Tumor Suppressive Effects of miR-124 and Its Function in Neuronal Development

MicroRNA-124 (miR-124) is strongly expressed in neurons, and its expression increases as neurons mature. Through DNA methylation in the miR-124 promoter region and adsorption of miR-124 by non-coding RNAs, miR-124 expression is known to be reduced in many cancer cells, especially with high malignancy. Recently, numerous studies have focused on miR-124 due to its promising tumor-suppressive effects; however, the overview of their results is unclear. We surveyed the tumor-suppressive effect of miR-124 in glial cell lineage cancers, which are the most frequently reported cancer types involving miR-124, and in lung, colon, liver, stomach, and breast cancers, which are the top five causes of cancer death. Reportedly, miR-124 not only inhibits proliferation and accelerates apoptosis, but also comprehensively suppresses tumor malignant transformation. Moreover, we found that miR-124 exerts its anti-tumor effects by regulating a wide range of target genes, most notably STAT3 and EZH2. In addition, when compared to the original role of miR-124 in neuronal development, we found that the miR-124 target genes that contribute to neuronal maturation share similarities with genes that cause cancer cell metastasis and epithelial-mesenchymal transition. We believe that the two apparently unrelated fields, cancer and neuronal development, can bring new discoveries to each other through the study of miR-124.


Introduction
MicroRNAs (miRNAs) are short, single-stranded RNAs consisting of approximately 20 nucleotides. The first miRNA discovered was the lin-4 in Caenorhabditis elegans [1,2]. Since then, many miRNAs have been identified in many species. miRNAs play a role in regulating post-transcriptional gene expression by forming an RNA-inducible silencing complex, which binds complementarily to the 3 UTR of mRNAs and cleaves or represses translation of the mRNA. Genes whose expression is regulated by miRNAs are called target genes, and a single miRNA could potentially regulate thousands of target genes. Therefore, miRNAs contribute to a wide range of processes, from morphogenesis to disease development, carcinogenesis, and its progression. miRNAs that promote cancer development are called oncomirs, while miRNAs that suppress cancer development are called tumor suppressive miRNAs (ts-miRNAs). Although many ts-miRNAs exist [3,4], we are focusing on one microRNA, microRNA-124 (miR-124), which is important in neurogenesis. Elsewhere, epigenetic changes of miRNAs in cancer cells can also be useful as biomarkers; however, this is outside the scope of this text and is summarized in other literatures [5][6][7]. miR-124 is derived from three independent genes (miR-124-1, miR-124-2, and miR- , and is one of the most highly expressed miRNAs in the central nervous system [8]. It is particularly expressed in neurons and is involved in their maturation and function [9,10]. Aside from being extremely abundant in neurons, miR-124 is thought to also play an important role in suppressing the oncogenic transformation of normal cells in other tissues, even if the miR-124 expression level is considerably lower than that in neurons. In recent years, miR-124 has been increasingly reported as an miRNA with antitumor activity. However, many reports with unclear overview of their results exist. Therefore, in this review, we summarize the anti-tumor effects of miR-124. We then discuss the functions of miR-124 with that of its original role in neurons.

Suppression of miR-124 Functions in Cancer Cells
miRNAs are downregulated globally in human cancers, suggesting their role as general tumor suppressors [11]. Among them, the expression level of miR-124 is decreased in cancer cells compared to that in normal tissues, and is presumably involved in the pathogenesis of cancer. In 2007, the decreased expression of miR-124 was discovered to be caused by methylation of the CpG islands of the genomic region encoding miR-124 in colorectal cancer cells HCT-116 [12]. This finding led to the further discovery of the tumor-suppressive effect of miR-124. Genes whose expression is regulated by miRNAs are called target genes. The CDK6 gene in HCT-116 cells, which is involved in cell cycle progression, was identified as a target gene of miR-124 [12,13]. Subsequently, miR-124 expression was also found to be reduced in glioblastoma multiforme and medulloblastoma compared to normal tissues, and exogenous miR-124 expression was found to be effective in inhibiting cancer cell growth by targeting CDK6 [14,15].
Thus, a great variety of cancers create a more favorable environment for tumor growth either by suppressing miR-124 expression, by adsorbing miR-124, or in combination ( Figure 1).

