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A review on the importance of miRNA-135 in human diseases

MicroRNA-135 (miR-135) is a microRNA which is involved in the pathoetiology of several neoplastic and non-neoplastic conditions. Both tumor suppressor and oncogenic roles have been reported for this miRNA. Studies in prostate, renal, gallbladder and nasopharyngeal cancers as well as glioma have shown down-regulation of miR-135 in cancerous tissues compared with controls. These studies have also shown the impact of miR-135 down-regulation on enhancement of cell proliferation and aggressive behavior. Meanwhile, miR-135 has been shown to be up-regulated in bladder, oral, colorectal and liver cancers. Studies in breast, gastric, lung and pancreatic cancers as well as head and neck squamous cell carcinoma have reported dual roles for miR-135. Dysregulation of miR-135 has also been noted in various non-neoplastic conditions such as Alzheimer’s disease, atherosclerosis, depression, diabetes, Parkinson, pulmonary arterial hypertension, nephrotic syndrome, endometriosis, epilepsy and allergic conditions. In the current review, we summarize the role of miR-135 in the carcinogenesis as well as development of other disorders.


Introduction
MicroRNAs (miRNAs) represent a group of small-sized transcripts with high impact on the regulation of gene expression. For many years the role of miRNA is mostly associated with translation arrest. miRNAs have crucial roles in the developmental processes and have various biological functions (Fu et al., 2013;Ghafouri-Fard et al., 2021a;Hussen et al., 2021). The process of miRNA synthesis contains multiple steps with the final step being incorporation of either the 5p or 3p strands of the mature miRNA duplex into a complex, namely miRNA-induced silencing complex (miRISC). Notably, shuttling of this complex inside the cell has an essential role in the extent of miRNAmediated modulation of gene expression (O'Brien et al., 2018;Ghafouri-Fard et al., 2021b;Ghafouri-Fard et al., 2021c;Ghafouri-Fard et al., 2021d). miRNAs regulate gene expression in a dynamic manner which results in buffering of expression levels to reach a stable state (O'Brien et al., 2018). Most remarkably, accessibility and relative abundance of miRNAs and their targets can define the genes that are modulated by miRNAs. Moreover, miRNA-mediated inhibition of target transcripts is not universal among different kinds of cells. This effect is modulated by alternative splicing/polyadenylation events and the amounts of cell type-specific factors that change secondary structure of target transcripts (O'Brien et al., 2018).
The impact of several miRNAs in normal developmental and pathological conditions has been investigated unraveling a wide variety of different functions for these transcripts. Most notably, circulating miRNAs have recently attracted attention of researchers for their application in diagnosis of human disorders, particularly cancers . Although in some cases alterations in the levels of miRNAs in the peripheral blood might be solely a by-products of the underlying condition, many of these miRNAs have been shown to participate in the occurrence and development of cancer through direct or indirect routes. They can be used as tools for subtype classification of tumors, detection of resistance to chemo-or radiotherapy and clinical outcome . However, there is an urgent need for conduction of large scale studies to enhance the sensitivity, specificity, and applicability of these markers. miR-135 is an example of miRNAs with diverse roles in both neoplastic and non-neoplastic conditions and possible application as a maker for both kinds of disorders. This miRNA is encoded by three different loci, namely MIR135A1 (3p21.2), MIR135A2 (12q23.1) and MIR135B (1q32.1). While MIR-135A1 and MIR-135A2 are located at different chromosomes in humans, they are transcribed into miR-135a. MIR-135B is the only gene accountable for miR-135b expression in humans.
miR-135b has been found to be expressed in brain, cerebellum, artery, colon, lung, stomach, esophagus, thyroid, salivary gland, breast, ovary, prostate and testis. miR-135a has a broader range of tissue expression. In addition to these tissues, it is expressed in whole blood, spinal cord, tibial nerve, heart, skeletal muscle, small intestine, adipocyte, kidney, liver, lung, spleen and a number of other tissues (GeneCards, 2017).
In the current review, we summarize the role of miR-135 in the carcinogenesis as well as development of other disorders.

