Radioresistance Of Hepatocellular Carcinoma Research Articles

UBAP2 contributes to radioresistance by enhancing homologous recombination through SLC27A5 ubiquitination in hepatocellular carcinoma

Radiotherapy stands as an effective method in the clinical treatment of hepatocellular carcinoma (HCC) patients. However, both primary and acquired radioresistance limit its clinical application in HCC. Therefore, investigating the mechanism of radioresistance may provide other options for treating HCC. Based on single-cell RNA sequencing (scRNA-seq) and HCC transcriptome datasets, 227 feature genes with prognostic value were selected to establish the tSNE score. The tSNE score emerged as an independent prognostic factor for HCC and correlated with cell proliferation and radioresistance-related biological functions. UBAP2 was identified as the most relevant gene with the tSNE score, consistently elevated in human HCC samples, and positively associated with patient prognosis. Functionally, UBAP2 knockdown impeded HCC development and reduced radiation resistance in vitro and in vivo. The ectopic expression of SLC27A5 reversed the effects of UBAP2. Mechanically, we uncovered that UBAP2, through the ubiquitin-proteasome system, decreased the homologous recombination-related gene RAD51, not the non-homologous end-joining (NHEJ)-related gene CTIP, by degrading the antioncogene SLC27A5, thereby generating radioresistance in HCC. The findings recapitulated that UBAP2 promoted HCC progression and radioresistance via SLC27A5 stability mediated by the ubiquitin-proteasome pathway. It was also suggested that targeting the UBAP2/SLC27A5 axis could be a valuable radiosensitization strategy in HCC.

CircROBO1 knockdown improves the radiosensitivity of hepatocellular carcinoma by regulating RAD21

Introduction and ObjectivesRadioresistance is a common problem in the treatment of many cancers, including hepatocellular carcinoma (HCC). Previous studies have shown that circROBO1 is highly expressed in HCC tissues and acts as a cancer promoter to accelerate the malignant progression of HCC. However, the role and mechanism of circROBO1 in HCC radioresistance remain unclear. Materials and MethodsCircROBO1, microRNA (miR)-136-5p and RAD21 expression levels were analyzed by quantitative real-time PCR. Cell function and radioresistance were evaluated by colony formation assay, cell counting kit 8 assay, EdU assay and flow cytometry. Protein expression was determined using western blot analysis. RNA interaction was analyzed by dual-luciferase reporter assay and RNA pull-down assay. In vivo experiments were performed by constructing mice xenograft models. ResultsCircROBO1 was highly expressed in HCC, and its knockdown inhibited HCC cell proliferation and promoted apoptosis to enhance cell radiosensitivity. On the mechanism, circROBO1 could serve as miR-136-5p sponge to positively regulate RAD21. MiR-136-5p inhibitor or RAD21 overexpression reversed the regulation of circROBO1 knockdown on the radiosensitivity of HCC cells. Also, circROBO1 interference improved the radiosensitivity of HCC tumors in vivo. ConclusionsCircROBO1 might be a promising target for treating HCC radioresistance.

TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) stands as the fifth most prevalent malignant tumor on a global scale and presents as the second leading cause of cancer-related mortality. DNA damage-based radiotherapy (RT) plays a pivotal role in the treatment of HCC. Nevertheless, radioresistance remains a primary factor contributing to the failure of radiation therapy in HCC patients. In this study, we investigated the functional role of transketolase (TKT) in the repair of DNA double-strand breaks (DSBs) in HCC. Our research unveiled that TKT is involved in DSB repair, and its depletion significantly reduces both non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair. Mechanistically, TKT interacts with PARP1 in a DNA damage-dependent manner. Furthermore, TKT undergoes PARylation by PARP1, resulting in the inhibition of its enzymatic activity, and TKT can enhance the auto-PARylation of PARP1 in response to DSBs in HCC. The depletion of TKT effectively mitigates the radioresistance of HCC, both in vitro and in mouse xenograft models. Moreover, high TKT expression confers resistance of RT in clinical HCC patients, establishing TKT as a marker for assessing the response of HCC patients who received cancer RT. In summary, our findings reveal a novel mechanism by which TKT contributes to the radioresistance of HCC. Overall, we identify the TKT-PARP1 axis as a promising potential therapeutic target for improving RT outcomes in HCC.

