Efficacy and safety of laparoscopic common bile duct exploration with primary closure and intraoperative endoscopic nasobiliary drainage for choledocholithiasis combined with cholecystolithiasis.

BACKGROUND: The need for intraoperative endoscopic nasobiliary drainage during laparoscopic cholecystectomy and laparoscopic common bile duct exploration with primary closure is controversial in the treatment of cholecystolithiasis combined with choledocholithiasis. The aim of this study was to evaluate the safety and efficacy of laparoscopic cholecystectomy + laparoscopic common bile duct exploration + intraoperative endoscopic nasobiliary drainage + primary closure (LC + LCBDE + IO-ENBD + PC). The safety of different intubation methods in IO-ENBD was also evaluated. METHOD: From January 2018 to January 2022, 168 consecutive patients with cholecystolithiasis combined with choledocholithiasis underwent surgical treatment in our institution. Patients were divided into two groups: group A (n = 96) underwent LC + LCBDE + IO-ENBD + PC and group B (n = 72) underwent LC + LCBDE + PC. Patient characteristics, perioperative indicators, complications, stone residual, and recurrence rates were analyzed. Group A was divided into two subgroups. In group A1, the nasobiliary drainage tube was placed in an anterograde way, and in group A2, nasobiliary drainage tube was placed in an anterograde-retrograde way. Perioperative indicators and complications were analyzed between subgroups. RESULTS: No mortality in the two groups. The operation success rates in groups A and B were 97.9% (94/96) and 100% (72/72), respectively. In group A, two patients were converted to T-tube drainage. The stone clearance rates of group A and group B were 100% (96/96) and 98.6% (71/72), respectively. Common bile duct diameter was smaller in group A [10 vs. 12 mm, P < 0.001] in baseline data. In perioperative indicators, group A had a longer operation time [165 vs.135 min, P < 0.001], but group A had a shorter hospitalization time [10 vs.13 days, P = 0.002]. The overall complications were 7.3% (7/96) in group A and 12.5% (9/72) in group B. Postoperative bile leakage was less in group A [0% (0/96) vs. 5.6% (4/72), P = 0.032)]. There were no residual and recurrent stones in group A. And there were one residual stone and one recurrent stone in group B (all 1.4%). The median follow-up time was 12 months in group A and 6 months in group B. During the follow-up period, 2 (2.8%) patients in group B had a mild biliary stricture. At subgroup analysis, group A1 had shorter operation time [150 vs. 182.5 min, P < 0.001], shorter hospitalization time [9 vs. 10 days, P = 0.002], and fewer patients with postoperative elevated pancreatic enzymes [32.6% (15/46) vs. 68% (34/50), P = 0.001]. CONCLUSION: LC + LCBDE + IO-ENBD + PC is safer and more effective than LC + LCBDE + PC because it reduces hospitalization time and avoids postoperative bile leakage. In the IO-ENBD procedure, the antegrade placement of the nasobiliary drainage tube is more feasible and effective because it reduces the operation time and hospitalization time, and also reduces injury to the duodenal papilla.

[Clinical Characteristics and Risk Factors Analysis for Visceral Pleural Invasion in 
Mixed Ground-glass Nodular Lung Adenocarcinoma].

BACKGROUND: Lung cancer is still the malignant tumor with the highest morbidity and mortality in China. Lung adenocarcinoma is the most common subtype, and the number of lung cancer presenting as mixed ground glass nodule (mGGN) in imaging is gradually increasing. Visceral pleural invasion (VPI) is an important factor affecting the prognosis of mGGN type lung adenocarcinoma. The aim of the study is to explore and analyze the risk factors for VPI in mGGN type lung adenocarcinoma. METHODS: From November 2016 to November 2019, 128 patients with mGGN lung adenocarcinoma underwent radical surgical resection in the First Affiliated Hospital of Nanjing Medical University. Their clinical data, including imaging, pathological and biological features, were collected and analyzed retrospectively. There were 40 males and 88 females, aged 60.3±9.3 years ranging from 30 to 81 years. Single factor Chi-square test and multivariate Logistic regression were used to analyze the risk factors of VPI in mGGN type lung adenocarcinoma. RESULTS: Among 128 mGGN patients who met the inclusion criteria, 57 cases were pathologically confirmed with pleural invasion. Between the VPI (+) and VPI (-) group (P<0.05), there were significant differences in gender, maximum diameter of solid component, consolidation tumor ratio (CTR), spicule sign, history of lung disease, family history of hypertension, relation of lesion to pleura (RLP), coursing relationship between bronchi and nodules. In multivariate Logistic regression analysis, RLP (OR=3.529, 95%CI: 1.430-8.713, P=0.006) and coursing relationship between bronchi and nodules (OR=3.993, 95%CI: 1.517-10.51, P=0.005) were found to be independent risk factors for VPI (P<0.05). CONCLUSIONS: The possibility of VPI in m GGN lung adenocarcinoma should be evaluated by combining these parameters in clinical diagnosis and treatment. As independent risk factors, RLP and coursing relationship between bronchi and nodules are instructive to identify VPI in mGGN type lung adenocarcinoma.