miR-135 in cancers Digestive tract cancers
In silico analyses using the Cancer Genome Atlas (TCGA) data on gastrointestinal cancers (including those originated from colon, esophagus, liver, pancreas, rectum and stomach) has shown that over-expression of miR-135 in the cancerous tissues is associated with a poor overall survival in these types of cancers. These significant findings were based on the receiver operating characteristic curves and Kaplan-Meier analyses which included 1,488 patients with gastrointestinal cancers whose data on survival and miR-135 expression was available in the TCGA (Chao et al., 2019).

Colon cancer
In a pioneer study in this field, Nagel et al. have shown that miR-135a and miR-135b target the 3′ UTR of APC to decrease expression of this tumor suppressor gene and enhance activity of Wnt pathway. Authors have also reported significant overexpression of miR-135a and miR-135b in colorectal adenomas and carcinomas in correlation with down-regulation of APC transcripts (Nagel et al., 2008). Subsequently, Valeri et al. have shown that APC loss induces up-regulation of miR-135b leading to dysregulation of PTEN/PI3K pathway and up-regulation of SRC which in turn promotes cell transformation and progression of colorectal cancer. Over-expression of miR-135b has been found to be a common finding in sporadic and inflammatory bowel disease-associated human colorectal cancers. Moreover, its over-expression is correlated with tumor stage and poor patients' survival. Suppression of miR-135b in animal models of colorectal cancer could reduce tumor growth through modulation of genes participating in proliferation, invasion, and apoptosis (Valeri et al., 2014). Bai et al. have shown that the exosome-mediated delivery of miR-135b to gastric cancer cells enhances angiogenic processes in these cells both in vitro and in vivo. Tumor cells-derived miR-135b could suppress expression of FOXO1 transcription factor that regulates gluconeogenesis and glycogenolysis by insulin signaling. This miRNA can also increase development of blood vessels (Bai et al., 2019). Huangfu et al. have shown that up-regulation of miR-135b enhances cell proliferation, migratory aptitude and invasive properties of gastric cancer cells through targeting the tumor suppressor gene CAMK2D which is a serine/threonine protein kinase pertaining to the subfamily of Ca2+/calmodulindependent protein kinases. Notably, in vivo administration of miR-135b antagonist has suppressed tumor growth and metastatic potential of tumors in xenograft models (Huangfu et al., 2021). Another study in early gastric cancer has reported down-regulation of miR-135a in about one-third of patients. Notably, these patients have exhibited advanced TNM stage and higher possibility of lymph node metastasis compared with patients having high levels of miR-135a. Functional studies have revealed that miR-135a suppresses viability of cells, EMT, invasive properties, and their migration. The rho-associated, coiled-coil-containing protein kinase 1 ROCK1 has been identified as the target of miR-135a in gastric cancer cells (Shin et al., 2014a). Similar to this report, He et al. have demonstrated down-regulation of miR-135 in gastric cancer tissues compared with adjacent tissues. Downregulation of this miRNA has been associated with lower overall survival rate of patients. The impact of miR-135 on reduction of proliferation, invasion and migration of gastric cancer cells has also been confirmed in BGC-823 and SGC-7901 cell lines. Moreover, it has been revealed that this miRNA has a regulatory role on expression of the GTP exchange factor SMAD2 . On the other hand, Han et al. have found that miR-135b is the mostly up-regulated miRNA in gastric tissues from K19-C2mE and Gan mice. Moreover, expression of this miRNA has been shown to be elevated during the early stages of gastritis-associated carcinogenesis. This miRNA has also been shown to be up-regulated in gastric tumor tissues from gp130 F/F mice and human clinical samples. Interleukin 1 could enhance expression of this miRNA in gastric organoids and immortalized cell lines. Oncogenic effects of miR-135b in gastric cancer have been exerted through targeting FOXN3 and RECK tumor suppressors (Han et al., 2019). Zhou et al. have shown up-regulation of miR-135b in pancreatic cancer tissues and pancreatic cancer stem cells (CSCs). This miRNA has been shown to target the apoptosis and differentiation gene JADE-1. Up-regulation of miR-135b has increased proliferation, migratory potential, and invasion of pancreatic CSCs, suppressed their apoptosis and surged levels of stemness-related factors. Furthermore, miR-135b has been shown to enhance phosphorylated levels of AKT and mTOR. In vivo studies have also confirmed the impact of miR-135b up-regulation on acceleration of tumor growth . Contrary to this study, Zhang et al. have shown down-regulation of miR-135a in pancreatic cancer tissues and cell lines compared with the corresponding controls. Luciferase activity assay has shown the interaction between long non-coding RNA (lncRNA) UCA1 and miR-135a. Notably, miR-135a could reverse the impact of UCA1 on apoptosis and viability of pancreatic cancer cells (Zhang et al., 2017a).

