[Construction and quality evaluation of strawberry seedling index model]. / 草莓种苗壮苗指数模型的构建与质量评价.

With the development of strawberry cultivated area in China, the demands for high-quality strawberry seedlings are increasing year by year. The research on the evaluation index of strawberry seedlings is needed to ensure the quality of the seedlings. This study aimed to establish an optimal index model of strawberry seedlings to improve the accuracy of seedlings evaluation. In this study, 320 seedlings of 'Benihoppe' strawberry seedlings growing for 40 days were taken as the materials. Based on the determination of 16 individual indicators including the growth of aboveground and underground parts, fresh weight, and dry weight, we firstly conducted the membership function corresponding to single indicator. Then the comprehensive evaluation index of strawberry seedlings was calculated using weighted fuzzy evaluation method. Furthermore, the key indicators out of the 16 indicators which were filtered out by means of the principal component analysis method were combined into different index models of strawberry seedlings. The correlation analysis between the comprehensive evaluation index and seedling index models was done and finally the optimal seedling index model was selected and verified. The results showed that there were significant differences in 16 indices of 320 randomly selected strawberry seedlings. The comprehensive evaluation index of strawberry seedlings was in the range of 0.165-0.817, indicating that the comprehensive evaluation index could totally reflect the quality of seedlings and could be used as the evaluation basis. The 16 individual indices of strawberry seedlings were classified into three principal components, including aboveground related indicators, underground related indicators, and the pigment indicators. The cumulative contribution rate of three principal components was 79.7%. Twenty-seven seedlings index models were combined by randomly selecting three indices with a large contribution value from each principal component. Five strawberry seedlings index mo-dels were selected from 27 models due to the highest correlation with the comprehensive evaluation index. Among them, the model "aboveground dry weight×root surface area×chlorophyll a" was identified as the optimal one to evaluate the quality of strawberry seedlings, due to the highest correlation with the comprehensive evaluation index. The correlation coefficient of between strong seedling index and comprehensive evaluation index in three strawberry variety 'Benihoppe', 'Kantoseika' and 'Sweet Charlie' were 0.879, 0.924, and 0.975, respectively. According to the comprehensive evaluation index, the quality of strawberry seedlings were classified into three grades: grade Ⅰ (comprehensive evaluation index ≥0.5, seedling index ≥4.0) with high-quality seedlings; grade Ⅱ (comprehensive evaluation index 0.3-0.5, seedling index 0.5-4.0) with qualified seedlings; grade Ⅲ (comprehensive evaluation index ≤0.3, seedling index ≤0.5) with weak seedlings. The results provided a theoretical basis and scientific method for the evaluation of the health status of strawberry seedlings or other fruits and vegetable seedlings.

Correction to: MiR-612 regulates invadopodia of hepatocellular carcinoma by HADHA-mediated lipid reprogramming.

An amendment to this paper has been published and can be accessed via the original article.

miR-17-5p and miR-20a-5p suppress postoperative metastasis of hepatocellular carcinoma via blocking HGF/ERBB3-NF-κB positive feedback loop.

Dysregulation of microRNA (miRNA) is a frequent event in hepatocellular carcinoma (HCC), but little is known whether it is a bystander or an actual player on residual HCC metastasis during liver microenvironment remodeling initiated by hepatectomy. Methods: The differently expressed miRNAs and mRNAs were identified from RNA-seq data. Western blot, qRT-PCR, fluorescence in situ hybridization, immunofluorescence and immunohistochemical were used to detect the expression of miRNA and mRNA in cell lines and patient tissues. The biological functions were investigated in vitro and in vivo. Chromatin immunoprecipitation, proximity ligation and luciferase reporter assay were used to explore the specific binding of target genes. The expression of HGF/ERBB3 signaling was detected by Western blot. Results: In this study, HGF induced by hepatectomy was shown to promote metastasis of residual HCC cells. miR-17-5p and miR-20a-5p were confirmed to play inhibitory roles on HCC metastasis. And ERBB3 was found to be the common target of miR-17-5p and miR-20a-5p. HCC cells with lower levels of miR-17-5p and miR-20a-5p or higher level of ERBB3 were often more sensitive to response HGF stimuli and to facilitate metastatic colonization both in vitro and in vivo experimental systems. Furthermore, HGF reinforced ERBB3 expression by NF-κB transcriptional activity in a positive feedback loop. Of particular importance, HCC patients with lower levels of miR-17-5p and miR-20a-5p or higher level of ERBB3 had significantly shorter OS and PFS survivals after surgical resection. Conclusion: miR-17-5p and miR-20a-5p could suppress postoperative metastasis of hepatocellular carcinoma via blocking HGF/ERBB3-NF-κB positive feedback loop and offer a new probable strategy for metastasis prevention after HCC resection.

MiR-612 regulates invadopodia of hepatocellular carcinoma by HADHA-mediated lipid reprogramming.