Value of multidisciplinary team (MDT) in minimally invasive treatment of complex intrahepatic bile duct stones.

This study aimed to investigate the value of multidisciplinary team (MDT) management in minimally invasive treatment of complex intrahepatic bile duct stones (IHDs) by laparoscopy, choledochoscopy and percutaneous choledochoscopy. The characteristics, perioperative index, complication rate and minimally invasive rate of patients in MDT group (n = 75) and non-MDT group (n = 70) were compared. The members of MDT include doctors in ultrasound, imaging, hepatobiliary and pancreatic surgery, anaesthesia and intensive care medicine. The results showed that minimally invasive surgery reduced the incidence of postoperative residual stones, OR (95% CI) = 0.365 (0.141-0.940) (p = 0.037). MDT reduced the operation time, OR (95% CI) = 0.406 (0.207-0.796) (p = 0.009). Minimally invasive surgery significantly reduced intraoperative bleeding, OR (95% CI) = 0.267 (0.133-0.534) (p < 0.001). Minimally invasive surgery also reduced hospitalization time, OR (95% CI) = 0.295 (0.142-0.611) (p = 0.001). The stone clearance rates of MDT group and non-MDT group were 81.33% and 81.43% respectively. In the MDT group, the operative time was less than that in the non-MDT group (p = 0.010); the intraoperative bleeding volume was significantly less than that in the non-MDT group (p < 0.001); the hospitalization time was less than that in the non-MDT group (p = 0.001). Minimally invasive operation rate:48 cases (64.00%) in MDT group were significantly higher than 17 cases (24.29%) in non-MDT group (p < 0.001). In conclusion, minimally invasive procedures can be selected more through MDT. MDT can shorten the operation time, and minimally invasive surgery can reduce the incidence of residual stones, reduce intraoperative bleeding, and may shorten hospital stay. Therefore, MDT management model can provide personalized and minimally invasive surgical protocol for patients with complex IHD, which has high application value.

The Gene Signature Associated with Hepatocellular Carcinoma in Patients with Nonalcoholic Fatty Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is becoming a critical risk of hepatocellular carcinoma (HCC). As both NAFLD and HCC are heterogeneous diseases, this study aims to identify the similarity between the subtypes of NAFLD and HCC based on gene modules. METHODS: Coexpressed gene modules were extracted for both NAFLD and HCC. The association between the coexpressed gene modules of NAFLD and HCC was evaluated by Fisher's exact test. The overlapping coexpressed gene module was validated in three independent human NAFLD datasets. Furthermore, the preserved gene module was assessed in four independent NAFLD mouse datasets. The significantly enriched motifs within the gene module were inferred from upstream sequences. RESULTS: Four coexpressed gene modules were extracted from NAFLD. Of the four coexpressed gene modules, one was significantly overlapping with a module of HCC. This overlapping gene module was regarded as the HCC-associated NAFLD gene module (HANM). Enrichment analysis of biological processes revealed inflammatory response in HANM. Specifically, within the inflammatory response biological process, IL-17, TNF-α, and NF-κB signaling pathways were enriched. HANM was found to be strongly or moderately conserved across four mouse NAFLD datasets. Motif analysis of the upstream genomic sequences of HANM revealed nine transcription factors (FLI1, NRF1, ZBTB33, ELK1, YY1, ZNF143, TAF1, SF1, and E2F1), of which three transcription factors (YY1, E2F1, and ZNF143) were significantly highly expressed in the NAFLD patients and exhibited survival significance in HCC. CONCLUSION: This study demonstrated a robust way to identify the sharing gene signature between subtypes of NAFLD and HCC, which contributed to a comprehensive understanding of pathogenesis from NAFLD to HCC.

Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is severely affecting the health and lives of patients. Clarifying the composition and regulatory factors of tumor immune microenvironment (TIME) is helpful for the treatment of PDAC. We analyzed the unique TIMEs and gene expression patterns between PDAC and adjacent normal tissue (ANT) using Gene Expression Omnibus (GEO) to find new immunotherapy targets. The Cancer Genome Atlas (TCGA) datasets were used to elucidate the possible mechanism of which tumor-associated macrophages (TAMs) changed in PDAC. We found that the composition of TAMs subtypes, including M0, M1, and M2, was different between PDAC and ANT, which was validated in recently published single-cell RNA-seq data. Many immune cells interacted with each other to affect the TIME. There were many DEGs enriched in some pathways that could potentially change the immune cell composition. KRT6A was found to be a DEG between PDAC and ANT that overlapped with DEGs between the M0-high group and the M0-low group in TCGA datasets, and it might alter and regulate TAMs via a collection of genes including COL5A2, COL1A2, MIR3606, SPARC, and COL6A3. TAMs, which could be a target of immunotherapy, might be influenced by genes through KRT6A and indicate an undesirable prognosis in PDAC.

Long non-coding RNA snaR is involved in the metastasis of liver cancer possibly through TGF-ß1.

It was previously demonstrated that the long non-coding RNA (lncRNA) small NF90-associated RNA (snaR) served an oncogenic role in human colon cancer, although its roles in other types of cancer remain unknown. To investigate the potential involvement of lncRNA snaR in hepatocellular carcinoma (HCC), expression of snaR in liver biopsies and plasma of patients with HCC and healthy controls was detected by reverse transcription-quantitative polymerase chain reaction. ELISA was used to determine the protein expression levels of transforming growth factor-ß1 (TGF-ß1). A snaR expression vector was transfected into HCC cells, and the effects on cell migration and invasion were analyzed by Transwell migration and Matrigel invasion assays, respectively. The protein expression levels of TGF-ß1 in HCC cells were detected by western blotting. The expression of snaR and TGF-ß1 was significantly increased in the patients with HCC compared with the healthy controls. The plasma expression levels of snaR and TGF-ß1 were positively correlated in patients with HCC; however, not in healthy controls. snaR overexpression significantly promoted cancer cell migration and invasion, and additionally increased TGF-ß1 expression. Treatment with TGF-ß1 did not significantly affect snaR expression. A TGF-ß1 inhibitor attenuated the effects of snaR overexpression in cancer cell migration and invasion. snaR may promote the metastasis of liver cancer through TGF-ß1.

Using Tree Shrews (Tupaia belangeri) as a Novel Animal Model of Liver Transplantation.

Liver transplantation (LT) is most effective and promising approach for end-stage liver disease. However, there remains room for further improvement and innovation, for example, to reduce ischemic reperfusion injury, transplant rejection and immune tolerance. A good animal model of LT is essential for such innovation in transplant research. Although rat LT model has been used since the last century, it has never been an ideal model because the results observed in rat may not be applied to human because these two species are genetically distinct from each other. In this study, we for the first time performed LT using the tree shrew (Tupaia belangeri), a species in the Order Scandentia which is closely related with primates, and evaluated the possibility to adopt this species as a new model of LT. We performed LT on 30 animals using the two-cuff technique, examining the success rate, the survival rate and the immunological reaction. The recipient operation time was 60 min averagely, and we limited the time of the anhepatic phase within 20 min. Twenty-seven (90%) of the animals survived for at least 3 days after the transplantation. Thirteen animals that did not receive any immunosuppressive drug died in 8 days mostly because of acute rejection effect (n=9), similar to the reaction in human but not in experimental rat. The rest 14 animals that were given rapamycin survived significantly longer (38 days) and half of them survived for 60 days until the end of the study. Our results suggest that performing LT in tree shrews can yield high success rate and high survival rate. More importantly, the tree shrews share similar immunological reaction with human. In addition, previous genomics study found that the tree shrews share more proteins with human. In sum, the tree shrews may outperform the experimental rats and could be used as a better and cost-effective animal model for LT.

