The tumor suppressor role and ceRNA network of miR-1294 in cancer

miRNAs are endogenous small RNAs that are important regulators of gene expression. miR-1294 was found to be significantly down-regulated in 15 cancers and regulated by 21 upstream regulators. miR-1294 affects the proliferation, migration, invasion, and apoptosis of cancer cells. The target genes of miR-1294 are involved in the PI3K/AKT/mTOR, RAS, and JAK/STAT signaling pathways. Six target genes of miR-1294 are the targets of a variety of drugs. Low expression of miR-1294 is associated with resistance to cisplatin and TMZ and a poorer prognosis in patients with ESCC, GC, EOC, PDAC, or NSCLC. Therefore, this work outlines the molecular mechanisms and provides a basis for the clinical significance of the tumor suppressor miR-1294 in cancer.


Introduction
As endogenous small RNAs, microRNAs (miRNAs) bind to the 3′ UTRs of their target messenger RNAs (mRNAs) to inhibit their expression, thereby affecting the development, differentiation, and progression of diseases [1,2]. Competitive endogenous RNAs (ceRNAs) such as long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) can compete with miRNAs [3], and thus regulate the expression of miRNAs and their targeted inhibition of protein-coding genes [4]. There are at least 19 target genes of miR-1294. The regulation of miR-1294 by ceRNA in various cancers can affect the expression of downstream target genes and various cellular behaviors of cancer cells. The downstream genes of miR-1294 are involved in the regulation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mechanistic target of rapamycin kinase (mTOR), RAS, and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways. Six target genes of miR-1294 are the targets of a variety of known drugs. This work provides a comprehensive summary of miR-1294, which provides potential directions for future research.
Notably, ceRNAs of miR-1294 have not been found in Breast cancer (BC), ccRCC, OC, and low expression of miR-1294 can relax the repression of HOXA6 [29], IGF1R [30], thereby promoting cancer risk. In addition, low expression of circLDLR in ovarian fluid significantly upregulated the expression of miR-1294, which was associated with the risk of polycystic ovary syndrome (PCOS) [31].
Pan-cancer analysis showed that miR-1294 was downregulated in TCGA-LUAD and TCGA-THCA (Figs. 1a and 1b), which further validated the anticancer effect of miR-1294. Notably, miR-1294 was upregulated in TCGA-UCEC. Due to the small number of noncancerous samples involved (n = 10), the cancer-promoting effect of miR-1294 in TCGA-UCEC needs to be treated with caution.
Studies have shown that the expression of miR-1294 is significantly down-regulated in 7 cancers including BC, ESCC, EC, GC, ccRCC, HCC, and NSCLC. However, there was no significant association of miR-1294 expression with cancer risk among the corresponding TCGA cancer types (BRCA, ESCA, STAD, KIRC, LIHC, and LUSC) ( Table 3).
These inconsistencies may be due to the following reasons. First, miR-1294-related studies are mostly based on cell lines with controlled heterogeneity. However, the heterogeneity of the cancerous and paracancerous tissues in TCGA was high. The proportion of cancer cells also varied among TCGA cancer tissue samples. Second, the expression level of miR-1294 was lower in various cancer types of TCGA (Q2-Q3). The expression of miR-1294 in TCGA was detected by RNA-seq technology. However, the existing miR-1294-related research generally uses qRT-PCR technology to amplify the target gene, and this method can detect very low expression of miR-1294. In addition, cell line-based studies involve more target RNA content and are more suitable for studying miR-1294, which is less expressed. Third, there may be highly expressed tissuespecific regulatory factors or ceRNAs, which significantly inhibit the expression level of miR-1294. And this affects the differential analysis of miR-1294 expression between cancerous and paracancerous tissues in TCGA. Taken together, the differences in the association results between miR-1294 expression and cancer risk may be related to different cancer tissue samples, gene expression detection methods, differences in sample numbers, and the presence of tissue-specific regulators such as ceRNAs. The anticancer effect of miR-1294 in more samples needs to be further verified in the future.

