Global profiling of protein lysine lactylation and potential target modified protein analysis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, often metastasizes to the lungs. The implications of lysine lactylation (Kla), a recently identified histone post-translational modification (PTM), in the pathology of HCC remain unclear. Here, we report the first proteomic survey of this specific modification in HCC (with no metastasis during 3 years of follow-up), normal liver tissues, and lung metastasis samples of HCC. Of the 2045 modification sites detected on 960 proteins, 1438 sites on 772 proteins contained quantitative information. Subsequently, we analyzed the differentially modified proteins among the different groups. Differentially lactylated proteins were found to be involved in several biological processes, including-but not limited to-amino acid metabolism, ribosomal protein synthesis, and fatty acid metabolism. In addition, we identified numerous highly valuable lactate-modified proteins from the literature. Among them, we verified the lactate modification levels of the following two tumor-related proteins and obtained similar results: USP14 and ABCF1. These two modified proteins will be further investigated in our future studies. This paper is the first report on the lactylome of HCC and it provides a reliable foundation for further research on Kla in HCC.     

The competing endogenous circular RNA ADAMTS14 suppressed hepatocellular carcinoma progression through regulating microRNA-572/regulator of calcineurin 1

Emerging evidence have discovered that circular RNAs (circRNAs) may serve as diagnostic or tumor promising biomarkers. This study aimed to investigate how circular RNA ADAMTS14 (circADAMTS14) regulates microRNA-572/ regulator of calcineurin 1(miR-572/ RCAN1) in hepatocellular carcinoma (HCC). The expression profiles of circRNA/microRNA (mRNA) between HCC tissues and paired adjacent tissues were analyzed via microarray analysis. The expressions of circADAMTS14, miR-572, and RCAN1 were measured by real-time polymerase chain reaction (PCR). The protein expression level of RCAN1 in HCC cells was detected by western blot. The viability and apoptosis levels of HCC cell lines were measured by the cell counting Kit-8 (CCK-8) assay and fluorescence-activated cell sorter. The invasiveness and migration of cells were detected based on the transwell and wound-healing assay, respectively. The dual-luciferase reporter assays were used to reveal circADAMTS14 and RCAN1 as a potential target of miR-572, which was predicted by TargetScan and miRBase. The effect of circADAMTS14 on HCC cells was demonstrated by tumor formation in nude mice in vivo. CircADAMTS14 and RCAN1 were lowly expressed in HCC clinical specimens and cell lines using microarrays and qRT-PCR, but miR-572 inversely. Our study further verified the direct interaction between circADAMTS14 and RCAN1 with miR-572 via the dual-luciferase reporter gene assay. Overexpressed circADAMTS14 and RCAN1 induced apoptosis of HCC cells and inhibited cell proliferation and invasion. But overexpressed miR-572 could decrease apoptosis of HCC cells and promote proliferation and invasion. In vivo, circADAMTS14 inhibited the tumor growth, correlated positively with the protein expression levels of RCAN1. Our results demonstrated that circADAMTS14 might suppress HCC progression through regulating miR-572/ RCAN1 as the competing endogenous RNA.     

Tumor-derived secretory clusterin induces epithelial–mesenchymal transition and facilitates hepatocellular carcinoma metastasis

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Metastasis is the major concern that causes death in HCC. The goal of this study was to identify tumor-derived proteins in serum during HCC metastasis using an orthotopic xenograft tumor model and explore the role of key protein in HCC metastasis. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Twenty tumor-derived proteins were identified through human specific peptides. Secretory clusterin (sCLU), which was significantly upregulated during cancer progression and metastasis, was chosen for further study. The expression of sCLU was significantly higher in metastatic HCC cell lines and samples from metastatic HCC patients. ShRNA-mediated down-regulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis in vivo. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. Further study found that sCLU contributed to HCC migration and epithelial–mesenchymal transition (EMT) in vitro, and metastasis in vivo. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling. These findings suggest that sCLU may promote HCC metastasis via the induction of EMT process and may be a candidate target for HCC therapy.     

