Succinylation Inhibits the Enzymatic Hydrolysis of the Extracellular Matrix Protein Fibrillin 1 and Promotes Gastric Cancer Progression.

Extracellular matrix (ECM) remodeling is crucial in the regulation of gastric cancer (GC) progression. This work aims to reveal novel posttranslational modifications and their relevant mechanisms in GC. In 3D matrix culture and animal models, it is found that fibrillin 1 (FBN1) expression is increased in advanced GC and has succinylation modification. The succinylation modification of FBN1 blocks its degradation by matrix metalloproteinases (MMPs). The long-term accumulation and deposition of FBN1 enhance tumor progression by activating TGF-ß1 and intracellular PI3K/Akt pathway. The FBN1 succinylation site monoclonal antibody can effectively intervene the effect of succinylation modification and inhibit GC progression. FBN1 is specifically upregulated in the progression of GC compared with other tumors. In conclusion, FBN1 is widely present in the form of K672-succinylated modifications in GC. Besides, the succinyl group of FBN1 blocks its binding to MMP2, inhibits its degradation by MMP2, and leads to the accumulation of FBN1, which poses a long-term risk to the poor prognosis of GC.

Proactive exploration of inferior parathyroid gland using a novel meticulous thyrothymic ligament dissection technique.

INTRODUCTION: The inferior parathyroid gland (IPTG) is widely distributed; effective techniques for its safe exploration and protection during thyroid surgery have not been documented. The thyrothymic ligament (TTL) is a connective tissue located between the thymic tongue and thyroid. This study aims to introduce a novel meticulous thyrothymic ligament dissection technique and assess its role in proactive exploration and situ preservation of IPTG. MATERIALS AND METHODS: 737 patients undergoing initial thyroid surgery between 2017 and 2021 in the Department of General Surgery of the First Affiliated Hospital of Nanjing Medical University were retrospectively recruited for this clinical study. In 391 of the recruited patients, the TTL was dissected, and the number and location of IPTG were recorded. Among them, 214 patients underwent total/near-total thyroidectomy (TT) plus central neck dissection (CND) were assigned to the observation group. The control group included 346 consecutive patients who underwent conventional TT plus CND. After 1:1 propensity score matching, each group contained 206 patients. The incidence of postoperative hypoparathyroidism was recorded. RESULTS: Among the 391 patients, 596 sides were dissected, out of which 436 sides (73.2%) had TTL, and approximately 90.1% of IPTG were located and identified. A statistically significant difference in incidence of temporary (27.7 vs. 49.0%, P < 0.001) and permanent hypoparathyroidism (0 vs. 8.2%, P = 0.047) was noted between the observation group and the control group. CONCLUSION: The meticulous thyrothymic ligament dissection technique helps to protect IPTG in situ and reduce the incidence of postoperative hypoparathyroidism.

LncRNA DLEU1 Contributes to the Growth and Invasion of Colorectal Cancer via Targeting miR-320b/PRPS1.

Growing evidences suggest that long non-coding RNAs (lncRNAs) are closely correlated to the development of human cancer, such as colorectal cancer (CRC). A previous report suggested that DLEU1 accelerated CRC development. However, DLEU1's underlying mechanism in CRC remains unclear. In our study, the level of DLEU1 in CRC tissues is investigated by qRT-PCR. Our data exhibited that DLEU1 level was observably increased in CRC tissues and CRC cell lines and was closely associated with bad prognosis of CRC patients. CRC cell proliferation was repressed by sh-LncRNA DLEU1, whereas cell apoptosis was markedly stimulated. Moreover, knockdown of DLEU1 inhibited cell migration and invasion. Mechanistically, through interacting with miR-320b in CRC, DLEU1 promoted the level of PRPS1 which was a target of miR-320b. The rescue experiment confirmed that knockdown of DLEU1 repressed cell proliferation, migration and invasion while stimulated cell apoptosis via miR-320b/phosphoribosyl pyrophosphate synthetase 1 (PRPS1) axis. Meanwhile, the data of xenograft model exhibited that inhibition of DLEU1 suppressed tumor growth in vivo. In summary, DLEU1 knockdown may repress PRPS1 expression via miR-320b, and then repress cell proliferation, migration and invasion while stimulate cell apoptosis. Our research may provide a novel target for the treatment of CRC.

