Deregulated miRNA clusters in ovarian cancer: Imperative implications in personalized medicine.

OC is an extremely fatal gynecological cancer and poses significant challenges to its clinical management. Genetic and epigenetic profiling of various histological subtypes of OC has identified specific molecular changes with a potential to be used as a marker for diagnosis, prognosis, and clinical management of OC. Herein, we present a comprehensive overview of the potential applications of miRNA clusters for the clinical management of OC ( Fig. 3 ). 63 Figure 3 Clinical utility of miRNA cluster expression in OC. Both oncogenic and tumor-suppressive functions of miRNA clusters are reported in OC. Altered expression levels of these miRNA clusters can be used as biomarkers in ovarian cancer diagnosis, prognosis, cancer staging, in the prediction of drug resistance and disease recurrence. Fig. 3 Clinical utility of miRNA cluster expression in OC. Both oncogenic and tumor-suppressive functions of miRNA clusters are reported in OC. Altered expression levels of these miRNA clusters can be used as biomarkers in ovarian cancer diagnosis, prognosis, cancer staging, in the prediction of drug resistance and disease recurrence.

This is one of the largest clusters in the human genome located at chr19q13.42 and it contains about 46 miRNAs. 131 C19MC is a primate specific cluster and has been shown to play both oncogenic as well as tumor-suppressive roles. 132 Reduced expression of miR-498 was observed in OC, which in turn is correlated with poor prognosis and OS (overall survival). 133 Ectopic expression of this tumor-suppressive miRNA in OC cells repressed tumor proliferation by targeting FOXO3 expression. 134 miR-519d targets XIAP and suppresses OC cell proliferation and is shown to reduce cisplatin resistance. 135 Another miRNA of this cluster, miR-520a-3p, negatively regulates EOC and inhibits tumorigenesis by suppressing SUV39H1 (suppressor of variegation 39H1). 136 miR-522–3p is associated with PTX resistance in OC cells. Forced expression of this miRNA downregulates the expression of E2F2 , in turn mitigating PTX resistance. 137 miR-520g and miR-520h were reported to have an oncogenic function in EOC via targeting DAPK2 (death-associated protein kinase-2) and SMAD7 . 138 , 139 Based on the various published data, most of the miRNAs of C19MC act as tumor suppressors in OC. However, only a few members of this family have been studied in OC and there is a lack of data pertaining to the majority of the members belonging to this cluster.

miRNA based treatment opportunities for OC management involve inhibition or supplementation of miRNAs with the use of complementary nucleic acids. 178 Few studies have demonstrated the potential of miRNA-based therapeutics in OC. A study has revealed that targeted delivery of miR-29a (miR-29a/b cluster) chimera to OC cells induced apoptosis by increasing PTEN expression. 175 Ohyagi-Hara et al have found that transfection with miR-92a (miR-17/92 cluster) diminished ITGA5 (integrin α5) expression and thus repressed peritoneal metastasis of OC cells. 179 Further, recovery of miR-200c (miR-200c/141 cluster) in OC cells significantly reduced tumor burden and enhanced PTX sensitivity suggesting the use of miR-200c restoration along with chemotherapy to improve treatment response in OC subjects. 180 Additional studies are essential to establish MC based therapy for OC.

AK and DA wrote the manuscript; VD, PS, SC, and RR helped in the critical revision; SPK conceived the study and edited the manuscript.

The study is supported by 10.13039/501100001843 Science and Engineering Research Board , 10.13039/501100006143 Department of Science and Technology , Government of India (No. EMR/2016/002314), 10.13039/501100001411 Indian Council of Medical Research , Government of India (No. 2020-3173).

All authors declare no conflict of interests.

This cluster is located on 13q31.3 within C13orf25 (chromosome 13 open reading frame-25), and is often dysregulated in neurodegenerative disorders, immune and cardiovascular diseases. 85 This miRNA cluster is also denoted as oncomiR-1 and encodes for 6 miRNAs, namely miR-19a, miR-19b, miR-17, miR-20a, miR-18a, and miR-92a. 86 The members of this cluster participate in pathways, notably cell cycle, tumor cell proliferation, apoptosis, and EMT. 85 Many studies have revealed the oncogenic as well as tumor-suppressive nature of this MC. In PTX resistant OC cell lines, elevated level of miR-17/92 was observed. 87 The over-expression of miR-92 in OC is involved in immune suppression, which is known to be regulated via LATS2/YAP1/PD-L1 pathway. 88 Further, a study by Liu et al has reported miR-19b to be significantly upregulated in OC and enhances tumor migration and invasion by suppressing PTEN/AKT pathway. 89 miR-20a enhanced tumor development by activating EMT in OVCAR3/DDP cells. 90 Gong et al confirmed that miR-17 overexpression in OC cells suppressed adhesion and invading ability and impeded peritoneal metastasis in SKOV3 xenograft model via targeting ITGA5 (integrin α5) and ITGB1 (integrin-β1). 91 miR-18a suppresses proliferation and promotes apoptosis in OC cells by targeting IPMK (inositol phosphate multikinase) and TRIAP1 (tumor protein p53-regulating inhibitor of apoptosis gene-1). 92 Collectively, these data suggest the oncogenic and tumor-suppressive role of miR-17/92 cluster, and the regulation of this cluster could be valuable in OC therapy.

