Prevalence and molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common intestinal pathogens that infect humans and animals. To date, research regarding these three protozoa in the Ningxia Hui Autonomous Region (Ningxia) has mostly been limited to a single pathogen, and comprehensive data on mixed infections are unavailable. This study aimed to evaluate the zoonotic potential of these three protozoa. In this study, small subunit ribosomal RNA (SSU rRNA) and 60 kDa glycoprotein (gp60) genes of Cryptosporidium; internal transcribed spacer (ITS) gene of E. bieneusi; and SSU rRNA, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis were examined. DNA extraction, polymerase chain reaction, and sequence analysis were performed on fecal samples collected from 320 dairy cattle at three intensive dairy farms in Ningxia in 2021 to determine the prevalence and genetic characteristics of these three protozoa. The findings revealed that 61.56% (197/320) of the samples were infected with at least one protozoan. The overall prevalence of Cryptosporidium was 19.38% (62/320), E. bieneusi was 41.56% (133/320), and G. duodenalis was 29.38% (94/320). This study identified four Cryptosporidium species (C. bovis, C. andersoni, C. ryanae, and C. parvum) and the presence of mixed infections with two or three Cryptosporidium species. C. bovis was the dominant species in this study, while the dominant C. parvum subtypes were IIdA15G1 and IIdA20G1. The genotypes of E. bieneusis were J, BEB4, and I alongside the novel genotypes NX1-NX8, all belonging to group 2, with genotype J being dominant. G. duodenalis assemblages were identified as assemblages E, A, and B, and a mixed infection involving assemblages A + E was identified, with assemblage E being the dominant one. Concurrently, 11 isolates formed 10 different assemblage E multilocus genotypes (MLGs) and 1 assemblage A MLG and assemblage E MLGs formed 5 subgroups. To the best of our knowledge, this is the first report on mixed infection with two or three Cryptosporidium species in cattle in Ningxia and on the presence of the C. parvum subtype IIdA20G1 in this part of China. This study also discovered nine genotypes of E. bieneusis and novel features of G. duodenalis assemblages in Ningxia. This study indicates that dairy cattle in this region may play a significant role in the zoonotic transmission of Cryptosporidium spp., E. bieneusi, and G. duodenalis.

First report of Giardia duodenalis in dairy cattle and beef cattle in Shanxi, China.

BACKGROUND: Giardia duodenalis is an important intestinal parasitic protozoan that infects several vertebrates, including humans. Cattle are considered the major source of giardiasis outbreak in humans. This study aimed to investigate the prevalence and multilocus genotype (MLG) of G. duodenalis in Shanxi, and lay the foundation for the prevention and control of Giardiosis. METHODS AND RESULTS: DNA extraction, nested polymerase chain reaction, sequence analysis, MLG analysis, and statistical analysis were performed using 858 bovine fecal samples from Shanxi based on three gene loci: ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). The overall prevalence of G. duodenalis was 28.3%, while its prevalence in Yingxian and Lingqiu was 28.1% and 28.5%, respectively. The overall prevalence of G. duodenalis in dairy cattle and beef cattle was 28.0% and 28.5%, respectively. G. duodenalis infection was detected in all age groups evaluated in this study. The overall prevalence of G. duodenalis in diarrhea and nondiarrhea samples was 32.4% and 27.5%, respectively, whereas that in intensively farmed and free-range cattle was 35.0% and 19.9%, respectively. We obtained 83, 53, and 59 sequences of bg, gdh, and tpi in G. duodenalis, respectively. Moreover, assemblage A (n = 2) and assemblage E (n = 81) by bg, assemblage A (n = 1) and assemblage E (n = 52) by gdh, and assemblage A (n = 2) and assemblage E (n = 57) by tpi were identified. Multilocus genotyping yielded 29 assemblage E MLGs, which formed 10 subgroups. CONCLUSIONS: To the best of our knowledge, this is the first study to report cattle infected with G. duodenalis in Shanxi, China. Livestock-specific G. duodenalis assemblage E was the dominant assemblage genotype, and zoonotic sub-assemblage AI was also detected in this region.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy and beef cattle in Shanxi, China.