Hexokinase 2 confers radio-resistance in hepatocellular carcinoma by promoting autophagy-dependent degradation of AIMP2

With technological advancements, radiotherapy (RT) has become an effective non-surgical treatment for hepatocellular carcinoma (HCC), comprehensively improving the local control rate of patients with HCC. However, some patients with HCC still experience radio-resistance, cancer recurrence, and distant metastasis following RT. Our previous study has revealed that hexokinase 2 (HK2), a potent oncogene, was overexpressed in radio-resistant HCC cell lines; however, its role in HCC radio-resistance remains elusive. Here, we confirmed the upregulation of HK2 in HCC tissue, which is related to unfavorable prognosis in patients with HCC, and demonstrated that HK2 exerts a radio-resistant role by attenuating apoptosis and promoting proliferation in HCC cell lines. HK2 downregulation combined with ionizing radiation showed an excellent synergistic lethal effect. Mechanistically, HK2 alleviated ionizing radiation-mediated apoptosis by complexing with pro-apoptotic protein aminoacyl tRNA synthetase complex interacting multifunctional protein 2 (AIMP2) while enhancing its autophagic lysosomal-dependent degradation, thereby increasing radio-resistance of HCC. Pharmacologically, ketoconazole, an FDA-approved antifungal drug, served as an inhibitor of HK2 and synergistically enhanced the efficacy of RT. Our results indicated that HK2 played a vital role in radio-resistance and could be a potential therapeutic target for improving RT efficacy in HCC.

Statement of Retraction: Down-regulated lncRNA TP73-AS1 reduces radioresistance in hepatocellular carcinoma via the PTEN/Akt signaling pathway

Statement of Retraction: Down-regulated lncRNA TP73-AS1 reduces radioresistance in hepatocellular carcinoma via the PTEN/Akt signaling pathway

Deficiency of Carbamoyl Phosphate Synthetase 1 Engenders Radioresistance in Hepatocellular Carcinoma via Deubiquitinating c-Myc

Tumor radiation resistance is the main obstacle to effective radiation therapy for patients with hepatocellular carcinoma (HCC). We identified the role of urea cycle key enzyme carbamoyl phosphate synthetase 1 (CPS1) in radioresistance of HCC and explored its mechanism, aiming to provide a novel radiosensitization strategy for the CPS1-deficiency HCC subtype. The expression of CPS1 was measured by western blot and immunohistochemistry. Cell growth assay, EdU assay, cell apoptosis assay, cell cycle assay, clone formation assay, and subcutaneous tumor assay were performed to explore the relationship between CPS1 and radioresistance of HCC cells. Lipid metabonomic analysis was used for investigating the effects of CPS1 on lipid synthesis of HCC cells. RNA sequencing and coimmunoprecipitation assay were carried out to reveal the mechanism of CPS1 participating in the regulation of HCC radiation therapy resistance. Furthermore, 10074-G5, the specific inhibitor of c-Myc, was administered to HCC cells to investigate the role of c-Myc in CPS1-deficiency HCC cells. We found that urea cycle key enzyme CPS1 was frequently lower in human HCC samples and positively associated with the patient's prognosis. Functionally, the present study proved that CPS1 depletion could accelerate the development of HCC and induce radiation resistance of HCC in vitro and in vivo, and deficiency of CPS1 promoted the synthesis of some lipid molecules. Regarding the mechanism, we uncovered that inhibition of CPS1 upregulated CyclinA2 and CyclinD1 by stabilizing oncoprotein c-Myc at the posttranscriptional level and generated radioresistance of HCC cells. Moreover, inactivation of c-Myc using 10074-G5, a specific c-Myc inhibitor, could partially attenuate the proliferation and radioresistance induced by depletion of CPS1. Our results recapitulated that silencing CPS1 could promote HCC progression and radioresistance via c-Myc stability mediated by the ubiquitin-proteasome system, suggesting that targeting c-Myc in CPS1-deficiency HCC subtype may be a valuable radiosensitization strategy in the treatment of HCC.