The accurate detection and quantification of miRNAs has been challenging due to their unique features. 36 Technological advancements have substantially improved the methods for miRNA isolation, amplification and profiling. miRNAs can be isolated from a wide range of samples including cell lines, serum, plasma, fresh or fixed tissue samples. 36 Techniques such as immunoprecipitation with AGO2, 37 electrophoresis-based size purification, 38 and crosslinking immunoprecipitation (CLIP) 39 have been employed for the isolation and analysis of miRNAs. Reverse transcription-quantitative PCR (RT-qPCR), 40 microarray, 41 and RNA sequencing (RNA-Seq) 42 are the popular methods used for the profiling of miRNAs. For targeted miRNA analysis, RT-qPCR projects as a gold standard technique and offers absolute quantification even with low RNA inputs. 43 Microarray analysis was the commonly used high-throughput technique for parallel examination of large number of miRNAs. 44 This hybridization-based approach is best suited for the analysis of relative abundance of particular miRNAs among different group of samples. 45 However, it does not facilitate absolute quantification and detection of novel miRNAs and isomiRs. Further, microarray-based analysis generally needs high RNA inputs and due to its inadequate specificity, initial findings need to be validated by other methods such as Northern blot or RT-qPCR. 36 The advent of small RNA sequencing platforms has allowed simultaneous identification and quantification of novel miRNAs, isomiRs and other small RNA species. 43 High cost, complex workflow, and the requirement of computational infrastructure for data interpretation are the limitations of this approach. 43 Recent techniques such as single molecule real-time sequencing (SMRT) assures less biased and faster analysis than other approaches. 46 However, cost and high error rate hampers their usage. Though they have been employed in analyzing short RNA species, SMART approach is yet to be used in miRNA profiling. 36