Cryptosporidium spp. are key gastrointestinal protists in humans and animals worldwide. Infected cattle are considered the main source of cryptosporidiosis outbreaks in humans. However, little is known about the genetic makeup of Cryptosporidium populations in Shanxi province, China. We analyzed 858 fecal samples collected from farms in Shanxi. The presence of Cryptosporidium spp. was determined via polymerase chain reaction and subsequent sequence analysis of the small subunit rRNA gene as well as restriction fragment length polymorphism analysis. Cryptosporidium parvum was subtyped following sequence analysis of the 60 kDa glycoprotein gene (gp60). The overall prevalence of Cryptosporidium in cattle was 11.19%, with a prevalence of 13.30% and 8.67% in Lingqiu and Yingxian, respectively. The overall prevalence of Cryptosporidium in dairy and beef cattle was 10.78% and 11.50%, respectively. Cryptosporidium infection was detected across all analyzed age groups. The overall prevalence of Cryptosporidium in diarrhea and nondiarrhea samples was 18.24% and 9.72%, respectively, whereas that in intensively farmed and free-range cattle was 17.40% and 3.41%, respectively. We identified five Cryptosporidium species, with C. andersoni being the dominant species. Further, two cases of mixed infections of Cryptosporidium species were detected. All identified C. parvum isolates belonged to the subtype IIdA17G1.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China.

BACKGROUND: Cryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia. METHODS: We focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium. RESULTS: SSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1. CONCLUSIONS: To the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

Prevalence and molecular characterization of Giardia duodenalis in dairy cattle in Central Inner Mongolia, Northern China.

Giardia duodenalis is a gastrointestinal protozoan ubiquitous in nature. It is a confirmed zoonotic pathogen, and cattle are considered a source of giardiasis outbreaks in humans. This study aimed to evaluate the prevalence and multilocus genotype (MLG) of G. duodenalis in dairy cattle in Central Inner Mongolia. This study was based on the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed on 505 dairy cattle fecal samples collected in 2021 from six sampling sites and four age groups in Central Inner Mongolia to determine the prevalence and MLG distribution of G. duodenalis. The PCR results of SSU rRNA revealed that the overall prevalence of G. duodenalis was 29.5% (149/505) and that the overall prevalence of the diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.5% (103/359), respectively; the difference was not significant (p > 0.05). SSU rRNA sequence analysis revealed that G. duodenalis assemblage E (91.1%, 133/146) was primarily detected and that assemblage A (8.9%, 13/146) was detected in 13 samples. The G. duodenalis-positive samples were PCR amplified and sequenced for gdh, tpi, and bg, from which 38, 47, and 70 amplified sequences were obtained, respectively. A combination of G. duodenalis assemblages A and E were detected in seven samples. Multilocus genotyping yielded 25 different assemblage E MLGs, which formed six subgroups. To the best of our knowledge, this is the first report regarding G. duodenalis infection in dairy cattle in Inner Mongolia, China. This study revealed that Inner Mongolian cattle pose a risk of giardiasis transmission to humans and that the distribution of local cattle G. duodenalis assemblage E MLGs is diverse. The findings of this study can bridge the knowledge gap in the molecular epidemiological investigation of giardiasis in Central Inner Mongolia.

Aucubin enhances the antitumor activity of cisplatin through the inhibition of PD-L1 expression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality in the world. However, the anticancer effects of aucubin against HCC have yet to be reported. Cisplatin often decreased CD8+ tumor-infiltrating lymphocytes in the tumor microenvironment through increasing programmed death-ligand 1 (PD-L1) expression, which seriously affected the prognostic effect of cisplatin in the treatment of patients with HCC. Therefore, it is necessary to identify a novel therapeutic avenue to increase the sensitivity of cisplatin against HCC. PURPOSE: This study aims to evaluate the anti-tumor effect of aucubin on HCC, and also to reveal the synergistic effects and mechanism of aucubin and cisplatin against HCC. STUDY DESIGN AND METHODS: An H22 xenograft mouse model was established for the in vivo experiments. Cancer cell proliferation was detected by MTT assay. RT-qPCR was performed to analyze CD274 mRNA expression in vitro. Western blotting was employed to determine the expression levels of the PD-L1, p-Akt, Akt, p-ß-catenin, and ß-catenin in vitro. Immunofluorescence was carried out to examine ß-catenin nuclear accumulation in HCC cells. Immunohistochemistry was used to detect tumoral PD-L1 and CD8α expression in xenograft mouse model. RESULTS: Aucubin inhibits tumor growth in a xenograft HCC mouse model, but did not affect HCC cell viability in vitro. Aucubin treatment significantly inhibited PD-L1 expression through inactivating Akt/ß-catenin signaling pathway in HCC cells. Overexpression of PD-L1 dramatically reversed aucubin-mediated tumoral CD8+ T cell infiltration and alleviated the antitumor activity of aucubin in xenograft mouse model. Moreover, Cisplatin could induce the expression of PD-L1 through the activation of the Akt/ß-catenin signaling pathway in HCC cells, which can be blocked by aucubin in vitro. In xenograft mouse model, cisplatin treatment induced PD-L1 expression and alleviated the infiltration of CD8+ T lymphocytes in the tumor microenvironment. Aucubin not only abrogated cisplatin-induced PD-L1 expression but also enhanced the antitumor efficacy of cisplatin in a mouse xenograft model of HCC. CONCLUSION: Aucubin exerts antitumor activity against HCC and also enhances the antitumor activity of cisplatin by suppressing the Akt/ß-catenin/PD-L1 axis.