NUPR1 contributes to radiation resistance by maintaining ROS homeostasis via AhR/CYP signal axis in hepatocellular carcinoma

BackgroundRadiotherapy (RT) is one of the major therapeutic approaches to hepatocellular carcinoma (HCC). Ionizing radiation (IR) inducing the generation of reactive oxygen species (ROS) leads to a promising antitumor effect. However, the dysregulation of the redox system often causes radioresistance and impairs the efficacy of RT. Increasing evidence indicates that nuclear protein 1 (NUPR1) plays a critical role in redox reactions. In this study, we aim to explore the role of NUPR1 in maintaining ROS homeostasis and radioresistance in HCC.MethodsThe radioresistant role of NUPR1 was determined by colony formation assay, comet assay in vitro, and xenograft tumor models in vivo. Probes for ROS, apoptosis assay, and lipid peroxidation assay were used to investigate the functional effect of NUPR1 on ROS homeostasis and oxidative stress. RNA sequencing and co-immunoprecipitation assay were performed to clarify the mechanism of NUPR1 inhibiting the AhR/CYP signal axis. Finally, we analyzed clinical specimens to assess the predictive value of NUPR1 and AhR in the radiotherapeutic efficacy of HCC.ResultsWe demonstrated that NUPR1 was upregulated in HCC tissues and verified that NUPR1 increased the radioresistance of HCC in vitro and in vivo. NUPR1 alleviated the generation of ROS and suppressed oxidative stress, including apoptosis and lipid peroxidation by downregulating cytochrome P450 (CYP) upon IR. ROS scavenger N-acetyl-L-cysteine (NAC) and CYP inhibitor alizarin restored the viability of NUPR1-knockdown cells during IR. Mechanistically, the interaction between NUPR1 and aryl hydrocarbon receptor (AhR) promoted the degradation and decreased nuclear translation of AhR via the autophagy-lysosome pathway, followed by being incapable of CYP’s transcription. Furthermore, genetically and pharmacologically activating AhR abrogated the radioresistant role of NUPR1. Clinical data suggested that NUPR1 and AhR could serve as novel biomarkers for predicting the radiation response of HCC.ConclusionsOur findings revealed the role of NUPR1 in regulating ROS homeostasis and oxidative stress via the AhR/CYP signal axis upon IR. Strategies targeting the NUPR1/AhR/CYP pathway may have important clinical applications for improving the radiotherapeutic efficacy of HCC.

Methyltransferase 1 is required for nonhomologous end-joining repair and renders hepatocellular carcinoma resistant to radiotherapy.

Radiotherapy is an increasingly essential therapeutic strategy in the management of hepatocellular carcinoma (HCC). Nevertheless, resistance to radiotherapy is one of the primary obstacles to successful treatment outcomes. Hence, we aim to elucidate the mechanisms underlying radioresistance and identify reliable biotargets that would be inhibited to enhance the efficacy of radiotherapy in HCC. From a label-free quantitative proteome screening, we identified transfer RNA (tRNA; guanine- N [7]-) methyltransferase 1 (METTL1), a key enzyme for N7-methylguanosine (m 7 G) tRNA modification, as an essential driver for HCC cells radioresistance. We reveal that METTL1 promotes DNA double-strand break (DSB) repair and renders HCC cells resistant to ionizing radiation (IR) using loss-of-function and gain-of-function assays in vitro and in vivo. Mechanistically, METTL1-mediated m 7 G tRNA modification selectively regulates the translation of DNA-dependent protein kinase catalytic subunit or DNA ligase IV with higher frequencies of m 7 G-related codons after IR treatment, thereby resulting in the enhancement of nonhomologous end-joining (NHEJ)-mediated DNA DSB repair efficiency. Clinically, high METTL1 expression in tumor tissue is significantly correlated with poor prognosis in radiotherapy-treated patients with HCC. Our findings show that METTL1 is a critical enhancer for HCC cell NHEJ-based DNA repair following IR therapy. These findings give insight into the role of tRNA modification in messenger RNA translation control in HCC radioresistance.

LncRNA LINC01134 Contributes to Radioresistance in Hepatocellular Carcinoma by Regulating DNA Damage Response via MAPK Signaling Pathway.