Similar to protein-coding genes, MC expression is regulated by a variety of gene regulatory mechanisms, which include epigenetic changes, genetic alterations, transcription factors, and miRNA processing genes ( Fig. 1 ). Current experimental evidences clearly describe the association between abnormal epigenetic changes and OC ( Table 1 ). Altered DNA methylation, histone tail modifications, and miRNA expression have been reported during OC. 24 The promoter regions of tumor-suppressive MC are hypermethylated, and downregulated in OC. For example, the miRNAs in the chromosome 14 cluster are identified as down regulated as a result of epigenetic modifications in EOC (epithelial ovarian cancer). 25 Additionally, miRNA regulation of miRNA expression is reported in OC. Li et al have described the regulation of miR-133b (miR-1/133a cluster) expression by miR-145 (miR-143/145 cluster) via targeting c-MYC and DNMT3A . 26 Numerous MCs are found colocalized along with breakpoint regions, fragile sites, loss of heterozygosity (LOH) regions, regions with amplifications or deletion, all of which add up to its abnormal expression 27 ( Table 1 ). Dysregulation of miR-7a and miR-30c is linked to genomic imbalances in OC. 28 Laddha et al have reported the loss of miR-379/miR-656 cluster in 14% of serous epithelial ovarian cancer (SEOC). 29 On the contrary, miR-182 of miR-183/96/182 cluster was amplified in 28.9% of EOC. 25 Genetic polymorphisms in MCs such as rs17147016 at miR-224, rs10771184 at miR-544, rs2075993 at miR-630 were strongly linked with OC. 30 A strong association between the miR-17/92 cluster expression and rs3814113 polymorphism were linked to familial OC risk. 31 Abnormal expression of TF-miRNA cluster axis participates in OC progression ( Table 1 ). miR-199A2/214-TWIST1 axis critically regulates stemness of EOC cells. 32 In EOC, the suppression of miR-222–3p of miR-221/222 cluster by SNAI2 induced EMT (epithelial–mesenchymal transition) by upregulating PDCD10 (programmed cell death-10). 33 A few reports have described the significance of miRNA processing genes in modulating miRNA expression in OC conditions ( Table 1 ). Guo et al have revealed miR-27b as a highly downregulated miRNA in OC cells lacking DGCR8 expression. 34 DDX1 (dead-box helicase-1) gene was found to be upregulated in OC and was shown to promote miR-200 family miRNAs (miR-200a, −200b, −200c, miR-429, and miR-141) expression. 35 These data suggest that genetic and epigenetic changes can significantly alter miRNA cluster expression in OC. Figure 1 Diagrammatic representation of distinct factors that regulate miRNA cluster expression in OC. Fig. 1 Table 1 Regulators of miRNA clusters in OC. Table 1 miRNA Cluster Name No. of miRNAs Alterations/Regulated by Reference Epigenetic Regulation miR-382 C14MC 52 miRNAs Promoter methylation 181 let-7a-3 let-7a-3/let-7b 3 miRNAs (let-7a-3, miR-4763, let-7b) Hypermethylation 182 miR-133b miR-1/133a 2 miRNAs (miR-1-2, miR-133a-1) Hypermethylation 26 miR-15a/16 miR-15a/16 2 miRNAs (miR-15a, miR-16-1) Promoter methylation 181 miR-34b/c miR-34b/c 2 miRNAs (miR-34b and miR-34c) Hypermethylation 183 miR-432 C14MC 52 miRNAs Hypermethylation 25 miR-424/503 miR-424/503 6 miRNAs (miR-424, miR-503, miR-542, miR-450a-2, miR-450a-1 and miR-450b) Hypermethylation 184 miR-199a-3p miR-199/214 3 miRNAs (miR-199a-5p, miR-199a-3p and miR-214) Hypermethylation 185 miR-130b miR-130b/301b 2 miRNAs (miR-130b and miR-301b) Hypermethylation 186 miR-203a miR-203a/b 2 miRNAs (miR-203a and miR-203b) Hypermethylation 186 , 187 miR-127 C14MC 52 miRNAs Hypermethylation 187 miR-137 miR-137/2682 2 miRNAs (miR-137 and miR-2682) Hypermethylation 187 miR-29b miR-29a/b 2 miRNAs (miR-29a and miR-29b-1) Hypermethylation 188 miR-125b miR-99a/let-7c/miR-125b 3 miRNAs (miR-99a, miR-125b, let-7c) Hypermethylation 189 miR-497 miR-497/195 2 miRNAs (miR-195, miR-497) Hypermethylation 190 miR-199b-5p miR-199b/3154 2 miRNAs (miR-199b and miR-3154) Hypermethylated 191 miR-191 miR-191/425 4 miRNAs (miR-191–3p, miR-191–5p, miR-425–3p, miR-425–5p) Hypomethylated 187 miR-133b miR-206/133b 2 miRNAs (miR-206, miR-133b) miR-145 26 Genetic Regulation miR-379/656 miR-379/656 52 miRNAs Loss 29 let-7a-3, let-7b let-7a-3/let-7b 3 miRNAs (let-7a-3, miR-4763, let-7b) CNV- Loss 192 miR-30d miR-30b/-30d 2 miRNAs (miR-30b, miR-30d) CNV- Gain miR-143/145 miR-143/145 2 miRNAs (miR-143, miR-145) Loss of heterogeneity 193 miR-15a/16-1 miR-15a/16-1 2 miRNAs (miR-15a, miR-16-1) Deletion 194 miR-17/92 miR-17/92 6 miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-92a) Deletion 194 miR-182 miR-183/96/182 3 miRNAs (miR-183, miR-96, miR-182) Amplification 25 miR-224 miR-224/452 2 miRNAs (miR-224, miR-452) SNP (rs17147016) 30 miR-544 miR-379/544 38 miRNAs SNP (rs10771189) miR-17/92 miR-17/92 6 miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-92a) SNP (rs3814113) 31 Transcription Factors miR-199A2/214 miR-199a/214 3 miRNAs (miR-199a-5p, miR-199a-3p and miR-214) TWIST1 32 miR-199a FOXD3 195 miR-212/132 miR-212/132 2 miRNAs (miR-212, miR-132) SOX4/EZH2 80 miR-222–3p miR-221/222 2 miRNAs (miR-221, miR-222) SNAI2 33 miR-19a-3p miR-17/92 6 miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-92a) NF-κB 196 miR-92a STAT3 197 miR-17/92 MYC 87 miRNA Processing Genes miR-27b miR-23b/24–1/27b 3 miRNAs (miR-23b, miR-27b, miR-24-1) DGCR8 34 23 miRNAs – – DICER 198 miR-200a, −200b, −200c, miR-429, miR-141 miR-200b/200a/429 3 miRNAs (miR-200b, miR-200a, miR-429) DDX1 35 miR-200c/141 2 miRNAs (miR-200c, miR-141) Diagrammatic representation of distinct factors that regulate miRNA cluster expression in OC. Regulators of miRNA clusters in OC.

