Asparagine synthetase modulates glutaminase inhibitor sensitivity through metabolic reprogramming and serves as a prognostic biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major global health burden and its treatment options are limited. Spermatogenesis associated serine rich 2(SPATS2), a recent defined oncogene, was found to be a prognostic biomarker in HCC. However, the explicit mechanism underlying SPATS2 was urged to be elucidated. In vitro, knockdown of SPATS2 hampered the proliferation, invasion and migration of HCC cells. Moreover, phosphorylation of signal transducer and activator of transcription 3 (STAT3) and its downstream oncogenes were dramatically suppressed by SPATS2 knockdown. In addition, tripartite motif containing 44 (TRIM44) was found to be positively associated with SPATS2 in TCGA and declined after SPATS2 knockdown in HCC cells. Overexpression of TRIM44 rescued the effect of SPATS2 silencing on p-STAT3 and its downstream oncogenes. In vivo, SPATS2 silencing was confirmed to impede HCC tumor development in nude mice. In our own cohort containing 112 HCC patients, high SPATS2 protein level is indicative of an unfavorable clinicopathological feature and poor prognosis and could serve as an independent risk factor. Collectively, the present study is the first to propose the mechanism of significance of SPATS2-TRIM44-p-STAT3 in HCC and provide a new theoretical basis for targeted therapy.

BackgroundIncreasing evidences demonstrate that miRNAs contribute to development and progression of hepatocellular carcinoma (HCC). Underexpression of miR-1296 is recently reported to promote growth and metastasis of human cancers. However, the expression and role of miR-1296 in HCC remain unknown.MethodsThe levels of miR-1296 in HCC tissues and cells were detected by qRT-PCR. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. Transwell assays were performed to determine migration and invasion of HCC cells. A lung metastasis mouse model was used to evaluated metastasis of HCC in vivo. The putative targets of miR-1296 were disclosed by public databases and a dual-luciferase reporter assay.ResultsWe found that the expression of miR-1296 was reduced in HCC tissues and cell lines, and it was associated with metastasis and recurrence of HCC. Notably, miR-1296 overexpression inhibited migration, invasion and EMT progress of HCCLM3 cells, while miR-1296 loss facilitated these biological behaviors of Hep3B cells in vitro and in vivo. In addition, miR-1296 inversely regulated SRPK1 abundance by directly binding to its 3′-UTR, which subsequently resulted in suppression of p-AKT. Either SRPK1 re-expression or PI3K/AKT pathway activation, at least partially, abolished the effects of miR-1296 on migration, invasion and EMT progress of HCC cells. Furthermore, miR-1296 and SRPK1 expression were markedly correlated with adverse clinical features and poor prognosis of HCC patients. We showed that hypoxia was responsible for the underexpression of miR-1296 in HCC. And the promoting effects of hypoxia on metastasis and EMT of HCC cells were reversed by miR-1296.ConclusionsUnderexpression of miR-1296 potentially serves as a prognostic biomarker in HCC. Hypoxia-induced miR-1296 loss promotes metastasis and EMT of HCC cells probably by targeting SRPK1/AKT pathway.

BackgroundAs one of the most frequently seen malignancies, hepatocellular carcinoma (HCC) serves as the second largest contributor to malignancy‐specific mortality worldwide. MicroRNA‐1225‐5p (miR‐1225) exerts an essential impact on the growth and metastasis of many malignancies. However, the contribution of miR‐125 to HCC and the molecular mechanism of cancer cell viability and apoptosis are still unclear. We focused our research on exploring the function and molecular mechanism of miR‐1225 in regulating HCC cell growth, migration, and invasion.MaterialQuantitative PCR data showed that miR‐1225 expression was repressed in HCC cell lines and in the tissues of HCC patients, compared to that in normal human hepatic cells and tissues. Transfection of a miR‐1225 mimic inhibited cell viability and proliferation as indicated by CCK‐8 staining and MTT assay. Transwell invasion, wound healing assay, and Western blotting were performed to assess whether miR‐1225 repressed the metastasis and invasion of HCC cells, and decreased matrix metalloproteinase 9 (MMP9) expression. Further bioinformatic prediction and dual‐luciferase reporter assay suggested that miR‐1225 targeted the 3′‐UTR of NFκB p65.ResultsOverexpression of p65 protein counteracted the repressive impact of miR‐1225 on invasion, migration, and proliferation of HCC cells.ConclusionThis research provided new evidences that miR‐1225 inhibits the viability, migration, and invasion of HCC cells by downregulation of p65.