Ecto-5′-nucleotidase (CD73) is a potential target of hepatocellular carcinoma

High expression of ecto-5′-nucleotidase (CD73) has been reported in a number of epithelium origin malignancies. Here, we hypothesize that CD73 promotes hepatocellular carcinoma (HCC) growth and metastasis and that the effect is mediated by epithelial growth factor receptor (EGFR). HCC cells with different malignancies and Tissue microarrays of the tumor and peritumoral liver tissues from 30 independent patients were used to examine CD73 and EGFR expression. Then, MTT and Ki67 detection, together with cell adhesion, invasion, and migration assays were used to evaluate the effects of CD73 on cell growth and metastasis. The expression of EGFR in HCC cells was also tested after suppressing or overexpressing CD73. Lastly, tumor tissues from nude mice, which had been injected subcutaneously with HCC cells, were transplanted subcutaneously into CD73^−/− and wild-type (WT) C57 mice. CD73 expression was higher in HCC cells with greater metastatic potentials and tumor tissues compared with low metastatic cells and peritumor tissues. CD73 and EGFR were coexpressed and positively correlated in tumor and peritumor liver tissues in HCC tissue microarrays. Up-regulationof CD73 by plasmid transfection or by pharmacological agents promoted EGFR expression in HCC cells, whereas suppression of CD73 inhibited these effects. The growth of transplanted tumor tissues was dramatically slower in CD73^−/− mice than in WT type mice in the in vivo experiments. CD73 promotes HCC growth and metastasis and upregulated the expression of EGFR in HCC. Thus, CD73 and EGFR are potential targets in the treatment of HCC.     

肝细胞肝癌组织NTS、SPNS2、Mortalin表达与上皮间质转化标志物、临床病理特征和预后的关系

摘要 目的：探讨肝细胞癌（HCC）癌组织神经降压素（NTS）、鞘氨醇-1-磷酸转运体2（SPNS2）、热休克蛋白75（Mortalin）表达与上皮间质转化（EMT）标志物、临床病理特征和预后的关系。方法：选取2010年1月～2017年1月联勤保障部队第九〇〇医院仓山院区收治的90例HCC患者,采用免疫组化法检测患者癌组织和对应癌旁组织中NTS、SPNS2、Mortalin及EMT标志物N-钙粘蛋白（N-Cad）、E-钙粘蛋白（E-Cad）表达情况。分析NTS、SPNS2、Mortalin表达与HCC患者EMT标志物、临床病理特征和预后的关系。结果：HCC癌组织中NTS、SPNS2、Mortalin、N-Cad阳性表达率高于癌旁组织,E-Cad阳性表达率低于癌旁组织（P＜0.05）。Pearson相关性分析显示,HCC癌组织中NTS、SPNS2、Mortalin表达水平与N-Cad表达水平呈正相关,与E-Cad表达水平呈负相关（P＜0.05）。 HCC癌组织中NTS、SPNS2、Mortalin表达与Child-Pugh分级、血管侵犯、巴塞罗那临床肝癌（BCLC）分期、淋巴结转移、远处转移有关（P＜0.05）。90例HCC患者术后5年总生存率为48.89％（44/90）。Kaplan-Meier生存曲线分析显示,NTS、SPNS2、Mortalin阳性组总生存率分别低于NTS、SPNS2、Mortalin阴性组（P＜0.05）。结论：HCC癌组织中NTS、SPNS2、Mortalin表达上调,与EMT、Child-Pugh分级、血管侵犯、BCLC分期、淋巴结转移、远处转移和预后有关,可作为HCC病情及预后的辅助评估指标。     

MICA/B蛋白在不同宫颈病变组织中的表达及意义

目的:探讨主要组织相容性复合物I类相关蛋白A/B(MICA/B)在不同宫颈病变组织及宫颈细胞系中的表达及定位。方法:采用免疫组织化学SP法检测宫颈炎症组织、高级别鳞状上皮内病变(high-grade squamous intraepithelial lesions, HSIL)及宫颈鳞癌(cervical squamous cell carcinoma, CSCC)组织中MICA/B蛋白的表达情况。采用免疫荧光化学与激光共聚焦显微术结合的方法研究3种宫颈癌细胞系C33a(HPV-)、Siha(HPV16+)、Hela(HPV18+)及正常宫颈上皮细胞系H8中MICA/B的表达和定位。结果:MICA/B蛋白主要表达定位于细胞浆,部分细胞核,在宫颈鳞癌组织中阳性表达率(83.3%、81.8%)高于宫颈炎症组织(39.3%、44.0%),差异具有统计学意义(均有P0.001);MICA蛋白在HSIL组织的阳性表达率(81.8%)高于宫颈炎症组织(39.3%),差异具有统计学意义(P=0.002);与分化程度、临床分期、淋巴结转移等临床病理参数之间比较无统计学差异(P0.05)。结论:MICA蛋白随着宫颈组织病变的加重阳性表达率逐渐增高,MICB蛋白在宫颈癌组织的表达高于宫颈炎症组织。提示MICA/B蛋白可为宫颈癌的诊断及靶向治疗提供新方向。     