Since the discovery of miRNA clusters, several studies have explored its implications in different cancers. There has been a plethora of evidences suggestive of dysregulation of these miRNA clusters in different cancers including OC. By transcriptional regulation and post-transcriptional repression, miRNAs fine tune the gene regulatory networks to control the function of every cell. OC is one of the fatal gynecological malignancies with high morbidity and mortality rate. The high rate of fatality in OC is mainly due to its diagnosis at a late stage and lack of efficient detection modalities. Both clinical and model system-based studies have proposed that altered expression of miRNA clusters has the potential to be used as a diagnostic and prognostic indicator for better management of OC. Moreover, therapeutic modalities based on the manipulation of these clusters could successfully sensitize the OC cells to these treatment systems. Herein, we have performed a comprehensive review of literature and provided evidences and acuities to the role of miRNA clusters in OC and their possible therapeutic application for better management of OC ( Fig. 4 ). One of the key challenges to develop miRNA cluster-based markers for diagnostic and prognostic application is to recognize the best miRNA cluster as they show significant heterogeneity in their expression. Besides, majority of the studies have analyzed the significance of individual members of the miRNA clusters than looking at the entire cluster. Towards these, comprehensive studies are necessitated to understand the role of complete clusters. Since, members of the miRNA clusters show similar trend of expression and can target multiple genes belonging to the same or different pathways, manipulation of miRNA clusters may show a more significant impact than targeting individual miRNAs. Towards this, more comprehensive mapping of miRNA clusters in OC, manipulation studies and their impact needs to be investigated. Another careful consideration and challenge may be concerning the targets of miRNAs. Since miRNAs can target multiple genes and pathway which can involve both oncogene and tumor suppressor genes. Thus, one should be very careful while selecting the miRNA cluster and target genes for clinical applications in cancer in general and OC in particular. While selecting the miRNA cluster for therapy, one should also consider their target genes, signaling and other interactome. Detection of abnormal expression of miRNA clusters in the blood may provide a unique opportunity to use miRNA profiling as a minimally invasive method for diverse clinical application in OC. In this direction, more detailed and comprehensive studies are required. Figure 4 miRNA cluster biogenesis, functions, role in OC progression and their clinical application. Upon getting transcribed from the miRNA gene, pri-miRNAs/pri-miRNA clusters are processed into pre-miRNAs. These pre-miRNAs are then exported to cytosol, wherein they are further processed to form mature miRNAs. Members of miRNA clusters (MCs) functions to regulate mRNA degradation, deadenylation and gene transcription. Derailed regulation or aberrant expression of these MCs can foster ovarian cancer progression by triggering different cancer hallmarks and by modulating cellular signaling pathways. Differential expression of MC in normal and ovarian cancer tissues can be employed in OC diagnosis, prognosis, cancer staging, classification, in the prediction of therapy resistance and disease relapse. miRNA replacement therapy using different miRNA delivery systems such as exosomes, ultrasound-targeted microbubble destruction (UTMD), nanoparticles, and nanoparticles–chimera complex may serve as a potential strategy in ovarian cancer treatment. Fig. 4 miRNA cluster biogenesis, functions, role in OC progression and their clinical application. Upon getting transcribed from the miRNA gene, pri-miRNAs/pri-miRNA clusters are processed into pre-miRNAs. These pre-miRNAs are then exported to cytosol, wherein they are further processed to form mature miRNAs. Members of miRNA clusters (MCs) functions to regulate mRNA degradation, deadenylation and gene transcription. Derailed regulation or aberrant expression of these MCs can foster ovarian cancer progression by triggering different cancer hallmarks and by modulating cellular signaling pathways. Differential expression of MC in normal and ovarian cancer tissues can be employed in OC diagnosis, prognosis, cancer staging, classification, in the prediction of therapy resistance and disease relapse. miRNA replacement therapy using different miRNA delivery systems such as exosomes, ultrasound-targeted microbubble destruction (UTMD), nanoparticles, and nanoparticles–chimera complex may serve as a potential strategy in ovarian cancer treatment.

This MC is located at 5q33.1, encodes for miR-143 and miR-145, and is widely studied for its function in vascular biology and pathology related to cardiovascular disease. 101 Members of this MC are reported to regulate genes associated with cell propagation, cell cycle progression, migration, invasion and apoptosis in several cancer types. 102 miR-145 and miR-143 are downregulated in OC tissue samples and cell lines. Downregulation of miR-145 and miR-145/Sp1/CDK6/Pgp/pRb axis is proposed as a key mechanism of chemoresistance in OC. 103 Clinically, low expressions of miR-145 were linked to poor prognosis. 104 Biologically, miR-145 hinders proliferation, migration and cancer dissemination by targeting MTDH (metadherin) in HGSOCs (high grade serous ovarian cancer) via p53-miR-145-MTDH axis. 105 Similar to miR-145, miR-143 expression is also considerably reduced in OC cell lines and tissues. Forced expression of this miRNA reduced the cancer hallmarks in SKOV3, ES2, and OVCAR3 cells via targeting TAK1 (transforming growth factor beta-activated kinase-1). 106 Another study proposed that targeting TGF-β/miR-143–3p/CSTB axis can be used for clinical management of OC. 107 These data demonstrate that the down regulation of members of this cluster is critical in OC. Targeting the network regulated by miR-143/145 cluster may have a significant impact on the clinical management of OC.

miR-132 and miR-212 of miR-212/132 cluster are highly conserved vertebrate miRNAs mapped to 17p13.3 and are important for the morphogenesis of neurons, synaptic transmission, and angiogenesis. 79 miR-212/132 locus was initially described to target CREB (cAMP-response element binding protein) in neuronal cells. 79 Subsequently, the expression of miR-212/132 was reported in non-neuronal cells. Abnormal expression of this cluster participates in oncogenesis. 79 Majority of findings have analyzed the downregulation and tumor-suppressive function of miR-212/132 in OC. In vitro studies have shown that downregulation of miR-212/132 modulates the induction of EMT. 80 In SKOV3 cells, miR-212 by targeting HBEGF (heparin binding EGF like growth factor) inhibits cell propagation, migration, and invasion. 81 Forced expression of miR-212 suppressed PEA15 to inhibit OC cell proliferation via induction of apoptosis. 82 miR-132, when upregulated, halts tumor migration and proliferation via downregulating E2F5. 83 Interestingly, a study reported that the forced expression of miR-132 reversed cisplatin resistance. 84 Collectively, miR-212/132 is a tumor-suppressive miRNA cluster and its re-expression could be attempted to overcome therapy resistance in OC.