It has previously been reported that glycosylphosphatidylinositol anchor attachment 1 (GPAA1) is overexpressed in hepatocellular carcinoma (HCC); however, its role in regulating the development of HCC remains unknown. The present study aimed to examine the potential role of GPAA1 in HCC and to characterize the associated mechanism. The expression of GPAA1 was first examined using the Gene Expression Profiling Interactive Analysis 2 database, and was then determined using reverse transcription-quantitative PCR and western blotting. The effects of GPAA1 silencing on the proliferation, colony formation, migration and invasion of HuH-7 cells were measured using Cell Counting Kit-8, colony formation, wound healing and Transwell assays, respectively. The interaction between splicing factor (SF)3B4 and GPAA1 was predicted by starBase and confirmed using RNA immunoprecipitation. The results of the present study demonstrated that GPAA1 was upregulated in HCC cells, and silencing GPAA1 markedly inhibited the proliferation, migration and invasion of HCC cells, which was accompanied by reduced levels of MMP2 and MMP9. In addition, it was observed that SF3B4 could bind to GPAA1. Furthermore, to confirm whether SF3B4 binds to GPAA1 to modulate HCC cell behavior, GPAA1 was knocked down and SF3B4 was overexpressed. Overexpression of SF3B4 reversed the effects of GPAA1 knockdown on the proliferation, migration and invasion of HCC cells. In conclusion, SF3B4 may promote the proliferation, invasion and migration of HCC cells by binding to GPAA1. The present study provided novel insight into the pathogenesis of HCC.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. This study aimed to analyze the prognostic value of microRNA-1266-5p (miR-1266-5p) in HCC patients and investigate its biological function in HCC progression. The expression of miR-1266-5p in tissues and cells was measured by quantitative real-time PCR (qRT-PCR). Cell counting kit-8 (CCK-8) assay was used to detect HCC cell proliferation. Transwell assay was performed to evaluate the migration and invasion of HCC cells. Kaplan-Meier methods and Cox regression analysis were used to assess the prognostic value of miR-1266-5p in HCC patients. The relationship between miR-1266-5p and DAB2IP was evaluated by luciferase reporter assay. Relative expression of miR-1266-5p in tumor tissues, tissues from patients with advanced TNM stage (III-IV) and HCC cells was increased compared with that in corresponding control group. MiR-1266-5p expression was significantly associated with tumor size and TNM stage in HCC patients. Elevated expression of miR-1266-5p was associated with poor prognosis of HCC patients and served as an independent prognostic factor for HCC patients. Overexpression of miR-1266-5p significantly promoted, while miR-1266-5p knockdown significantly inhibited the proliferation, migration and invasion of HCC cells. DAB2IP could directly bind to the miR-1266-5p. Our findings indicated that elevated expression of miR-1266-5p can predict the poor prognosis of HCC patients, and promotes the proliferation, migration and invasion of HCC cells. Therefore, we predict that miR-1266-5p may be a novel biomarker and therapeutic target for the treatment of HCC.

BackgroundLong non-coding RNAs (lncRNAs) are involved in the development of hepatocellular carcinoma (HCC). We aimed to investigate the function of LINC01146 in HCC.MethodsThe expression of LINC01146 in HCC tissues was explored via The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and was verified using quantitative real-time polymerase chain reaction (qRT–PCR) in our HCC cohort. Kaplan–Meier analysis was used to assess the relationship between LINC01146 and the prognosis of HCC patients. Cell Counting Kit 8, colony formation assays, Transwell assays, flow cytometric assays, and tumour formation models in nude mice were conducted to reveal the effects of LINC01146 on HCC cells both in vitro and in vivo. Bioinformatic methods were used to explore the possible potential pathways of LINC01146 in HCC.ResultsLINC01146 was significantly decreased in HCC tissues compared with adjacent normal tissues and was found to be related to the clinical presentations of malignancy and the poor prognosis of HCC patients. Overexpression of LINC01146 inhibited the proliferation, migration, and invasion of HCC cells in vitro, while promoting their apoptosis. In contrast, downregulation of LINC01146 exerted the opposite effects on HCC cells in vitro. In addition, overexpression of LINC01146 significantly inhibited tumour growth, while downregulation of LINC01146 promoted tumour growth in vivo. Furthermore, the coexpressed genes of LINC01146 were mainly involved in the “metabolic pathway” and “complement and coagulation cascade pathway”.ConclusionLINC01146 expression was found to be decreased in HCC tissues and associated with the prognosis of HCC patients. It may serve as a cancer suppressor and prognostic biomarker in HCC.