Hepatocellular carcinoma (HCC) is a highly mortal cancer that could be treated by radiotherapy. DNA damage response (DDR) is a vital factor affecting cancer development after radiotherapy. Long non-coding RNAs (lncRNAs) have been revealed to regulate DNA damage response and repair in cancer cells. Nevertheless, the function of long intergenic non-protein coding RNA 1134 (LINC01134) has not been explored in DDR. In this study, we targeted digging into the function of LINC01134 in DDR and exploring the underlying mechanism in HCC cells. RT-qPCR was employed to measure LINC01134 expression, and we found LINC01134 was significantly upregulated in HCC cells. Functional analysis suggested that LINC01134 depletion attenuated radioresistance of HCC cells by facilitating DNA damage. In vivo assays demonstrated LINC01134 depletion hindered HCC tumor growth. Mechanism assays unveiled LINC01134 sequestered microRNA-342-3p (miR-342-3p) and recruited insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) protein to modulate mitogen-activated protein kinase 1 (MAPK1) expression, consequently activating MAPK signaling pathway. Rescue assays validated the LINC01134/miR-342-3p/MAPK1 axis in the radio-resistant HCC cells. In conclusion, LINC01134 might be identified to be a useful biomarker for the therapy of HCC.

COMMD10 inhibits HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe balance in hepatocellular carcinoma

Copper (Cu) is an essential trace element whose serum levels have been reported to act as an effective indicator of the efficacy of radiotherapy. However, little is known about the role of Cu in radiotherapy. In this study we aimed to determine this role and investigate the precise mechanism by which Cu or Cu-related proteins regulate the radiosensitivity of hepatocellular carcinoma (HCC). The expression and function of Cu and copper metabolism MURR1 domain 10 (COMMD10) were assessed via a Cu detection assay, immunostaining, real-time PCR, western blot, a radiation clonogenic assay and a 5-ethynyl-2'-deoxyuridine assay. Ferroptosis was determined by detecting glutathione, lipid peroxidation, malondialdehyde and ferrous ion (Fe) levels. The invivo effects of Cu and COMMD10 were examined with Cu/Cu chelator treatment or lentivirus modification of COMMD10 expression in radiated mouse models. We identified a novel role of Cu in promoting the radioresistance of HCC cells. Ionizing radiation (IR) induced a reduction of COMMD10, which increased intracellular Cu and led to radioresistance of HCC. COMMD10 enhanced ferroptosis and radiosensitivity invitro and invivo. Mechanistically, low expression of COMMD10 induced by IR inhibited the ubiquitin degradation of HIF1α (by inducing Cu accumulation) and simultaneously impaired its combination with HIF1α, promoting HIF1α nuclear translocation and the transcription of ceruloplasmin (CP) and SLC7A11, which jointly inhibited ferroptosis in HCC cells. In addition, elevated CP promoted HIF1α expression by reducing Fe, forming a positive feedback loop. COMMD10 inhibits the HIF1α/CP loop to enhance ferroptosis and radiosensitivity by disrupting Cu-Fe homeostasis in HCC. This work provides new targets and treatment strategies for overcoming radioresistance in HCC. Radiotherapy benefits patients with unresectable or advanced hepatocellular carcinoma (HCC), but its effectiveness is hampered by radioresistance. Herein, we uncovered a novel role for copper in promoting the radioresistance of HCCs. This work has revealed new targets and potential treatment strategies that could be used to sensitize HCC to radiotherapy.

Pre-mRNA processing factor 19 functions in DNA damage repair and radioresistance by modulating cyclin D1 in hepatocellular carcinoma

Pre-mRNA processing factor 19 (PRP19) is elevated in hepatocellular carcinoma (HCC); however, little is known about its function in DNA damage repair in HCC. In this study, analysis of The Cancer Genome Atlas data and our tumor models after ionizing radiation (IR) treatment indicated that increased expression of PRP19 was positively correlated with DNA damage repair. Gain of PRP19 expression induced by plasmids resulted in decreases in apoptosis and double-strand breaks (DSBs), and an increase in cell survival after IR. Loss of PRP19 expression induced by small interfering RNAs resulted in the accumulation of apoptosis and DSBs, and a decrease in cell survival. Mechanistically, the effect of PRP19 on DNA damage repair was mediated by the modulation of cyclin D1 expression in HCC. PRP19 controlled the translation of cyclin D1 by modulating eukaryotic initiation factor 4E. PRP19 affected the DNA damage repair ability of cyclin D1 by interacting with the WD40 domain. The combination of PRP19 and cyclin D1 was more valuable than each single marker for predicting the prognosis of patients. Taken together, the present results demonstrate that PRP19 promotes DNA damage repair by modulating cyclin D1 expression and function, thereby contributing to the radioresistance in HCC.