miR-379/656 cluster is also called chromosome 14 cluster or C14MC and is one of the largest MCs. miR-379/656 cluster harbors a total of 52 miRNAs and is located at 14q32.31 within the DLK1-DIO3 locus. 113 , 114 In a genome-wide analysis on the expression profiles of miR-379/656 in different cancers, it was reported that about 14% of the miRNAs of this cluster were downregulated in ovarian serous cystadenocarcinoma. This study has provided evidence for this cluster to be tumor-suppressive. 29 miR-127 is usually downregulated in OC and exerts its tumor-suppressive role by regulating its target gene BAG5 (BAG cochaperone-5). 115 Many studies have reported the expression of miR-134 to be lower in PTX resistant OC cells. 116 , 117 , 118 , 119 A study has reported the role of miR-136 in PTX resistance and identified the oncogene NOTCH3 as its potential target. 120 miR-370 demonstrates tumor-suppressive nature via regulating ENG (endoglin) in endometroid OC. 121 Ectopic expression of miR-377 was revealed to negatively target CUL4A (cullin-4A) and reduce the metastatic ability of the cells. Additionally, it is also known to affect the activity of Wnt/β-catenin signaling by modulating the expression of MMP2 and MMP9. 122 By targeting c-MYC, FGFR2 (fibroblast growth factor receptor 2) , CUL4A and IGF1R , miR-494 impeded OC cell growth, proliferation, and migration. 123 , 124 , 125 , 126 While the majority of the miRNAs of this cluster are reported to be downregulated and function as tumor suppressors in OC, a few of the members display an upregulated expression and function as oncomiRs. miR-299 displayed significantly higher expression levels in OC and it could facilitate tumor cell proliferation and migration by regulating OCT4 . 127 Likewise, miR-376a targets KLF15 (Kruppel like factor-15) and Caspase-8 and aids tumor progression. 128 miR-485–5p and miR-539–3p target SRC and SPARCL1 (SPARC like-1) respectively, and promote OC progression. 129 , 130

This cluster comprises of 7 miRNAs, namely miR-506, -507, −508, −509, −510, −513, and miR-514 and is located at Xq27.3. 93 The members of this cluster are usually downregulated and play a tumor-suppressive role in OC. Two studies have shown that the lncRNAs, DQ786243 and MALAT-1 (metastasis-associated lung adenocarcinoma transcript-1) regulate miR-506 expression, and modulates OC cell growth via targeting CREB1 and iASPP (inhibitor of apoptosis stimulating protein of p53), respectively. 94 , 95 Liu and colleagues showed that this miRNA induced senescence and repressed the proliferation of OC cells by targeting CDK4/6-FOXM1 axis. 96 miR-508 suppresses EMT, invasion, and migration through blocking the MAPK1/ERK pathway. 97 This miRNA also targets CCNA2 (cyclin A2) and MMP7 , thus inhibiting OC development. 98 miR-509–3p re-sensitizes OC cells to cisplatin treatment via targeting BCL2, MCL1 (MCL1 apoptosis regulator) , BCL2L2 (BCL2 like-2) and XIAP (X-linked inhibitor of apoptosis) resulting in apoptosis. 99 , 100 Taken together, the miR-506/514 cluster exhibits anti-tumor properties in OC, and modulation of this axis could be significant towards OC therapeutics.