Comparative analysis of microbial communities in different growth stages of Dermacentor nuttalli.

Ticks were identified as arthropods that are pathogenic vectors. Dermacentor nuttalli is one of the dominant tick species in Inner Mongolia, and it carries and transmits a wide range of pathogenic microorganisms. However, at present, only the detection of D. nuttalli adult ticks and D. nuttalli different developmental stages carrying one specific pathogen, or the next-generation sequencing of D. nuttalli adult ticks were available. In this study, we investigated the microbial community structures of D. nuttalli in different growth stages under laboratory artificial feeding conditions. Total DNA was extracted from seven growth stages (female adult ticks, eggs, larval ticks, engorged larval ticks, nymphal ticks, engorged nymphal ticks, and second-generation adult ticks) obtained from laboratory artificial feeding of engorged D. nuttalli female ticks in Inner Mongolia. Then, the 16S rDNA V3-V4 hypervariable region was amplified to construct an Illumina PE250 library. Finally, 16S rRNA sequencing was performed on Illumina Novaseq 6000 platform. The sequencing data were analyzed using molecular biology software and platforms. The Illumina PE250 sequencing results showed that the egg stage had the highest diversity and number of species (28.74%, 98/341), while the engorged nymph stage had the lowest diversity and number of species (9.72%, 21/216). A total of 387 genera of 22 phyla were annotated in D. nuttalli, with 9 phyla and 57 genera found throughout all 7 growth stages. The dominant phylum was Proteobacteria; the dominant genera were Arsenophonus and Rickettsia; and the genera with the highest relative abundance in the 7 growth stages were Pseudomonas, Paenalcaligenes, Arsenophonus, Arsenophonus, Pseudomonas, Arsenophonus, and Rickettsia, respectively. Among the 23 exact species annotated, Brucella melitensis exhibits pathogeny that poses a serious threat to humans and animals. In this study, the microbial community composition at different growth stages of D. nuttalli was comprehensively analyzed for the first time.

First determination of DNA virus and some additional bacteria from Melophagus ovinus (sheep ked) in Tibet, China.

Melophagus ovinus (sheep ked) is one of the common ectoparasites in sheep. In addition to causing direct damage to the host through biting and sucking blood, sheep ked is a potential vector of helminths, protozoa, bacteria, and viruses. Sheep M. ovinus samples from three regions in Tibet were selected for DNA extraction. The 16S rDNA V3-V4 hypervariable region was amplified, after genomic DNA fragmentation, Illumina Hiseq libraries were constructed. The 16S rRNA sequencing and viral metagenomics sequencing were separately conducted on the Illumina Novaseq 6000 platform and molecular biology software and platforms were employed to analyze the sequencing data. Illumina PE250 sequencing results demonstrated that the dominant bacteria phylum in M. ovinus from Tibet, China was Proteobacteria, where 29 bacteria genera were annotated. The dominant bacterial genera were Bartonella, Wolbachia, and Arsenophonus; Bartonella chomelii, Wolbachia spp., and Arsenophonus spp. were the dominant bacterial species in M. ovinus from Tibet, China. We also detected Kluyvera intermedia, Corynebacterium maris DSM 45190, Planomicrobium okeanokoites, and Rhodococcus erythropolis, of which the relative abundance of Kluyvera intermedia was high. Illumina Hiseq sequencing results demonstrated that 4 virus orders were detected in M. ovinus from Tibet, China, and 3 samples were annotated into 29 families, 30 families, and 28 families of viruses, respectively. Virus families related to vertebrates and insects mainly included Mimiviridae, Marseilleviridae, Poxviridae, Ascoviridae, Iridoviridae, Baculoviridae, Hytrosaviridae, Nudiviridae, Polydnaviridae, Adomaviridae, Asfarviridae, Hepeviridae, Herpesviridae, and Retroviridae; at the species level, the relative abundance of Tupanvirus_soda_lake, Klosneuvirus_KNV1, and Indivirus_ILV1 was higher. African swine fever virus and many poxviruses from the family Poxviridae were detected, albeit their relative abundance was low. The dominant bacterial phylum of M. ovinus from Tibet, China was Proteobacteria, and the dominant bacterial genera were Bartonella, Wolbachia, and Arsenophonus, where 23 out of 29 annotated bacteria genera were first reported in M. ovinus. Kluyvera intermedia, Corynebacterium maris DSM 45190, Planomicrobium okeanokoites, and Rhodococcus erythropolis were detected for the first time. All DNA viruses detected in this study have been reported in M. ovinus for the first time.

Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate ß-catenin in vitro and in vivo.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of ß-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of ß-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of ß-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating ß-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.

EDDM3A drives gastric cancer progression by promoting HIF-1α-dependent aerobic glycolysis.

Epididymal protein 3A (EDDM3A) is a protein involved in sperm maturation. It has been demonstrated that EDDM3A expression is upregulated and promotes cell proliferation in non-small cell lung cancer (NSCLC). However, the role of EDDM3A in other types of human cancers, including gastric cancer (GC), is still unexplored. Here, we show that the expression of EDDM3A is significantly upregulated in gastric cancer (GC) tissues and its upregulation correlates with poorer survival in patients with gastric cancer. Knockdown of EDDM3A inhibited growth and metastasis of GC cells, whereas overexpression of EDDM3A exhibited the opposite effect. Mechanistically, enhanced aerobic glycolysis mediated by upregulation of HIF-1α and subsequently increased target glycolytic genes and decreased mitochondrial biogenesis was found to contribute to the promotion of tumor growth and metastasis by EDDM3A in GC cells. Additionally, upregulation of EDDM3A in GC is at least partially mediated by downregulation of miR-618. In conclusion, elevated EDDM3A plays a pivotal oncogenic role in gastric carcinogenesis, suggesting it as a potential therapeutic target for treatment of GC.

SLC25A1 promotes tumor growth and survival by reprogramming energy metabolism in colorectal cancer.

Abnormal lipid metabolism has been commonly observed in various human cancers, including colorectal cancer (CRC). The mitochondrial citrate carrier SLC25A1 (also known as mitochondrial citrate/isocitrate carrier, CIC), has been shown to play an important role in lipid metabolism regulation. Our bioinformatics analysis indicated that SLC25A1 was markedly upregulated in CRC. However, the role of SLC25A1 in the pathogenesis and aberrant lipid metabolism in CRC remain unexplored. Here, we found that SLC25A1 expression was significantly increased in tumor samples of CRC as compared with paired normal samples, which is associated with poor survival in patients with CRC. Knockdown of SLC25A1 significantly inhibited the growth of CRC cells by suppressing the progression of the G1/S cell cycle and inducing cell apoptosis both in vitro and in vivo, whereas SLC25A1 overexpression suppressed the malignant phenotype. Additionally, we demonstrated that SLC25A1 reprogrammed energy metabolism to promote CRC progression through two mechanisms. Under normal conditions, SLC25A1 increased de novo lipid synthesis to promote CRC growth. During metabolic stress, SLC25A1 increased oxidative phosphorylation (OXPHOS) to protect protects CRC cells from energy stress-induced cell apoptosis. Collectively, SLC25A1 plays a pivotal role in the promotion of CRC growth and survival by reprogramming energy metabolism. It could be exploited as a novel diagnostic marker and therapeutic target in CRC.

Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China.

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

Long non-coding RNA Down syndrome cell adhesion molecule-anti-sense 1 promotes gastric carcinoma cell proliferation and migration by regulating the miR-204/TPT1 axis.