Tumor Suppressive Effects by miR-124 in Glial Lineage Cancers
Although miR-124 is downregulated in various types of cancers and exhibits tumorsuppressive effects through overexpression, the most frequently reported cancers associated with miR-124 are those of the glial cell lineage, such as glioblastomas and astrocytomas [15,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. Reportedly, expression of the target gene of miR-124 is consistent with malignancy and prognosis [38,41,51]. Many miR-124 target genes have been identified, including CDK6, and miR-124 can not only suppress cell proliferation and progress apoptosis but also inhibits cell invasion, metastasis, and angiogenesis by reducing the expression of the target genes. Thus miR-124 is expected to be effective in almost all processes of antitumor effects including tumor malignant transformation (Table 1). Moreover, interestingly, it has been reported that miR-124 expression affects not only the glioma stem cells but also the cancer-immune system by activating T cells through the glioma stem cells [39]. Temozolomide (TMZ) is an anticancer drug that damages DNA by methylating guanine, induces cell cycle arrest, and causes cell death due to cellular stress [52]. TMZ is the first-line chemotherapeutic agent in glioblastoma, and its effect is reportedly reinforced by miR-124 [45,48]. Therefore, the combination of miR-124 with anticancer drugs is expected to be more effective in glioma chemotherapy. In human cancer cells, miRNA expression is generally downregulated, triggering the progression of cancer pathology. miR-124 transcription is repressed by methylation of CpG islands in the genomic region encoding pri-miR-124. In addition, miR-124 is adsorbed and removed by circular RNAs and long non-coding RNAs that have sequences complementary to miR-124. Abbreviations: pri-miR-124, primary microRNA-124; circRNA, circular RNA; lncRNA, long non-coding RNA; EMT, epithelial-mesenchymal transition; AGO, Argonaute.

Tumor Suppressive Effects by miR-124 in Glial Lineage Cancers
Although miR-124 is downregulated in various types of cancers and exhibits tumorsuppressive effects through overexpression, the most frequently reported cancers associated with miR-124 are those of the glial cell lineage, such as glioblastomas and astrocytomas [15,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. Reportedly, expression of the target gene of miR-124 is consistent with malignancy and prognosis [38,41,51]. Many miR-124 target genes have been identified, including CDK6, and miR-124 can not only suppress cell proliferation and progress apoptosis but also inhibits cell invasion, metastasis, and angiogenesis by reducing the expression of the target genes. Thus miR-124 is expected to be effective in almost all processes of anti-tumor effects including tumor malignant transformation (Table 1). Moreover, interestingly, it has been reported that miR-124 expression affects not only the glioma stem cells but also the cancer-immune system by activating T cells through the glioma stem cells [39]. Temozolomide (TMZ) is an anticancer drug that damages DNA by methylating guanine, induces cell cycle arrest, and causes cell death due to cellular stress [52]. TMZ is the first-line chemotherapeutic agent in glioblastoma, and its effect is reportedly reinforced by miR-124 [45,48]. Therefore, the combination of miR-124 with anticancer drugs is expected to be more effective in glioma chemotherapy. In human cancer cells, miRNA expression is generally downregulated, triggering the progression of cancer pathology. miR-124 transcription is repressed by methylation of CpG islands in the genomic region encoding pri-miR-124. In addition, miR-124 is adsorbed and removed by circular RNAs and long non-coding RNAs that have sequences complementary to miR-124. Abbreviations: pri-miR-124, primary microRNA-124; circRNA, circular RNA; lncRNA, long non-coding RNA; EMT, epithelial-mesenchymal transition; AGO, Argonaute.

AURKA
Inhibition of cell proliferation and potentiation of the temozolomide TMZ-based chemosensitivity. [45]

CAPNS1
Inhibition of cell migration and invasion.

EZH2
Expression of miR-124/128/137 of artificial miRNA clusters, reduction of cell proliferation both in vitro and in vivo, and prolongation of survival in a mouse model. [50] KITLG, NRP2, SEMA6D, and THBS1 Regulation of self-renewal, apoptosis, and invasion. [44]

NRP1
Arresting of cell cycle, inhibition of cell proliferation and migration, inhibition of tumor angiogenesis, and induction of apoptosis. [49]

PIM1
Inhibition of cell proliferation, invasion, and aerobic glycolysis and promotion of apoptosis. [43]

Tumor Suppressive Effects of miR-124 in Major Cancers
According to a WHO study in 2020, lung, colorectal, liver, gastric, and breast cancers account for half of all worldwide cancer deaths. Many studies and reports exist that prove miR-124 is effective against all five of these cancers.

