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Sphingomyelin phodiesterase acid-like 3A promotes hepatocellular carcinoma growth through the enhancer of rudimentary homologSphingomyelin phodiesterase acid-like 3A promotes hepatocellular carcinoma growth through the enhancer of rudimentary homolog

ABSTRACT: Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, with unclear pathogenesis. Sphingomyelin phodiesterase acid-like 3A (SMPDL3A) affects cell differentiation and participates in immune regulation. However, its molecular biological function in HCC has not yet been elucidated. Methods：Data from 180 HCC patients were analyzed the relationship between the expression of SMPDL3A in liver cancer tissues and the prognosis of liver cancer patients. Crispr-Cas9 dual vector lentivirus was used to knock out SMPDL3A in HCC cell lines. The effects of SMPDL3A on cell viability were determined by CCK8 assay, clone formation experiment, cell cycle assay, cell scratch, TUNEL experiment and flow cytometry. Xenograft tumor assays in BALB/c nude mice confirmed that SMPDL3A promoted tumor growth and in vivo. Preliminary exploration of SMPDL3A interacting protein by mass spectrometry analysis and co-immunoprecipitation. Results: This study showed that the expression of SMPDL3A in HCC tissue differed from that in tumor-adjacent tissues. Moreover, the overall survival rate and tumor-free survival rate of patients with high-SMPDL3A expression were significantly lower than those with low-SMPDL3A expression. SMPDL3A expression was closely related to the level of protein induced by PIVKA-II, liver cirrhosis, tumor diameter, microvascular invasion, and Barcelona clinic liver cancer staging. Thus, SMPDL3A is an independent risk factor that affects the tumor-free survival rate and overall survival rate of HCC patients. In vitro study using Crispr-Cas9 genome editing technology revealed the knockout effect of SMPDL3A on cell proliferation, apoptosis, and migration. Cell counting kit-8 assay and clone formation experiment showed that sgSMPDL3A inhibited tumor cell proliferation and migration. Flow cytometry and TUNEL assay showed that sgSMPDL3A promoted apoptosis in tumors. Moreover, sgSMPDL3A inhibited tumor growth during subcutaneous tumor formation in nude mice. Immunohistochemistry of Ki67 and PNCA also indicated that sgSMPDL3A inhibited subcutaneous tumor proliferation in tumor-bearing nude mice. Further experiments showed that SMPDL3A interacts with the enhancer of rudimentary homolog (ERH). Conclusions: High-SMPDL3A expression was related to poor prognosis of patients with HCC. Knockout of SMPDL3A inhibited the proliferation and migration and accelerated the migration of HCC cells. SMPDL3A interacted with ERH to affect the tumorigenesis and progression of HCC.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Liver Cancer

SUBMITTER: Yu Zhang

LAB HEAD: Yu Zhang

PROVIDER: PXD032824 | Pride | 2022-05-23

REPOSITORIES: Pride

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Project description:Background: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. Methods: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. Findings: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. Interpretation: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC. Fund: This work was supported in part by the financial support from the China Natural Science Foundation (No. 81972642, No. 81601122 and No. 81770389), Hunan Natural Science Foundation (No. 2017JJ3205), Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), and the Scientific Research Fund of Hunan Provincial Education Department (17B162).

Project description:BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and mRNAs with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta , or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in-silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched non-tumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 non-tumor liver samples using the Taqman assay. RESULTS: Levels of the microRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with non-tumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of AKT1-MYC-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans.

Project description:Hepatectomy generally offers the best chance of long-term survival for patients with hepatocellular carcinoma (HCC). Many studies have shown that hepatectomy accelerates tumor metastasis, but the mechanism remains unclear. In this study, palliative hepatectomy was performed in an orthotopic nude mice model of HCC (MHCC97H) with high metastatic potential. Using Human Tumor Metastasis Microarray, we screened the metastasis-related genes in tumor tissues following palliative resection, and found that Metastasis suppressor 1 (MTSS1) located in the central position of gene function net of residual HCC; MTSS1 was up-regulated in residual tumor after palliative resection. Further studies found that MTSS1 enhanced the metastasis of residual HCC. In hepatitis B-related HCC patients undergone palliative hepatectomy, those with higher MTSS1 mRNA expression accompanied by the activation of matrix metalloproteinase 2 (MMP2) in residual HCC, had earlier residual HCC detection after hepatectomy and poorer survival when compared to those with lower MTSS1 level. In different cell lines, the levels of MTSS1 mRNA increased in parallel with metastatic potential. MTSS1 down regulation via siRNA decreased MMP2 activity, reduced invasive potentials of HCC by 28.9% in vitro, and averted the deteriorated lung metastatic extent in vivo. In conclusion, the poor prognosis of hepatitis B-related HCC patients following palliative hepatectomy associates with elevated MTSS1 mRNA expression; therefore, MTSS1 may provide a new research field for HCC diagnosis and treatment. Eighteen nude mice bearing HCC xenografts were randomized into three groups 14 days after orthotopic implantation: palliative resection group (mice received partial HCC resection with preservation of 2 mm tumor pedicles), sham operation group (mice only undergone an exposure of liver but without resection), and blank control group (mice without further surgical intervention). All mice were sacrificed by cervical dislocation 14 days following palliative resection based on pre-experimental results. The first time surgically removed HCC tissues were named tumor tissues T1; the second time surgically removed HCC tissues from sham operation group were named tumor tissues T2; Tissues from blank control group were named tumor tissues T3; Tissues from palliative resection group were named tumor tissues T4 that was residual HCC. Randomly selected 5 tumor specimens from each group were used for the screen of genes related to metastasis by microarray techniques.

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Sphingomyelin phodiesterase acid-like 3A promotes hepatocellular carcinoma growth through the enhancer of rudimentary homologSphingomyelin phodiesterase acid-like 3A promotes hepatocellular carcinoma growth through the enhancer of rudimentary homolog

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