MiR-942 Promotes Cancer Stem Cell-like Traits In Esophageal Squamous Cell Carcinoma Through Activation Of Wnt/β-catenin Signalling Pathway

Cancer stem cells (CSCs), a small population of cancer cells that possess the ability to self-renew and differentiate, are generally responsible for cancer initiation and poor prognosis. It has been reported that CSCs exhibit both stem cell-like traits and cancer properties. Meanwhile, CSCs exhibit greater resistance to cancer treatment than cancer cells, such as chemotherapy and radiotherapy. Therefore, targeting CSCs could be an effective treatment for cancer therapy.Esophageal carcinoma (EC) is one of the most common gastrointestinal cancer, whose incidence and mortality remains high. Among ECs,90 percent belongs to esophageal squamous cell carcinoma (ESCC). Several studies have reported that CSCs play a vital role in esophageal squamous cell carcinoma progression. Therefore, targeting CSCs could be an effective treatment for ESCC therapy. And it is vital to understand the molecular mechanisms which control CSCs in ESCC.The Wnt/β-catenin signalling pathway is a well-known pathway involved in regulating self-renewal and oncogenesis in many systems. In normal physiological state, the Wnt/β-catenin signalling pathway regulates downstream genes involved in basic cellular processes, such as cell proliferation, differentiation, migration and cell death. However, extensive findings have shown that the Wnt/β-catenin pathway is constitutively activated in CSCs. Dysregulation of Wnt/β-catenin pathway negative regulators could also contribute to constitutive activation of the Wnt/β-catenin signalling in cancer. However, how these negative Wnt/β-catenin signaling pathway regulators regulate tumors is largely unclear.microRNAs (miRNAs) are a class of endogenous non-coding RNA, located in the genome associated with tumor fragile site, containing 21-25 nucleotides, 21-25 nucleotides, which regulate the translation and degraduation of target mRNAs by direct interaction with 3’-untranslated region (3’UTR) of the target mRNAs. It has been reported that miRNAs play an important role in ontogeny, apoptosis, proliferation and differentiation of life activities. New studies indicated that miRNAs are abnormally expressed in CSCs in a variety of tumors, which play an important role in regulation of tumor differentiation and self-renewal and other biological functions.Several studies have reported that dysregulated miRNAs play a vital role in ESCC progression. miR-941 dysregulation has been demonstrated to be involved in many tumors. In this study, we firstly used PCR.to examine the expression of miR-942 between ESCC and adjacent normal tissues, the results indicate a possible link between miR-942 overexpression and human ESCC progression. Moreover, miR-942 overexpression promotes the stem cell-like traits of ESCC cells. To further understand the functions of miR-942 in ESCC, using publicly available algorithms to predict the potential targets and to be validated. As shown, individually silencing sFRP4, GSK3ê, or TLE1 increased the tumour sphere formation and enhanced the TOP flash/FOP flash activity of antagomir-942 cells. Thus, our study may deeply understand the pathogenesis of ESCC and may help to introduce a new area of research on miR-942-based therapeutic strategies targeting CSCs.Part 1 Expression of miR-942 in ESCC tussues and the effect of miR-942 in ESCC cells biological behavior Objective:Several studies have reported that dysregulated miRNAs play a vital role in ESCC progression. miR-941 dysregulation has been demonstrated to be involved in many tumors. In this part, we firstly used qPCR.to examine the expression of miR-942 between ESCC and adjacent normal tissues, then establish cell and animal models to further study the functions of miR-942 in ESCC, which may provide a scientific basis to study the pathogenesis of ESCC.Methods:● Using qPCR to analysis the expression of miR-942 in ESCC and adjacent normal tissues, and then analyzed the relationship between miR-942 expression and pathological parameters and prognosis.● Using qPCR to analysis the expression of miR-942 in ESCC cells, then establish ESCC cells stably transduced and silenced endogenous miR-942 models.● Using MTS cell proliferation assays, tumour sphere formation assays and flow cytometric analysis to analyze the alterations of ESCC biological behaviors.● Using BALB/c nude mice to detect the effect of miR-942 during the tumor formation in vivo.Results:● miR-942 expression is significantly increased in ESCC. miR-942 expression was positively correlated with clinical stage (P<0.001), tumour-node-metastasis (TNM) classification (T:P=0.005; N:P<0.001; M:P=0.004), and histologic differentiation (P=0.045) in patients with ESCC. Importantly, patients with higher miR-942 expression had a shorter survival time, whereas patients with lower miR-942 expression had a longer survival time (P=0.01; Fig. 1E). Moreover, Univariate and multivariate analyses indicated that miR-942 expression and clinical stage were independent prognostic factors in ESCC. Taken together, these results indicate a possible link between miR-942 overexpression and human ESCC progression.