AKT2
Arresting of cell cycle at the G0/G1 phase and inhibition of cell growth, colony formation, and tumor growth without apoptosis.Inhibition of migratory and invasive abilities. [66] BECN1, RELA, and SQSTM1 Disruption of autophagy and reduction of cell survival. [59]

Inhibition of cell proliferation and invasion. [68]
EZH2 Inhibition of tumor cell proliferation and inhibition of the EMT process. [67]

MYO10
Inhibition of migration and metastatic ability.

STAT3
Inhibition of cell proliferation and induction of apoptosis. [55]

STAT3
Inhibition of cell growth and colony formation and induction of apoptosis. [32]

STAT3
Inhibition of cell growth and colony formation and induction of apoptosis. Increasing radio sensitivity. [63]
Epithelial-mesenchymal transition (EMT) is a differentiation mechanism that results in the acquisition of undifferentiated traits during carcinogenesis. Reportedly, miR-124 suppresses EMT in NSCLC cells. According to these studies, miR-124 suppresses enhancer of zeste homolog 2 (EZH2) and zinc finger E-box binding homeobox 1 (ZEB1) [57,67], which are transcription factors that promote EMT, and also targets N-cadherin (CDH2) [68]. Furthermore, in cells with KRAS mutations that cause EMT, miR-124 leads to cell death by suppressing autophagy, which is not observed in cancer cells with wild-type KRAS [59]. The same miR-124 target genes may or may not be targeted by miR-124 depending on the presence of KRAS mutation, which is interesting from the viewpoint of the gene targeting mechanism of miRNAs.
In colorectal cancer, miR-124 inhibits tumor formation by suppressing the proliferation of cancer cells and inhibiting metastasis [69][70][71]. Additionally, miR-124 expression increases oxidative stress and induces apoptosis [72]. Malignant tumors are known to have a glycolytic bias in glucose metabolism, known as the Warburg effect, which is thought to result from the adaptation of malignant tumors to a hypoxic environment [73]. miR-124 has also been reported to suppress the Warburg effect in a study on colorectal cancer [74] (Table 3). Table 3. Anti-tumor function of miR-124 in colorectal cancer.

STAT3
Induction of apoptosis and inhibition of tumor growth. [76] In liver cancer, miR-124 is expected to have tumor-suppressive effects, such as arrest of cell proliferation, induction of apoptosis, and inhibition of invasion and metastasis by inhibiting EMT [77][78][79][80][81][82][83] (Table 4). The chloride intracellular channel 1 (CLIC1) is a chloride intracellular channel, and its expression is upregulated in many cancer cells. Many studies have focused on changes in cytoskeleton-related genes in the suppression of EMT, but in liver cancer, a channel called CLIC has been identified as a target of miR-124 and is effective in suppressing metastasis and invasion [83]. Table 4. Anti-tumor function of miR-124 in liver cancer.

TARGET GENES EFFECTS REF. CASC3
Inhibition of tumor growth.

CLIC1
Inhibition of cell proliferation, migration and invasion. [83] EZH2 and ROCK2 Inhibition of cell motility and invasion, and suppression of intrahepatic and pulmonary metastasis. Inhibition of EMT with impaired formation of stress fibers, filopodia, and lamellipodia. [79] ITGAV and SP1 Inhibition of migration and tumor metastasis.
It is known that miR-124 is also downregulated in gastric cancer cell lines, and the expression of miR-124 has been reported to inhibit growth, decrease colony-forming ability, induce apoptosis, and suppress metastasis and invasion [84][85][86][87][88] (Table 5). In gastric cancer cells, the transfection of miR-124 can reportedly enhance the anticancer effect of 5-fluorouracil [88].

