].
However, more questions also need to be answered as translated into clinical strategies. Firstly, given the complex networks in which cancer cells are located, the roles of miR-221/222 in various cells' background as well as its target genes need more micromesh clarifications. For example, a laboratory announced that miR-222 promotes the proliferation of human non-small cell lung cancer cell line H460 [128]; in contrast, at almost the same time, Yamashita and colleagues [129] demonstrated that miR-221/222 promoted the growth of H460 cell lines while miR-221 inhibited the growth of the other four NSCLC cell lines. Another example is that the low expression of miR-221/222 inhibited the malignant proliferation of vascular smooth muscle cells [130] but also inhibited the growth of myocardial cells and the increase of muscle cell proliferation markers induced by exercise [131]. It is worth noting that the model miRNA analyzed and transfected in tumor samples may have isomiR properties, which may cause some of the results to be contradictory.
Secondly, with the deepening of the research, the expression level of miR-221 and miR-222 is not proportional to the development of tumor, while its functions are abundant. erefore, the ratio of miR-221 and miR-222 expression level is expected to become a new research field [66].
Finally, it is expected that a single miRNA could act as a therapeutic inhibitor towards the cancer cellular pathway by regulating the different genes involved in the network. For example, miR-221/222 could inhibit the expression of ER-α and FOXO3A transcription factors, which results in the overall change of gene expression through the expression inhibition of ER-α and FOXO3A at posttranscriptional stage.
e suppressed ER-α has the ability to negatively regulate miR-221/222, and the suppressed FOXO3A blocked the transcriptional activation of p27 and Bim [63]. is shows that the regulation of individual miRNA may have great therapeutic potential towards various kinds of cancers. Back to the clinical strategy, for effectively suppressing onco-miRNA, locked nucleic acid-(LNA-) based oligonucleotides has been demonstrated to have great potential as inhibitors of small RNA targets [132].

Data Availability
Some or all data, models, or code generated or used during the study are available from the corresponding author by request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.