GPNMB+ Gal-3+ hepatic parenchymal cells promote immunosuppression and hepatocellular carcinogenesis.

Hepatocellular carcinoma (HCC) formation is a multi-step pathological process that involves evolution of a heterogeneous immunosuppressive tumor microenvironment. However, the specific cell populations involved and their origins and contribution to HCC development remain largely unknown. Here, comprehensive single-cell transcriptome sequencing was applied to profile rat models of toxin-induced liver tumorigenesis and HCC patients. Specifically, we identified three populations of hepatic parenchymal cells emerging during HCC progression, termed metabolic hepatocytes (HCMeta ), Epcam+ population with differentiation potential (EP+Diff ) and immunosuppressive malignant transformation subset (MTImmu ). These distinct subpopulations form an oncogenic trajectory depicting a dynamic landscape of hepatocarcinogenesis, with signature genes reflecting the transition from EP+Diff to MTImmu . Importantly, GPNMB+ Gal-3+ MTImmu cells exhibit both malignant and immunosuppressive properties. Moreover, SOX18 is required for the generation and malignant transformation of GPNMB+ Gal-3+ MTImmu cells. Enrichment of the GPNMB+ Gal-3+ MTImmu subset was found to be associated with poor prognosis and a higher rate of recurrence in patients. Collectively, we unraveled the single-cell HCC progression atlas and uncovered GPNMB+ Gal-3+ parenchymal cells as a major subset contributing to the immunosuppressive microenvironment thus malignance in HCC.

A Low-Current and Multi-Channel Chemiresistor Array Sensor Device.

This paper describes the design of a low-current, multichannel, handheld electronic device integrated with nanostructured chemiresistor sensor arrays. A key design feature of the electronic circuit board is its low excitation current for achieving optimal performance with the arrays. The electronics can rapidly acquire the resistances for different sensors, not only spanning several orders of magnitude, but also as high as several hundreds of megaohms. The device tested is designed using a chemiresistor array with nanostructured sensing films prepared by molecularly-mediated assemblies of gold nanoparticles for detection. The low-current, wide-range, and auto-locking capabilities, along with the effective coupling with the nanostructured chemiresistor arrays, meet the desired performances of a low excitation current and low power consumption, and also address the potential instability of the sensors in a complex sensing environment. The results are promising for potential applications of the device as a portable sensor for the point-of-need monitoring of air quality and as a biosensor for point-of-care human breath screening for disease biomarkers.

Prognostic value of tumor stromal collagen features in patients with hepatocellular carcinoma revealed by second-harmonic generation microscopy.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide. The search for new biomarkers that predict the outcome of HCC patients is ongoing. We propose the second harmonic generation-based quantitative assessment approach to evaluate the prognostic value of tumor stromal collagen in HCC. MATERIALS AND METHODS: We evaluated tumor stromal collagen in paraffin-embedded specimens from 109 HCC patients by second-harmonic generation imaging. The parameters and quantitative assessment of collagen were obtained using a fiber network extraction algorithm. The relationships between collagen features and clinical pathological features and overall survival were statistically analyzed. RESULT: Among the collagen features, some parameters of aggregated collagen correlated well with clinical pathological features, especially the aggregated collagen cross-linked density. Cross-linked collagen fibers form a fiber network in moderately and poor differentiated HCCs. Kaplan-Meier analyses and the multivariate Cox proportional hazard model showed that high aggregated collagen cross-linked density was associated with poor overall survival. The chi-squared test showed that aggregated cross-link density was significantly associated with histological grade and tumor recurrence. CONCLUSION: Our results indicate the prognostic value of the quantitative evaluation of tumor stromal collagen using second harmonic generation imaging of patients with HCC.

Targeted inhibition of ACK1 can inhibit the proliferation of hepatocellular carcinoma cells through the PTEN/AKT/mTOR pathway.

