Downregulation of hsa-microRNA-204-5p and identification of its potential regulatory network in non-small cell lung cancer: RT-qPCR, bioinformatic- and meta-analyses

The worldwide morbidity and mortality of pulmonary cancer has remained high for decades in both genders, reflecting an increase in contributory factors like tobacco use and air pollution [1‐5]. The two primary categories of pulmonary neoplasms are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for approximately 80% of all pulmonary cancers. NSCLC includes adenocarcinoma, squamous cell lung carcinoma, undifferentiated large cell carcinoma, adenosquamous carcinoma and bronchioalveolar carcinoma; the first three are the best known types [6]. The survival of patients with NSCLC is still bleak due to delayed diagnosis, undisciplined treatment, incident chemoresistance, and frequent tumor recurrence [7‐9]. Thus, thorough investigation of the molecular mechanisms underlying lung carcinogenesis remains an urgent task, for establishing new and effective guidelines for cancer screening and for identifying novel genetic targets for treatments.

One potential class of molecular targets are the microRNAs (miRNAs). These are small non-coding RNA molecules, with approximately 20 nucleotides in length, that negatively modulate expression of target genes by completely or incompletely binding to the 3′ untranslated region (UTR) of messenger RNAs (mRNAs) [10‐13]. The miRNAs have been proposed as novel diagnostic biomarkers and prognostic indicators for tumorigenic processes, as they play indispensable roles in cancer cell differentiation, proliferation, and apoptosis, and in metastasis and recurrence of numerous malignant tumors [10, 14]. One miRNA, hsa-microRNA-204-5p (also known as miR-204-5p, or miR-204), has attracted attention in NSCLC research, because its low expression in NSCLC tumors is associated with advanced progression, poor prognosis and severe metastatic potential [15‐17].

Previous studies on the mechanisms of miR-204-5p on NSCLC has mainly focused on the repression of specific mRNAs, so knowledge about its multilateral functions or its clinical prospects remains limited. Aberrant expression of miR-204-5p is now a well-established feature of pulmonary carcinogenesis; however, what is still unclear is the clinical contribution of miR-204-5p and particularly its potential role in the early detection of NSCLC. The mechanism by which miR-204-5p mediates its target mRNA-protein signaling networks to regulate tumor progression is also not yet established.

The current work describes distinctive features of miR-204-5p expression in NSCLC by integrative analysis of results from real-time quantitative polymerase chain reaction (RT-qPCR) and from sequence and genechip data from the cancer genome atlas (TCGA), Gene Expression Omnibus (GEO), and the current literature, in addition to relevant prediction materials from online tools. Our goals were to explore the possibility that miR-204-5p might be a promising indicator for NSCLC process and to identify our perspective on other underlying regulatory mechanisms at the molecular level (Fig. 1).

The data presented here verified the decrease in miR-204-5p expression in NSCLC by comprehensive analysis of RT-qPCR, microarrays, sequencing data, and publications and revealed an obvious decrease in cancerous tissues and the LUAD subtype. An auxiliary role for miR-204-5p was also identified by in NSCLC, particularly in tissues and LUAD, which was verified by the meta-analysis. Unfortunately, the prognostic implications for miR-204-5p were weak and showed no statistical significance in the meta-analysis, due to a shortage of large-scale investigations. Nevertheless, the information provided by GO annotation and KEGG analysis indicated that the target genes of miR-204-5p were associated with neuron projection, transcription factor activity, RNA polymerase II transcription regulation, extracellular matrix (ECM) metabolism, and ion channel activity, as well as were connected with microRNAs in cancer, cell adhesion molecules (CAMs) and signaling pathways regulating pluripotency of stem cells. Three of the six hub genes, GDNF, EPHB2 and DLG1, were selected for continued research due to their distinct characteristics in NSCLC. TF prediction revealed speculatively functional relations of MAX, MYC, and RUNX1 between miR-204-5p and these three genes, although only MAX demonstrated the TFBS sequences connected with miR-204-5p upon further query.

At present, miR-204-5p has aroused considerable interest in cancer research for its dual function as an oncogene and tumor suppressor [14]. MiR-204-5p is clearly attenuated in NSCLC, and its expression is negatively linked with tumor size, clinical stage, and metastasis [28‐30, 59, 61]. Downregulation of miR-204 occurs in part due to its hypermethylation in the promoter region [59]. Elevated expression of miR-204-5p depresses NSCLC migration and invasion by targeting Janus kinase 2 (JAK2) [17], restrains proliferation of NSCLC cells by regulating SIX homeobox 1 (SIX1) and attenuates LUAD angiogenesis potentially by JAK2-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway [16]. In addition, miR-204-5p serves as a cancer suppressor gene by modulating oncogenic Wnt/FZD signaling pathways [62], inhibiting NUAK family kinase 1 (NUAK1) in NSCLC [59], and mediating a long-noncoding RNA (lncRNA) MALAT1 effect on the epithelial-to-mesenchymal transition (EMT) and cells invasion [63]. Our results confirmed the downregulation of miR-204-5p expression in NSCLC and revealed a constant level of decline in LUAD.