Ovarian cancer (OC) is the 7th most common cancer worldwide and is the most lethal type of gynecological malignancies. 1 The GLOBOCAN estimates had reported 313, 959 new victims and 207, 252 fatalities due to OC in 2020. 2 According to the American Cancer Society, in 2020, there were a total of 21, 750 new OC cases and 13, 940 deaths in the United States. 3 Furthermore, in Europe, approximately 29, 000 deaths were predicted due to OC in 2020. 4 The Australian Institute of Health and Welfare estimated an incidence of 1,337 cases and 1,010 casualties in 2020 ( http://www.aihw.gov.au/reports/cancer/cancer-data-in-australia ). The high mortality rate of OC is attributed to its detection at an advanced stage, making it highly fatal. 5 OC is very rarely detected in women below the age of 30 years, and the risk is greater with increasing age. OC is very frequently observed in postmenopausal women. 6 The risk factors associated with OC include family history, ethnicity, genetic syndrome, BRCA1/ 2 (breast cancer 1/2), and MMR (mismatch repair) gene mutations, endometriosis, age, obesity, nonsteroidal anti-inflammatory drugs, dietary factors, postmenopausal hormone therapy and smoking. 6 , 7 , 8 The three prominent types of OC include sex-cord-stromal, germ cell and epithelial. 6 Based on the histopathological characters, there are five primary subtypes of epithelial OC, which are low- and high-grade serous carcinoma, endometrioid, mucinous and clear cell. 9 Owing to the vague array of symptoms, OC screening is very challenging. 6 Physical examination, ultrasound, biopsy, blood tests, PET (positron emission tomography), CT (computerized tomography), and MRI (magnetic resonance imaging) scans are commonly used for the detection and diagnosis of OC. Blood-based biomarkers in combination with transvaginal ultrasound imaging have been utilized for the screening of OC. 10 CA125 (cancer antigen 125) is a well-known tumor biomarker that has been used for OC detection. 11 But, this blood-based biomarker has very low sensitivity as well as specificity, and because of this, CA125 is not recommended as a stand-alone screening method. 6 , 11 However, the combination of transvaginal ultrasound coupled with screening for CA125 and HEP-4 (human epididymis protein 4) has been proposed as an efficient OC screening tool. 6 Long term use of oral contraceptives has proven to be effective in preventing the risk of OC. 12 On the other hand, the use of oral contraceptives should be equalized to prevent the possible risk of cervical and breast cancer. 13 Despite the availability of diagnostic tools and preventive interventions, the prognosis of OC is poor. Besides, the high mortality rate in OC is attributed to the nebulous nature of symptoms, diagnosis at an advanced stage, tumor cell heterogeneity, therapeutic resistance, metastasis, and relapse. 14 Many previous studies have suggested that the use of molecular markers can significantly improve the clinical management of OC. This suggests the need to understand the disease at the molecular level and identify clinically relevant markers for diagnosis, prognosis and management of OC. 15 , 16 Towards this, expression profiling of miRNAs can be employed for the clinical management of OC. miRNAs were first discovered in Caenorhabditis elegans in 1993 and are single-stranded, non-coding RNAs with an approximate length of 22 nt. 17 , 18 , 19 The mature miRNAs critically modulate target gene expression by translation repression or mRNA cleavage. 20 Adjacently located miRNA genes get transcribed together to result in miRNA clusters (MC). 21 These clusters contain 2 or more miRNA genes transcribed together in the same orientation. 22 Many of the MCs are evolutionarily conserved and regulate diverse biological pathways by controlling the expression of protein-coding genes. 22 Several studies have highlighted the involvement of MCs in OC progression by facilitating the acquisition of cancer hallmarks. Both the tumor-suppressive and tumor promoting roles of MCs are reported in OC. 23 Various mechanistic studies have suggested that the MC members' abnormal expression can result in ovarian tumorigenesis and metastasis. Towards this, the current review provides a comprehensive overview of the role of MCs in OC development. More specifically, the present review article focuses on the regulation and biological function of miRNA clusters in OC.

This X-chromosome based MC encodes for 6 miRNAs, namely miR-18b, miR-19b-2, miR-106a, miR-92a, miR-20b, and miR-363. The members of this cluster exhibit a prominent role in facilitating the propagation of ovarian tumors and are also implicated in controlling multi-drug resistance in OC cells. In both OC cells and tissues, miR-106a expression was found to be significantly elevated and was shown to promote tumor growth, proliferation, and intrusion in OC cells by negatively regulating PTEN . 108 Increased expression of miR-106a mediated proliferation and differentiation of HGSOC via targeting p130 (RBL2). 109 This miRNA showed upregulated expression in PTX resistant OC cell lines and reduced BCL10 and Caspase-7 expression. 110 Another member of this cluster, miR-18b, whose upregulated expression boosts migratory as well as invasive properties of OC cells by directly targeting PTEN . 111 Reduced level of miR-363 was observed in OC cells and tissues. The overexpression of this miRNA resulted in the decreased expression of NOB1 [NIN1 (RPN12) binding protein-1 homolog], which in turn suppressed tumor growth and proliferation. 112 However, the rest of the members of miR-106a/363 cluster aren't very well studied in OC. These data suggest oncogenic as well as tumor-suppressive functions of this MC in OC, and have a role in regulating multidrug resistance.

miR-199a/214, located at 1q24, encodes for miR-214, miR-199a-3p and miR-199a-5p, and is transcribed as a part of the transcription of DNM3OS (dynamin 3 opposite strand) using E-Box promoters. 61 The members of the cluster participate in the development of various tissues, notably heart, bone, muscle, pancreatic, nervous system, nephrogenesis, and vascularization. 62 At the cellular level, miR-199a/214 cluster controls proliferative, migratory, invasive ability, and cell cycle progression in OC cells. 63 A study reported 56% and 53% of OC tissues showed upregulation of miR-214 and miR-199a, respectively. 64 Interestingly, miR-214 and miR-199a have the potential to differentiate ovarian cancer stem cells into OC cells. OC cells possessing cell surface markers such as CD44 + /CD117 + are known to render stemness and chemotherapeutic resistance. 65 By transfecting CD44 + /CD117 + cells with miR-199a, there is a substantial decrease in both protein and mRNA levels of CD44 with a concomitant decrease in ABCG2 (ATP binding cassette subfamily G member-2) expression. 66 In OC, miR-214 displays an oncogenic function by targeting PTEN to activate AKT signaling and stimulates cisplatin resistance, cell survival, 67 and radio-resistance. 64 , 68 On the contrary, by targeting CTNNB1 (β-catenin), miR-214 acts as a tumor suppressor in OC. Collectively, abnormal expression of miR-199a/214 may promote stemness and therapeutic resistance in OC. 32 Thus, modulation of miR-199a/214 expression may offer an opportunity to reverse the stemness and chemotherapeutic resistance in OC.