Activated Cdc42-associated kinase 1 (ACK1) expression is upregulated in hepatocellular carcinoma (HCC) tissues and other tumour tissues. However, the function and regulatory mechanism of ACK1 in HCC remains unclear. In this study, the expression of pTyr284-ACK1, pSer473-AKT and PTEN in HCC was detected by immunohistochemistry, and its clinicopathological significance was analysed. Then, ACK1-targeted small molecule inhibitors AIM-100 and Dasatinib were used to treat cells SK-Hep-1 and HepG2, and changes in activity and biological behaviours of PTEN/AKT/mTOR signalling pathway were observed. The results showed that pTyr284-ACK1 protein was highly expressed in HCC tissues and was related to the poor prognosis of patients; the expression of pTyr284-ACK1 protein was positively correlated with pSer473-AKT and negatively correlated with PTEN. In addition, after treatment either with AIM-100 or Dasatinib, both proliferation of two cells and migration, invasion of SK-Hep-1 cells were all significantly inhibited. Meanwhile, ACK1, pTyr284-ACK1, pSer473-AKT, mTOR and EGFR were down-regulated; PTEN was up-regulated when analysed by western-blot in SK-Hep-1 cells. These results demonstrated that ACK1 may promote HCC development via PTEN/AKT/mTOR pathway. Targeted inhibition of ACK1 may be a novel therapeutic strategy for HCC. SIGNIFICANCE OF THE STUDY: Hepatocellular carcinoma (HCC) is a common malignant tumour with high mortality. Our study showed that ACK1 and pTyr284-ACK1 are highly expressed in HCC and may promote HCC development through the PTEN/AKT/mTOR signalling pathway. Targeted inhibition of ACK1 expression with small inhibitors AIM-100 and Dasatinib may weaken tumour cells ability of proliferation, migration and invasion. Our results suggested that downregulation of ACK1 may be a potential therapeutic strategy for HCC.

Effects of IL-10- and FasL-overexpressing dendritic cells on liver transplantation tolerance in a heterotopic liver transplantation rat model.

Acute rejection is the major determinant for the long-term survival of donor liver after liver transplantation (LT). The aim of this study was to examine the therapeutic potential of interleukin (IL)-10-FasL-overexpressing immature dendritic cells (imDCs) to induce local immunosuppression in liver grafts. imDCs derived from donors were transduced by lentiviral vectors expressing human IL-10 and/or Fas ligand (FasL) gene(s), and the expression of surface molecules and the ability to induce T-cell proliferation were measured. imDCs were intraperitoneally injected into recipient rats as a model of LT to examine the rejection grade [Banff rejection activity index (RAI)], liver functions [Alanine aminotransferase, Aspartate aminotransferase (AST) and total bilirubin (TBIL)] and post-transplant survival. IL-10 and FasL co-transduction of imDCs induced a greater reduction in CD80, CD86 and major histocompatibility complex class II (MHC II) expression, as well as T-cell proliferation, but increased levels of IL-10 and FasL in culture supernatants compared with mono-transduced or untransduced imDCs (P < 0.05). The infusion of co-transduced imDCs in LT recipients reduced RAI scores, decreased plasma AST and TBIL, and prolonged survival compared with mono-transduced or untransduced imDC-treated liver allografts. These findings demonstrated that the transfusion of IL-10-FasL/imDCs enhanced immune tolerance and prolonged the survival of liver allografts after LT. The immunomodulatory activity of IL-10- and FasL-modified imDCs might be a new therapeutic approach to prevent organ rejection in clinical transplantation.

Tumor-infiltrating CD8+ T cells combined with tumor-associated CD68+ macrophages predict postoperative prognosis and adjuvant chemotherapy benefit in resected gastric cancer.