EZH2
Inhibition of cell proliferation and colony formation and induction of apoptosis. Increased sensitization of 5-FU. [87] EZH2 and JAG1 Inhibition of cell growth, migration, invasion, and tumor growth. [88]

ROCK1
Inhibition of cell proliferation, migration, and invasion.
[84] SPHK1 Suppression of cell proliferation and invasion. [85] 5-FU: 5-fluorouracil. miR-124 is also known to be downregulated in breast cancer cells and has been reported to have anticancer activity [89][90][91][92][93][94][95][96][97][98] (Table 6). Although apoptosis is not induced in breast cancer [90], cell cycle arrest and inhibition of invasion and metastasis are the major tumor suppressive effects of miR-124. In breast cancer cells, miR-124 expression can suppress metastasis by targeting and regulating genes that contribute to cytoskeletal dynamics, such as connective tissue growth factor (CTGF), ras homolog family member G (RHOG), integrin beta-1 (ITGB1), and rho-associated coiled-coil-containing protein kinase (ROCK1) [89]. Bone is a highly favorable environment for the colonization and growth of metastatic tumors, and breast cancer patients are particularly prone to skeletal metastasis. Osteolysis is triggered by cancer cells to invade the bone [99]. Reportedly, miR-124 also inhibits bone metastasis by suppressing interleukin 11 (IL11) expression in breast cancer cells, regulating osteoclastogenesis, and reducing osteolysis [96]. The mechanism of inhibiting cancer progression by regulating the differentiation of other cells through the target gene represents the diverse anticancer effects of miR-124. Table 6. Anti-tumor function of miR-124 in breast cancer.

CD151
Inhibition of proliferation via cell cycle arrest but does not induce apoptosis. Reduction of invasive and metastatic potential. [90]

CDK4
Inhibition of cell proliferation.

SNAI2
Inhibition of cell colony formation and pulmonary metastasis. [92]

Inhibition of cell proliferation and invasion. [97] STAT3
Improving sensitization of doxorubicin.
Thus, miR-124 has tumor-suppressive effects on various tumors, such as inhibition of cell growth, invagination, migration, metastasis, invasion, and EMT. Moreover, miR-124 also alters the metabolism of cancer cells, thereby suppressing the Warburg effect. It also causes a decrease in autophagy function. In addition, miR-124 expression enhances the efficacy of established therapies, such as improving the sensitivity of treatment-resistant cancer cells (Figure 2). We believe that the effect of miR-124 will have a positive impact on cancer immunotherapy as well, spreading to the cells surrounding the cancer cells expressing miR-124. Through the suppression of target gene expression, miR-124 prevents cancer progression and enhances therapeutic effects. These factors collectively lead to direct tumor suppression effects, such as cancer cell death. Abbreviations: ROS, reactive oxygen species; EMT, epithelial-mesenchymal transition

Target Genes Responsible for the Tumor Suppressor Effect of miR-124, STAT3, and EZH2
The detailed mechanism of gene targeting by miRNAs remains unclear, and the same target gene may be targeted, or not targeted, in different cell types [59]. Therefore, we examined the target genes of miR-124 commonly responsible for tumor suppression in many cancer cells.
Among the many target genes of miR-124, signal transducer and activator of transcription 3 (STAT3) is the most popular gene shared by many types of cancer cells and is reportedly a target gene in glioma [39], lung cancer [32,55,63,65], colorectal cancer [75,76], hepatocellular carcinoma [80], breast cancer [97,100], endometrial cancer [101], esophageal cancer [102,103], nasopharyngeal carcinoma [104], retinoblastoma [105], prostate cancer [106], and cholangiocarcinoma [107]. In gastric cancer, miR-124 and STAT3 have already been used to evaluate drug efficacy in anticancer activity [108]. STAT3 is a transcription factor that is activated by phosphorylation which in turn activates the expression of anti-apoptosis-related genes [109]. Moreover, malignant transformation of cells is mediated by the activation of STAT3, and targeting STAT3-signaling reduces the susceptibility of many cell types to malignant transformation [110,111]. Therefore, the fact that miR-124 targets STAT3 is an excellent explanation for the molecular mechanism of the antitumor effect of miR-124. Note that STAT3 suppresses apoptosis, but can also promote apoptosis [112,113], which may be the cause of the difference in the induction of apoptosis by miR-124 in different cancer cell types and environments.