Lysine-222 succinylation reduces lysosomal degradation of lactate dehydrogenase a and is increased in gastric cancer.

BACKGROUND: Lysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains underexplored. METHODS: Proteomic quantification of lysine succinylation was performed in human GC tissues and adjacent normal tissues by mass spectrometry. The mRNA and protein levels of lactate dehydrogenase A (LDHA) in GC and adjacent normal tissues were analyzed by qRT-PCR and western blot, respectively. The expression of K222-succinylated LDHA was measured in GC tissue microarray by the K222 succinylation-specific antibody. The interaction between LDHA and sequestosome 1 (SQSTM1) was measured by co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The binding of carnitine palmitoyltransferase 1A (CPT1A) to LDHA was determined by co-IP. The effect of K222-succinylated LDHA on tumor growth and metastasis was evaluated by in vitro and in vivo experiments. RESULTS: Altogether, 503 lysine succinylation sites in 303 proteins were identified. Lactate dehydrogenase A (LDHA), the key enzyme in Warburg effect, was found highly succinylated at K222 in GC. Intriguingly, this modification did not affect LDHA ubiquitination, but reduced the binding of ubiquitinated LDHA to SQSTM1, thereby decreasing its lysosomal degradation. We demonstrated that CPT1A functions as a lysine succinyltransferase that interacts with and succinylates LDHA. Moreover, high K222-succinylation of LDHA was associated with poor prognosis in patients with GC. Finally, overexpression of a succinylation-mimic mutant of LDHA promoted cell proliferation, invasion, and migration. CONCLUSIONS: Our data revealed a novel lysosomal pathway of LDHA degradation, which is mediated by the binding of K63-ubiquitinated LDHA to SQSTM1. Strikingly, CPT1A succinylates LDHA on K222, which thereby reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation. This study thus uncovers a new role of lysine succinylation and the mechanism underlying LDHA upregulation in GC.

Helicobacter pylori infection promotes Aquaporin 3 expression via the ROS-HIF-1α-AQP3-ROS loop in stomach mucosa: a potential novel mechanism for cancer pathogenesis.

Helicobacter pylori (H. pylori) is the major stomach carcinogen, but the molecular mechanism responsible for the pathogenesis of cancer development mediated by H. pylori infection is still unclear. Aquaporin 3 (AQP3), overexpressed in gastric carcinoma, has a crucial role in gastric carcinogenesis and progression. However, the triggers and precise regulations for AQP3 upregulation during gastric carcinogens also remain unknown. Here we report that H. pylori infection-mediated carcinogenesis may be mechanistically depended on the upregulation of AQP3 expression via reactive oxygen species (ROS) pathway activation in the stomach. The retrospective analyses of clinical samples from patients with gastric carcinoma and other different stages of gastric diseases indicated that AQP3 expression was positively associated with gastric mucosal disease progression and H. pylori infection status as well. Furthermore, H. pylori infection significantly upregulated AQP3 and HIF-1α expression and increased ROS amount in human gastric epithelial AGS and GES-1 cells. Blockage of ROS with inhibitors, NAC and DPI, markedly decreased the expression of AQP3 and HIF-1α in both AGS and GES-1 cells simultaneously. Furthermore, the increased AQP3 in cells was mechanistically due to the transcriptional regulation by HIF-1α. In addition, H. pylori infection exerted production of proinflammatory cytokines IL-6, IL-8, and TNF depending on AQP3 level. Importantly, these in vitro novel findings were further investigated in vivo in a mouse H. pylori infectious model. Our current studies identify the mechanistic link between H. pylori infection and AQP3 upregulation in the pathogenesis of gastric carcinoma, which involves the activation of the ROS-HIF-1α axis and the exacerbated ROS-HIF-1α-AQP3-ROS loop.

Prognostic role of long non-coding RNA LINC00152 in Chinese cancer patients: a meta-analysis.