Ferroptosis is an iron-dependent programmed non-apoptotic cell death characterized by the accumulation of free iron ions and lipid peroxidation. It is associated with the inactivation of glutathione peroxidase (GPX) and the accumulation of lipid peroxides within cells. Ferroptosis is closely related to the occurrence and development of hepatocellular carcinoma (HCC). Chlorogenic acid (CGA), an important bioactive component found in 61 traditional Chinese medicines such as Eucommia ulmoides, has been extensively studied for its effects on various malignant tumors. However, the specific role and potential mechanism of CGA in HCC remain unclear. To elucidate the anti-tumor characteristics and potential mechanisms of CGA in inducing ferroptosis in HCC cells. The effects of CGA on the proliferation, migration, and invasion of HCC cells were evaluated through in vitro experiments. Bioinformatics analysis combined with network pharmacology was used to study the potential targets and molecular mechanisms of CGA intervention in HCC ferroptosis. In vitro experiments were conducted to verify and explore the anti-HCC effects and mechanisms of CGA through the ferroptosis pathway. In vitro experiments showed that CGA dose-dependently inhibited the proliferation, invasion, and migration of HCC cells. Bioinformatics analysis combined with network pharmacology revealed that the pathway of CGA intervention in HCC cell ferroptosis was mainly enriched in the prostaglandin endoperoxide synthase 2 (PTGS2)/aldo-keto reductase family 1 member C3 (AKR1C3)/GPX4 signaling pathway, which was associated with arachidonic acid. In vitro experiments further confirmed that CGA-induced ferroptosis in HCC cells was related to mitochondrial damage through the reprogramming of arachidonic acid metabolism by regulating the PTGS2/AKR1C3/GPX4 signaling pathway. This study demonstrates that CGA inhibits HCC cell proliferation, migration, and invasion by inducing ferroptosis through the PTGS2/AKR1C3/GPX4 axis, suggesting its potential as a novel ferroptosis inducer or anti-HCC drug.

Background:Asparagine synthetase (ASNS) is associated with drug resistance in leukaemia, and the function of this enzyme in the context of hepatocellular carcinoma (HCC) is not clear. In this study, the relationship between ASNS expression and clinical outcomes after surgical resection was investigated, and the therapeutic value of ASNS was also evaluated.Methods:The expression of ASNS was evaluated in HCC samples by real-time PCR and immunohistochemistry assays. The correlation between ASNS expression and clinicopathological features was investigated. Potential clinicopathological prognostic factors were examined by univariate and multivariate survival analysis. Asparagine synthetase was overexpressed and knocked down in HCC cell lines to assess the influence of the enzyme on cell proliferation, migration and tumourigenicity. L-asparaginase was used to treat HCC cells with high or low levels of ASNS in vitro and in vivo to examine the therapeutic efficacy.Results:The expression of ASNS was higher in HCC tumour tissues and was closely correlated with the serum AFP level, tumour size, microscopic vascular invasion, tumour encapsulation, TNM stage and BCLC stage. Patients with low ASNS expression levels had a poor prognosis with respect to overall survival (OS). The multivariate survival analysis indicated that ASNS is an independent prognostic factor for OS. Furthermore, functional studies demonstrated that ASNS significantly inhibits the proliferation, migration and tumourigenicity of HCC cells. The knockdown of ASNS markedly increased sensitivity to L-asparaginase, indicating that cells with different ASNS protein levels have different sensitivities to L-asparaginase.Conclusion:The expression of ASNS is an independent factor affecting the survival of HCC patients, and low ASNS expression in HCC was correlated with worse surgical outcomes. The ASNS may be a promising therapeutic target for the treatment of HCC.