BACKGROUND: Tumor-infiltrating immune cells are present in various malignant tumors, but their clinical significance in gastric cancer (GC) remains unclear. This study aimed to investigate the prognostic significance of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). METHODS: Using a prospective database containing 401 cases of GC, we evaluated TIL (cluster of differentiation 8 (CD8) expression) and TAM (cluster of differentiation 68 (CD68) expression) statuses via immunohistochemical staining. RESULTS: Compared with CD8+ TIL-negative cases (n = 196, 48.6%), CD8+ TIL-positive cases (n = 205, 51.1%) showed significantly better recurrence-free survival (RFS) [log-rank p<0.001; multivariate HR: 0.372; 95% confidence interval (CI): 0.239-0.579, p<0.001]. In contrast, compared with CD68+ TAM-negative cases (n = 217, 54.1%), CD68+ TAM-positive cases (n = 184, 45.9%) had significantly poor RFS [log-rank p<0.001; multivariate HR: 2.182; 95% CI: 1.435-3.318, p<0.001]. Thus, patients with a positive CD8+ TIL and negative CD68+ TAM status exhibited significantly increased RFS. Multivariate analysis demonstrated that CD8+ TILs and CD68+ TAMs may serve as independent prognostic markers for RFS. Incorporating CD8+ TIL and CD68+ TAM statuses into the AJCC TNM system generated a predictive model with better predictive accuracy for RFS. More importantly, patients with a positive TIL and negative TAM status showed a tendency of improved RFS after postoperative adjuvant chemotherapy (PAC). Similar results were obtained by overall survival (OS) analysis. CONCLUSIONS: CD8+ TIL and CD68+ TAM statuses were identified as independent prognostic factors that may be integrated into the current TNM staging system to refine risk stratification and to better predict the survival benefit from PAC in patients with GC. TRIAL REGISTRATION: The current controlled trial was registered at ClinicalTrials.gov (ID: NCT02327481 ) on December 30, 2014.

Somatostatin Octapeptide Inhibits Cell Invasion and Metastasis in Hepatocellular Carcinoma Through PEBP1.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a major threat to human health. The condition carries a high risk of death; 45% of new cases occur in China. Surgical resection is the first choice for treatment of HCC, but 30.9% of patients experience recurrence within 6 months after the operation. To improve patient survival, we must determine how to reduce the probability of recurrence and metastasis and elucidate the underlying mechanism of disease. We therefore studied the effect of somatostatin octapeptide (octreotide) on the invasion and metastasis of HCC. METHODS: The migration and invasion cytological tests were used to detect the effect of octreotide on liver cancer cells (SK-Hep-1 and HepG2). PEBP1 RNAi was used to knockdown expression. Invasion and metastasis were measured with transwell migration and wound-healing assays. Western blotting was used to detect changes in levels of PEBP1 and invasion pathway proteins after octreotide treatment. The effect of octreotide was studied in vivo by establishing a pulmonary metastasis model using SK-Hep-1 cells in nude mice. In-vivo bioluminescence imaging and hematoxylin and eosin staining of lung tissue were used to verify the results. RESULTS: Increasing concentrations of octreotide were progressively more effective in halting the invasion and metastasis of liver cancer cells. Octreotide may upregulate PEBP1, TIMP-2, and E-cadherin while downregulating MMP-2 and Twist to inhibit cell invasion and metastasis. And downregulation of PEBP1 would also change the expression of MMP-2, TIMP-2 and Twist. The in-vivo experiments showed no cancer cell metastasis in 4 of the 6 mice in the octreotide-treatment group, while all of the mice in the control group displayed pulmonary metastasis of human HCC cells. And the survival period of the mice in the octreotide-treatment group was significantly prolonged. CONCLUSIONS: Octreotide may weaken invasion and metastasis through the upregulation of PEBP1. Octreotide may reduce the risk of recurrence and metastasis after surgery for liver cancer.

Effects of sorafenib on lung metastasis in rats with hepatocellular carcinoma: the role of microRNAs.