Target Genes Responsible for the Tumor Suppressor Effect of miR-124, STAT3, and EZH2
The detailed mechanism of gene targeting by miRNAs remains unclear, and the same target gene may be targeted, or not targeted, in different cell types [59]. Therefore, we examined the target genes of miR-124 commonly responsible for tumor suppression in many cancer cells.
Among the many target genes of miR-124, signal transducer and activator of transcription 3 (STAT3) is the most popular gene shared by many types of cancer cells and is reportedly a target gene in glioma [39], lung cancer [32,55,63,65], colorectal cancer [75,76], hepatocellular carcinoma [80], breast cancer [97,100], endometrial cancer [101], esophageal cancer [102,103], nasopharyngeal carcinoma [104], retinoblastoma [105], prostate cancer [106], and cholangiocarcinoma [107]. In gastric cancer, miR-124 and STAT3 have already been used to evaluate drug efficacy in anticancer activity [108]. STAT3 is a transcription factor that is activated by phosphorylation which in turn activates the expression of antiapoptosis-related genes [109]. Moreover, malignant transformation of cells is mediated by the activation of STAT3, and targeting STAT3-signaling reduces the susceptibility of many cell types to malignant transformation [110,111]. Therefore, the fact that miR-124 targets STAT3 is an excellent explanation for the molecular mechanism of the anti-tumor effect of miR-124. Note that STAT3 suppresses apoptosis, but can also promote apoptosis [112,113], which may be the cause of the difference in the induction of apoptosis by miR-124 in different cancer cell types and environments.

Comparison with Neuronal Development, the Original Function of miR-124
Thus far, we have summarized the tumor-suppressive effects of miR-124 in cancer cells. However, miR-124 is strongly expressed in neurons typically and plays an important role in neuronal development. In addition, the expression level of miR-124 increases with neuronal maturation [9,118]. Therefore, it is thought that miR-124 plays various roles at each step in the development of the nervous system. During the development of the central nervous system, nascent neurons migrate to appropriate locations during maturation to form neurocircuits and stabilize them. In addition, neurons generally do not proliferate after their fate has been determined. Considering these features, many of the genes that have been identified as molecular mechanisms involved in miR-124mediated tumor suppression are not specific to cancer cells but are also important in neuronal differentiation. Here, we linked the tumor suppressor function of miR-124 to actual neuronal development (Figure 3).  miR-124 is strongly expressed in neurons, and its expression increases as the neurons mature. miR-124 is thought to act as a gatekeeper during the generation of neurons from non-neuronal cells, thus preventing the reversal of cell fate. In the process of neuronal maturation, miR-124 targets genes that contribute to proper neuronal migration, neurite formation, and synapse formation. Genes that contribute to neuronal maturation also contribute to EMT and metastasis in cancer cells. Abbreviations: RPC, retinal progenitor cell; NPC, neural progenitor cell; EMT, epithelial-mesenchymal transition.