Gallbladder cancer
Zhou et al. have found that expression of miR-135a-5p is repeatedly diminished in gallbladder cancer tissues in correlation with histologic grade. Forced over-expression of miR-135a-5p has suppressed proliferation of gallbladder cancer cells in vitro and in vivo. Furthermore, the cell surface protein participating in receptor-mediated endocytosis VLDLR has been identified as a direct target of miR-135a-5p. The p38 MAPK pathway has been found to participate in miR-135a/VLDLR downstream signaling (Zhou et al., 2014). Consistently, miR-135a has been shown to inhibit invasiveness and metastasis of gallbladder cancer cells and induce their apoptosis through regulation of ROCK1, HOXA10 and BCL-2 expression levels. miR-135a-loaded liposomes adapted with Anti-EGFR antibodies could decrease tumor growth in xenograft models (Feng et al., 2020).

FIGURE 1
In breast cancer, miR-135 is regarded as a tumor suppressor miRNA, since over-expression of miR-135 could suppress cell proliferation, migration, invasion and EMT by inhibition of Wnt/βcatenin axis. In fact, expression of miR-135 has been positively associated with expression of p-GSK3, but inversely associated with expressions of Wnt and β-catenin. Up-regulation of miR-135 has increased p-GSK3 levels . Mechanistically, GSK-3β participates in the formation of a complex with APC and Axin which has a role in β-catenin phosphorylation and its degradation (Wu and Pan, 2010).