The integrative meta-analysis also indicated a promising role for miR-204-5p for NSCLC screening, as did subgroup SROC curves, even though the sample origins were different. The variation in miR-204-5p expression in tissues was helpful in diagnosis of LUAD than of LUSC. Considering the invasive work, high cost, and cumbersome procedure of tissue biopsy, analysis of blood circulating miR-204-5p was considered an attractive screening indicator. However, low sensitivity, high specificity, and small sample sizes of the currently available data mean that more research and detailed profiling at all levels are needed to provide information to confirm the effectiveness of blood screening.

The correlation between the low miR-204-5p and high risk of death in patients with NSCLC [15, 59] was evident in our study but failed to reach statistical significance. The subsequent integrative meta-analysis was conducted to gain insights into the potential usefulness of miR-204-5p in NSCLC prognosis, but the data from the literature and from the present study regarding the ability of miR-204-5p to predict survival times of patients with NSCLC are conflicting. Consequently, no conclusion can be made in terms of miR-204-5p for NSCLC, at least for now.

The GO analysis indicated that the estimated target genes were mainly enriched in neuron projection, transcription factor activity, RNA polymerase II transcription regulation, ECM metabolism and ion channel activity, suggesting a potential involvement of miR-204-5p in the molecular function and signal modulation associated with NSCLC biological processes. The KEGG pathway analysis indicated that some of candidate genes were participated in microRNAs in cancer, CAMs, and signaling pathways regulating pluripotency of stem cells. Like other miRNAs, miR-204-5p plays an indispensable role in cancer proliferation, migration, and metastasis by regulating the tumor microenvironment, such as ECM structure and CAM metabolism [64]. Cancer stem cells (CSCs) attain stemness by complicated processes and signaling pathways, such as JAK-STAT, nuclear factor kappa B, Sonic hedgehog, transforming growth factor beta, Wnt/β-catenin, and PI3K/AKT [65, 66]. Many miRNAs take part in processes that maintain a balance between differentiation and quiescence of pulmonary CSCs, adjust the tumor microenvironment and affect cell cycle progression via regulation of these signaling pathways [67]. Consequently, miR-204-5p and its target genes could serve as important determinants of NSCLC pathogenesis and development.

Continued investigation of the hub genes involved in GO enrichment and KEGG Pathway analysis identified six leading relevant genes that were screened out due to binding to 3’UTR of miRNA. However, only GDNF has been investigated for a direct relationship with miR-204-5p in NSCLC [60].

GDNF, also called ATF or ATF2, is a well-characterized oncogene that promotes tumor growth, invasion, and metastasis, in addition to tumor microenvironment alterations [68]. GDNF expression occurs high level in NSCLC, though a significant difference exists with regard to factors such as race, gender, age, smoking status, and histologic subtype [69]. GDNF is upregulated at the transcript level in LUSC [70], and is hypermethylated in tumor tissues [71]. It also facilitates demethylation of the fibromodulin promoter and promotes subsequent angiogenesis in human glioblastomas [72]. Nerve-derived GDNF increases programmed death ligand 1 (PD-L1) levels in head and neck squamous cell carcinoma cells by activating the JAK2-STAT1 signaling pathway, which in turn promotes the evasion of cancer cells from immune system surveillance in the nerve-cancer microenvironment [73]. Recent research in colorectal cancer (CRC) has indicated that miR-196a-5p exerts its function in cell proliferation and migration by regulating GDNF expression [74], while miR-451 influences drug resistance in renal cell carcinoma by targeting GDNF [75]. GDNF is also targeted and regulated by miR-204-5p which inversely affects GDNF mRNA and protein levels, to inhibit NSCLC growth, migration, and cell cycle alteration and promote apoptosis [60]. Therefore, the interaction between miR-204-5p and GDNF appears to be critical in the development and progression of NSCLC and requires thorough research.