BACKGROUND: MicroRNA-612 (miR-612) has been proven to suppress EMT, stemness, and tumor metastasis of hepatocellular carcinoma (HCC) via PI3K/AKT2 and Sp1/Nanog signaling. However, its biological roles on HCC progression are far from elucidated. METHODS: We found direct downstream target of miR-612, hadha by RNA immunoprecipitation and sequencing. To explore its biological characteristic, potential molecular mechanism, and clinical relevance in HCC patients, we performed several in-vitro and in-vivo models, as well as human tissue chip. RESULTS: Ectopic expression of miR-612 could partially reverse the level of HADHA, then suppress function of pseudopods, and diminish metastatic and invasive potential of HCC by lipid reprogramming. In detail, miR-612 might reduce invadopodia formation via HADHA-mediated cell membrane cholesterol alteration and accompanied with the inhibition of Wnt/ß-catenin regulated EMT occurrence. Our results showed that the maximum oxygen consumption rates (OCR) of HCCLM3miR-612-OE and HCCLM3hadha-KD cells were decreased nearly by 40% and 60% of their counterparts (p < 0.05). The levels of acetyl CoA were significantly decreased, about 1/3 (p > 0.05) or 1/2 (p < 0.05) of their controls, in exogenous miR-612 or hadha-shRNA transfected HCCLM3 cell lines. Besides, overexpression of hadha cell lines had a high expression level of total cholesterol, especially 27-hydroxycholesterol (p < 0.005). SREBP2 protein expression level as well as its downstream targets, HMGCS1, HMGCR, MVD, SQLE were all deregulated by HADHA. Meanwhile, the ATP levels were reduced to 1/2 and 1/4 in HCCLM3miR-612-OE (p < 0.05) and HCCLM3hadha-KD (p < 0.01) respectively. Moreover, patients with low miR-612 levels and high HADHA levels had a poor prognosis with shorter overall survival. CONCLUSION: miR-612 can suppress the formation of invadopodia, EMT, and HCC metastasis and by HADHA-mediated lipid programming, which may provide a new insight of miR-612 on tumor metastasis and progression.

miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling.

BACKGROUND: Accumulation of evidence indicates that miRNAs have crucial roles in the regulation of EMT-associated properties, such as proliferation, migration and invasion. However, the underlying molecular mechanisms are not entirely illustrated. Here, we investigated the role of miR-296-5p in hepatocellular carcinoma (HCC) progression. METHODS: In vitro cell morphology, proliferation, migration and invasion were compared between HCC cell lines with up- or down-regulation of miR-296-5p. Immunofluorescence and Western blot immunofluorescence assays were used to detect the expression of EMT markers. Bioinformatics programs, luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-296-5p. Xenograft nude mouse models were established to observe tumor growth and metastasis. Immunohistochemical assays were conducted to study the relationships between miR-296-5p expression and Neuregulin-1 (NRG1)/EMT markers in human HCC samples and mice. RESULTS: miR-296-5p was prominently downregulated in HCC tissues relative to adjacent normal liver tissues and associated with favorable prognosis. Overexpression of miR-296-5p inhibited EMT along with migration and invasion of HCC cells via suppressing NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling in vitro. More importantly, miR-296-5p disrupted intrahepatic and pulmonary metastasis in vivo. NRG1, as a direct target of miR-296-5p, mediates downstream biological responses. In HCC tissues from patients and mice, the levels of miR-296-5p and NRG1 also showed an inverse relationship. CONCLUSIONS: miR-296-5p inhibited EMT-related metastasis of HCC through NRG1/ERBB2/ERBB3/RAS/MAPK/Fra-2 signaling.

miR-106b-5p promotes stem cell-like properties of hepatocellular carcinoma cells by targeting PTEN via PI3K/Akt pathway.

BACKGROUND: The miRNA miR-106b-5p has been previously reported to be increased in hepatocellular carcinoma (HCC) tissues compared to cirrhotic tissues. The purpose of this study was to detect its expression in HCC cell lines with distinct metastatic potentials and to explore the molecular mechanisms underlying HCC stemness and migration. METHODS: miR-106b-5p expression was studied in HCC tissues and cell lines. In vitro cancer stem cell (CSC)-like properties, cell migration and invasion were compared between HCC cell lines with upregulation or downregulation of miR-106b-5p. In vivo tail vein injection models were established to evaluate the role of miR-106b-5p in lung metastasis. Bioinformatics programs, luciferase reporter assay and rescue experiments were used to validate the downstream targets of miR-106b-5p. The relationship between the expression of the targeted gene and clinicopathological parameters was also analyzed. RESULTS: miR-106b-5p expression was higher in HCC tissues and cell lines than that in non-tumor tissues and hepatocyte Chang liver, respectively. Upregulation of miR-106b-5p exhibited a promoting role in CSC properties, cell migration and activation of phosphatidylinositol-3 kinase (PI3K)/Akt signaling in vitro, as well as in lung metastasis in vivo. However, downregulation of miR-106b-5p exhibited the opposite effect. Furthermore, PTEN was verified as a direct target of miR-106b-5p. Upon clinicopathological analysis, lower level of PTEN was significantly associated with more aggressive characteristics. Patients with high PTEN expression had longer overall survival and disease-free survival. CONCLUSION: miR-106b-5p promotes HCC stemness maintenance and metastasis by targeting PTEN via PI3K/Akt pathway. Inhibition of miR-106b-5p might be effective therapeutic strategies to treat advanced HCC.