The role of long intergenic non-coding RNA 152 (LINC00152) in predicting the prognosis of cancer has been investigated but results remain inconclusive and inconsistent. A meta-analysis was performed to explore the effect of LINC00152 on cancer prognosis. PubMed and ScienceDirect were searched for suitable studies and the results of 10 studies with a total of 775 patients were pooled. Pooled hazard ratios (HRs) and odds ratios (ORs) were calculated to assess the prognostic value of LINC00152. The results revealed that tumour patients with high LINC00152 expression were more likely to have lymph node metastasis (OR = 2.94, 95% CI 1.97-4.40, P < 0.001) and unfavourable tumour-node-metastasis stage (grade III/IV vs. I/II: OR = 3.07, 95% CI 1.69-5.59, P < 0.001). In addition, high LINC00152 expression levels were significantly associated with poor overall survival (pooled HR = 1.99, 95% CI 1.54-2.56, P < 0.001). The results suggest that high LINC00152 expression may serve as a predictive biomarker for the poor prognosis of various cancers in the Chinese population.

Identification of AQP3 and CD24 as biomarkers for carcinogenesis of gastric intestinal metaplasia.

Gastric intestinal metaplasia (GIM) is a precancerous gastric carcinoma (GC) lesion with pivotal roles in carcinogenesis. CD24, LGR5 and Ki67 are expressed in GIM; we previously demonstrated that aquaporin 3 (AQP3) is expressed in goblet cells and is positively correlated with GIM severity. However, the relationships of AQP3 with GIM classification and with other proteins, and their roles in the transition from GIM to gastric carcinoma (GC) remain unknown. Sixteen patients with intestinal-type GC were enrolled in this study. GIM was determined according to the updated Sydney system; GIM classification was determined via HID-AB staining, and AQP3, CD24, LGR5 and Ki67 expression were determined by immunohistochemistry. Type III GIM was more prevalent around the GC and displayed a positive association with GIM severity. CD24 was found in GIM, but LGR5 and Ki67 were found in tissues regardless of GIM. AQP3 expression showed significant correlation to type III GIM. CD24 expression was correlated with the marked GIM and incomplete GIM, while LGR5 expression decreased with GIM aggravation and did not have relationship with classification of GIM. However, Ki67 presented no association with GIM grade or classification. These observations identify AQP3 and CD24 as biomarkers for carcinogenesis of GIM, and may provide a precise strategy for screening at-risk candidates with GIM.

Aquaporin 3 facilitates chemoresistance in gastric cancer cells to cisplatin via autophagy.

Cisplatin (cDDP) remains one of the first-line chemotherapeutic agents for gastric cancer (GC) treatment, and resistance to cDDP is the major limitation in its clinical application. Mechanisms of cDDP resistance have been shown to be varied and complicated. Aquaporin 3 (AQP3) has been demonstrated to be overexpressed in GC tissues and is thought to be involved in GC carcinogenesis and progression. However, the role of AQP3 in chemosensitivity of GC to cytotoxic agents remains unknown. In this study, we show that AQP3 overexpression induced resistance to cDDP in AGS cells (P<0.05), and AQP3 knockdown increased the chemosensitivity in MGC803 and SGC7901 cells (P<0.05). Moreover, cDDP treatment enhanced AQP3 expression in MGC803, SGC7901 and AGS cells. AQP3 overexpression promoted the conversion of LC3-I to LC3-II in AGS cells, whereas AQP3 knockdown inhibited this conversion in MGC803 and SGC7901 cells. AQP3 upregulation increased Atg5 and Beclin-1 expression, and inhibited P62 expression in AGS cells, whereas AQP3 knockdown showed the opposite results in MGC803 and SGC7901 cells. Chloroquine (CQ), an autophagy inhibitor, enhanced the cytotoxicity of cDDP in GC cells, and CQ reversed the chemoresistance to cDDP caused by AQP3 overexpression in GC cells. Together, our data demonstrate that AQP3 facilitates cisplatin resistance in gastric cancer cells via autophagy, and suggest that the development of AQP3-based tumor therapeutics could play a key role in future GC treatment strategies.

Aquaporin 3 promotes the stem-like properties of gastric cancer cells via Wnt/GSK-3ß/ß-catenin pathway.