Background: Several recent studies have suggested that the long non-coding RNA (lncRNA) DSCAM-AS1 (Down syndrome cell adhesion molecule - anti-sense 1) is aberrantly expressed in many malignancies. Purpose: In this study, we aimed to explore the the role of DSCAM-AS1 in gastric carcinoma. Research Design: Expression of DSCAM-AS1 mRNA, miR-204, and TPT1 (Tumor Protein, Translationally-Controlled 1) were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Proliferation and apoptosis of GC cells were determined using the CCK-8 cell counting assay and flow cytometry. The rate of cell migration and invasion was determined using a transwell assay. The relationships between DSCAM-AS1, miR-204, and TPT1 were predicted and confirmed using a dual-luciferase reporter assay. Expression of TPT1 protein was quantified by Western blot. Results: In this study, we found that DSCAM-AS1 was significantly overexpressed in GC tissues and cell lines. Functional experiments indicated that GC cells with DSCAM-AS1 silencing exhibited a dynamic reduction in proliferation and migration. We identified miR-204 as a target of DSCAM-AS1 and found that it targeted TPT1 in GC cells, which further led to decreased expression of miR-204 in GC tissues and cell lines. A rescue assay revealed that knocked-down DSCAM-AS1 hindered GC progression, which was reversed upon miR-204 downregulation or TPT1 overexpression. Conclusion: We conclude that DSCAM-AS1 is expressed as a tumor oncogene in GC progression, modulated via the miR-204/TPT1 axis. These findings indicate the potential of DSCAM-AS1 as a therapeutic target for GC prevention.

Corrigendum: PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

[This corrects the article DOI: 10.3389/fphar.2020.577108.].

A naphthalimide-polyamine conjugate preferentially accumulates in hepatic carcinoma metastases as a lysosome-targeted antimetastatic agent.

Disseminated tumors lead to approximately 90% of cancer-associated deaths especially for hepatocellular carcinoma (HCC), indicating the imperative need of antimetastatic drugs and the ineffectiveness of current therapies. Recently polyamine derivatives have been identified as a promising prospect in dealing with metastatic tumors. Herein, a novel class of naphthalimide-polyamine conjugates 8a-8d, 13a-13c, 17 and 21 were synthesized and the mechanism was further determined. The polyamine conjugate 13b displayed remarkably elevated anti-tumor and anti-metastatic effects (76.01% and 75.02%) than the positive control amonafide (46.91% and 55.77%) at 5 mg/kg in vivo. The underlying molecular mechanism indicated that in addition to induce DNA damage by up-regulating p53 and γH2AX, 13b also targeted lysosome to modulate polyamine metabolism and function in a totally different way from that of amonafide. Furthermore, the HMGB1/p62/LC3II/LC3I and p53/SSAT/ß-catenin pathways were mainly involved in the inhibition of 13b-induced HCC metastasis by targeting polyamine transporters (PTs) overexpressed in HCC. At last, 13b down-regulated the concentrations of Put, Spd and Spm by modulating polyamine metabolism key enzymes SSAT and PAO, which favored the suppression of fast growing tumor cells. Taken together, our study implies a promising strategy for naphthalimide conjugates to treat terminal cancer of HCC by targeting autophagy and tumor microenvironment with reduced toxicities and notable activities.

Short-Term and Quality of Life Outcomes of Patients Using Linear or Circular Stapling in Esophagojejunostomy after Laparoscopic Total Gastrectomy.

BACKGROUND: Studies comparing the incidence of reflux esophagitis (RE) and patients' quality of life (QoL) when using circular stapler (CS) and linear stapler (LS) in esophagojejunostomy (EJS) after laparoscopic total gastrectomy (LTG) are rare, and certainly there are not enough to make a definitive decision on best practice. Presented herein is a study on the comparison of the short-term outcomes, QoL of the patients with the focus on the incidence of RE after both linear and circular stapling in LTG. METHODS: From January 2014 to October 2018, 120 patients were analyzed; of these, 42 patients underwent laparoscopy-assisted total gastrectomy (LATG) with CS (CS group) and 78 patients who underwent totally laparoscopic total gastrectomy (TLTG) with LS (LS group). We examined the results obtained in terms of perioperative outcomes, reflux-related assessments (GerdQ questionnaire and endoscopy findings with all cases; 24-h pH monitoring with limited cases), and EORTC QLQ-C30 and QLQ-STO22. In addition, questionnaires were also supplied to patients and the results were recorded. RESULTS: The incidence of anastomotic stenosis (7.1% vs. 0; P < 0.05) and the median intraoperative blood loss (180.0 vs. 100.0 mL; P < 0.05) of the CS group were higher than the LS group. The factor aside, no significant differences were observed between the two groups with regard to the incidence of RE assessed by the QLQ-STO22 reflux scale, the GerdQ scores, endoscopy (in all cases), or the percent time of pH > 7 (in limited cases) (P > 0.05). In the EORTC QLQ-C30 and QLQ-STO22, it was noted that the score of constipation [0 (0, 0) vs. 0 (0, 33.3); P = 0.028] and postoperative dysphagia [0 (0, 0) vs. 0 (0, 22.2); P = 0.046] of the LS group in a 1-year follow-up were lower than the CS group. CONCLUSIONS: TLTG with LS generated better results than LATG with CS in terms of the incidence of anastomotic stenosis, intraoperative blood loss, and postoperative constipation and dysphagia. Furthermore, when compared with circular stapling, linear stapling in EJS did not increase the incidence of RE assessed by the QLQ-STO22 reflux scale, the GerdQ scores, endoscopy (in all cases), or the percent time of pH > 7 (in limited cases).