Many patients with advanced hepatocellular carcinoma (HCC) develop lung metastasis and available treatments are limited. The anticancer drug sorafenib has opened a window of hope for patients with advanced hepatocellular carcinoma. However, the effect of sorafenib is limited by drug resistance. MicroRNAs have been reported to play an important role in HCC, but the effect of sorafenib on microRNAs (miRNAs) and on lung metastasis is not clear. This report employed a high-throughput deep sequencing technique to detect the difference of miRNAs and immunohistochemical technique to detect the difference of protein in implanted primary tumors and in metastatic HCC tumors after treatment with sorafenib. Among the detected miRNAs, we identified rno-miR-122-3p and rmo-miR-122-5p that were downregulated and rno-miR-383-5p and rno-miR-34a-5p that were upregulated and one novel miRNAs is reported as downregulated here for the first time. Immunohistochemical analysis of known miRNAs identified CMYC protein expression as inhibited, MDM2 protein was expressed, and NM23 and GST protein were upregulated. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of novel miRNA found that the targeted genes were concentrated in pathways of metabolism, especially in cytochrome P450. These results indicate that these miRNAs are likely to be involved in the treatment response of lung metastases of HCC to sorafenib. They may be useful as biomarkers to predict the clinical treatment response of sorafenib.

Galectin-4 serves as a prognostic biomarker for the early recurrence / metastasis of hepatocellular carcinoma.

Galectin-4 is a multifunctional lectin found at both intracellular and extracellular sites. It could serve as a tumor suppressor intracellularly and promote tumor metastases extracellularly during colorectal cancer development. However, galectin-4 expression and its prognostic value for patients with hepatocellular carcinoma (HCC) have not been well investigated. Here we report that galectin-4 was significantly downregulated in early recurrent/metastatic HCC patients, when compared to non-recurrent/metastatic HCC patients. Low expression of gelectin-4 was well associated with larger tumor size, microvascular invasion, malignant differentiation, more advanced TNM stage, and poor prognosis. Cancer cell migration and invasion could be significantly reduced through overexpression of galectin-4, but upregulated by knocking down of galectin-4 in vitro. Moreover, the serum galectin-4 level could be significantly elevated solely by hepatitis B virus infection. Combined with clinicopathological features, the higher serologic level of galectin-4 was well associated with more aggressive characteristics of HCC. Taken together, galectin-4 expression closely associates with HCC progression and might have potential use as a prognostic biomarker for HCC patients.

Cotransfection with IL-10 and TGF-ß1 into immature dendritic cells enhances immune tolerance in a rat liver transplantation model.

Dendritic cells transfected with interleukin (IL)-10 and transforming growth factor-ß1 (TGF-ß1) enhance T cell immunity and tolerance. However, no quantitative studies have investigated the suppressive functions of immature dendritic cells (imDC) cotransfected with IL-10 and TGF-ß1. The effects of imDC cotransfected with IL-10 and TGF-ß1 (IL-10-TGF-ß1-imDC) on immune tolerance induction in a rat transplantation model were investigated. In addition, effects of IL-10-TGF-ß1-imDC relative to IL-10-transfected imDC (IL-10-imDC) and TGF-ß1-transfected imDC (TGF-ß1-imDC) were compared. The infusion of IL-10-TGF-ß1-imDC into recipients prolonged liver graft survival, which was sustained for >90 days. IL-12 serum levels decreased, whereas alanine transaminase and total bilirubin slightly increased in rats infused with IL-10-TGF-ß1-imDC compared with the IL-10-imDC and TGF-ß1-imDC groups. Furthermore, a higher percentage of terminal transferase-mediated UTP nick end-labeling-positive cells was observed, and histological analysis of the allografts indicated a rejection activity index of mild acute rejection. Our results suggest infusion of IL-10 and TGF-ß1 cotransfected imDC induces alloantigen-specific T cell hyporesponsiveness, inhibits antigen-specific immunological responses to liver allografts, prolongs liver allograft survival, and enhances the immune tolerance. This approach may provide a promising alternative for enhancing donor-specific tolerance during liver transplantation.

