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Hepatocellular carcinoma (HCC) is most common malignant tumor worldwide, and one of the most lethal malignancies. MEX3A, an RNA-binding protein, is profoundly implicated in tumor initiation and progression. But its role and potential mechanism in HCC remains fully unclear. In this study, MEX3A expression was upregulated in HCC tissue and cell lines. Knockdown or overexpression of MEX3A disturbed the proliferation, migration and apoptosis of HCC cells by modulating the activation of Hippo signaling pathway. The expression of MEX3A was negatively associated with sorafenib sensitivity and upregulated in sorafenib resistant HCC cells. MEX3A knockdown facilitated the expression of WWC1, a negative modulator of Hippo signaling pathway, and led to increase of the phosphorylation of LATS1 and YAP1. Pharmacological inhibition of LATS1 or WWC1 overexpression alleviated the proliferative and migrated suppression and increased sorafenib sensitivity, whereas WWC1 inhibition using genetic interference strategy showed opposite trend in MEX3A knockdown HCC cells. Importantly, MEX3A knockdown led to growth and lung metastasis inhibition using xenograft model established by means of subcutaneous or tail vein injection. In addition, a combination of MEX3A knockdown and WWC1 overexpression dramatically enhances the growth inhibition of sorafenib in vivo. Collectively, our results demonstrated that MEX3A may facilitate HCC progression and hinder sorafenib sensitivity via inactivating Hippo signaling. The present study suggested that targeting MEX3A can be served as a novel therapeutic strategy for HCC.

Background: Cholangiocarcinoma (CCA) is a rare malignant carcinoma characterized by high mortality, challenging diagnosis, and poor prognosis. A powerful prediction biomarker is urgently needed for the early diagnosis and individualized treatment of CCA patients. Methods: A systematic bioinformatics analysis was conducted based on mRNA expression data and clinical information from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and National Genomics Data Center (NGDC) datasets. Differentially expressed genes (DEGs) between tumor tissues and adjacent counterpart controls were identified in the TCGA and GSE107943 datasets. A nine-gene prediction model was constructed, and its effects on CCA prognosis were analyzed using univariate, multivariate and LASSO Cox proportional hazards regression models, Kaplan-Meier plotter, CIBERSORT and OncoPredict in the discovery and validation cohorts. Additionally, the expression profiles of the target genes were determined via qRT-PCR and DEG analyses in an independent cohort. Results: A nine-gene signature (HELLS, HOXC6, MFSD2A, OTX1, PTGES, PYGB, SMOC1, TEX30 and ZBTB12) displayed excellent predictive performance for the overall survival of CCA. According to the prognostic signature, CCA patients were classified into high-risk and low-risk groups, with obvious differences in overall survival probabilities. The low-risk group had a significantly better prognosis than the high-risk group in both the discovery cohort (n = 66) and replication cohort (n = 255) (P < 0.0001, P < 0.0001). Additionally, five cancer drugs (Erlotinib, ML323, AGI-6780, Gallibiscoquinazole and AZD3759) presented clearly specific sensitivities for high-risk and low-risk group patients. Moreover, according to tumor microenvironment analyses, high-risk group patients had a higher level of M0 macrophage infiltration than low-risk group patients (P = 0.025, P = 0.0048). In replicating the expression patterns of the nine genes, eight of the nine genes (except TEX30) were found to have significant expression profiles between 15 tumor tissues and adjacent counterpart controls; moreover, the qRT-PCR results validated the abnormal expression pattern of the target genes in CCA. Conclusions: Collectively, we established an effective prognostic model for different populations of CCA patients based on nine DEGs. These findings may provide potential benefits for the development of new prognostic biomarkers and therapeutic targets for CCA.

Background: The regulatory loop between circular RNAs and microRNAs has a vital role in cell death. Ferroptosis is the form of iron-dependent cell death, which is distinct from necroptosis and apoptosis. Increasing evidences showed that ferroptosis is an important form of cell death in hepatocellular carcinoma.Methods: Real-time PCR were used to examine the expression level of circ_0013731 in hepatocellular carcinoma tissues. Edu and colony formation were performed to detect the cell proliferation. A luciferase reporter assay was used to determine the relationship between circ_0013731, miR-877-3p and SLC7A11. ChIP-qPCR assays were performed to examine the potential binding of E2F1 to the circ_0013731 promoter. Iron Assay Kit (Sigma Aldrich) was used to detect total iron or Fe2+. C11 BODIPY 581/591 staining and flow cytometer were used to examine the Lipid ROS. The role of circ_0013731 was examined in xenograft tumors model. Results: We revealed that the expression level of circ_0013731 was elevated in hepatocellular carcinoma. Moreover, E2F1 promote the transcription of circ_0013731. Overexpression of circ_0013731 promoted cell growth and inhibited ferroptosis in SMMC-7721 and QGY-7703 in vitro. miR-877-3p was proved as the direct target of circ_0013731. Then, inhibition of miR-877-3p enhanced cell growth and inhibited ferroptosis. Further mechanism research demonstrated that circ_0013731 upregulated the expression level of SLC7A11 by sponging miR-877-3p. Finally, circ_0013731 promoted HCC growth via miR-877-3p/ SLC7A11 axis in vivo.Conclusions: Our data reveal that circ_0013731 mediated by E2F1 facilitates cell growth and suppressed the ferroptosis via miR-877-3p/ SLC7A11 axis in hepatocellular carcinoma. Therefore, circ_0013731 could be acted as potential therapeutical target for hepatocellular carcinoma.