This cluster, located at 12p13.31, encodes for miR-141 and miR-200c that are often abnormally expressed in various cancers including OC. 47 The members of miR-200c/141 cluster regulate the expression of genes associated with growth, proliferation, migration, invasion, EMT, stemness, apoptosis, and chemosensitivity. Both tumor-suppressive and oncogenic functions have been assigned to miR-200c/141 cluster in cancers. 48 In OC cells miR-200c/141 cluster increases cell proliferation and induces drug resistance via regulating the expression of KEAP1 (Kelch like ECH associated protein-1). 49 The activation of EMT signaling cascade significantly fosters metastasis 50 and may contribute to high mortality rate in OC. Forced expression of miR-200c in OC cells substantially reduced migratory and invasive potential by enhancing CDH1 (E cadherin) expression. 51 Due to its ability to reduce tumorigenicity and invasion of OC cells, miR-200c is proposed as an important therapeutic target. 52 The list of validated targets and signaling pathways are shown in Table 3 . miR-141 enhances anoikis resistance via targeting KLF12/SP1/survivin axis in OC. Besides, members of this cluster also target metastatic pathway genes. 53 By targeting SIK1 (salt-inducible kinase 1) and KEAP1 , miR-141 promotes cell proliferation and cisplatin resistance in OC. 54 Together, these results suggest that miR-200c/141 cluster functions as both tumor-suppressive and oncogenic in OC and is vividly reported to regulate EMT and metastatic axis. Further, it may serve as a valuable therapeutic target and a prominent diagnostic marker. Table 3 miRNAs cluster expression and their target in OC. Table 3 miRNA cluster miRNAs in cluster Expression Regulated by Signaling pathways Targets Reference miR-200c/141 miR-200c, miR-141 Downregulated – EMT NF-κB ZEB1/pSMAD JAK-STAT3 KEAP1 CDH1 SNAI1 ZEB2 KLF12 SIK1 KEAP1 50 , 52 , 54 , 55 , 208 , 209 , 210 miR-200b/200a/429 miR-200b, miR-200a, miR-429 Upregulated – Wnt/β-catenin PI3K/AKT ING5 PCDH9 PTEN KIAA0101 ZEB1 ATAD2 55 , 56 , 57 , 59 , 209 , 210 , 211 miR-199a/214 miR-199a-5p, miR-199a-3p and miR-214 Downregulated TWIST1 DNMT3 FOXD3 IKKβ/NF-κB PTEN/AKT TGF-β PI3K/AKT CTNNB1 PTEN NF-κB1 DDR1 TGF-β2 ABCG2 Sema 4D 32 , 66 , 67 , 185 , 195 , 212 , 213 miR-183/182/96 miR-183, miR-96, miR-182 Upregulated DNMT3A Leptin TGF-β/SMAD4 AKT SMAD4 PDCD4 BRCA1 MTSS1 HMGA2 FOXO3 CAV1 71 , 72 , 73 , 74 , 214 , 215 miR-23a/24–2/27a miR-23a, miR-24-2, miR-27a Upregulated – NF-κB WNT/MAPK WNT/β-catenin IKKα ST7L DLG2 NANOG OCT4 BCL2 CUL5 FOXO1 HIPK2 BTG1 FBLN5 76 , 77 , 78 , 216 , 217 , 218 , 219 miR-23b/24–1/27b miR-23b, miR-27b, miR-24-1 Up/Downregulated – – CCNG1 RUNX2 DGCR8 VE- cadherin CXCL1 34 , 220 , 221 , 222 , 223 miR-106b/25 miRNA-106b, miR-93, miR-25 Up/Downregulated – PTEN/AKT RHOC BIM LATS2 PTEN 224 , 225 , 226 , 227 miR-212/132 miR-212, miR-132 Downregulated – – SOX4 HBEGF MAP3K3 PEA15 E2F5 BMI1 80 , 81 , 82 , 83 , 84 , 228 miR-221/222 miR-221, miR-222 Up/Downregulated SNAI2 PI3K/AKT Wnt/β-catenin PTEN BMF APAF1 ARF4 p27 Kip1 GNAI2 PDCD10 33 , 153 , 154 , 155 , 229 , 230 , 231 , 232 miR-302/367 miR-367, 302d, 302c-5p, 302c-3p, 302a-5p, 302a-3p, 302b-5p, 302b-3p Downregulated – STAT3 signaling CTNNB1 RUNX1 ATAD2 RAB22A 233 , 234 miR-17/92 miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-92a Upregulated NF-κB PTEN/AKT Hippo-YAP ITGA5 ITGB1 LATS2 TRIAP1 IPMK IGFBP-3 YES1 PTEN 88 , 91 , 92 , 196 , 235 , 236 miR-506/514 miR-506, -507, −508, −509, −510, −513, miR-514 Downregulated DQ786243 , MALAT1 AKT/FOXO3A MAPK1/ERK CDK4/6–FOXM1 MTMR6 CREB1 CDK4 CDK6 iASPP SIRT1 MAPK1 CCNA2 MMP7 BCL2 MCL1 BCL2L2 XIAP 94 , 95 , 96 , 97 , 98 , 99 , 100 , 237 , 238 miR-143/145 miR-143, miR-145 Downregulated – TGF-β Hippo signaling SP1 CDK6 MTDH TRIM2 TAK1 CSTB 103 , 104 , 105 , 106 , 107 , 239 miR-106a/363 miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92-2, miR-363 Up/Downregulated – Hippo signaling PTEN p130 BCL-10 Caspase-7 MCL1 PDCD4 NOB1 LATS2 108 , 109 , 110 , 111 , 112 , 240 , 241 , 242 miR-1-1/133a-2 and miR-1-2/133a-1 miR-1-1, miR-133a-2, miR-1-2, miR-133a-1 Downregulated – Wnt/β-catenin c-MET IGFR1 PYGB 243 , 244 , 245 miR-371/373 miR-371, miR-372, miR-373 Up/Downregulated – – LATS2 ATAD2 DKK1 CCNA1 p62 RAB22A 142 , 156 , 246 , 247 miR-379/656 52 miRNAs Up/Downregulated – PI3K/AKT Wnt/β-catenin APK/ERK1/2 NOTCH3 WNT AKT MYC BAG5 PNAS-105 C16orf72 F-box protein 2 SRM GAPDH RPL41 PRPF6 VIM PAK2 FEN1 TAB1 NOTCH3 PIK3CA PIK3CB ENG CUL4A YY1 NOTCH1 c-MYC FGFR2 CUL4A IGF1R CDCP1 PLAGL2 RAB1A OCT4 KLF15 Caspase-8 SRC SPARCL1 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 248 , 249 , 250 , 251 , 252 C19MC 46 miRNAs Up/Downregulated TGF-β FOXO3 XIAP SUV39H1 RNF216 E2F2 DAPK2 SMAD7 134 , 135 , 136 , 137 , 138 , 139 , 253 miRNAs cluster expression and their target in OC.

