Bispecific GPC3/PD­1 CAR­T cells for the treatment of HCC.

Constantly stimulated by the tumor microenvironment (TME), programmed death 1 (PD­1) is elevated, and it interacts with PD ligand 1 (PD­L1), rendering chimeric antigen receptor (CAR)­T cells dysfunctional. Hence, CAR­T cells immune to PD­1­induced immunosuppression were constructed to improve the function of CAR­T cells in hepatocellular carcinoma (HCC). Double­target CAR­T cells, targeting glypican­3 (GPC3) [a tumour-associated antigen (TAA)] and hindering PD­1­PD­L1 binding, were established. The expression of GPC3, PD­L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR­T cells were determined using lactate dehydrogenase release assay, enzyme­linked immunosorbent assay, and flow cytometry, respectively. HCC cells were targeted and eliminated by double­target CAR­T cells. These double­target CAR­T cells limit PD­1­PD­L1 binding and sustain cytotoxicity to PD­L1+ HCC cells. The relatively low IR expression and differentiation level in double­target CAR­T cells in tumour tissues induced tumour­suppression and extended survival in PD­L1+ HCC TX models, as opposed to their single­target counterparts. The results of the present study suggested that the newly constructed double­target CAR­T cells exhibit stronger tumour­suppressing effects in HCC than their single­target counterparts, which are common, suggesting the potential of strengthening CAR­T cell activity in HCC treatment.

Silencing of TMED5 inhibits proliferation, migration and invasion, and enhances apoptosis of hepatocellular carcinoma cells.

BACKGROUND: Transmembrane P24 trafficking protein 5 (TMED5) is highly expressed in cervical and bladder cancer cell lines. Moreover, TMED5 promotes nuclear autophagy and the malignant behavior of cervical cancer cells. However, the role of TMED5 in hepatocellular carcinoma (HCC) has not been extensively reported. OBJECTIVES: To investigate the role of TMED5 in HCC cells. MATERIAL AND METHODS: Bioinformatics was used to analyze the messenger-ribonucleic acid (mRNA) expression of TMED5 in HCC and its relationship with overall survival and disease-free interval of HCC patients. After TMED5 was decreased in SMMC-7721 and Hep3B cells, they were assayed for proliferation, cell cycle, apoptosis, migration, and invasion. RESULTS: The expression of TMED5 mRNA in HCC tissues was higher than in adjacent normal tissues, and the overall survival of HCC patients with high TMED5 transcription levels was worse. Moreover, the overexpression of TMED5 was associated with HCC progression. The downregulation of TMED5 suppressed cell proliferation, migration and invasion, and enhanced apoptosis. Therefore, TMED5 may be involved in the regulation of the cell cycle, the mammalian target of rapamycin signaling pathway, and the transforming growth factor beta (TGF-ß) signaling pathway. CONCLUSIONS: The TMED5 has the potential to promote HCC progression. Therefore, lowering TMED5 levels could represent a potential strategy for the treatment of HCC.

Anti-aging effect of ß-carotene through regulating the KAT7-P15 signaling axis, inflammation and oxidative stress process.

BACKGROUND: Research on aging is growing as the elderly make up a greater share of the population, focusing on reversing and inhibiting the aging process. The exhaustion and senescence of stem cells are the fundamental drivers behind aging. ß-Carotene has been depicted to have many biological functions, and we speculate that it may have an anti-aging effect. METHODS: Firstly, the anti-aging property of ß-carotene was investigated in vitro using mesenchymal stem cells (MSCs) induced by H2O2. The anti-aging effect was characterized using Western-bloting, confocal laser scanning microscopy, indirect immunofluorescence, and immunohistochemistry. The anti-aging property was also tested in vivo using aged mice. RESULTS: The in vitro experiment revealed that ß-carotene could relieve the aging of MSCs, as evidenced by a series of aging marker molecules such as p16 and p21. ß-Carotene appeared to inhibit aging by regulating the KAT7-P15 signaling axis. The in vivo experiment revealed that ß-carotene treatment has significantly down-regulated the aging level of tissues and organs. CONCLUSIONS: In this work, we explored the anti-aging effect of ß-carotene in vivo and in vitro. The experimental results indicate that ß-carotene may be an important potential anti-aging molecule, which can be used as a drug or in functional food to treat aging in the future.

microRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3.