Lupeol-Loaded Nanoparticles Enhance the Radiosensitivity of Hepatocellular Carcinoma by Inhibiting the Hyperactivation in Raf/Mitogen-Activated Protein Kinase and Phospatidylinositol-3 Kinase/mTOR Pathways.

Radioresistance limits the effectiveness of radiotherapy for hepatocellular carcinoma. Raf and PI3K signaling cascades promote the formation of radioresistance in hepatocellular carcinoma (HCC). Lupeol has anticancer activity despite itspoor solubility in water and is toxic effect on normal tissue. In this study, nanoparticles (lupeol-NPs) were constructed using PEG-PLGA diblock copolymer vector, and results revealed that Lupeol-NPs reversed the radioresistance of hepatocellular carcinoma by inhibiting cellular proliferation and cell-cycle progression and promoting cellular apoptosis through blocking Raf/MAPK and PI3K/Akt signal transduction in radioresistant Huh-7R cells. In vivo, Lupeol-NPs combined with radiotherapy inhibited the growth of radioresistant hepatocellular carcinoma in a xenogenic nude mouse model. Ki-67 proliferation index decreased significantly (p < 0.05). We conclude that Lupeol-NPs can increase the sensitivity of radioresistant hepatocellular carcinoma to radiotherapy through inhibiting the Raf and PI3K signal cascades.

UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation

BackgroundRadioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood.MethodsWe obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T.ResultsHere, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy.ConclusionOur results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.

PEX5, a novel target of microRNA-31-5p, increases radioresistance in hepatocellular carcinoma by activating Wnt/β-catenin signaling and homologous recombination.

Rationale: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with high recurrence and metastasis rates. Although radiation is an effective treatment for tumors, it is often limited by intrinsic radioresistance in HCC. The contributions of dysregulated microRNAs, including miR-31-5p, to HCC progression have been recently reported. However, the role of miR-31-5p in the radiation response of HCC is unknown. In this study, we aimed to investigate the impact of miR-31-5p on HCC radiosensitivity.Methods: miR-31-5p expression in HCC tissues, paired adjacent tissues, and HCC cell lines was measured using quantitative real-time polymerase chain reaction and in situ hybridization. Bioinformatic analyses, gain- and loss-of-function experiments, and luciferase reporter assays were performed to validate peroxisomal biogenesis factor 5 (PEX5) as a direct target of miR-31-5p. The biofunctions of PEX5 and miR-31-5p in HCC were determined by Transwell, wound-healing, and Cell Counting Kit-8 (CCK8) assays. A colony formation assay was used to evaluate the radiosensitivity of HCC cells. The interaction among PEX5, β-catenin, Rac1, and JNK-2 was confirmed by coimmunoprecipitation. A xenograft tumor model was established to validate the effects of miR-31-5p and PEX5 on HCC progression and radiosensitivity in vivo.Results: Low expression of miR-31-5p in HCC specimens, as observed in this study, predicted a poor clinical outcome. However, the expression pattern of PEX5, as a direct target of miR-31-5p, was opposite that of miR-31-5p, and high PEX5 expression indicated poor prognosis in HCC patients. Ectopic expression of PEX5 increased the proliferation, migration, and invasion abilities and enhanced the radioresistance of HCC cells in vitro and in vivo; however, these phenotypes were inhibited by miR-31-5p. Mechanistically, PEX5 stabilized cytoplasmic β-catenin and facilitated β-catenin nuclear translocation to activate Wnt/β-catenin signaling. Moreover, upon radiation exposure, PEX5 reduced excessive reactive oxygen species (ROS) accumulation and activated the homologous recombination (HR) pathway, which protected HCC cells from radiation-induced damage.Conclusions: Our findings demonstrated a novel role for PEX5 as a miR-31-5p target and a mediator of the Wnt/β-catenin signaling and HR pathways, providing new insights into studying HCC radiation responses and implicating PEX5 and miR-31-5p as potential therapeutic targets in HCC.