Background: Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis, with no effective therapy other than surgical resection. Circular RNAs (circRNAs) serve as a brand-new class of transcription products among abundant cancer processes. Nevertheless, the mechanisms account for their modification in CCA remain unknown. Methods: First, microarray sequencing was applied to detect the difference of circRNAs expression between CCA and corresponding non-tumor tissues. We utilized qRT-PCR to measure circ-0006302 levels in CCA cells and specimens. Gain/loss of-function assays and animal model of CCA were performed for the purpose of revealing the functions of circ-0006302 on the invasion, migration, and proliferation of CCA. We performed dual luciferase reporter, RNA-FISH and rescue assays for clarifying the mechanism behind. Results: In CCA tissues and cell lines circ-0006302 was highly expressed relatively. In vitro, overexpression of circ-0006302 intensifies the epithelial-to-mesenchymal transition (EMT) and the invasion, migration, and growth of CCA cells; and intensifies the growth as well as metastasis of tumors in a CCA mouse model. Furthermore, it was elucidated that circ-0006302 sponged miR-1299 to upregulate PD‐L1 expression. Through the process above, circ-0006302 binds to miR-1299 and emancipates PD-L1, facilitating the invasion, migration, and proliferation in CCA cells. Momentously, the results obtained revealed that circ-0006302 silencing elevated the expression of interferon (IFN)‐γ, and interleukin (IL)‐4 but diminished the IL-10 expression, while these effects could be reversed by miR-1299 inhibitor.Conclusion: circ-0006302 silence blocked the CCA progression via intensifying miR‐1299‐targeted downregulation of PD‐L1. Our conclusion provides novel therapeutic tactics for treating this fatal disease.

Background Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR). Results Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes de through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provide novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

Background: Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods: Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR).Results: Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes KIF1Bβ through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion: These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provided novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

Background Emerging evidence suggests that circular RNAs play critical roles in disease development especially in cancers. Previous genome-wide RNA-seq studies found that a circular RNA derived from SOD2 gene was highly upregulated in hepatocellular carcinoma (HCC), however, the role of circSOD2 in HCC remains largely unknown. Methods The expression profiling of circSOD2 and microRNA in HCC patients were assessed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). SiRNA or CRISPR-CAS9 were used to silence gene expression. The biological function of circSOD2 in HCC was investigated using in vitro and in vivo studies including, trans-well cell migration, cell apoptosis, cell cycle, CCK8, siRNA interference, western blots, and xenograft mouse model. The underlying molecular mechanism was determined by Chromatin Immunoprecipitation quantitative real time PCR (ChIP-qPCR), bioinformatic analysis, biotin-pull down, RNA immunoprecipitation, 5-mc DNA pulldown and luciferase assays. Results In accordance with previous sequencing results, here, we demonstrated that circSOD2 was highly expressed in HCC tumor tissues compared with normal liver tissues. Mechanically, we showed that histone writer EP300 and WDR5 bind to circSOD2 promoter and trigger its promoter H3K27ac and H3K4me3 modification, respectively, which further activates circSOD2 expression. SiRNA mediated circSOD2 suppression impaired liver cancer cell growth, cell migration, prohibited cell cycle progression and in vivo tumor growth. By acting as a sponge, circSOD2 inhibits miR-502-5p expression and rescues miR-502-5p target gene DNMT3a expression. As a DNA methyltransferase, upregulated DNMA3a suppresses SOCS3 expression by increasing SOCS3 promoter DNA methylation. This event further accelerates SOCS3 downstream JAK2/STAT3 signaling pathway activation. In addition, we also found that activated STAT3 regulates circSOD2 expression in a feedback way. Conclusion The novel signaling axis circSOD2/miR-502-5p/DNMT3a/JAK2/STAT3/circSOD2 provides a better understanding of HCC tumorigenesis. The molecular mechanism underlying this signaling axis offers new prevention and treatment of HCC.

BackgroundIn this study, we comprehensively analyzed genes related to ferroptosis and iron metabolism to construct diagnostic and prognostic models and explore the relationship with the immune microenvironment in HCC.MethodsIntegrated analysis, cox regression and the least absolute shrinkage and selection operator (LASSO) method of 104 ferroptosis- and iron metabolism-related genes and HCC-related RNA sequencing were performed to identify HCC-related ferroptosis and iron metabolism genes.ResultsFour genes (ABCB6, FLVCR1, SLC48A1 and SLC7A11) were identified to construct prognostic and diagnostic models. Poorer overall survival (OS) was exhibited in the high-risk group than that in the low-risk group in both the training cohort (P ConclusionsThe prognostic and diagnostic models based on the four genes indicated superior diagnostic and predictive performance, indicating new possibilities for individualized treatment of HCC patients.Graphical abstract