Programmed death ligand 1 (PD-L1) plays an essential role in the development or progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression during normal and pathophysiological events. Here, we explored the functions and detailed mechanisms of miR-378a-3p and PD-L1 in HCC progression. First, miR-378a-3p was selected by analyzing miRNA levels in two HCC Gene Expression Omnibus datasets. We found that miR-378a-3p levels exhibited a downward trend in HCC and were negatively correlated with PD-L1 levels. Additionally, a dual luciferase assay predicted that miR-378a-3p directly targets PD-L1. Moreover, the transfection of miR-378a-3p mimics into Li-7 and HuH-7 cells effectively decreased the PD-L1 mRNA and protein expression levels, and inhibited Treg differentiation in co-culture models by modulating the expression levels of certain cytokines. Furthermore, the overexpression of miR-378a-3p hindered cell proliferation and migration but facilitated apoptosis by repressing STAT3 signaling in HCC cells. In conclusion, miR-378a-3p appears to inhibit HCC tumorigenesis by regulating PD-L1 and STAT3 levels. Thus, miR-378a-3p may be a potential target for HCC therapy.

Different isoforms of growth hormone (20 kD-GH and 22 kD-GH) shows different biological activities in mesenchymal stem cell (MSC).

There are two main types of growth hormone (GH) in the circulatory system. One is 22 kD-GH, which is the predominant isoform in the circulating system, 90% GH is present as a 22 kD protein, and 10% of GH is present as a 20 kD protein. Amino acid sequences are identical between 20 kD-GH and 22 kD-GH protein, except that 20 kD-GH lacks 15 amino acid residues 32 to 46. Studies have shown that GH has many important biological effects on mesenchymal stem cells (MSCs). However, so far, the cellular characteristics of the two types of GH have not been studied in BM-MSCs. Furthermore, the biological activity of 20 kD-GH has not been explored in BM-MSCs. For this, in the current work, BM-MSCs were used as in vitro cell model. We have carried out the current research using a series of experimental techniques (such as Western-blot and indirect immunofluorescence). Firstly, we explored the cell behavior of two types of GH in the Bm-MSC model and found that they showed different biological characteristics; Secondly, we investigated the biological characteristics of 20 kD-GH and 22 kD-GH, and results showed that 22 kD-GH and 20 kD-GH exhibited different signaling profiles; Thirdly, we found that the 20 kD-GH and 22 kD-GH Gexhibited different regulatory effects on the osteogenic differentiation of BM-MSCs. The current research lays a solid foundation for further studies on the regulatory effects of GH on MSCs.

Objective to identify and verify the regulatory mechanism of DTNBP1 as a prognostic marker for hepatocellular carcinoma.

Although the overall survival of hepatocellular carcinoma (HCC) patients has been significantly improved, prognostic clinical evaluation remains a substantial problem owing to the heterogeneity and complexity of tumor. A reliable and accurate predictive biomarker may assist physicians in better monitoring of patient treatment outcomes and follow the overall survival of patients. Accumulating evidence has revealed that DTNBP1 plays functional roles in cancer prognosis. Therefore, the expression and function of DTNBP1in HCC was systematically investigated in our study. The expression and prognostic value of DTNBP1 were investigated using the data from Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) cohorts and clinical samples. A series of cellular function assays were performed to elucidate the effect of DTNBP1 on cellular proliferation, apoptosis and metastasis. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment and Protein-protein interaction (PPI) network construction were performed to screen the genes with highest interaction scores with DTNBP1. Finally, the underlying mechanism was also analyzed using Gene Set Enrichment Analysis (GSEA) and confirmed using RT-qPCR and western blotting. DTNBP1 was upregulated in many types of cancers, especially in HCC. The DTNBP1 expression levels is associated with clinicopathologic variables and patient survival status. The differential expression of DTNBP1 could be used to determine the risk stratification of patients with HCC. DTNBP1 deficiency inhibited cell proliferation and metastasis, but promoted cell apoptosis. Mechanistically, DTNBP1 regulated the cell cycle progression through affecting the expression of cell cycle-related genes such as CDC25A, CCNE1, CDK2, CDC20, CDC25B, CCNB1, and CDK1. DTNBP1, which regulates the cell cycle progression, may be used as a prognostic marker for HCC.

