Background: N6-methyladenosine (m 6 A) modification is an emerging layer of epigenetic regulation which is widely implicated in the tumorigenicity of hepatocellular carcinoma (HCC), offering a novel perspective for investigating molecular pathogenesis of this disease. The role of AlkB homolog 5 (ALKBH5), one of the m 6 A demethylases, has not been fully explored in HCC. Here we clarify the biological profile and potential mechanisms of ALKBH5 in HCC. Methods: Expression of ALKBH5 and its correlation with clinicopathological characteristics of HCC were evaluated using tissue microarrays and online datasets. And biological effects of ALKBH5 in HCC were determined in vitro and in vivo. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq), and following m 6 A dot blot, MeRIP-qPCR, RIP-qPCR or dual luciferase reporter assays were employed to screen and validate the candidate targets of ALKBH5. Results: We demonstrated that ALKBH5 was down-regulated in HCC, and decreased ALKBH5 expression was an independent prognostic factor of worse survival in HCC patients. Functionally, ALKBH5 suppressed the proliferation and invasion capabilities of HCC cells in vitro and in vivo. Mechanistically, ALKBH5-mediated m 6 A demethylation led to a post-transcriptional inhibition of LY6/PLAUR Domain Containing 1 (LYPD1), which could be recognized and stabilized by the m 6 A effector IGF2BP1. In addition, we identified that LYPD1 induced oncogenic behaviors of tumors in contrast to ALKBH5. Dysregulation of ALKBH5/LYPD1 axis impelled the progression of HCC. Conclusion: Our study reveals that ALKBH5, characterized as a tumor suppressor, attenuates the expression of LYPD1 via an m 6 A-dependent manner in HCC cells. Our findings enrich the landscape of m 6 A-modulated tumor malignancy, and provide new insights into potential biomarkers and therapeutic targets of HCC treatment.
Exosomes play an important role in intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). However, cellular communication between heterogeneous HCC cells with different metastatic potentials and the resultant cancer progression are not fully understood in HCC. Here, HCC cells with high-metastatic capacity (97hm and Huhm) were constructed by continually exerting selective pressure on primary HCC cells (MHCC-97H and Huh7). Through performing exosomal miRNA sequencing in HCC cells with different metastatic potentials (MHCC-97H and 97hm), many significantly different miRNA candidates were found. Among these miRNAs, miR-92a-3p was the most abundant miRNA in the exosomes of highly metastatic HCC cells. Exosomal miR92a-3p was also found enriched in the plasma of HCC patient-derived xenograft mice (PDX) model with high-metastatic potential. Exosomal miR-92a-3p promotes epithelial-mesenchymal transition (EMT) in recipient cancer cells via targeting PTEN and regulating its downstream Akt/Snail signaling. Furthermore, through mRNA sequencing in HCC cells with different metastatic potentials and predicting potential transcription factors of miR92a-3p, upregulated transcript factors E2F1 and c-Myc were found in high-metastatic HCC cells promote the expression of cellular and exosomal miR-92a-3p in HCC by directly binding the promoter of its host gene, miR17HG. Clinical data showed that a high plasma exosomal miR92a-3p level was correlated with shortened overall survival and disease-free survival, indicating poor prognosis in HCC patients. In conclusion, hepatoma-derived exosomal miR92a-3p plays a critical role in the EMT progression and promoting metastasis by inhibiting PTEN and activating Akt/Snail signaling. Exosomal miR92a-3p is a potential predictive biomarker for HCC metastasis, and this may provoke the development of novel therapeutic and preventing strategies against metastasis of HCC.
MicroRNAs (miRNAs) have been validated to play prominent roles in the occurrence and development of many kinds of malignant cancer. MiR-424-5p has been reported to participate in various tumors proliferation and metastasis as a suppressor. On the contrary, miR-424-5p would promote cell proliferation in some tumors. However, the expression of miR-424-5p in intrahepatic cholangiocarcinoma (ICC) is rarely reported and its mechanism remains unclear. Here, we discover that miR-424-5p is frequently downregulated in ICC tissues compared with adjacent normal tissues and in ICC cells. Over-expression of miR-424-5p significantly inhibits the invasion and migration of ICC cells in vitro. Importantly, miR-424-5p is found to be a suppressor of ARK5, by binding to 3'-UTR of ARK5 mRNA and then inhibiting mTOR phosphorylated, thus deregulating epithelial-mesenchymal transition (EMT) of ICC. Furthermore, ARK5 is found to play a role in ICC metastasis and regulating EMT. Knockdown of ARK5 inhibits invasion and migration of ICC, while the over-expression gives an opposite effect. Besides, high-expression of ARK5 is also associated with poor prognosis. In conclusion, our study reveals that miR-424-5p is critical to the invasion, migration and EMT progression in ICC cells. Targeting the pathway described here may be a novel approach to inhibit metastasis of ICC and the restoration of miR-424-5p expression may be a promising strategy for ICC therapy.