Quantitative proteomics analysis of early recurrence/metastasis of huge hepatocellular carcinoma following radical resection.

BACKGROUND: Hepatic resection is the preferred treatment for huge hepatocellular carcinoma (>10 cm in diameter; H-HCC). However, the patients with H-HCC suffer from poor prognosis due to the early recurrence/metastasis. The underlying mechanism of H-HCC's early recurrence/metastasis is currently not well understood. RESULTS: Here, we describe an Isobaric Tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics approach to analyze the early recurrence/metastasis related proteins of H-HCC after radical resection through multidimensional chromatography coupled with tandem mass spectrometry (2DLC-MS/MS). The different protein expression profiles between the early recurrence/metastasis within 6 months(R/M≤6months) and late recurrence/metastasis within 6-12 months after surgery (R/M6-12months) were confirmed and might reveal different underlying molecular mechanisms. We identified 44 and 49 significantly differentially expressed proteins in the R/M≤6months group and the R/M6-12months group compared to the group who had no recurrence within 2 years post surgery (the NR/M group), respectively. Moreover, among those proteins, S100A12 and AMACR were down regulated in the R/M≤6months group but up-regulated in the R/M6-12months group; and this regulation was further confirmed in mRNA and protein level by Q-PCR, Western-Blot and Immunohistochemistry (IHC). CONCLUSIONS: This current study presents the first proteomic profile of the early recurrence/metastasis of H-HCC. The results suggest that S100A12 and AMACR might be potential prognostic markers for predicting the early recurrence/metastasis of H-HCC after hepatectomy.

Decreased expression of liver-type fatty acid-binding protein is associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND/AIMS: The purpose of this study was to assess liver-type fatty acid-binding protein (L-FABP) expression and its association with clinicopathological features in hepatocellular carcinoma (HCC). METHODOLOGY: L-FABP mRNA expression in 57 samples of HCC and corresponding adjacent liver tissue and 8 normal liver tissue samples were examined by real-time reverse transcriptase (RT)­PCR analyses. Tissue microarray technique and immunohistochemistry (IHC) were used to detect the expression of L-FABP in 163 HCCs. The association between L-FABP expression and the clinicopathological factors and prognosis was analyzed. RESULTS: The average expression of L-FABP mRNA was 0.233 in the HCC tissues, 1.407 in the peri-carcinoma tissues, and 1.0 in the normal liver tissues. IHC analysis showed that there were 47% (76/163) HCCs exhibited weak or even no immunoreactivity of L-FABP. The L-FABP expression in HCC showed significant associations with preoperative levels of AFP (p=0.039), tumor size (p=0.026), histological grade (p=0.000), differential degree (p=0.000), vascular invasion (p=0.016), capsular invasion (p=0.029) and recurrence (p=0.004). Patients with L-FABP high-expression showed better prognosis than patients with L-FABP low-expression (p=0.008). CONCLUSIONS: L-FABP was downregulated in HCC and could be served as a promising prognostic marker for HCC patients.

Expression and effect of miR­27b in primary liver cancer.