High-throughput assessment of CpG site methylation for distinguishing between HCV-cirrhosis and HCV-associated hepatocellular carcinoma

Methylation of promoter CpG islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. Therefore, some postulate that aberrantly methylated CpG regions may be important biomarkers indicative of cancer development. In this study we used the Illumina GoldenGate Methylation BeadArray Cancer Panel I for simultaneously profiling methylation of 1,505 CpG sites in order to identify methylation differences in 76 liver tissues ranging from normal to pre-neoplastic and neoplastic states. CpG sites for ESR1, GSTM2, and MME were significantly differentially methylated when comparing the pre-neoplastic tissues from patients with concomitant hepatocellular carcinoma (HCC) to the pre-neoplastic tissues from patients without HCC. When comparing paired HCC tissues to their corresponding pre-neoplastic non-tumorous tissues, eight CpG sites, including one CpG site that was hypermethylated (APC) and seven (NOTCH4, EMR3, HDAC9, DCL1, HLA-DOA, HLA-DPA1, and ERN1) that were hypomethylated in HCC, were identified. Our study demonstrates that high-throughput methylation technologies may be used to identify differentially methylated CpG sites that may prove to be important molecular events involved in carcinogenesis.     

MiR-490-5p inhibits the stemness of hepatocellular carcinoma cells by targeting ECT2

To explore the targeting relationship between miR-490-5p and ECT2 in hepatocellular carcinoma (HCC) and the influences of miR-490-5p and ECT2 on the stemness of HCC cells. The expressions of miR-490-5p and ECT2 in HCC tissues and adjacent tissues were identified by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between the expression levels of miR-490-5p/ ECT2 and the overall/disease-free survival (OS/DFS) of patients with HCC were evaluated using correlative curves. In addition, the targeting relationship between miR-490-5p and ECT2 was predicted by TargetScan and verified by dual-luciferase reporter assay. Plasmid transfection was used for overexpression of ECT2 in HepG2 cells, and transfection efficiency was verified by qRT-PCR. Cell Counting Kit-8 assay and cell sphere-formation assay were conducted to detect the proliferation and sphere-formation ability of HCC cells, respectively. Cell populations with different cell transfections were sorted using flow cytometry. The expression levels of proteins in the stem cell signaling pathway were determined using Western blot analysis. MiR-490-5p was remarkably downregulated, yet ECT2 was upregulated in HCC tissues compared with adjacent tissues. MiR-490-5p expression was positively correlated with OS and DFS of patients with HCC, which were otherwise negatively correlated with ECT2 expression. ECT2 was validated to be the downstream target of miR-490-5p. Overexpression of miR-490-5p restrained the sphere formation ability, stemness, and proliferation of HCC cells. MiR-490-5p repressed the stemness of HCC cells through inhibiting the expression of ECT2. MiR-490-5p may be an underlying therapeutic target in HCC treatment.     

The underlying pathophysiology association between the Type 2-diabetic and hepatocellular carcinoma

Type 2-diabetic (T2D) disease has been reported to increase the incidence of liver cancer, however, the underlying pathophysiology is still not fully understood. Here, we aimed to reveal the underlying pathophysiology association between the T2D and hepatocellular carcinoma (HCC) and, therefore, to find the possible therapeutic targets in the occurrence and development of HCC. The methylation microarray data of T2D and HCC were extracted from the Gene Expression Omnibus and The Cancer Genome Atlas. A total of 504 differentially methylated genes (DMGs) between T2D samples and the controls were identified, whereas 6269 DMGs were identified between HCC samples and the control groups. There were 336 DMGs coexisting in diabetes and HCC, among which 86 genes were comethylated genes. These genes were mostly enriched in pathways as glycosaminoglycan biosynthesis, fatty acid, and metabolic pathway as glycosaminoglycan degradation and thiamine, fructose and mannose. There were 250 DMGs that had differential methylation direction between T2D DMGs and HCC DMGs, and these genes were enriched in the Sphingolipid metabolism pathway and immune pathways through natural killer cell-mediated cytotoxicity and ak-STAT signaling pathway. Eight genes were found related to the occurrence and development of diabetes and HCC. Moreover, the result of protein-protein interaction network showed that CDKN1A gene was related to the prognosis of HCC. In summary, eight genes were found to be associated with the development of HCC and CDKN1A may serve as the potential prognostic gene for HCC.     