Doublecortin-Like Kinase 1 (DCLK1) Regulates B Cell-Specific Moloney Murine Leukemia Virus Insertion Site 1 (Bmi-1) and is Associated with Metastasis and Prognosis in Pancreatic Cancer.

BACKGROUND/AIMS: Cancer stem cells (CSCs) are largely responsible for tumor relapse and metastatic behavior. Doublecortin-like kinase 1 (DCLK1) was recently reported to be a biomarker for gastrointestinal CSCs and involved in the epithelial-mesenchymal transition (EMT) and tumor progression. B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) is a crucial regulator of CSC self-renewal, malignant transformation and EMT, and a previous study from our group showed that Bmi-1 is upregulated in pancreatic cancer progression and participates in EMT. However, it remains unclear whether DCLK1 is involved in pancreatic cancer or whether DCLK1 is associated with the altered level of Bmi-1 expression. METHODS: The correlation of DCLK1 expression and clinical features of pancreatic cancer was analyzed in 210 paraffin-embedded archived pancreatic cancer specimens by immunohistochemical analysis. The biological effects of DCLK1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays, cell migration by wound healing assay and cell invasion by Transwell invasion assay. We further investigated the effect of therapeutic siRNA targeting DCLK1 on pancreatic cancer cell growth in vivo. Moreover, the molecular mechanism by which DCLK1 upregulates Bmi-1 expression was explored using real-time PCR, western blotting and Co-immunoprecipitation assay. RESULTS: DCLK1 is overexpressed in pancreatic cancer and is related to metastasis and prognosis. Knockdown of DCLK1 markedly suppressed cell growth in vitro and in vivo and also inhibited the migration and invasion of pancreatic cancer cells. Furthermore, we found that DCLK1 silencing could inhibit EMT in cancer cells via downregulation of Bmi-1 and the mesenchymal markers Snail and Vimentin and upregulation of the epithelial marker E-cadherin. Moreover, high DCLK1 expression in human pancreatic cancer samples was associated with a mesenchymal phenotype and increased cell proliferation. Further co-immunoprecipitation indicated that DCLK1 did not interact with Bmi-1 directly. CONCLUSION: Our data suggest that upregulation of DCLK1 may contribute to pancreatic cancer metastasis and poor prognosis by increasing Bmi-1 expression indirectly. The findings indicate that inhibiting DCLK1 expression might be a novel strategy for pancreatic cancer therapy.

Microarray expression profile analysis of circular RNAs in pancreatic cancer.

Pancreatic cancer remains one of the most malignant tumors with a poor prognosis. Despite advances in diagnosis and treatment, no reliable biomarkers are available for clinical practice. Circular RNAs (circRNAs) are a novel class of endogenous non­coding RNA, which are abundant, stable and conserved, and serve crucial roles in disease, particularly in cancer. The purpose of the present study was to investigate the expression profile of circRNAs in 20 pancreatic cancer tissues and corresponding paracancerous tissues using arraystar human circRNA array analysis, high­throughput circRNA microarray, bioinformatic analysis and reverse transcription­quantitative polymerase chain reaction. It was revealed that the circRNAs expression profile was significantly different between pancreatic cancer tissue and paracancerous tissue, which indicates a potential role in pancreatic cancer. It was predicted that circRNAs may act as a micro RNA sponge to modulate gene expression in pancreatic cancer. Additionally, microarray expression analysis data was submitted to the Gene Expression Omnibus under accession no. GSE79634. The present study revealed that circRNAs expression was visibly diverse in pancreatic cancer compared with paracancerous tissue and provides more reliable biomarkers and new insights into the mechanisms of pancreatic cancer.

Discs large homolog 5 decreases formation and function of invadopodia in human hepatocellular carcinoma via Girdin and Tks5.