● The proliferative rates were slightly altered in both miR-942-transduced and inhibited ESCC cells. However, miR-942-transduced cells formed more and larger spheres than vector-tranduced cells in tumour sphere formation assays. Additionally, CD90 positive cells were dramatically increased in miR-942-transduced cells. Furthermore, miR-942 overexpression significantly upregulated the mRNA expression levels of multiple pluripotency factors, including ABCG2, KLF4, SOX2, OCT4, and NANOG. Similarly, when miR-942 was inhibited, the cells formed fewer and smaller spheres, CD90 population was dramatically decreased. miR-942 inhibition significantly decreased the mRNA expreesion of ABCG2, KLF4, SOX2, OCT4, and NANOG. These results indicated miR-942 might act as a cancer stem cell inducer which promotes ESCC stem cell-like traits.● In vivo study showed the tumours formed by Eca109/miR-942 cells were larger, in both size and weight, than the tumours formed from vector control cells. In contrast, when endogenous expression of miR-942 was inhibited using antagomir-942, the tumours were obviously smaller and lighter than those formed by control cells. These results indicated that miR-942 strongly promotes ESCC tumourigenesis in vivo.Conclusions:miR-942 expression was positively correlated with clinical stage, TNM classification and histologic differentiation in patients with ESCC, and patients with higher miR-942 expression had a shorter survival time, whereas patients with lower miR-942 expression had a longer survival time. miR-942-transduced cells formed more and larger spheres and dramatically increased CD90 positive cells and upregulated the mRNA expression levels of multiple pluripotency factors, including ABCG2, KLF4, SOX2, OCT4, and NANOG In vivo study showed the tumours formed by Eca109/miR-942 cells were larger, in both size and weight.Taken together, our results indicated that miR-942 may play an important role in ESCC through maintaining cancer stem cell-like traits.Part 2 miR-942 promotes cancer stem cell-like traits in ESCC through activation of Wnt/p-catenin signalling pathwayObjective:Wnt/β-catenin signalling pathway regulates downstream genes involved in basic cellular processes, such as cell proliferation, differentiation, migration and cell death. The Wnt/p-catenin pathway has been shown to be constitutively activated in various types of cancer, leading to cell reprogramming and generation of a stem-like phenotype, which has been indicated to play an important role in CSCs. miRNAs regulate the translation and degraduation of target mRNAs by direct interaction with 3’-untranslated region (3’UTR) of the target mRNAs, which play an important role in regulation of tumor differentiation and self-renewal and other biological functions. To further understand the functions of miR-942 in ESCC, using publicly available algorithms to predict the potential targets and to be validated. Our results may help to introduce a new area of research on miR-942-based therapeutic strategies targeting CSCs.Methods:● Using TOP-Flash/FOP-Flash dual luciferase reporter gene assay to detect the intracellular Wnt signaling activity.● Using bioinformatics analysis to predict the potential targets of miR-942.● Using western blotting assay to detect the protein levels of sFRP4, GSK3|3and TLE1.● Using luciferase reporter assay to effect on the 3’UTRs of sFRP4, GSK3P, and TLE1 luciferase activity.● Using small interfering RNA to inhibit the expressions of sFRP4, GSK3P, and TLE1 in miR-942 knockdown ESCC cells● Using tumour sphere formation assays and flow cytometric analysis to analyze the alterations of ESCC biological behaviors.Results:● miR-942 overexpression markedly increased the luciferase activity of the TOP flash/FOP flash reporter. Conversely, transfection of antagomir-942 reduced the luciferase activity, and mutant miR-942 had no effect, indicating that miR-942 activates Wnt/p-catenin signaling.● Using publicly available algorithms (TargetScan6.2 and miRanda), we found that sFRP4, GSK3β, and TLE1, the multiple negative regulators of Wnt/β-catenin pathway, might be the potential targets of miR-942.● Western blot analysis revealed that the expressions of sFRP4, GSK3β, and TLE1 was drastically decreased in miR-942-transduced cells but pronouncedly elevated in antagommir-942 cells, compared with their corresponding control cells.● The luciferase reporter activities, which linked to the 3’UTRs of sFRP4, GSK3P, and TLE1, indicated that miR-942 overexpression significantly repressed, whereas inhibition of endogenous miR-942 increased, the luciferase activity of 3’UTRs, and ectopically expressing mutant miR-942 had no inhibitory effect on the 3’UTRs luciferase activity. The results indicate that sFRP4, GSK3β, and TLE1 are the bona fide targets of miR-942.● Individually silencing sFRP4, GSK3β, or TLE1 increased the tumour sphere formation and enhanced the TOP flash/FOP flash activity of antagomir-942 cells. These results demonstated that sFRP4, GSK3β, and TLE1 are important for miR-942-induced stem cell-like traits and indicated that Wnt/β-catenin signaling is a functional mediator for miR-942-induced function in ESCC cell lines.Conclusions:miR-942 overexpression markedly increased the luciferase activity of the TOP flash/FOP flash reporter, drastically decreased the protein levels of sFRP4, GSK3β, and TLE1, and significantly repressed the luciferase activity of 3’UTRs of sFRP4, GSK3β, and TLE1. Moreover, individually silencing sFRP4, GSK3β, or TLE1 increased the tumour sphere formation and enhanced the TOP flash/FOP flash activity of antagomir-942 cells.Taken together, our results indicated miR-942 promotes stem cell-like traits in ESCC by directly suppressing sFRP4, GSK3β, and TLE1, which are multiple negative regulators of Wnt/β-catenin signaling.