LINC00205 promotes proliferation, migration and invasion of HCC cells by targeting miR-122-5p

BACKGROUNDPrevious studies have shown that the Shi-pi-xiao-ji (SPXJ) herbal decoction formula is effective in suppressing hepatocellular carcinoma (HCC), but the underlying mechanisms are not known. Therefore, this study investigated whether the antitumor effects of the SPXJ formula in treating HCC were mediated by acetyl-coA acetyltransferase 1 (ACAT1)-regulated cellular stiffness. Through a series of experiments, we concluded that SPXJ inhibits the progression of HCC by upregulating the expression level of ACAT1, lowering the level of cholesterol in the cell membrane, and altering the cellular stiffness, which provides a new idea for the research of traditional Chinese medicine against HCC.AIMTo investigate the anti-tumor effects of the SPXJ formula on the malignant progression of HCC.METHODSHCC cells were cultured in vitro with SPXJ-containing serum prepared by injecting SPXJ formula into wild-type mice. The apoptotic rate and proliferative, invasive, and migratory abilities of control and SPXJ-treated HCC cells were compared. Atomic force microscopy was used to determine the cell surface morphology and the Young’s modulus values of the control and SPXJ-treated HCC cells. Plasma membrane cholesterol levels in HCC cells were detected using the Amplex Red cholesterol detection kit. ACAT1 protein levels were estimated using western blotting.RESULTSCompared with the vehicle group, SPXJ serum considerably reduced proliferation of HCC cells, increased stiffness and apoptosis of HCC cells, inhibited migration and invasion of HCC cells, decreased plasma membrane cholesterol levels, and upregulated ACAT1 protein levels. However, treatment of HCC cells with the water-soluble cholesterol promoted proliferation, migration, and invasion of HCC cells as well as decreased cell stiffness and plasma membrane cholesterol levels, but did not alter the apoptotic rate and ACAT1 protein expression levels compared with the vehicle control.CONCLUSIONSPXJ formula inhibited proliferation, invasion, and migration of HCC cells by decreasing plasma membrane cholesterol levels and altering cellular stiffness through upregulation of ACAT1 protein expression.

To explore how lncRNA SNHG14 modulates the biological features of hepatocellular carcinoma (HCC) cells by regulating SOX9 via mediating miR-206. HCC tissues were collected to perform the quantitative reverse transcriptase polymerase chain reaction to determine the expressions of SNHG14, miR-206, and SOX9. HCC cell line SMCC7721 was selected for co-transfection by si-SNHG14/miR-206 inhibitor/si-SOX9, followed by the measurement of cell proliferation using Cell Count Kit-8 (CCK-8) assay and clone formation assay. The migration and invasion were evaluated by wound healing test and Transwell assay. The apoptotic rate was determined by flow cytometry. Levels of the apoptosis-related proteins were measured through Western blotting. SNHG14 and SOX9 were up-regulated in HCC tumor tissues compared with adjacent normal tissues, with decreased miR-206 expression. Moreover, SNHG14 expression was significantly associated with the TNM stage, lymphatic metastasis, and histological differentiation of HCC patients. Besides, inverse correlations between SNHG14 and miR-206, as well as between miR-206 and SOX9, were noted. The dual luciferase reporter gene assay, RIP, and RNA pull-down experiments also revealed the targeting relationship between SNHG14 and miR-206 or between miR-206 and SOX9. Silencing SNHG14 and SOX9 inhibited the proliferation, invasion, and migration of HCC cells, with increased apoptosis, which was all abolished by silencing miR-206. Inhibition of SNHG14 suppresses SOX9 by up-regulating miR-206, to further inhibit the proliferation, migration, and invasion of HCC cells with the promoted apoptosis, which is a novel target for the treatment of HCC.

IntroductionAlthough long non-coding RNA SNHG1 (lncRNA SNHG1) action on cell proliferation and invasion of hepatocellular carcinoma (HCC) cells has been reported, the effects of lncRNA SNHG1 on migration of HCC cells and the mechanisms are still unclear. The present study aimed to investigate the influence of lncRNA SNHG1 on metastasis in HCC cells and the possible mechanisms underlying this phenotype.Material and methodsExpression of lncRNA SNHG1 and miR-195 was determined using qRT-PCR in both HCC cell lines Huh7 and HepG2. Si-RNA was used to silence SNHG1 and miR-195 inhibitor was used to inhibit expression of miR-195. Luciferase reporter assay was conducted to confirm whether miR-195 was the direct binding target of SNHG1.ResultslncRNA SNHG1 was significantly up-regulated and miR-195 was significantly down-regulated in HCC cell lines. When transfected with si-SNHG1, migration and invasion of HCC cells, as well as expression of astrocyte elevated gene 1 (AEG-1) protein, were significantly inhibited compared with the control cells. Results of dual luciferase reporter assay showed that lncRNA SNHG1 acted as an endogenous sponge of miR-195. On the other hand, the expression of miR-195 in tumor tissue was much lower than that of miR-195 in the corresponding normal tissue. Furthermore, the correlation analysis showed a strong negative relationship between lncRNA SNHG1 and miR-195 expression in HCC tissues.ConclusionsSNHG1 may promote cell invasion and migration in HCC cells by sponging miR-195. These results can provide deeper understanding of SNHG1 in hepatocellular cancer and give new potential targets for treatment of HCC.