Discussion
Each of the papers discussed in this review addresses several target genes as a mechanism for the anticancer effects of miR-124. However, since each study focuses on a unique gene, unidentified targets might be missed. We think transcriptome analysis is a necessary tool, although it may be difficult to find all of them, since repression of the target genes also occurs at the translational repression level. In addition, each single cancer cell may have different characteristics, even if they are from the same strain. Therefore, the diversity of the effects of miR-124 might be determined by analyzing it at the single-cell level. Furthermore, although there have been many reports on the anticancer effects of miR-124, it is thought that there are cancers for which miR-124 does not work. We believe that analysis of such cancers is also necessary for the application of miR-124 in the treatment of cancer.
Many experimental methods express miRNAs into cancer cells using gene transfer During brain development, neurons are derived from neural progenitor cells called radial glia. In contrast, in the retina, both neurons and glial cells are produced from common retinal progenitor cells. STAT3 is an important target gene of miR-124 for its tumor suppressor effect, while in nervous system development, it is known to contribute to astrocyte differentiation [119]. In addition, P19 cells, an embryonic-derived teratoma cell line, can also differentiate into neurons and astrocytes. miR-124 reportedly targets EZH2 to allow differentiation of P19 cells into neurons and suppresses their differentiation into astrocytes [120]. Therefore, miR-124 is also thought to play a role in suppressing glial cell differentiation. Overexpression of miR-124 in mouse retinal progenitor cells decreases the percentage of glial cell differentiation and increases the percentage of neuronal differentiation [121]. Thus, it is possible that STAT3 and EZH2 are also targeted in the active fate determination of neurons from retinal progenitor cells in actual in vivo conditions. Furthermore, overexpression of miR-124 can induce the conversion of non-neuronal cells to neurons [122]. As well as Ezh2 [123], among the target genes of miR-124 in cancer cell, polypyrimidine tract-binding protein 1 (Ptbp1; PTB1 gene in humans) is known to be associated with such an effect [124]. PTBP1/PTB1 regulates cell type-specific alternative splicing. In neural stem cells, PTBP1/PTB1 functions to produce non-neuronal splicing isoform proteins. Suppression of Ptbp1 by miR-124 results in the production of neuralspecific isoforms in mice [125]. Such a difference in splicing isoforms is also known to affect the metabolism of cancer cells. Pyruvate kinase muscles (PKMs) have two splicing isoforms, the PKM1 and PKM2. In cancer cells, especially colon cancer cells, the amount of PKM2 increases due to the regulation of splicing by PTB1, and PMK1 increases when PTB1 expression is suppressed by miR-124. This alters the metabolic pathways of cancer cells and leads to a decrease in the Warburg effect of miR-124 [74]. Although it has not been found at the present time, it may be discovered in the future that PTB1-mediated metabolic control is also important for neuronal differentiation.
Newborn neurons then undergo a maturation process. Studies on miR-124-1 knockout mice have shown that miR-124 is essential for neuronal maturation [9]. miR-124 targets LIM-homeobox domain 2 (Lhx2), which is thought to regulate neuronal maturation [9]. The transcription factor LHX2 is expressed in neural stem cells and immature neurons, and is essential for neuronal production, maturation, and normal axonal projection [126][127][128]. Interestingly, in NSCLC, it is reported miR-124 targets LHX2 and that its reduction is associated with inhibition of metastasis and invasion [64]. Furthermore, miR-124 reportedly targets RHOG and promotes dendritic branching during neuronal maturation [129]. In breast cancer, RHOG has been identified as a target gene of miR-124 in suppressing the metastasis of cancer cells [89]. Besides these, genes that contribute to neuronal maturation [130][131][132][133] appear to be linked to genes that contribute to malignant transformation, such as migration and invasion, in cancer cells [70,79,83,86,89,91,98] (Figure 3). Reportedly, zinc finger E-box binding homeobox 2 (ZEB2) is a target of miR-124 in breast cancer [98], and reduction of ZEB2 suppresses EMT and metastasis. Although there are no clear examples regarding the important functional regulation of miR-124 to ZEB2 in neurons, we believe that targeting of ZEB2 by miR-124 is important in neuronal maturation as ZEB2 promotes axonal branching and regulates normal migration of interneurons [133].

Discussion
Each of the papers discussed in this review addresses several target genes as a mechanism for the anticancer effects of miR-124. However, since each study focuses on a unique gene, unidentified targets might be missed. We think transcriptome analysis is a necessary tool, although it may be difficult to find all of them, since repression of the target genes also occurs at the translational repression level. In addition, each single cancer cell may have different characteristics, even if they are from the same strain. Therefore, the diversity of the effects of miR-124 might be determined by analyzing it at the single-cell level. Furthermore, although there have been many reports on the anticancer effects of miR-124, it is thought that there are cancers for which miR-124 does not work. We believe that analysis of such cancers is also necessary for the application of miR-124 in the treatment of cancer.
Many experimental methods express miRNAs into cancer cells using gene transfer of pre-miRNAs by plasmid or viral vectors, and induction of these miRNAs by the Tet-ON system. For clinical methods, miRNAs themselves, or chemically modified miRNAs called miRNA mimics, packaged into nanoparticles and delivered to cancer cells for uptake may be a more realistic means of miRNA-based cancer therapy [134].

Conclusions
miR-124 showed anticancer effects in various stages of cancer progression, and was not only a replacement for existing treatment methods by itself, but also enhanced the effects of the existing treatment methods, probably due to the characteristic of miRNAs to regulate multiple target genes simultaneously.
In this review, we compared the anticancer effects of miR-124 with its effect on neural development. Despite the different functions of anticancer and neurogenic regulation, there are many common genes between the two. This means that neurodevelopmental functions may lead to new anticancer targets and anticancer effects may lead to new and important neurodevelopmental discoveries. Both fields can be made more progressive by paying attention to each other.