DLG1 is a vital participant in the control of cellular processes like polarity, proliferation and migration, so its dysregulation and mutation give rise to pathologies that include oncogenic processes [76]. DLG1 is mainly identified as a tumor suppressor, since overexpression is observed early in the onset of cervical cancer (CeCa) [77] and elevated DLG1 promotes intestinal tumorigenesis [78], predicts poor prognosis in people with CRC [79] and increases the invasiveness of NSCLC cell lines [80]. Increased phosphorylation of the DLG1 SH3-Hook region promotes interaction with the PDZ ligand of PKCα and accelerates cell migration [80]. The lncRNA DLG1-AS1 acts as a competitive inhibitor that influences the activity of miR-107 on its target gene ZHX1, thereby inducing cancer cell proliferation [81]. Moreover, DLG1 deficiency results in incorrect spindle polarity and a delay in cells transiting orientation [78], which disrupts cellular structure and distribution [82]. Interestingly, DLG1 protein levels are significantly lower in NSCLC and hepatocellular carcinoma (HCC) than in the corresponding normal tissues [83, 84], but are nearly undetectable in poorly differentiated stages of colon adenocarcinoma [85], in contrast to our findings and the existing literature. One possible reason is that DLG1 dysregulation in advanced tumor progression or in more malignant forms depends on its spatial/temporal distribution. Future research should focus on this possibility.

A series of studies have reported a direct correlation between EPHB2 expression and numerous human malignancies, including NSCLC. EPHB2 activates bidirectional signaling cascades and its upregulation predicts poor survival in LUAD [86], CRC [87], breast cancer [88] and malignant mesothelioma [89]. One study indicated that EPHB2 enhances cellular growth, migration and invasion in CeCa by a competitive inhibition that counteracts the miR-204 effect on cell cycle arrest, Bax overexpression and PI3K/AKT signaling pathway deactivation via competitive inhibition [90]. Expression of miRNAs also significantly suppresses EPHB2 expression, resulting in a decrease of tubulogenesis and angiogenesis [91, 92]. EPHB2 affects cell viability in medulloblastoma in part by promotion of the G2/M phase of the cell cycle [93]. Activation of EPHB2 promotes the progression of cutaneous squamous cell carcinoma cells by accelerating the production of invasive proteinases like MMP13 and MMP1 [94]. Nevertheless, some publications highlight EPHB2 declines in CRC, which was supposedly attributable to EMT modulation [95] and epigenetic modification of promoter [96, 97]. Future research could identify where whether specific differences in the interaction of EPHB2 and miR-204-5p are associated with NSCLC.

As discussed above, the function of miR-204-5p in NSCLC is also influenced by TFs as well, but an unanswered question is whether TFs regulate miR-204-5p or whether TFs could be adjusted or controlled by this miRNA in some way. Although great achievements have been made in understanding the biological behavior of miR-204-5p and its mRNA targets, integrative analysis of miRNA-TF-gene regulatory networks is still needed, as TFs are undoubtedly involved in pulmonary cancer initiation, progression, dissemination, recurrence, and even drug resistance [98]. At least ten kinds of TF-miRNA synergistic regulatory networks apparently function in NSCLC [99]. In addition to combining with and regulating its target genes, miR-204-5p also attenuates some angiogenic inducers like hypoxia inducible factor-1α (HIF-1α) to impair angiogenesis in LUAD [16]. Another study has demonstrated a dependence of miR-204-5p level on promoter hypermethylation and support by positive feedback of three TFs, c-MYB, ETS1 and RUNX2 [100]. Osterix, a transcription factor that is essential and specific for osteogenesis, coordinately modulates miR-204-5p and its endogenous competitors, as well as ultimately establishing a feed-forward loop (FFL) ultimately [101]. Activation of STAT3 suppresses miR-204-5p activities, in turn affecting proliferation and apoptotic resistance in human pulmonary arterial hypertension and nasopharyngeal carcinoma [102, 103]. A positive FFL between Hepatitis B virus, miR-204-5p, and STAT3 appears to contribute to HCC incidence [104].

Other work has suggested that miR-204-5p reciprocally represses TrkB expression; however, TrkB expression noticeably increases JAK2 and STAT3 phosphorylation. The phospho-STAT3 then directly binds to promoter sequence of miR-204-5p, resulting in increased clonogenic proliferation, migration and invasion in endometrial carcinoma, via a feed-backward-loop (FBL) motif of TF-miRNA-target gene [105]. The activity of the IL-6R/STAT3/miR-204-5p FBL also leads to chemosensitivity [106]. The possibility exists that, at molecular level, the cellular activities involved in NSCLC progression, including tumor cell proliferation, differentiation, invasion, apoptosis, recurrence, and even drug resistance, are associated with downregulation of miR-204-5p and account for the direct upregulation of its target genes as well as unidirectional or bidirectional activation of TFs.