Synthesis and characterization of anaerobic degradation biomarkers of n-alkanes via hydroxylation/carboxylation pathways.

Metabolite profiling is a powerful method in research on anaerobic biodegradation of hydrocarbons. Hydroxylation and carboxylation are proposed pathways in anaerobic degradation but very little direct evidence is available about metabolites and signature biomarkers. 2-Acetylalkanoic acid is a potential signature metabolite because of its unique and specific structure among possible intermediates. A procedure for the synthesis of four homologues with various carbon chain lengths was proposed and the characteristics of 2-acetyl- alkanoic acid esters were investigated using four derivatization processes, namely methyl, ethyl, n-butyl and trimethylsilyl esterification. Four intermediate fragments observed were at m/z 73 + 14n, 87 + 14n, 102 + 14n (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester, respectively) and [M - 42]+ for three of the derivatization methods. For silylation, characteristic ions were observed at m/z 73, 117, [M - 42](+) and [M - 55](+). These are basic and significant data for the future identification of potential intermediates of the hydroxylation and carboxylation pathways in hydrocarbon degradation.

Insights into the Anaerobic Biodegradation Pathway of n-Alkanes in Oil Reservoirs by Detection of Signature Metabolites.

Anaerobic degradation of alkanes in hydrocarbon-rich environments has been documented and different degradation strategies proposed, of which the most encountered one is fumarate addition mechanism, generating alkylsuccinates as specific biomarkers. However, little is known about the mechanisms of anaerobic degradation of alkanes in oil reservoirs, due to low concentrations of signature metabolites and lack of mass spectral characteristics to allow identification. In this work, we used a multidisciplinary approach combining metabolite profiling and selective gene assays to establish the biodegradation mechanism of alkanes in oil reservoirs. A total of twelve production fluids from three different oil reservoirs were collected and treated with alkali; organic acids were extracted, derivatized with ethanol to form ethyl esters and determined using GC-MS analysis. Collectively, signature metabolite alkylsuccinates of parent compounds from C1 to C8 together with their (putative) downstream metabolites were detected from these samples. Additionally, metabolites indicative of the anaerobic degradation of mono- and poly-aromatic hydrocarbons (2-benzylsuccinate, naphthoate, 5,6,7,8-tetrahydro-naphthoate) were also observed. The detection of alkylsuccinates and genes encoding for alkylsuccinate synthase shows that anaerobic degradation of alkanes via fumarate addition occurs in oil reservoirs. This work provides strong evidence on the in situ anaerobic biodegradation mechanisms of hydrocarbons by fumarate addition.

Synthesis of anaerobic degradation biomarkers alkyl-, aryl- and cycloalkylsuccinic acids and their mass spectral characteristics.

Anaerobic biodegradation of petroleum hydrocarbons has been reported to proceed predominantly via fumarate addition to yield substituted succinate metabolites. These metabolites, commonly regarded as signature biomarkers, are specific indicators of anaero- bic hydrocarbon degradation by microbial activity. To the best of our knowledge, mass spectrometry information for 2-(1-methylalkylj succinic acids, 2-arylsuccinic acids, 2-cycloalkylsuccinic acids and/or their derivatives is still incomplete, especially for the analysis of environmental samples. Here, a novel approach is proposed for the successful synthesis of five hydrocarbon-derived succinic acids. The characteristic fragments of 2-[1-methylalkyllsuccinic acid diesters were investigated by four derivatization processes (methyl, ethyl, n-butyl and trimethylsilyl esterification], some of which are not available in official Libraries. Under electron ionization mass spec- trometry conditions, informative fragments of various molecular masses have been obtained. Results confirmed characteristic differ- ences among the derivatization processes of the chemically synthesized compounds. In the case of 2-[cyclo)alkylsuccinate esters, four intermediate fragments were observed at m/z 114 + 14n, 118 + 28n, [M - [17 + 14n1]]+ and [M - (59 + 14n)]+ (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester]. However, for silylation the abundant fragment ions are at m/z 262, 217, 172, 147, 73 and [M - 15]+. These data provide information for the identification of hydrocarbon-derived succinic acids as anaerobic biodegradation intermediates in hydrocarbons- rich environments.