RETRACTED ARTICLE: Down-regulated lncRNA TP73-AS1 reduces radioresistance in hepatocellular carcinoma via the PTEN/Akt signaling pathway

Statement of Retraction We, the Editors and Publisher of the journal Cell Cycle, have retracted the following article: Wei Song, Jingjing Zhang, Qingxin Xia and Miaomiao Sun. Down-regulated lncRNA TP73-AS1 reduces radioresistance in hepatocellular carcinoma via the PTEN/Akt signaling pathway. Cell Cycle. 2019;18(22):3177-3188. doi: 10.1080/15384101.2019.1671089 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors have been unable to address the concerns raised and have not been able to provide sufficient supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. The corresponding author listed in this publication has been informed. The authors agree with the retraction. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Downregulation of lncRNA NEAT1_2 radiosensitizes hepatocellular carcinoma cells through regulation of miR-101-3p/WEE1 axis.

Radioresistance is a major obstacle in hepatocellular carcinoma (HCC) radiotherapy. Aberrant expression of long non-coding RNA (lncRNA) has been postulated to be implicated in the development of HCC radioresistance. We investigated the role of lncRNA nuclear enriched abundant transcript 1_2 (NEAT1_2) in radioresistance of HCC and its molecular mechanism in this study. We found that NEAT1_2 and WEE1 were upregulated, and miR-101-3p was downregulated in HCC tissues, as well as HCC cell lines. Downregulation of WEE1 sensitized the radiosensitivity of HCC cells, as evidenced by decreased survival fractions of Huh7 and PLC5 cells and increased percentage of apoptotic cells. Also, knockdown of NEAT1_2 exerted a reinforcing effect on the radiosensitivity of HCC cells. In addition, WEE1 was confirmed as a direct target of miR-101-3p. Upregulation of miR-101-3p obviously decreased the mRNA and protein levels of WEE1 compared with that in the miR-NC group, while transfection of anta-miR-101-3p presented the opposite effects. In parallel, NEAT1_2 was identified to interact with miR-101-3p, and NEAT1_2 upregulated the expression of WEE1 in Huh7 cells through sponging miR-101-3p. Besides, the reinforcing effect of NEAT1_2 silencing could be attenuated by downregulation of miR-101-3p. To conclude, our results support the concept that downregulation of lncRNA NEAT1_2 radiosensitizes hepatocellular carcinoma cells through regulation of miR-101-3p/WEE1 axis.

MiR-92b targets p57kip2 to modulate the resistance of hepatocellular carcinoma (HCC) to ionizing radiation (IR) -based radiotherapy

Hepatocellular carcinoma (HCC) is one of the most common digestive system malignant tumors. Due to the resistance to radiotherapy, the prognosis in patients with HCC is poor. Based on previous studies and online tools prediction, we hypothesized that miR-92b, which was reported to promote HCC cell proliferation, might bind to p57kip2, a well-known tumor suppressor, to modulate the radioresistance of HCC to ionizing radiation (IR) -based radiotherapy. In the present study, a higher miR-92b expression in HCC tissues and cell lines was observed; a high miR-92b expression was correlated with poorer prognosis in patients with HCC. The overexpression of miR-92b enhanced the radioresistance of HCC to IR treatment by promoting cancer cell proliferation, attenuating cell apoptosis and remove IR-induced cell cycle at G2/M phase. Through directly binding to the 3′-UTR of p57kip2, miR-92b negatively regulated the protein levels of p57kip2; miR-92b inhibition enhanced the cell effect of IR on HCC cells, which could be attenuated by the p57kip2 knockdown, in other words, miR-92b modulated the radioresistance of HCC to IR-based radiotherapy through p57kip2. Taken together, miR-92b inhibits p57kip2 expression in HCC tissues and cell lines, thus enhancing the radioresistance of HCC to IR-based radiotherapy; targeting miR-92b to rescue p57kip2 expression in HCC might help sensitive HCC cells to IR-based radiotherapy.

RNF6 facilitates metastasis and radioresistance in hepatocellular carcinoma through ubiquitination of FoxA1

Ring finger protein 6 (RNF6), a RING-domain E3 ubiquitin Ligase, functions as a potential tumor promoter in several caners. However, the function and clinical significance of RNF6 in hepatocellular carcinoma (HCC) remains unclear. Here, we found that high RNF6 expression was detected frequently in primary HCC tissues, and was closely associated with malignant phenotype and shorter survival among the HCC patients. Multivariate analyses also revealed that RNF6 overexpression was independent prognostic factors for poor outcome of patients with HCC. Further, RNF6 knockdown notably inhibited the metastasis abilities of HCC in vivo and in vitro. RNF6 silence also suppressed the epithelial-mesenchymal transition (EMT) and radioresistance in HCC. Mechanistically, our results demonstrated that RNF6 directly bound and ubiquitylated Forkhead box protein A1 (FoxA1), an important transcriptional repressor of EMT process. Additionally, our data shown that the oncogenic effect of RNF6 in HCC is partially dependent on FoxA1 degradation. Collectively, our data suggest that RNF6 plays a crucial oncogenic role in HCC tumorigenesis, and we provide a novel evidence that RNF6 may be serve as a prognostic and therapeutic target for HCC progression.