Hormone-dependent cancers
Breast cancer Jiang et al. have reported a tumor suppressor role for miR-135 in human breast cancer cell lines as well as mice models. They have reported that forced over-expression of miR-135 suppresses growth, migratory aptitude, invasiveness and epithelial-mesenchymal transition (EMT) of MDA-MB-468 and MCF-7 cell lines. Mechanistically, miR-135 can suppress activity of Wnt/β-catenin pathway ( Figure 1) .
Similarly, Yang et al. have reported down-regulation of miR-135-5p in breast cancer samples compared to neighboring breast tissues with a more prominent reduction in patients having lymph node involvement. On the other hand, expression of SMAD3 has been shown to be increased in cancerous samples. Functional studies have shown that miR-135-5p inhibits TGF-β-mediated EMT, thus reducing metastatic ability of breast cancer cells in vivo. Besides, SMAD3 silencing has led to a similar phenotype with miR-135-5p up-regulation in breast cancer cells. Additional mechanistical assays have revealed that SMAD3, a fundamental modulator of TGF-β/SMAD signaling is directly targeted by miR-135-5p (Yang et al., 2020a).
In an attempt to find miRNAs regulating expression of the bone-metastasis promoting factor Runx2, Taipaleenmäki et al. have reported the inhibitory role of miR-135 on this factor in breast tissues. While miR-135 has been reported to be highly expressed in normal breast epithelial cells, it has not been expressed in metastatic breast cancer cells and clinical samples with high expression of Runx2. Forced over-expression of miR-135 in metastatic MDA-MB-231-luc cells has decreased expression of Runx2 and levels of pro-metastatic targets of Runx2, namely IL11, MMP-13, and PTHrP. These effects have been accompanied with reduction of tumor growth and bone metastasis in animal models (Taipaleenmäki et al., 2015).
Through a bioinformatics approach, Bertoli et al. have identified miR-135b as one of miRNAs with essential roles in regulation of the reformed functional pathways in basal type of breast cancer. With a degree centrality of 12, miR-135b has been shown to control expression of 12 over 35 genes inside 1 couple pathways (ethanol degradation X and Mismatch repair in eukaryotes). Experiments have confirmed over-expression of miR-135b in BT20 and MDA-MB-231 breast cancer cell lines compared to normal-like cell line of breast, i.e. MCF10A (Bertoli et al., 2021). Consistent with this study, Uva et al. have reported that expression levels of miR-135b are firmly correlated with triple negative breast cancer (TNBC) with basal-like phenotype. Target analyses of miR-135b have shown impact of this miRNA on TGF-β, WNT and ERBB pathways. Moreover, miR-135b expression has been correlated with neoplastic proliferative index (Uva et al., 2018). Aakula et al. have shown that miR-135b has binding sites for 3′ UTR of androgen receptor (AR), estrogen receptor (ER) and HIF1AN and acts as a regulator of these genes. This miRNA has higher expressions in patients with ER negative breast tumors and in prostate cancer patients with low levels of AR (Aakula et al., 2015). In addition, Tribollet et al. have discovered a negative relationship between ER with miR-135 expression in breast and prostate cancers. miR-135a has an ability to suppress invasion and aggressiveness of these malignant cells (Tribollet et al., 2016).

Genitourinary cancers
Bladder cancer miR-135 has been shown to be increased in bladder cancer cell lines and clinical samples. Up-regulation of miR-135a has enhanced proliferation of bladder cancer cells, while suppression of miR-135a has reversed this effect. PHLPP2 and FOXO1 have been identified as direct targets of miR-135a whose expressions are decreased by miR-135a. Cumulatively, miR-135a can promote proliferation in bladder cancer cells through decreasing levels of PHLPP2 and FOXO1 (Mao et al., 2015). Consistent with this study, Mao et al. have reported upregulation of miR-135a, β-catenin, cyclin D1 and vimentin in bladder cancer samples compared with non-cancerous controls. Moreover, they have reported down-regulation of GSK3β and E-cadherin in cancerous samples. Functional studies have shown the role of miR-135a in acceleration of EMT, invasion and migratory potential of bladder cancer cells through enhancing activity of Wnt/β-catenin signaling. These effects are mediated via GSK3β down-regulation (Mao et al., 2018). Another study in bladder cancer cells has shown interaction between miR-135a and lncRNA MBNL1-AS1. In fact, the tumor suppressor role of MBNL1-AS1 is exerted through decreasing miR-135a levels and influencing activity of PHLPP2/FOXO1 axis (Wei et al., 2020).