P300-dependent acetylation of histone H3 is required for epidermal growth factor receptor-mediated high-mobility group protein A2 transcription in hepatocellular carcinoma.

High-mobility group protein A2 (HMGA2) is highly expressed in hepatocellular carcinoma (HCC) cells and contributes to tumor metastasis and poor patient survival. However, the molecular mechanism through which HMGA2 is transcriptionally regulated in HCC cells remains largely unclear. Here, we showed that the expression HMGA2 was upregulated in HCC, and that elevated HMGA2 could promote tumor metastasis. Incubation of HCC cells with epidermal growth factor (EGF) could promote the expression of HMGA2 mRNA and protein. Mechanistic studies suggested that EGF can phosphorylate p300 at Ser1834 residue through the PI3K/Akt signaling pathway in HCC cells. Knockdown of p300 can reverse EGF-induced HMGA2 expression and histone H3-K9 acetylation, whereas a phosphorylation-mimic p300 S1834D mutant can stimulate HMGA2 expression as well as H3-K9 acetylation in HCC cells. Furthermore, we identified that p300-mediated H3-K9 acetylation participates in EGF-induced HMGA2 expression in HCC. In addition, the levels of H3-K9 acetylation positively correlated with the expression levels of HMGA2 in a chemically induced HCC model in rats and human HCC specimens.

PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

High expression of programmed death-ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) cells usually inhibits the proliferation and functions of T cells, leading to immune suppression in tumor microenvironment. However, very little has been described regarding the mechanism of PD-L1 overexpression in HCC cells. In the present study, we found epidermal growth factor (EGF) stimulation promoted the expression of PD-L1 mRNA and protein in HCC cells. Inhibition of epidermal growth factor receptor (EGFR) could reverse EGF-induced the expression of PD-L1 mRNA and protein. Subsequently, we also observed that the phosphorylation level of Pyruvate kinase isoform M2 (PKM2) at Ser37 site was also increased in response to EGF stimulation. Expression of a phosphorylation-mimic PKM2 S37D mutant stimulated PD-L1 expression as well as H3-Thr11 phosphorylation in HCC cells, while inhibition of PKM2 significantly blocked EGF-induced PD-L1 expression and H3-Thr11 phosphorylation. Furthermore, mutation of Thr11 of histone H3 into alanine abrogated EGF-induced mRNA and protein expression of PD-L1, Chromatin immunoprecipitation (ChIP) assay also suggested that EGF treatment resulted in enhanced H3-Thr11 phosphorylation at the PD-L1 promoter. In a diethylnitrosamine (DEN)-induced rat model of HCC, we found that the expression of phosphorylated EGFR, PKM2 nuclear expression, H3-Thr11 phosphorylation as well as PD-L1 mRNA and protein was higher in the livers than that in normal rat livers. Taken together, our study suggested that PKM2-dependent histone H3-Thr11 phosphorylation was crucial for EGF-induced PD-L1 expression at transcriptional level in HCC. These findings may provide an alternative target for the treatment of hepatocellular carcinoma.

EGF promotes DKK1 transcription in hepatocellular carcinoma by enhancing the phosphorylation and acetylation of histone H3.

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of ß-catenin, a Wnt signaling effector. We investigated how DKK1 expression is induced in HCC and found that activation of the epidermal growth factor receptor (EGFR) promoted parallel MEK-ERK and PI3K-Akt pathway signaling that converged to epigenetically stimulate DKK1 transcription. In HCC cell lines stimulated with EGF, EGFR-activated ERK phosphorylated the kinase PKM2 at Ser37, which promoted its nuclear translocation. Also in these cells, EGFR-activated Akt phosphorylated the acetyltransferase p300 at Ser1834 Subsequently, PKM2 and p300 mediated the phosphorylation and acetylation, respectively, of histone H3 at the DKK1 promoter, which synergistically enhanced DKK1 transcription. The mechanism was supported with mutational analyses in cells and in a chemically induced HCC model in rats. The findings suggest that dual inhibition of the MEK and PI3K pathways might suppress the expression of DKK1 and, consequently, tumor metastasis in patients with HCC.