A20 Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells to 60Co-γ Ionizing Radiation

The radioresistance of hepatocellular carcinoma (HCC) cells is a critical obstacle for effectively applying radiotherapy (RT) in HCC treatment. NF-κB, an important transcription factor, can influence critical cell fate decisions by promoting cell survival or anti-apoptosis in response to cell-stress, e.g. chemotherapies or ionizing radiation (IR). A20, also named as tumor necrosis factor α induced protein 3 (TNFAIP3 ), is a dominant negative regulator of NF-κB pathway and its functions in HCC are largely unknown. Cell culture and adenovirus vectors L-02, HepG2, MHCC-97H (a highly aggressive HCC cell line) and MHCC-97L (a lowly aggressive HCC cell line) cells were purchased from cell resources center of Chinese Academy of Medical Sciences (Beijing, China). The adenovirus vectors containing full length sequence of A20 and its A20 siRNA was purchased from Vigene Company (Jinan, Shandong Province, China). The HCC specimens and the adjacent non-tumor tissues were obtained by routinely surgical operations and preserved by our lab. The collection of tissues and the study protocol was all approved by the Ethics Committee of Xi’an Jiaotong University School of Medicine, and with the informed consent of patients. Paired HCC and non-tumorous tissues (40 paired) were obtained from surgical resections from February 2015 to April 2016. Using such methods as qPCR; Western Blot analysis; 60Co-γ ionizing radiation and colony formation; TUNEL reaction; Apoptosis analysis; Transwell analysis; In vivo tumor growth. Higher expression of A20 was detected in hepatic non-tumor cell line or clinical specimens compared with HCC cell lines or clinical specimens. A20 decreased the expression of proteins mediating cellular stress/injury response or epithelial-mesenchymal transition (EMT) process. Overexpression of A20 via adenovirus enhanced the effect of 60Co-γ ionizing radiation (IR) on HCC cells’ injury, e.g. G2/M arrest or DNA double strands break (DSB). Moreover, A20 also enhanced the in vitro or in vivo survival inhibiting of HCC cells induced by IR. Our results showed A20 downregulates the protein level of cellular stress/injury response regulators and by then potentiate the susceptibility of HCC cell line to 60Co-γ IR. Overexpression of A20 via an adenoviral vector can be a useful strategy for increasing tumor radiosensitivity.

Long non-coding RNA ROR promotes radioresistance in hepatocelluar carcinoma cells by acting as a ceRNA for microRNA-145 to regulate RAD18 expression

Radiotherapy plays a limited role in the treatment of hepatocellular carcinoma (HCC) due to the development of resistance. Therefore, further investigation of underlying mechanisms involved in HCC radioresistance is warranted. Increasing evidence shows that long non-coding RNAs (linc-RNAs) are involved in the pathology of various tumors, including HCC. Previously, we have shown that long noncoding RNA regulator of reprogramming (linc-ROR) promotes HCC metastasis via induction of epithelial-mesenchymal transition (EMT). However, the roles of linc-ROR in HCC radioresistance and its possible mechanisms are unclear. Here, we established two radioresistant HCC cell lines (HepG2-R and SMMC-7721-R) and found that linc-ROR was significantly upregulated in radioresistant HCC cells. Knockdown of linc-ROR reduces in vitro and in vivo radiosensitivity of parental HCC cells by reducing DNA repair capacity, while ectopic expression of linc-ROR enhances radiosensitivity of radioresistant HCC cells. Further mechanistic investigations revealed that lincRNA-ROR exerted its biological effects by acting as a competing endogenous RNA (ceRNA) for miR-145 to regulate RAD18 expression, thereby promoting DNA repair. Collectively, our findings demonstrate that linc-ROR promotes HCC radioresistance and targeting it will be a promising strategy for enhancing the efficacy of radiotherapies in HCC.