Other types of cancers
Lung cancer Tian et al. have shown down-regulation of miR-135a in non-small cell lung cancer (NSCLC) cells compared with normal bronchial epithelium. miR-135a has been found to inhibit proliferation, invasiveness and metastatic ability of NSCLC cells. Notably, miR-135a could suppress expression of several molecules in the RAS signaling pathway via suppression of expression of RAB1B (Tian et al., 2020). On the other hand, Zhao et al. have reported a pro-oncogenic role for miR-135b in NSCLC. They have also reported association between up-regulation of miR-135b and poor prognosis in Frontiers in Genetics frontiersin.org 04 (Continued on following page) Frontiers in Genetics frontiersin.org 05 these patients. The pro-proliferative, pro-angiogenic and antiapoptotic effects of miR-135b have been verified in animal models as well. miR-135b has been shown to directly target the deubiquitinase CYLD transcript, thus controlling ubiquitination and activation of NF-κB pathway. Expression of this miRNA has been shown to be enhanced by IL-6/ STAT3 axis. Cumulatively, IL-6/STAT3/miR-135b/NF-κB constitutes a positive feedback circuit which contributes in the development of NSCLC (Zhao et al., 2021).
Studies in prostate, renal and nasopharyngeal cancers as well as glioma have shown down-regulation of miR-135 in cancerous tissues compared with controls. These studies have also shown the impact of miR-135 down-regulation on enhancement of cell proliferation and aggressive behavior. On the other hand, miR-135 has been shown to be up-regulated in oral, cervical and liver cancers as well as myxoid liposarcoma, multiple myeloma and melanoma. Supplementary Table S1 summarizes the role of miR-135 in diverse types of cancers.
*Expression of this miRNA, in tumoral tissues has been compared with its expression in normal/non-cancerous tissues of the same origin.
Frontiers in Genetics frontiersin.org 06 Frontiers in Genetics frontiersin.org 07 miR-135a has been shown to contribute to paclitaxel resistance in various tumor cells possibly through downregulation of APC (Holleman et al., 2011). On the other hand, miR-135 has been reported to suppress tumor growth and cell invasion, and increase sensitivity to 5-fluorouracil and doxorubicin drugs through targeting FAK (Golubovskaya et al., 2014). Table 1 shows the effects of miR-135 in regulation of response to chemotherapy, radiotherapy and other therapeutic modalities.

ANCT: Adjacent non-cancerous tissue, IPMN: Intraductal papillary mucinous neoplasm Clinical significance of miR-135
Several studies have verified correlations between serum/ tissue levels of miR-135 and patients' survival (Table 2). In a cohort of breast cancer patients, a positive correlation has been detected between miR-135b expression and ki67 expression, and a negative correlation has been reported between miR-135b expression and age and androgen receptor expression (Ghafouri-Fard et al., 2021b). Other studies have also reported association between expression of this miRNA and tumor size, tumor grade or other clinical data ( miR-135 has diverse roles in the pathogenesis of human disorders. miR-135b acts as a neuroprotective miRNA via targeting GSK3β, thus it could neutralize effect of MPP + on proliferation and apoptosis of cells (Zhang et al., 2017b). Moreover, experiments in Zebrafish model have shown that miR-135a shields neural crest cells against alcohol-induced apoptosis and craniofacial deformities through regulation of Siah1/p38/p53 axis (Yuan et al., 2020). miR-135 can also affect the process of atherosclerosis through different mecahnisms (Figure 2). Table 3 shows the role of miR-135 in non-cancerous disorders.

mI/R: Myocardial ischemia/ reperfusion injury, FSGS: Focal segmental glomerulosclerosis
According to Kim et al. study in mice models and RBL2H3, B16F1, B16F10 cell lines, miR-135-5p could prevent allergic inflammation through targeting P62 (Shan et al., 2020). Sung et al. have performed a series of assays in Neuro-2a cells and mice model. Their investigation has revealed that ischemic preconditioning as an endogenous

Frontiers in Genetics
frontiersin.org 08 neuroprotective process promotes expression of ATPbinding cassette subfamily A member 1 (ABCA1), suppresses miR-135-5p expression, decreases the Bax/ Bcl2 proportion and activates caspase-9 and caspase-3, thus protecting neural cells against mitochondria-dependent apoptosis and subsequent brain injuries (Sung et al., 2021). According to Xie et al. study, inhibition could protect cells separated from myocardial tissues of mice from apoptosis and reperfusion injury by activating JAK2/STAT3 signaling axis (Xie et al., 2017).
*Expression of this miRNA, in affected tissues/cells has been compared with its expression in normal tissues/cells of the same origin.
Frontiers in Genetics frontiersin.org In an interesting study, the relationship between physical exercise and miR-135 expression in old mice was identified. In this survey, authors have reported miR-135a-5p down-regulation via exercise. The consequent cell cycle progression and proliferation of neural precursor cells has led to neurogenesis. Phosphatidylinositol signaling protein IP3 has been recognized as a target of this miRNA, so miR-135a-5p/IP3 axis has been suggested as a potential target for treatment of age-related brain injuries (Pons-Espinal et al., 2019).
According to Honardoost et al. study, transfection to C2C12 cell line caused Insr gene down-regulation, glucose uptake reduction and development of insulin resistance phenotype (Honardoost et al., 2016).