Invadopodium formation is a crucial early event of invasion and metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying regulation of invadopodia remain elusive. This study aimed to investigate the potential role of discs large homolog 5 (Dlg5) in invadopodium formation and function in HCC. We found that Dlg5 expression was significantly lower in human HCC tissues and cell lines than adjacent nontumor tissues and liver cells. Lower Dlg5 expression was associated with advanced stages of HCC, and poor overall and disease-free survival of HCC patients. Dlg5-silencing promoted epithelial-mesenchymal transition, invadopodium formation, gelatin degradation function, and invadopodium-associated invasion of HepG2 cells. In contrast, Dlg5 overexpression inhibited epithelial-mesenchymal transition, functional invadopodium formation, and invasion of SK-Hep1 cells. Both Girdin and Tks5, but not the Tks5 nonphosphorylatable mutant, were responsible for the enhanced invadopodium formation and invasion of Dlg5-silenced HepG2 cells. Furthermore, Dlg5 interacted with Girdin and interfered with the interaction of Girdin and Tks5. Dlg5 silencing promoted Girdin and Tks5 phosphorylation, which was abrogated by Girdin silencing and rescued by inducing shRNA-resistant Girdin expression. Moreover, Dlg5 overexpression significantly inhibited HCC intrahepatic and lung metastasis in vivo. Taken together, our data indicate that Dlg5 acts as a novel regulator of invadopodium-associated invasion via Girdin and by interfering with the interaction between Girdin and Tks5, which might be important for Tks5 phosphorylation in HCC cells. Conceivably, Dlg5 may act as a new biomarker for prognosis of HCC patients.

Scutellarin suppresses migration and invasion of human hepatocellular carcinoma by inhibiting the STAT3/Girdin/Akt activity.

Scutellarin is an active flavone from Erigeron breviscapine (vant) Hand Mass. This study aimed to investigate the potential role of scutellarin in migration and invasion of human hepatocellular carcinoma (HCC) cells and its possible mechanism. In comparison with the vehicle-treated controls, treatment with scutellarin (50 mg/kg/day) for 35 days significantly mitigated the lung and intrahepatic metastasis of HCC tumors in vivo. Scutellarin treatment significantly reduced HepG2 cell viability in a dose-dependent manner, and inhibited migration and invasion of HCC cells in vitro. Scutellarin treatment significantly reduced STAT3 and Girders of actin filaments (Girdin) expression, STAT3 and Akt phosphorylation in HCC cells. Introduction of STAT3 overexpression restored the scutellarin-downregulated Girdin expression, Akt activation, migration and invasion of HCC cells. Furthermore, induction of Girdin overexpression completely abrogated the inhibition of scutellarin on the Akt phosphorylation, migration and invasion of HCC cells. Scutellarin can inhibit HCC cell metastasis in vivo, and migration and invasion in vitro by down-regulating the STAT3/Girdin/Akt signaling.

Leptin promotes proliferation and metastasis of human gallbladder cancer through OB-Rb leptin receptor.

Emerging evidence has shown that leptin, an adipocyte-derived cytokine that is closely associated with obesity, play a significant role in carcinogenesis and tumorigenesis. However, its impact on gallbladder cancer (GBC) remains unclear. In this study, we firstly found that leptin and its functional receptor OB-Rb were significantly co-expressed in human GBC tissues and cell lines, the content of which were higher than those in normal human gallbladder tissues. Treatment with leptin promoted the proliferation, migration and invasion of GBC cells, which were attenuated by OB-Rb shRNA. Blocking in the G2/M period of cell cycle, increasing of MMP3 and MMP9, increasing of VEGF-C/D, activation of SOCS3/JAK2/p-STAT3 pathway was demonstrated after treatment with leptin. All of these positive responses were attenuated by OB-Rb receptor shRNA. Taken together, our findings suggest that leptin promoted the proliferation, migration and invasion of GBC cells by increasing OB-Rb expression through the SOCS3/JAK2/p-STAT3 signal pathway. Targeting the leptin/OB-Rb axis could be an attractive therapeutic strategy for treatment of GBC.

FHIT overexpression in HepG2 hepatoma cells affects growth and cyclin D1 expression in vitro.