Cancer stem cells (CSCs) are believed to contribute to the tumor growth in gastric carcinoma (GC), a common lethal malignancy. This study investigated the effect of aquaporin 3 (AQP3) on stem-like properties of human GC cells. Elevated AQP3 expression was associated with CD44 expression in human GC specimens. Expression of AQP3 and that of CD44 positively correlated with Lauren classification, lymph node metastasis, and lymphovascular invasion. Altering the AQP3 expression had pronounced effects on the tumorigenic potential and self-renewal capacity of the gastric cancer cell lines SGC7901, MGC803, and AGS, both in vitro and in vivo. Overexpression of AQP3 induced CD44 expression and activation of the ß-catenin signaling pathway, whereas silencing AQP3 expression using short hairpin RNA had the opposite effect. Furthermore, pharmacological inhibition of GSK-3ß using LiCl impaired the effect of AQP3 knockdown in CSCs, whereas the inhibition of the Wnt/ß-catenin pathway by XAV939 blocked the effect of AQP3 overexpression. These results demonstrate that AQP3 promotes stem-like properties of human GC cells by activating the Wnt/GSK-3ß/ß-catenin signaling pathway.

Potential role of aquaporin 3 in gastric intestinal metaplasia.

Gastric intestinal metaplasia (GIM) is a pre-cancerous condition and a pivotal step in the formation of gastric cancer (GC). Aquaporin 3 (AQP3) has been found to be expressed in goblet cells rather than mucus-secreting glands. To investigate the characteristics of GIM in non-cancerous tissues adjacent to GC, as well as the expression and role of AQP3 in GIM tissues, 16 patients diagnosed with gastric adenocarcinoma of intestinal type located in the lesser curve of the antrum were consecutively enrolled in this study. A new pathological technology called "gastric mucosal sausage roll" was introduced. GIM was determined according to the updated Sydney system, and AQP3 expression in goblet cells was determined by immunohistochemistry. GIM was found in all stomach specimens, and its incidence increased with progression to GC (P < 0.001). GIM prevalence displayed remarkable association with the distance to GC in the anterior gastric wall tissues (P = 0.016) and tissues toward the cardia (P = 0.014), such that GIM was more common in the areas closer to GC (P < 0.001). AQP3 was found to be expressed in 67.71% of parts with GIM, and AQP3 immunoreactivity was identified more frequently in severe GIM areas (P < 0.001). In short, the incidence and severity of GIM correlated with the distance from GC, and AQP3 was differentially expressed in goblet cells, with most AQP3-positive goblet cells presenting in severe GIM. Together, this study suggests that AQP3 may play an important role in gastric carcinogenesis from GIM.

Meta-analysis of the efficacies of extended and standard pancreatoduodenectomy for ductal adenocarcinoma of the head of the pancreas.

BACKGROUND: The purpose of the present study was to evaluate the efficacy of extended pancreatoduodenectomy (EPD) and standard pancreatoduodenectomy (SPD) for ductal adenocarcinoma of the head of the pancreas via meta-analysis. METHODS: Relevant articles (published between 1995 and 2012) were compiled from online data sources. A total of nine studies satisfied the selection criteria, including a total of 973 patients (478 in the SPD group and 495 in the EPD group). Evaluation parameters included 1-, 3-, and 5-year survival, as well as mortality, morbidity, and specific morbidity outcomes. RESULTS: Meta-analysis revealed (1) differences in morbidity (Odds ratio [OR] = 1.740; 95 % confidence interval [CI], 0.840-3.600; P = 0.140), mortality (OR = 0.890; 95 % CI, 0.560-1.400; P = 0.620), 1-year overall survival (OS) rate (OR = 1.20; 95 % CI, 0.490-2.930; P = 0.69), 3-year OS rate (OR = 0.770; 95 % CI, 0.460-1.280; P = 0.190), and 5-year OS rate (OR = 1.12; 95 % CI, 0.690-1.810; P = 0.560) were not significant between EPD and SPD. (2) For bile leak (OR = 2.640; 95 % CI, 1.040-6.700; P = 0.040), pancreatic leak (OR = 1.740; 95 % CI, 1.040-2.91; P = 0.030), delayed gastric emptying (OR = 2.090; 95 % CI, 1.240-3.520; P = 0.006), and lymphatic fistula (OR = 6.120; 95 % CI, 1.06-35.320; P = 0.040) differences between EPD and SPD were significant, whereas other specific morbidities were not significantly different. CONCLUSIONS: Extended pancreatoduodenectomy does not improve 1-, 3-, 5-year OS rates compared to SPD and there is a trend toward increased bile leak, pancreatic leak, delayed gastric emptying, and lymphatic fistula after EPD.

Multi-target lentivirus specific to hepatocellular carcinoma: in vitro and in vivo studies.