LINC02362 attenuates hepatocellular carcinoma progression through the miR-516b-5p/SOSC2 axis.

Hepatocellular carcinoma (HCC) is one of the most death-related cancers worldwide. Identifying cancer-associated genes and uncovering the vital molecular mechanisms of HCC progression contribute greatly to the prognosis and novel therapeutic strategies for HCC patients. Although lncRNAs have been proved to be critical modulators of various cellular processes, the functions of lncRNAs in HCC progression are just emerging. Here, we found that a long non-coding RNA (lncRNA) named LINC02362, whose biological effects have yet been unveiled in cancers, was associated with a better prognosis in patients with HCC. Gain-of-function analyses showed that LINC02362 inhibited the survival, migration, invasion and epithelial-to-mesenchymal transition (EMT) of HCC cells. Moreover, miR-516b-5p was enriched as a target of LINC02362, which functioned as a sponge to regulate the endogenous levels of miR-516b-5p. Furthermore, we confirmed that SOSC2 served as a downstream target gene which was negatively controlled by miR-516b-5p. Importantly, a series of rescue experiments indicated that the tumor-suppressive effects of LINC02362 were achieved through the modulation of the miR-516b-5p/SOSC2 axis. In summary, we identified LINC02362 as a candidate tumor-inhibitory lncRNA that might serve as a biomarker for the prognosis of HCC and a promising therapeutic agent for patients with HCC.

22-kD growth hormone-induced nuclear GHR/STAT5/CyclinD1 signaling pathway plays an important role in promoting mesenchymal stem cell proliferation.

Growth hormone (GH) exhibited the important biological activities in the mesenchymal stem cell (MSCs). However, the cellular behavior and properties of GH/GHR in MSCs remain unclear. A series of experiments (such as confocal laser scanning microscope [CLSM] and Western-blot) were performed to systematically investigate the cellular behavior of GH/GHR in MSCs, and the results showed that GH/GHR not only internalized into the cytoplasm, but also transported into the cell nuclei of MSCs. Furthermore, we studied the molecular mechanism by which GH/GHR internalized into cell, and the results indicated that clathrin plays more important role in the process of GHR internalization. More importantly, it can be found that nuclear-targeted GHR has the important biological functions in MSCs, which could promote MSCs proliferation. We further revealed the molecular mechanism by which nuclear-localized GHR regulates MSCs proliferation, and found that nuclear-targeted GHR enhanced the phosphorylation of STAT5, and the activated STAT5 initiates the transcription of CyclinD1, after which, the complex of CyclinD1 and CDK4 further phosphorylates Rb, and the activated Rb releases E2F1, the released E2F1 ultimately realizes the biological function of GH promoting cell proliferation. In short, in the current study，we used MSCs as a model to study the cellular behavior and properties of GH/GHR, and found that GH/GHR can internalize into cell cytoplasm and transport into the cell nuclei. Further work showed that nuclear GHR could drive cell proliferation via GHR/STAT5/CyclinD1 signaling pathway. The current research has laid an important foundation for further study on the regulatory effect of GH on MSCs.

Uridine alleviates carbon tetrachloride-induced liver fibrosis by regulating the activity of liver-related cells.

At present, liver fibrosis is a major challenge of global health. When hepatocyte regeneration cannot compensate for hepatocyte death, it will develop into liver fibrosis in chronic liver disease. Initially, collagen produced by myofibroblasts plays a role in maintaining liver integrity, but excessive collagen accumulation can inhibit the residual liver function, leading to liver failure. At present, many scientists are actively looking for drugs to alleviate liver fibrosis. In the current study, we investigated the potential role of uridine in the treatment of liver fibrosis (uridine is a plant/animal-derived pyrimidine nucleoside, therefore uridine can also be ingested and absorbed by the body, accompanied by the process of food intake). For this, we systematically studied the effect of uridine on CCl4-induced liver fibrosis in vitro and in vivo through a series of technologies, such as Western blot, laser confocal scanning microscope, ELISA and immunohistochemistry. The experimental results showed that uridine can effectively reduce the accumulation of collagen in liver. Furthermore, uridine can improve the activity of liver cells and alleviate CCl4-induced liver injury. Furthermore, uridine can significantly alleviate the risk factors caused by hepatic stellate cell activation, uridine treatment significantly down-regulated the expression of α-SMA, collagen type-I and fibronectin. In conclusion, the current research shows that uridine can alleviate CCl4-induced liver fibrosis, suggesting that uridine can be used as a potential drug to alleviate liver fibrosis.