Molecular mechanisms of miR-1294 affecting cancer cell behaviors
The low expression of miR-1294 in cancer cells can relieve its inhibitory effect on downstream protein-coding genes, and then regulate the proliferation, apoptosis, invasion, and migration of cancer cells, and finally lead to the occurrence and development of cancer (Fig. 2).
Cell proliferation is an essential component of cell growth and differentiation [41]. Low expression of miR- circ_0018289/miR-1294/ICMT [28] Note: "Y" means that the biological behavior is inhibited, "[" means that the biological behavior is promoted. Please check the full names of the abbreviations in the list of abbreviations. Downregulation of miR-1294 plays an important role in the ceRNA regulatory networks by relaxing the repression of target genes.

miR-1294 and PI3K/AKT/mTOR signaling pathway
The PI3K/AKT/mTOR signaling pathway is a master regulator of cancer [44], which is frequently activated in various cancers and is considered a promising therapeutic target [45]. In cisplatin-resistant tissues and cell lines (SKOV3/DDP) of OC, low expression of miR-1294 can increase the expression level of IGF1R, thereby mediating the activation of the PI3K/AKT/mTOR signaling pathway and promoting the proliferation, migration, and invasion of   OC cells [30]. In HCC, CircUBAP2 acts as a sponge for miR-1294, upregulates c-Myc expression, and inhibits PI3P, thereby inhibiting the PI3K/AKT/mTOR signaling pathway and promoting tumorigenesis [22]. In GC, LncRNA NEAT1 increased the expression level of AKT1 by sponging miR-1294, mediated the activation of the PI3K/AKT/mTOR signaling pathway, promoted the proliferation and migration of GC cells, and inhibited apoptosis [9].

miR-1294 and RAS signaling pathway
The RAS signaling pathway can control cell growth, survival, and differentiation by integrating extracellular signals. Aberrant activation of the RAS pathway is a highly prevalent major oncogenic event [46]. Circ_0000885, which is highly expressed in OS, can restore the expression level of FGFR1 by targeting miR-1294, thereby mediating the activation of the RAS signaling pathway and promoting the progression of OS [14].