The occurrence and development of primary liver cancer is associated with microRNA. Specifically, the expression of microRNA-27b (miR-27b) is upregulated in four liver cancer drug-resistance cell lines. Despite that, the function of miR-27b in liver cancer is not clear yet. The aim of the present study was to investigate the effect of miR-27b expression during oncogenesis, cell proliferation, apoptosis and chemotherapy resistance development in a model of liver cancer. Expression of miR-27b was detected with reverse transcription-quantitative PCR. To establish stable overexpression of miR-27b and negative control liver cancer cell lines, a lentiviral pre-miR-27b overexpression vector and negative control vector were transfected into each cell line. Cell Counting Kit-8 assay, clone formation assay and immunohistochemical assay were used to detect cell proliferation. Apoptosis and drug sensitivity were detected by flow cytometry and MTT assay, respectively. The expression level of miR-27b in liver cancer tissues was also lower than in liver tissues adjacent to the tumor. Two stable miR-27b overexpression liver cancer cell lines (Huh-7/miR-27b and HepG2/miR-27b) and their control cell lines (Huh-7/NC and HepG2/NC) were successfully constructed. It was revealed that upregulation of miR-27b can suppress cell proliferation, promote cell apoptosis and chemotherapy resistance. In addition, the findings of the present study demonstrated that patients with cirrhosis expressed lower miR-27b compared with patients without cirrhosis. The expression level of miR-27b was significantly associated with the age, serum alpha-fetoprotein and alanine aminotransferase level of patients with liver cancer. Meanwhile, it was indicated that the disease survival time of the low miR-27b expression group was longer than that of the high miR-27b expression group. The present study suggested that miR-27b functions as a liver cancer suppressor. Moreover, miR-27b can act as a biomarker to estimate drug sensitivity to chemotherapy in patients with liver cancer.

Fusion of NY-ESO-1 epitope with heat shock protein 70 enhances its induced immune responses and antitumor activity against glioma in vitro.

Background: Glioma is the most common tumor originating in the brain and is difficult to cure. New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a promising cancer testis antigen (CTA) for tumor immunotherapy, and heat shock proteins (HSPs) can promote the antigen presentation of chaperoned peptides. This study investigates the therapeutic potential of HSP70 and NY-ESO-1 epitope fusion protein for glioma. Methods: Recombinant HSP70 protein was purified and fused to NY-ESO-1 epitope to generate HSP70/NY-ESO-1 p86-94. NY-ESO-1 expression was induced in U251 glioma cells via 5-Aza-2'-deoxycytidine (5-Aza-CdR) treatment. Dendritic cells (DCs) loaded with HSP70/NY-ESO-1 p86-94 or NY-ESO-1 protein stimulated NY-ESO-1-specific cytotoxic T lymphocytes (CTLs). The killing effect of NY-ESO-1 specific CTLs on U251 cells was detected by lactate dehydrogenase (LDH). Results: 5-Aza-CdR successfully induced NY-ESO-1 expression in U251 cells. NY-ESO-1-stimulated CTLs lysed more significantly with NY-ESO-1-positive U251 cells than with NY-ESO-1-negative cells. The immune response stimulated by a DC-based vaccine of HSP70/NY-ESO-1 p86-94 fusion protein was significantly enhanced compared with that induced by NY-ESO-1 alone. Conclusions: These findings indicate that the HSP70/NY-ESO-1 p86-94 may significantly enhance CTLs-mediated cytotoxicity and targeting ability against NY-ESO-1-expressing tumors in vitro. 5-Aza-CdR treatment with HSP70 binding to tumor antigen is a new strategy for immunotherapy of the tumors with poor CTA expression.

A novel aptasensor for the ultra-sensitive detection of adenosine triphosphate via aptamer/quantum dot based resonance energy transfer.

We designed a novel aptamer based biosensor (aptasensor) for ultrasensitive detection of adenosine triphosphate (ATP) through resonance energy transfer (RET). The ATP aptamer was modified with Cy3 at the 3' end, and a green quantum dot (525) was attached to the 5' end of its complementary sequence respectively. The ATP aptamer and its complementary sequence could assemble into a duplex structure in the absence of target ATP, and then decrease the distance between the quantum dot and Cy3 which could produce significant RET signal. Upon ATP binding, the ATP aptamer could dissociate with its complementary sequence and then increase the distance between the quantum dot and Cy3 which would significantly decrease the RET signal. Therefore, the ATP detection could be easily achieved through detection of the fluorescence intensity ratio between 525 nm and 560 nm. The results show that the emission fluorescence intensity ratio of 525/560 is linearly related to the logarithmic concentration of ATP. The linear range of this aptasensor is from 0.1 nM to 1 µM, and the detection limit is lower down to 0.01 nM. Excellent selectivity of this aptasensor for ATP has been demonstrated through the detection of thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and adenosine diphosphate (ADP) respectively as control. The method we described here could easily detect ATP with excellent selectivity, linearity and sensitivity down to the nanomolar range, as well as avoid photobleaching.