Among the three TFs expected to take part in miR-204-5p networks, MAX forms a dimer-complex system of transcriptional regulation with other family members, which include MYC, Mad and Mxi1, and is implicated in cell proliferation, differentiation and apoptosis [107]. The sequence similarity in MAX and MYC TFBS in the current work was predicted based on the existence of the multiprotein complex. An increased expression of miR-22 in leukemia cells reduces the MAX expression level, blocking cell cycle progression at the G1 phase [108]. MAX expression in HCC activates Linc00176, which is a competing endogenous lncRNA (ceRNA) of tumor-suppressive miRNA, resulting in cell cycle acceleration and reduction of apoptosis by reducing the levels of miR-9 and miR-185 [109]. In CRC, a MAX/MYC heterodimer induced by elevated HIF-2α mediates transcriptional repression of hypoxia-related miR-15-16, leading to tumor angiogenesis and hematogenous metastasis by further loss of post-transcriptional restriction towards fibroblast growth factor-2 [110]. MYC, also known as MYCC and c-Myc, is frequently amplified in numerous human cancers via transcriptional regulation of specific target genes, including miRNA and lncRNA [111]. MiR-296 − 3p directly targets PRKCA to impair FAK-Ras-MYC signaling, thereby accelerating its own transcription in a FBL that obstructs the EMT signal and progression through the cell cycle, following suppression of cell proliferation, metastasis and chemosensitivity in LUAD [112]. Another study has suggested that miR-342-3p is capable of indirectly adjusting MYC by directly repressing E2F1, a MYC-collaborating molecule [52]. In breast tumor, MYC expression correlates positively with miR-203b-3p and miR-203a-3p but negatively with BCL2L1 expression, resulting in formation of a TF-FFL [113]. MIR7-3HG restrains MYC dephosphorylation by downregulation of AMBRA1 to form a positive feedback loop for its own expression and further contributing significantly to autophagic control [114].

As a crucial hematopoietic transcription factor, RUNX1 is well-documented in chromosomal translocations and in several types of carcinogenesis processes [115]. RUNX1 is positioned in the center of miRNA circuits relevant for malignant hematopoiesis in transcriptional programs [116, 117]. A RUNX1-microRNA-139-HCP5 axis shows a positive FBL for mediating the tumor-suppressive effects of glioma cells [118]. A miR-18a-RUNX1-ZO-1 regulatory network also increases the permeability of the blood-tumor barrier (BTB), thereby providing novel potential targets for drug transportation across the BTB as an attractive strategy for glioma treatment [119]. By binding to the miRNA promoter, RUNX1 increases the transcriptional level of miR-27a in breast cancer and concomitantly the decreases expression of ZBTB10, a direct target gene of miR-27a, to promote endothelial differentiation and subsequent angiogenesis and tumor metastasis [120]. Conversely, reduced expression of Runx1 in breast cancer cells leads to elevated expression of both pre-miR-378 and PPARGC1B, which is a host gene of miR-378, to create a FBL on that reduces cell migration and invasion [121]. The miR-204-5p circuits and its hub genes and TFs still await identification, but TFBS prediction was capable of offering fresh perspectives, and likewise, assisting in new theoretical insights into potential regulatory mechanisms. Based on the available data, MAX would appear likely to be a vital TF involved in miR-204-5p-mRNA interactions, since it was the only assumed attachment that focused on the upstream region of genetic sequences in the current work.

This study had drawbacks and limitations. One limitation is that the findings indicate a great heterogeneity between the data sources, which were then explored via a random effects model and subgroup meta-analyses. The trial quality was also generally poor due to heterogeneity that remained above 50%. One possible cause of the statistical heterogeneity is that the data were generated using different sources, operating protocols, and detection metrics. Univariate survival analysis is also the only appraisal method for determining the prognostic significance of miR-204-5p. A carefully designed evaluation system should be developed to provide a more in-depth assessment of this issue. In particular, time limits and tight budgets have prevented a satisfactory generation of experimental proof to validate the function of miR-204-5p regulatory networks with the target genes and TFs. In addition, the biological progression and molecular regulation of NSCLC is complicated, so other mechanisms mediated by miRNA circuits should also be addressed with in-depth research.

As the most frequent type of pulmonary cancer, NSCLC deserves more effort in achieving the goal of early detection and timely treatment. The findings presented in the current research demonstrated an attenuation of miR-204-5p expression in NSCLC, this decrease was more frequently observed in cancerous tissues and in the LUAD subtype and was, in part, helpful for diagnosis. The activities of miR-204-5p as an anti-oncogene were induced by its regulatory axes or circuits with target genes and TFs that participated in specific genetic pathways and biological processes.