BACKGROUND & AIMS: We aimed at investigating the effects of the targeted transduction of the Wtp53-pPRIME-miR30-shRNA gene into liver cancer cells, under the mediation of anti-alpha fetoprotein scFv-directed lentivirus, and the inhibitory effect of this system on liver cancer cells. METHODS: The result of infection was observed by fluorescence microscopy. Polymerase chain reaction and Western blotting were used to demonstrate the successful transduction and transcription of the Wtp53-pPRIME-miR30-shRNA-IGF1R gene. Cell growth was observed via the Cell-Counting Kit-8 Method, and cell apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. To observe further the effects of AFP-Wtp53-pPRIME-miR30-shRNA-IGF1R therapy in animals, models of BALB-C nude mice bearing subcutaneous human hepatocellular carcinoma were established. The influence of the growth of subcutaneously transplanted tumor, expression of Wtp53 protein, apoptosis, and microvessel formation on the overall level of AFP-Wtp53 pPRIME-miR30-shRNA-IGF1R were also evaluated. RESULTS: Recombinant lentivirus was successfully constructed, and its functional plaque-forming unit titer was determined as 4.58 × 10(9)plaque-forming units/ml. A positive strand was detected by polymerase chain reaction and Western blotting. Lentiviral construction worked effectively in AFP-positive liver cancer cells. In vitro and in vivo experiments showed that the recombinant lentivirus was more efficacious in inhibiting the proliferation of Hep3B cells. CONCLUSIONS: The Wtp53-pPRIME-miR30-shRNA gene can be subjected to targeted transduction into liver cancer cells under the mediation of anti-alpha fetoprotein scFv-directed lentivirus. The Wtp53-pPRIME-miR30-shRNA system has targeting ability and lethal effects on liver cancer cells.

Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model.

Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4(+)CD25(+) regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were recipients. Rats were randomly assigned to the following four groups: control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body irradiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased significantly to (459.2±76.9) U L(-1), total bilirubin increased to (124.1±33.7) µmol L(-1) (P<0.05) and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly to 1.50%±0.50% (P<0.05) compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.

Effects of Epstein-Barr virus infection on the development of multiple myeloma after liver transplantation.

Reduced cellular immune function in patients after liver transplantation easily results in many types of viral infections, such as Epstein-Barr virus. Epstein-Barr virus is a Γ-herpesvirus and is related to many malignant diseases, especially epithelial and lymph tumors. The abnormal interaction of cluster of differentiation 40 with cluster of differentiation 40 ligand and expression of cluster of differentiation 40 ligand are considered closely related to the development of myeloma cells. This study explored the influence and mechanism of Epstein-Barr virus infection on the phenotype and biological behavior of myeloma cells after liver transplantation. Flow cytometry was used to detect coexpression of cluster of differentiation 40 and cluster of differentiation 40 ligand in 10 samples of freshly isolated multiple myeloma cells. Cluster of differentiation 40 and cluster of differentiation 40 ligand were coexpressed in sample Nos. 5, 8, 9, and 10, particularly in sample No. 5. Western blot analysis was used to detect the expression of the Epstein-Barr virus antigens latent membrane protein 1 and Epstein-Barr virus nuclear antigen 2 in the multiple myeloma cell line RPMI 8226 infected with Epstein-Barr virus. The antigen expression indicated that Epstein-Barr virus can infect multiple myeloma virus cells in vitro. Reverse transcription-polymerase chain reaction revealed upregulated expression of cluster of differentiation 40 ligand on the infected RPMI 8226 cells, which may be involved in the anti-apoptosis activity of the infected cells. Confocal microscopy showed that pairs of molecules of cluster of differentiation 40, cluster of differentiation 40 ligand, and latent membrane protein 1 were colocalized on the surface of the infected cells. CXC chemokine receptor 4 was upregulated on the RPMI 8226 cells after Epstein-Barr virus infection. The migratory ability of the infected cells improved in the presence of the chemokine stromal cell-derived factor-1α. Anti-apoptosis and migration are known important biological characteristics of malignant cells. Our results indicate the involvement of Epstein-Barr virus in the origin and development of multiple myeloma. The risk of multiple myeloma increases when Epstein-Barr virus infects B cells in the germinal center, which may result in an anti-apoptosis effect of B cells and an improved ability to migrate from the germinal center to peripheral blood.