A pH- and photothermal-responsive zeolitic imidazolate framework (ZIF-8) compound was designed for loading a dual-drug in the tumor site and improving their curative effects.
The function of the N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in hepatocellular carcinoma (HCC) has not been thoroughly investigated. Here we determined the clinical value, biological functions, and potential mechanisms of RBM15 in HCC. Expression of RBM15 was identified using tissue microarrays and online databases. A risk-prediction model based on RBM15 was developed and validated. We determined the biological role of RBM15 on HCC cells in vitro and in vivo. RNA sequencing was used to screen candidate targets of RBM15. Subsequently, the m6A dot blot assay, methylated RNA immunoprecipitation qPCR, dual-luciferase reporter assays, RNA decay assay, and RNA immunoprecipitation qPCR were employed to explore the mechanisms of RBM15. Our study showed that RBM15 was highly expressed in HCC, and overexpression of RBM15 indicated a worse outcome. A new nomogram combining RBM15 with age and TNM stage was developed and validated to predict the outcome of HCC patients; our nomogram increased the prediction accuracy of the TNM system. Functionally, RBM15 facilitates the proliferation and invasiveness of HCC. RBM15-mediated m6A modification contributed to a post-transcriptional activation of YES proto-oncogene 1 (YES1) in an insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)-dependent manner. In addition, YES1 was confirmed as an oncogene in HCC cells by activating the mitogen-activated protein kinase (MAPK) pathway. In conclusion, RBM15-mediated m6A modification might facilitate the progression of HCC via the IGF2BP1–YES1–MAPK axis. RBM15 may be a promising biomarker in the outcome prediction of HCC.
The high activity of Histone deacetylases (HDACs) in hepatocellular carcinoma (HCC) usually positively correlates with poor prognosis of patients. Accordingly histone deacetylases inhibitors (HDACis) are considered to be potential agents treating patients with HCC. In our study, we evaluated effect of quisinostat alone and in combination with sorafenib in HCC cells via inducing G0/G1 phase arrest through PI3K/AKT/p21 pathway and apoptosis by JNK/c-Jun/caspase3 pathway in vitro and in vivo. The proliferation assay and flow cytometry were used to measure the viability, cell cycle and apoptosis. And Western blot assay was carried out to determine expression alternations of related proteins. Moreover HCCLM3 xenograft was further performed to detect antitumor effect of quisinostat in vivo. Here, we found that quisinostat impeded cell proliferation, and remarkably induced G0/G1 phase arrest and apoptosis in HCC cells in a dose-dependent manner. G0/G1 phase arrest was observed by alterations in PI3K/AKT/p21 proteins. Meanwhile the JNK, c-jun and caspase-3 were activated by quisinostat in a dose-dependent manner. Correspondingly quisinostat facilitated G0/G1 cycle arrest and apoptosis in HCC cells through PI3K/AKT/p21 pathways and JNK/c- jun/caspase3 pathways. Moreover, the potent tumor-suppressive effects facilitated by quisinostat, was significantly potentiated by combination with sorafenib in vitro and vivo. The combination treatment of quisinostat and sorafenib markedly suppressed cell proliferation and induced apoptosis in a synergistic manner. Moreover the therapy of quisinostat combined with sorafenib could apparently decrease tumor volume of a HCCLM3 xenograft model. Our study indicated that quisinostat, as a novel chemotherapy for HCC, exhibited excellent antitumor activity in vitro and vivo, which was even enhanced by the addition of sorafenib, implying combination of quisinostat with sorafenib a promising and alternative therapy for patients with advanced hepatocellular carcinoma.
Background: Hepatocellular carcinomas (HCC) constantly rank among the malignancies with the highest death tolls on the global scale. Moreover, HCC are associated with a limited set of therapeutic options. This is particularly true in the case of advanced stage cancers, where long-term survival is uncommon. For the inoperable, advanced HCC patients, chemotherapy is the main modality of treatment. Due to the lack of known molecular targets, the efficacy of the chemotherapy is limited.Conclusion: These findings clearly indicate that DNA methylation plays a key role in regulating ACADS expression and that it can be a potential therapeutic target for treating HCC.Materials and methods: A thorough comparative analysis of 282 cancer samples with 47 normal samples from GEO datasets resulted in the observation that that the level of ACADS was significantly downregulated in HCC. Loss-of-function analyses were then conducted to understand the biological function of ACADS in HCC. It was noted that ACADS was involved in the proliferation and metastasis of HCC. Experiments involving the knockdown of DMNT expression led to the discovery that the expression of ACADS in the HCC cells was significantly increased. The TCGA database was then employed to identify tumor tissue samples which showed higher methylation levels at cg01535453, cg08618068, and cg10174836 (which are the target sites of the ACADS CpG islands) as compared with normal liver tissue samples. All these findings indicated that ACADS might be a novel methylation biomarker associated with HCC.
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