Causes and consequences of aberrant miR-135 expression
miR-135 is a miRNA which is involved in the pathoetiology of several neoplastic and non-neoplastic conditions. Both tumor suppressor and oncogenic roles have been reported for this miRNA. Two studies in animal models of spontaneous carcinogenesis has reported oncogenic roles for miR-135b in colorectal (Valeri et al., 2014) and gastric  cancers. Since these two studies have provided the strongest level of evidence, oncogenic function is supported for this miRNA.
Studies in prostate, renal, gallbladder and nasopharyngeal cancers as well as glioma have shown down-regulation of miR-135 in cancerous tissues compared with controls. Meanwhile, miR-135 has been shown to be up-regulated in bladder, oral, colorectal and liver cancers. Studies in breast, gastric, lung and pancreatic cancers as well as head and neck squamous cell carcinoma have reported dual roles for miR-135. These different effects of miR-135 cannot be explained either by tissue-dependent factor or by different genetic loci that encode this miRNA (MIR135A versus MIR135B). Other explanations for these observations are the presence of tissuedependent elements, abundance of RNA binding proteins in each tissue, impact of alternative splicing/polyadenylation events on miR-135 targets and the amounts of cell typespecific factors that change secondary structure of target transcripts.
Studies aimed at identification of the impact of miR-135 on EMT process have shown extremely contradictory results. For instance, miR-135-5p as a tumor suppressor miRNA inhibits this process (Yang et al., 2020a). On the contrary, in bladder and gastric cancer, miR-135a and miR-135b have been found to increase EMT (Mao et al., 2018;Huangfu et al., 2021).
Circulatory levels of miR-135 can be used as prognostic markers in different types of cancers. For instance, high expression of miR-135a-3p in serum samples of patients with ovarian cancer has been associated with good prognosis (Hu et al., 2014). Moreover, over-expression of miR-135a in blood samples of patients with colon cancer has been associated with good prognosis 83)). On the other hand, high serum levels of miR-135b in patients with multiple myeloma have been correlated with the severity of bone lesions (Hao et al., 2016).
Amplification or deletion of any of three mentioned loci for miR-135, namely MIR135A1 (3p21.2), MIR135A2 (12q23.1) and MIR135B (1q32.1) might be associated with dysregulation of certain members of this family. For instance, frequent deletion of the MIR135A1 locus has been found to be associated with poor prognosis in primary breast cancers. Mechanistically, deletion of this locus and subsequent down-regulation of miR-135a levels enhances progression of ERα+ breast cancers and their resistance to tamoxifen .
Differences in pri-and pre-miRNA sequences indicate that diverse phases might be rate-limiting for each precursor, enabling the fruition of extra regulatory mechanisms. Moreover, miR-135 might be subjected to distinctive modes of regulation through certain interactions with different RNAbinding proteins.
Dysregulation of miR-135 has also been noted in various non-neoplastic conditions such as Alzheimer's disease, atherosclerosis, depression, diabetes, Parkinson, pulmonary arterial hypertension, nephrotic syndrome, endometriosis, epilepsy and allergic conditions.

Conclusion
Regarding the different roles of miR-135 in different contexts, several issues should be addressed about the possible effects of tissue-dependent elements that affect expression of miR-135 in each tissue. Although manipulation of expression of miR-135 is a possible therapeutic option for cancer, it is not expected that miR-135-targeted therapies enter clinical settings in near future.
Frontiers in Genetics frontiersin.org Author contributions SF wrote the draft and revised it. BH designed and supervised the study. SK and SE collected the data and designed the tables and figures. All the authors read and approved the submitted version.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The handling editor (WCC) declared a past co-authorship with the author(s) (BMH).

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