The aim of this study was to investigate the methylation status of fragile histidine triad (FHIT) and the effects of FHIT on cell growth and cyclin D1 expression in hepatoma cells. The total proteins from the human hepatoma cell lines HepG2, Hep3B and Huh7 were collected and the expression levels of FHIT were analyzed. The methylation status in the promoter region of FHIT in the hepatoma cells was measured using methylation-specific polymerase chain reaction (PCR). The HepG2, Hep3B and Huh7 cells were subsequently treated with 5-aza-2'-deoxycytidine (5-azadc) and the restoration of FHIT expression was then examined. A p-hemagglutinin (HA)-FHIT plasmid was constructed and used to transfect the HepG2 cells, and the inhibitory effects of the transfection on cell growth were then assessed. In addition, HepG2 cells were cotransfected with the pHA-FHIT plasmid and a cyclin D1 luciferase reporter plasmid, and the effects of FHIT on the activity of cyclin D1 transcription factor were analyzed using a luciferase assay. FHIT was observed to be expressed at a low level in Hep3B and HepG2 cells; however, it was expressed at a relatively high level in Huh7 cells. The promoter region of FHIT in the Hep3B and HepG2 cells was partially methylated, and 5-azadc treatment induced an increased expression of FHIT. The increased expression of FHIT inhibited the growth of HepG2 cells. Cotransfection with the pHA-FHIT plasmid significantly inhibited the transcriptional activity of the cyclin D1 promoter and decreased the expression of cyclin D1 in HepG2 cells. In conclusion, FHIT was partially methylated in the HepG2 and Hep3B hepatoma cells. The overexpression of FHIT inhibited cell growth and decreased the expression of cyclin D1 in HepG2 cells.

WW domain containing oxidoreductase induces apoptosis in gallbladder-derived malignant cell by upregulating expression of P73 and PUMA.

Gallbladder cancer (GBC) is one leading cause of cancer-related death worldwide. WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene which can suppress proliferation of a variety of tumors. However, little was known about the relationships between WWOX and gallbladder cancer. In the current study, we intended to investigate the tumor suppressive role of WWOX in gallbladder malignant cells both in vitro and in vivo, and explore the potential mechanism of tumor toxic function of WWOX. Our results have shown that WWOX triggerred apoptosis in GBC cells and increased the expression of P73 and PUMA in cytoplasm. We also have found that Bax has been upregulated after overexpression of WWOX, whereas, Bcl-2 was downregulated by WWOX. To further validate the results in vivo, we evaluated the tumor suppressive role of WWOX in mouse model of gallbladder cancer. The results have shown that the proliferation of the tumor was inhibited after delivery of WWOX, and the expressions of P73 and PUMA were upregulated in target tissues. The mice models administrated with WWOX have shown better prognosis than mice in negative control groups. The results from our study indicated that WWOX could be used as a therapeutic agent in the gene therapy of gallbladder cancer.

Periumbilical laparoscopic surgery through triple channels using common instrumentation.

Single-port laparoscopic technology is effective in minimally invasive surgery. However, this technology requires expensive instrumentation. In the present study, an alternative minimally invasive technique, periumbilical laparoscopic surgery through triple channels using common instrumentation, is introduced. Increased use of this new technique may be worthwhile since its results are comparable with those of single-port laparoscopic cholecystectomy. Periumbilical laparoscopic cholecystectomy using common instruments through triple channels was performed in 78 cases of simple cholecystolithiasis and 30 of gallbladder polyposis which were compared with a control group consisting of 356 cases of traditional laparoscopic cholecystectomy. The surgery was successfully completed using common instrumentation without complications in 106 cases from the experimental group. However, in 2 cases the surgery was changed to the traditional laparoscopic cholecystectomy due to bleeding in the area of Calot's Triangle. No statistical differences in the amount of surgical bleeding, intestinal function restoration time, hospitalization time and cost were observed between the two groups. The mean surgery times of the experimental and control groups were 110.31±14.57 and 43.98±7.64 min, respectively. The difference in surgery times was statistically significant. Based on relevant experience of the process of laparoscopic cholecystectomy, the periumbilical triple channel technique is safe and feasible for use in basic-level medical units and does not produce abdominal scarring so an unblemished appearance is preserved. Moreover, this approach only requires common laparoscopic instruments.