Background Hepatocellular carcinoma (HCC) is recognized as the fourth in incidence and the third in mortality worldwide. The onset of HCC is insidious and often asymptomatic at the early stage. HCC is more prone to metastasis, recurrence, and drug resistance than other solid tumors owing to its feature of high heterogeneity. Therefore, what particularly important is to search for effective molecular markers in the occurrence and progression of HCC. Aim To probe into the therapeutic potential of circACTG1 (hsa_circ_0046144) in HCC cell migration and invasion, providing a new insight and molecular target to diagnose and cure HCC patients. Methods The circACTG1 expression in collected HCC cells was determined by quantitative polymerase chain reaction (qPCR). Assessment for circACTG1 diagnosing capability was analyzed by receiver operating characteristic (ROC) curves. Transwell assay, wound healing assay, and cell counting kit-8 assay were used for monitoring the effect of circACTG1 in HCC cell invasion, migration, and proliferation, respectively; qPCR, luciferase reporter assay, databases, and Western blot analysis were used for identifying the modulation mechanisms among circACTG1, miRNA-940, and RIF1. What is more, our study verified AKT-mTOR signaling after miR-940 mimic treatment or circACTG1 knockdown. Results circACTG1 was overexpressed in HCC cells and tissues. Knockdown of circACTG1 restrained 97H and Huh7 cell migration and invasion. Significantly, circACTG1 was discovered to serve as a miR-940 sponge. miR-940 activation rebated the circACTG1 level, and conversely, miR-940 inhibition boosted the circACTG1 level. However, this effect or relationship was not seen after circACTG1 mutation. Furtherly, miR-940-downregulated expression was also found in HCC patients, and importantly, miR-940 inhibition reversed circACTG1 expression in 97H cells with circACTG1 knockdown. Moreover, the expression of RIF1 was significantly reduced after inhibiting circACTG1 or overexpressing miR-940 but rescued when both circACTG1 and miR-940 were inhibited. Finally, circACTG1 and miR-940 played significant roles of regulating AKT-mTOR signaling. Conclusion circACTG1 expression remarkably ascended in HCC, which is of certain diagnostic value. Moreover, circACTG1 potentially regulates HCC cell proliferation, invasion, and migration via miR-940/RIF1/AKT/mTOR pathway.

BackgroundTo study the effect of microRNA‐383 (miR‐383) on cell proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells, and explore its mechanism.MethodsThe expressions of miR‐383 and plant homology domain that refers to protein 8 (PHF8) were detected in tissues and cells by quantitative real‐time polymerase chain reaction (qRT‐PCR) or western blot respectively. The miR‐383 group (transfected miR‐383 mimics), miR‐con group (transfected miR‐con), si‐con group (transfected si‐con), si‐PHF8 group (transfected si‐PHF8), miR‐383 + ctrl group (cotransfected miR‐383 mimics and pcDNA‐3.1), miR‐383 + PHF8 group (cotransfected miR‐383 mimics and pcDNA‐3.1‐PHF8) were transfected into HepG2 cells by liposome method. Cell proliferation, migration and invasion were measured by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) or trans‐well assays respectively. The luciferase activity of each group was detected by dual luciferase reporter gene assay.ResultsCompared with normal adjacent tissues, the expression of miR‐383 was significantly down‐regulated and the expression of PHF8 was significantly up‐regulated (p < .05). Compared with normal hepatocellular cell LO2, the expression of miR‐383 was significantly reduced (p < .05) in HCC cells. Moreover, overexpression of miR‐383 or silencing of PHF8 significantly inhibited the proliferation, migration, and invasion of HCC cells. In addition, PHF8 was targeted by miR‐383 and its restoration rescued the inhibitory effect of miR‐383 on cell proliferation, migration, and invasion of HCC cells.ConclusionmiR‐383 could inhibit the proliferation, migration, and invasion of HCC cells by targeting PHF8, which will provide a basis for miR‐383 targeted therapy for HCC.

MiR-23b-3p suppresses epithelial-mesenchymal transition, migration, and invasion of hepatocellular carcinoma cells by targeting c-MET