Overexpression of osteopontin in hepatocellular carcinoma and its relationships with metastasis,invasion of tumor cells

Osteopontin (OPN) plays an important role in metastasis and relapse of human cancer. However, the whole story of OPN relating to cancer has been far from clear untill now. To investigate the expression of OPN in hepatocellular carcinoma (HCC) and its relationships with recurrence and metastasis of HCC, normal and malignant liver tissues from patients with HCC were analyzed using immunohistochemical staining. OPN expression was inhibited by small interfering RNA (siRNA) in HCC cells lines, and then colony formation and matrigel invasion were examined. The results showed that expression of OPN was associated with metastasis of HCC with a positive rate of OPN in the tissue of HCC (70.00%), which was highly more obvious than those in paracarcinoma tissue and normal liver tissue (P < 0.01). In HCC cell lines, OPN depletion could reduce formed colony and metastasizing numbers in vitro. In conclusion, Expression of OPN in the tissue of HCC is related to metastasis or metastases. Specific siRNA could decrease expressions of OPN at both mRNA and protein levels, and abates the invasiveness of hepatocellular carcinoma cells, suggesting that OPN might be a promising agent for treatment of metastasis and recurrence of HCC.     

miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

Caveolin enhances hepatocellular carcinoma cell metabolism,migration, and invasion in vitro via a hexokinase 2-dependent mechanism

The development and progression of hepatocellular carcinoma (HCC) have been associated with abnormal cellular metabolism. Gene Expression Profiling Interactive Analysis RNA sequencing data revealed caveolin-1 (CAV-1) and hexokinase 2 (HK2) messenger RNA (mRNA) were significantly upregulated in human HCC compared with normal tissues, and high HK2 expression was associated with significantly poorer overall survival in HCC ( p < 0.05). CAV-1 and HK2 mRNA and protein expression were upregulated and positively correlated in 42 fresh human HCC tissues compared with tumor-adjacent normal tissues. Overexpression of CAV-1 or HK2 in SMMC-7721 and HepG2 HCC cells enhanced glucose and lactate metabolism and increased cell migration and invasion in transwell assays; knocking down CAV-1 or HK2 had the opposite effects. Overexpression of CAV-1 increased HK2 expression; overexpression of HK2 did not affect CAV-1 expression. Knocking down HK2 partially reversed the ability of CAV-1 to promote cellular metabolism, invasion, and migration in HCC, indicating CAV-1 enhances glycolysis, invasion, and metastasis in HCC cells via HK2-dependent mechanism. Further studies of the function and relationship between CAV-1 or HK2 expression are warranted to explore the potential of these proteins as metabolic targets for the treatment of HCC.     

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

Peritumoral monocytes induce cancer cell autophagy to facilitate the progression of human hepatocellular carcinoma

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B⁺ cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

NXN suppresses metastasis of hepatocellular carcinoma by promoting degradation of Snail through binding to DUB3

The poor prognosis of hepatocellular carcinoma (HCC) could be attributed to its high metastasis rate. Here, we report the role of nucleoredoxin (NXN), a multifunctional redox-active protein, in HCC metastasis. The expression of NXN in HCC tissues was measured by immunohistochemistry. The role of NXN on HCC proliferation was determined by CCK-8, EdU and colony formation assays in vitro and subcutaneous tumor formation model in vivo. Transwell and wound healing assays and tail vein injection model were performed to assess the function of NXN on HCC metastasis. Co-immunoprecipitation assay was performed to examine the interaction among NXN, Snail and DUB3. Our results showed that NXN was downregulated in HCC tissues compared to adjacent liver tissues. Patients with low NXN expression had shorter overall survival (OS) time (P < 0.001) than those with high NXN expression. Biologically, ectopic expression of NXN significantly inhibited the proliferation and metastasis of HCC cells both in vitro and in vivo by suppressing epithelial-mesenchymal transition (EMT). Mechanistically, NXN promoted the ubiquitin-proteasome-mediated degradation of Snail through interaction with DUB3. Further, depletion of Snail abolished NXN-inhibited cell proliferation and metastasis. In summary, NXN suppressed the proliferation and metastasis of HCC by inhibiting DUB3-mediated deubiquitylation of Snail protein. Our study demonstrates that NXN, DUB3 and Snail complex functioned as an important regulatory mechanism of HCC progression and indicates a potential therapeutic approach for the treatment of HCC metastasis.Subject terms: Tumour-suppressor proteins, Metastasis