A mathematical model to predict short-term recurrence and metastasis of primary hepatocellular carcinoma larger than 10 cm in diameter.

BACKGROUND/AIMS: To construct a mathematical model to predict short-term recurrence and metastasis of primary hepatocellular carcinoma (HCC) larger than 10 cm in diameter after radical resection and analyze the influencing factors. METHODOLOGY: A total of 166 patients with primary HCC larger than 10 cm were recruited from January 2002 to December 2010. Univariable and multivariable logistic regression analyses were performed and a mathematic model predicting the recurrence of HCC at different time points was constructed. RESULTS: Univariable analysis showed preoperative serum AFP of ≥1210 ng/mL (X4), intraoperative macroscopic tumor thrombus (X7) and postoperative pathological grade (X11) were related to the recurrence within 6 months after surgery. Preoperative serum AFP of ≥1210 ng/mL (X4) and microscopic tumor thrombus (X10) were associated with the recurrence within 6-12 months after surgery. Multivariable analysis revealed the risk factors of recurrence within 6 months and 6-12 months after surgery included preoperative serum AFP of ≥1210 ng/mL (X4) and intra-operative macroscopic tumor thrombus (X7); those within 6-12 months after surgery were preoperative serum AFP of ≥1210 ng/mL (X4) and microscopic tumor thrombus (X10). CONCLUSIONS: Preoperative serum AFP and blood vessel involvement are risk factors of short-term recurrence/metastasis of huge HCC after surgery. The equation for prediction of HCC recurrence at different time points varies.

[Morphology and microRNA expression profiles of drug-resistant cells in hepatocellular carcinoma].

OBJECTIVE: To compare morphological differences of three drug-resistant hepatocellular carcinoma (HCC) cell subclones (Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) and their parental Huh-7 cell line, to analyze differential microRNA (miRNA) expression profiles in these cells and, finally to screen for the abnormal expressed miRNAs in drug-resistant HCC cells. METHODS: Cellular morphology was observed by histology and transmission electron microscopy. MiRNA microarray was used to analyze the differential miRNA expression profiles in these cells (Huh-7, Huh-7/ADM, Huh-7/CBP, Huh-7/MMC) followed by real time quantitative PCR validation. RESULTS: The drug-resistant cells had more intracytoplasmic organelles and were larger in size along with increased cytological pleomorphism than the parental Huh-7 cells. Compared with the parental Huh-7 cells, 32 simultaneously up-regulated and 22 down-regulated miRNAs were found in three drug-resistant cells. Up-regulation of miR-15a, miR-16, miR-27b, miR-30b, miR-146a, miR-146b-5p, miR-181a, miR-181d and miR-194 was verified by RT-qPCR. CONCLUSION: Drug-resistant HCC cells have abnormal expressed miRNAs, which may be explored to further investigate the association of miRNA expressions with multidrugs resistance in HCC.

Evaluation of transcriptomic molecular classification, biological behavior, and clinicopathological features in hepatocellular carcinoma.

BACKGROUND: Studies in France, Korea, and Singapore found that G1-G6 transcriptomes are involved in hepatocellular carcinoma (HCC) carcinogenesis. However, the suitability of this method in Chinese HCC patients has remained unknown. METHODS: The correlation between the G1-G6 molecular classification and clinicopathological features were analyzed in 107 Chinese HCC patients through the retrospective cohort study. RNA sequencing and bioinformatics analysis were performed to screen related targets and molecular signaling pathways. RESULTS: We found that the G1-G3 subgroups were associated with high serum alpha-fetoprotein (AFP) level, high copy number of hepatitis B virus (HBV) DNA, complex histopathological structure, macrovascular invasion. The G1 subgroup was mainly related to liver cancer stemness, and G3 subgroup showed the worst prognosis. The G5 and G6 subgroups were associated with activation of the Wnt/ß-catenin pathway. Compared with the G4-G6 group, the G1-G3 group showed significantly higher expression levels of regenerating family member 1 beta (REG1B), regenerating family member 3 gamma (REG3G), and inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and enriched calcium signaling pathway. CONCLUSIONS: This study enhances our understanding of the heterogenicity of China HCC and indicates that the G1-G6 signatures can be used to identify predictive biomarkers against HCC patients in China.

Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum.

In this study, a laccase-like gene from Thermomicrobium roseum DSM 5159 (TrLac-like) (NCBI: WP_012642205.1) was recombinantly expressed in Bacillus subtilis WB600. The optimum temperature and pH for TrLac-like were 50 °C and 6.0, respectively. TrLac-like showed high tolerance to mixed systems of water and organic solvents, indicating its potential for large-scale application in various industries. It showed 36.81 % similarity with YlmD from Geobacillus stearothermophilus (PDB:6T1B) in sequence alignment; therefore, 6T1B was employed as the template for homology modeling. To improve catalytic efficiency, amino acid substitutions within 5 Å of the inosine ligand were simulated to reduce the binding energy and promote substrate affinity. Single and double substitutions (44 and 18, respectively) were prepared, and the catalytic efficiency of the mutant A248D was increased to approximately 110-fold that of the wild type, while the thermal stability was maintained. Bioinformatics analysis revealed that the significant improvement in catalytic efficiency could be attributed to the formation of new hydrogen bonds between the enzyme and substrate. With a further decrease in the binding energy, the catalytic efficiency of the multiple mutant H129N/A248D was approximately 14-fold higher than that of the wild type but lower than that of the single mutant A248D. This is possibly because kcat also decreased with the decrease of Km; consequently, the substrate could not be released in time owing to the enzyme with the combination mutation not being able to release the substrate at a high rate.

Investigation of hub ferroptosis-related genes and the immune landscape in recurrent pregnancy loss and unexplained infertility using bioinformatics analysis.

Background: Recurrent pregnancy loss (RPL) and unexplained infertility (UI) are common pregnancy disorders that affect women's physical and mental health and lack effective treatment. Endometrial factors are one factor that leads to RPL. The latest research indicates that ferroptosis and immunity are closely related to the normal physiological function of the endometrium and may play a role in the pathogenesis of RPL and UI. Therefore, the present study analyzed the relationship between ferroptosis genes and immune infiltration in RPL and UI. Methods: We downloaded the GSE165004 dataset and analyzed differences in ferroptosis-related genes (FRGs) between RPL and UI patients and healthy controls. Hub differentially expressed ferroptosis-related genes (DE-FRGs) were screened using the LASSO algorithm, the SVM-RFE algorithm and the protein-protein interaction (PPI) network. Differences in immune infiltration between healthy endometrium and RPL and UI endometrium was analyzed, and the relationship between hub DE-FRGs and immune cell infiltration was examined. Results: We extracted 409 FRGs and identified 36 up-regulated and 32 down-regulated DE-FRGs in RPL and UI. Twenty-one genes were screened using the LASSO regression algorithm, and 17 genes were screened using the SVM-RFE algorithm. We intersected the LASSO genes, SVM-RFE genes and PPI network proteins to obtain 5 hub DE-FRGs. Gene set enrichment analysis (GSEA) functional enrichment analysis results indicated that the cytokine-cytokine receptor interaction signaling pathway was the common pathway for hub DE-FRGs. T follicular helper cells were highly infiltrated in RPL and UI, and M1 and M2 macrophages were highly infiltrated. The expression levels of MAPK1 and RELA positively correlated with T follicular helper cells. Conclusions: Ferroptosis-related genes may disrupt endometrial functions and signaling pathways and lead to the occurrence of RPL and UI.