Flap Endonuclease 1-Assisted DNA Walkers for Sensitively and Specifically Sensing ctDNAs.

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

METTL3 facilitates the progression of hepatocellular carcinoma by modulating the m6A level of USP7.

OBJECTIVE: To explore whether METTL3 was involved in the pathogenesis of hepatocellular carcinoma (HCC) by modulating the m6A level of USP7. METHODS: We performed qRT-PCR and western blot assays to detect the expression level of METTL3 in HCC tissues and paired adjacent normal tissues, as well as HCC cell lines. The level of m6A in HCC tissues and cells was quantitatively analyzed by m6A RNA Methylation Quantitative Kit. We examined the effect of METTL3 on cell proliferation ability by CCK-8 and EdU assays, and examined cell migration and cell invasion ability by Transwell assay. It was predicted via bioinformatics tool that USP7 may undergo methylation in HCC. Subsequently, we performed qRT-PCR assay to detect the expression level of USP7 in HCC tissues and analyzed its correlation with the expression level of METTL3. We verified the regulatory relationship between METTL3/USP7 and transfected USP7 siRNA in cells to detect its effects on cell invasion, migration and proliferation. The regulatory effect of METTL3 on USP7 in HCC was analyzed by corresponding experiments. RESULTS: The qRT-PCR results indicated that METTL3 was highly expressed in HCC tissues and cell lines. The level of m6A was remarkably increased in HCC tissues and cell lines. Besides, the elevated METTL3 expression was related to worse overall survival. The abilities of cell invasion, migration and proliferation were remarkably attenuated by down-regulation of METTL3 expression. Through bioinformatics analysis, it was found that USP7 might be regulated by METTL3 to undergo methylation modification. The qRT-PCR results showed that the USP7 was highly expressed in HCC tissues, and was positively correlated with the level of METTL3. Further experiments showed that down-regulation of USP7 could reduce cell proliferation, migration, and invasion. METTL3 could positively regulate the malignant phenotype of USP7 in HCC. CONCLUSION: METTL3 might regulate the expression of USP7 through m6A methylation and facilitate the invasion, migration and proliferation of HCC cells.

Identification of Cooperative Gene Regulation Among Transcription Factors, LncRNAs, and MicroRNAs in Diabetic Nephropathy Progression.

The pathogenesis of diabetic nephropathy (DN) is accompanied by alterations in biological function and signaling pathways regulated through complex molecular mechanisms. A number of regulatory factors, including transcription factors (TFs) and non-coding RNAs (ncRNAs, including lncRNAs and miRNAs), have been implicated in DN; however, it is unclear how the interactions among these regulatory factors contribute to the development of DN pathogenesis. In this study, we developed a network-based analysis to decipher interplays between TFs and ncRNAs regulating progression of DN by combining omics data with regulatory factor-target information. To accomplish this, we identified differential expression programs of mRNAs and miRNAs during early DN (EDN) and established DN. We then uncovered putative interactive connections among miRNA-mRNA, lncRNA-miRNA, and lncRNA-mRNA implicated in transcriptional control. This led to the identification of two lncRNAs (MALAT1 and NEAT1) and the three TFs (NF-κB, NFE2L2, and PPARG) that likely cooperate with a set of miRNAs to modulate EDN and DN target genes. The results highlight how crosstalk among TFs, lncRNAs, and miRNAs regulate the expression of genes both transcriptionally and post-transcriptionally, and our findings provide new insights into the molecular basis and pathogenesis of progressive DN.

Glucosamine-6-Phosphate Isomerase 1 Promotes Tumor Progression and Indicates Poor Prognosis in Hepatocellular Carcinoma.