miR-183/182/96 located at 7q32.2 is a highly conserved MC that is expressed in the retina, sensory organs and pluripotent stem cells. miR-182, miR-183 and miR-96 are the members of this cluster and play a critical role during the pluripotent stem cell differentiation into sensory organs. 69 miR-183/182/96 is one of the upregulated MCs in OC 70 and modulates tumor growth, invasion, apoptosis and therapy resistance. Upregulated miR-183 has been shown to activate TGF-β/SMAD4 pathway by negative regulation of SMAD4 to promote OC. 71 Importantly, by negatively regulating PDCD4 (programmed cell death-4), miR-182 induces growth, invasion, resistance to apoptosis, and resistance to cisplatin and taxol. 72 Another study demonstrated that miR-182 overexpression induced apoptosis via caspase-3 and -9 activation in Caov-3 cells, thus demonstrating its tumor-suppressive function in OC. 73 Both miR-96 and miR-182 are upregulated by the effect of leptin. It has been shown that the tumor promoting function of miR-96–5p could be annulled as an effect of CAV1 overexpression. 74 Thus, deregulated expression of miR-183/182/96 may participate in pathways leading to growth, proliferation, invasion, apoptotic evasion, and induction of therapy resistance.

miR-200b/200a/429 located at 1p36.33 belongs to miR-200 family and comprises miR-200b, miR-200a, and miR-429. Both downregulation and upregulation of the members of miR-200b/200a/429 cluster are reported in OC. For example, in T80 cells, overexpression of miR-200b/200a/429 promoted tumor growth in vivo by regulating ING5 (inhibitor of growth family-5). 55 Moreover, this cluster targets Wnt/β-catenin and PI3K/AKT to promote its tumor regulatory functions. MiR-200a is often upregulated in OC tissues and participates in pathways facilitating tumor advancement. 56 On the contrary, low level of miR-429 were found in OC cells. KIAA0101, an oncogene, is the direct target of miR-429 and are negatively correlated with one another. 57 The ectopic miR-429 expression upsurges drug sensitivity and results in the induction of MET (mesenchymal to epithelial transition) in metastasizing OC cells. 58 On upregulation, miR-200b-5p targeted ATAD2 (ATPase family AAA domain containing-2) and inhibited OC cell proliferation via PI3K/AKT pathway. 59 Interestingly, nanoparticle mediated delivery of miR-200a and −200b reduced the metastatic burden with improved organization of the vasculature. 60 These data clearly indicate that targeting miR-200b/200a/429 may have a significant impact on the clinical management of OC.

This cluster is located in 19p13.12 and encodes for miR-23a, miR-24-2, and miR-27a. 75 Being an overexpressed MC in OC, the expression of members of this cluster is linked with clinical stage, lymph node metastasis and poor patient survival. By directly targeting ST7L (suppression of tumorigenicity-7 like), miR-23a activates Wnt/MAPK pathway and acts as anti-apoptotic and promoter of cell cycle progression in OC. 76 MiR-23a inhibits DLG2 (discs large homolog-2) expression to foster tumor cell propagation and invasion via release of NANOG , OCT4 (octamer binding transcription factor-4), and BCL2 (BCL2 apoptosis regulator). 77 miR-27a by targeting HIPK2 (homeodomain-interacting protein kinase 2) brings about paclitaxel (PTX) resistance in OC cells via MDR1 /P-gp axis (multidrug resistance mutation 1/P-glycoprotein). 78 Taken together, miR-23a/24–2/27a cluster functions as an oncogene and its upregulation stimulates growth, proliferation, and invasion via inhibition of apoptosis and induction of Wnt/MAPK and Wnt/β-catenin pathways to promote PTX resistance.