miR-1294 and JAK/STAT signaling pathway
The JAK/STAT signaling pathway is a mechanism by which extracellular factors regulate gene expression and is involved in many key biological processes such as cell proliferation, differentiation, apoptosis, and immune regulation [47]. Arsenic trioxide (ATO) is the most toxic compound in traditional Chinese medicine and has been shown to effectively inhibit cancer cell processes. In HCC, ATO induced the upregulation of miR-1294, decreased the expression level of PIM1, and inhibited the JAK/STAT signaling pathway, thereby promoting the apoptosis of HCC cells [39].
The clinical significance of miR-1294 As shown in Table 4, the abnormal expression of miR-1294 in cancer is not only correlated with tumor prognostic indicators but also closely related to the clinicopathological phenotype of cancer patients. Cancer therapeutic drugs can target cancer by targeting the downstream genes of miR-1294. In addition, studies have also shown that low expression of miR-1294 is also associated with resistance to cisplatin and TMZ.
The diagnostic and prognostic value of miR-1294 As shown in Table 4, miR-1294 was down-regulated in most cancers, and its abnormal expression correlated with prognostic indicators of tumors. In ESCC, the overall survival rate of the miR-1294-low-expression group was significantly lower than that of the miR-1294-highexpression group [33]. In GC, patients with low miR-1294 expression had significantly shorter overall survival [36,40] and disease-free survival (DFS) [40] than patients with high expression of miR-1294 [36]. In EOC, the overall survival rate of the miR-1294-low-expression group was lower compared with the miR-1294-high-expression group [48]. The expressions of CircEYA3 and Circ_0030235 were significantly up-regulated in PDAC tissues compared with adjacent normal tissues. Survival analysis showed that the overall survival rate of PDAC patients with high expression of CircEYA3 and Circ_0030235 group was lower [17,16], thus indicating that in PDAC, the group with low expression of miR-1294 had lower overall survival rate. In NSCLC, the overall survival rate was lower in the miR-1294-low-expression group compared with the miR-1294high-expression group [25].
The relationship between miR-1294 and clinicopathological characteristics As shown in Table 4, the expression level of miR-1294 was closely related to the clinicopathological phenotype of cancer patients. In ESCC, low expression of miR-1294 was associated with larger tumors, positive lymphatic infiltration, lymph node metastasis, and positive venous infiltration [33,10]. In GC, low expression of miR-1294 was associated with larger tumors, lymph node metastasis, and distant metastasis [40]. In EOC, low expression of miR-1294 was associated with advanced FIGO stage and lymph node metastasis [48]. In PDAC, low expression of miR-1294 was associated with advanced TNM stage [17], higher tumor stage, and positive lymph node invasion [16]. In NSCLC, low expression of miR-1294 was associated with advanced TNM staging and distant metastasis in NSCLC patients [25].     (Table 5).
miR-1294 and drug resistance miR-1294 was closely associated with cisplatin and TMZ resistance in cancer cells (Fig. 5). miR-1294 can affect the drug resistance of tumor cells by regulating targets, activating signaling pathways, or changing the normal behavior of molecules in two tumor cells. Cisplatin is a well-known chemotherapy drug that has been used to treat a variety of human cancers [50]. The development of cisplatin chemoresistance can lead to the failure of cisplatin therapy [51]. In OC, miR-1294 was significantly decreased in tissues of cisplatin-resistant patients compared with cisplatin-sensitive patients. In vitro, miR-1294 also showed low expression in cisplatin-resistant cell lines (SKOV3/DDP) compared with OC SKOV3 cells. Low expression of miR-1294 can restore the expression level of the target gene IGF1R and activate the PI3K/AKT/mTOR signaling pathway, thereby upregulating the cisplatin resistance of OC cells [30].
Temozolomide is used as an oral alkylating agent in the treatment of glioblastoma multiforme (GBM) and astrocytoma [52]. miR-1294 expression was lower in highgrade gliomas than in low-grade gliomas. Low-expressed miR-1294 upregulates the expression of TPX2, which promotes the proliferation, migration, and invasion of GM cells, and reduces the chemosensitivity of GM cells to temozolomide [34]. Meanwhile, the expression of Circ_0000936 in temozolomide-resistant GM tissues was higher than that in temozolomide-sensitive GM tissues. The highly expressed Circ_0000936 can down-regulate the expression level of miR-1294, thereby increasing the resistance of GM cells to TMZ [12].  (Fig. 6). Low expression of miR-1294 can promote the proliferation, apoptosis, invasion, and migration of cancer cells, and can participate in the activation of PI3K/AKT/mTOR, RAS, JAK/STAT signaling pathways, and promote the development of cancer. Downregulation of miR-1294 was associated with poorer prognosis in ESCC, GC, EOC, PDAC, and NSCLC. In addition, low expression of miR-1294 was also associated with resistance to cisplatin and TMZ.
Notably, the analysis of TCGA also found that miR-1294 was down-regulated in TCGA-LUAD and TCGA-THCA, while its expression was up-regulated in TCGA-UCEC. Furthermore, miR-1294 expression was upregulated in the noncancerous disease PCOS. The functional differences of miR-1294 may be related to mechanisms such as differences in samples, miRNA detection methods, differences in sample numbers, and the presence of tissue-specific regulators such as ceRNAs.
Low expression of miR-1294 in ovarian cancer and glioma is associated with TMZ and cisplatin resistance. Porous lyotropic liquid crystal nanoparticles are promising delivery vehicles for cancer therapy [19]. The use of targeted nanomedicine to deliver miR-1294 may have great potential for cancer therapy.
However, there are still many deficiencies in the current research on miR-1294. First, the number of current research samples is small, and relevant results need to be verified in larger samples and other populations. Secondly, some studies on the biological functions of miR-1294 are limited to in vitro cell experiments, and it is necessary to strengthen the verification of in vivo animal experiments in the future. Finally, the molecular mechanism of miR-1294 in disease is still not fully understood, and more in-depth research is needed in the future to provide a theoretical basis for miR-1294-targeted therapeutic regimens.

Conclusion
As a tumor suppressor, the low expression of miR-1294 has an important molecular regulatory mechanism in cancer cell behavior and carcinogenesis. In addition, the overview of miR-1294 in cancer diagnosis, prognosis, and treatment is expected to provide potential clues and directions for miR-1294-related clinical research.
Acknowledgement: The authors would like to thank the PubMed, TCGA, KEGG, and CADDIE databases for useful information, and BioRender (https://biorender.com/) for rendering Figs. 2, 4 and 5. Availability of Data and Materials: All data generated or analyzed during this study are included in the article.
Ethics Approval: Not applicable.