PURPOSE: Reprogramming of metabolic pathways is a hallmark of the pathological changes that occur in cancer cells. Under physiological conditions, glucosamine-6-phosphate isomerase 1 (GNPDA1) promotes the conversion of the hexosamine system to the glycolytic pathway and may, therefore, affect energy metabolism. Low expression of GNPDA1 has been reported in normal liver tissues, however, analysis of the hepatocellular carcinoma (HCC) database in The Cancer Genome Atlas (TCGA) revealed that GNPDA1 was highly expressed in HCC tissues. The purpose of this study was to explore the role of GNPDA1 in HCC. PATIENTS AND METHODS: We analyzed the expression of GNPDA1 in HCC tissues as well as the clinical outcomes of HCC patients with high or low expression of GNPDA1. Furthermore, the relationship between the expression of GNPDA1 and advanced tumor stage, TNM stage, grade, gender, or metastasis was assessed using high-throughput RNA sequencing data from TCGA HCC cohort and Kaplan-Meier survival analysis. The expression of GNPDA1 in HCC and normal liver cell lines was subsequently detected by qRT-PCR and Western blot analysis. Additionally, the effects of GNPDA1 knockdown in SMMC-7721 and Huh7 cell lines were examined. Cell proliferation, migration, invasion, and apoptosis following knockdown were investigated by the MTT assay and EdU, cell cycle, apoptosis, transwell, and wound healing analyses. RESULTS: There was a significant association between high GNPDA1 expression and advanced tumor stage, TNM stage or grade, but not with gender. High GNPDA1 expression was associated with poor prognosis in patients with HCC. Furthermore, the MTT assay and EdU, cell cycle, apoptosis, wound healing, and transwell analyses revealed that GNPDA1 promoted the proliferation, migration, and invasion of HCC cells and inhibited apoptosis. CONCLUSION: The results of this study suggest that GNPDA1 may serve as a novel prognostic biomarker and therapeutic target for HCC.

[Value of susceptibility weighted imaging in hepatic fibrosis staging by using MR in a rabbit model].

OBJECTIVE: To assess the feasibility of susceptibility weighted imaging (SWI) in staging hepatic fibrosis(HF). METHODS: Sixty healthy rabbits were divided into three groups: HF group(n=32), control group(n=16), supplementary group(n=12). Rabbits in HF group and supplementary group were injected subcutaneously with 50% CCl4 oil solution to establish hepatic fibrosis model. On the basis of preliminary test, eight rabbits from HF group and four rabbits from control group underwent liver conventional MR scans and SWI once a time at 4, 5, 6, 10 weeks after CCl4 administration.After MR scans at each time point, rabbits were killed to detect pathological staging with Scheuer staging.The liver signal intensity (SI) and liver-to-muscle SI ratios (SIR) were measured. According to the Scheuer classification of histological fibrosis stages, the correlation about the SI value, SIR value and the histological fibrosis stages was investigated by using the Spearman correlation test. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of SWI for staging HF on the basis of the histopathologic analysis of HF. RESULTS: There were fifteen rabbits in pathological staging F0, the value of SIR and SI was 0.974 ± 0.018, 374±44, SIR values of pathological staging from F1 to F4 were 0.963 ± 0.018, 0.796 ± 0.023, 0.786 ± 0.025, 0.512±0.024 respectively. SI values of pathological staging from F1 to F4 were 372±18, 376±22, 346±15, 288±19 respectively. In the early period of liver fibrosis, there were no statistical differences in the SI value between F0 and F1, F1 and F2 stage.With progression of hepatic fibrosis, from F2 to F4, SI value decreased, the difference was statistically significant (P<0.05). With the progress of liver fibrosis, SIR value was reduced. It was negatively correlated with the HF stages and SIR value(r=-0.896, P<0.05). ROC curve analysis showed that the efficiency of SI value diagnosis in liver fibrosis was high in the late stage of liver fibrosis, but it was low in the early stage.The performance of liver-to-muscle SI ratio on SWI was high in the early stage. Liver-to-muscle SI ratio had a higher diagnostic performance than SI in the diagnosis of liver fibrosis stages. CONCLUSION: SWI can be a safe, reliable method for staging hepatic fibrosis and provide quantitative imaging basis for clinical treatment.