Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.

MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway.

Survival after surgery for gallbladder cancer is generally poor. A number of inflammation-based prognostic scores have been established to help predict survival after surgery for several types of cancer. The objective of this study was to analyze and compare the utility of two inflammation-based prognostic scores, the Glasgow prognostic score (GPS) and the neutrophil-to-lymphocyte ratio (NLR), for predicting survival in patients with gallbladder cancer after surgery with curative intent. We retrospectively reviewed the medical records of 85 patients with histologically confirmed, resectable gallbladder carcinoma (GBC), who were to receive curative surgery in our department. Univariate and multivariate analyses were performed to evaluate the relationship between the variables to overall survival (OS). A significant difference was detected in OS in patients with low and high GPS and NLR scores. Univariate analyses using clinicopathological characteristics revealed that tumor differentiation; tumor invasion; lymph node metastasis; tumor, node, metastasis classification system stage; positive margin status; combined common bile duct resection; serum levels of C-reactive protein, albumin, carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen, and CA125; white blood cell count; and GPS and NLR were all associated with OS. Among these characteristics, multivariate analysis demonstrated that a high GPS was independently associated with poorer OS, together with tumor invasion, lymph node metastasis, and positive margin status. GPS is superior to NLR with respect to its prognostic value for patients with GBC after surgery with curative intent. GPS is not only associated with tumor progression but is also an independent marker of poor prognosis.

Evaluation of Two Inflammation-Based Prognostic Scores in Patients with Resectable Gallbladder Carcinoma

Neutrophil/lymphocyte ratio (NLR) and 
platelet/lymphocyte ratio (PLR) were immune response-related indicators. Preoperative NLR and PLR had been considered to be related to the prognosis of various cancers. The objective of this study was to evaluate the prognostic significance of NLR and PLR in patients with gallbladder carcinoma (GBC). From 2001 to 2013, 145 patients with GBC were recruited in this retrospective study. Cutoff values of NLR and PLR were determined by receiver operating characteristic curves (ROC). The correlation of clinical data, including tumor differentiation, nevin stage, TNM stage, operation margin, operation mode, NLR, PLR, hemoglobin, C reactive protein (CRP), carcinoembryonic antigen (CEA), and carbohydrate antigen 199 (CA199) with median survival period of patients was analyzed by univariate survival analysis. The multivariate prognosis analysis was performed to select the independent prognostic factors. The cutoff values of NLR and PLR were 1.94 and 113.34, respectively. Compared with low NLR and low PLR group, the 5-year survival rates in high NLR and high PLR group were reduced (P < 0.05). The degree of tumor differentiation, nevin stage, TNM stage, operation mode, NLR, PLR, CA199, total bilirubin, CRP and CEA were associated with the median survival period of patients (P < 0.01). The multivariate prognosis analysis showed that NLR, nevin stage, operation mode and hemoglobin were independent prognostic factors (P < 0.05). Preoperative NLR and PLR were closely related to prognosis of patients with GBC and might be useful for the evaluation of prognosis of patients with GBC.

Prognostic significance of preoperative neutrophil/lymphocyte ratio and platelet/lymphocyte ratio in patients with gallbladder carcinoma

Prognostic significance of combined preoperative fibrinogen and CA199 in gallbladder cancer patients

Gallbladder carcinoma is the most common malignancy of the biliary tract and is associated with a very poor outcome. The aim of the present study was to investigate the effects of oxymatrine (OM) on gallbladder cancer cells and the possible mechanism of its effects. The effects of OM on the proliferation of gallbladder cancer cells (GBC-SD and SGC-996) were investigated using cell counting kit-8 and colony formation assays. Annexin V/propidium iodide double staining was performed to investigate whether OM could induce apoptosis in gallbladder cancer cells. The mitochondrial membrane potential (ΔΨm) and expression of apoptosis-associated proteins were evaluated to identify a mechanism for the effects of OM. In addition, the RNA expression of relevant genes was measured by qRT-PCR using the SYBR Green method. Finally, a subcutaneous implantation model was used to verify the effects of OM on tumor growth in vivo. We found that OM inhibited the proliferation of gallbladder cancer cells. In addition, Annexin V/propidium iodide double staining showed that OM induced apoptosis after 48 h and the ΔΨm decreased in a dose-dependent manner after OM treatment. Moreover, the activation of caspase-3 and Bax and downregulation of Bcl-2 and nuclear factor κB were observed in OM-treated cells. Finally, OM potently inhibited in-vivo tumor growth following subcutaneous inoculation of SGC-996 cells in nude mice. In conclusion, OM treatment reduced proliferation and induced apoptosis in gallbladder cancer cells, which suggests that this drug may serve as a novel candidate for adjuvant treatment in patients with gallbladder cancer.

Effects of oxymatrine on the apoptosis and proliferation of gallbladder cancer cells.

Objective To establish an experimental liver metastatic model of gallbladder cancer and isolate the subpopulation with high metastatic potential from the model, which may serve as a reliable tool in research on liver metastasis of gallbladder cancer in vivo and in vitro. Methods Human gallbladder cancer cells of the line GBC-SD were cultured. Ten nude athymic BALB/c mice, aged 4 approximately 5 weeks, underwent inoculation of suspension of GBC-SD cells into the spleens and then their spleens were resected. Three weeks later laparotomy was performed to observe if liver metastasis occurred. Once liver metastasis was discovered, the mice were killed and the livers were taken out to undergo microscopy, tumor cells were isolated from the metastatic foci and cultured in vitro, and then inoculated into other 10 mice in the same way as their parents cells for the second round of selection. The similar steps were repeated, altogether for three rounds of selection and a total 30 mice were inoculated in 3 rounds so as to find subpopulation with high metastatic potential. Another 10 mice underwent subcutaneous inoculation, 90 days later the mice were killed to observe if pulmonary metastasis occurred. PCR was used to detect the 3 microsatellite sequences D14S68, D18S69, and D20S199 in the DNA samples of the gallbladder cancer cells of the parent cell line GBC-SD, the isolated cells of the subpopulation with high metastatic potential, and the liver of experimental animal. Results 90% of the mice inoculated subcutaneously with the GBC-SD cells developed subcutaneous tumors, however, no mouse in this group died and no pulmonary metastasis was found. The liver metastatic rate was 65% in the 10 mice undergoing intrasplenic inoculation. Thus a metastatic model of human gallbladder cancer in nude mice was established. The liver metastatic tumors were uniformly distributed throughout the liver parenchyma with predominance to the periphery. Despite multiple sites of involvement, the left lobes were most commonly affected in all experimental animals. Histological examination of the metastatic lesion demonstrated adenocarcinoma. Gross hepatic metastasis was detected 10, 7, and 5 weeks after the inoculation respectively in the first, second, and third round selection respectively with an incidence rate of metastases of 60%, 70%, and 90% respectively. From the third round metastatic model a subpopulation with high metastatic potential was isolated and designated as GBC-SD/M, which exhibited the similar histological characteristics as its parent cell line GBC-SD under inverted light microscopy. The three amplified bands at the sites 14S68, D18S69, and D20S199, amplified with the three pairs of specific primers for the three homo-specific microsatellites, were detected in the GBC-SD cells and GBC-SD/M cells, but not in the liver of tumor-bearing animal. Conclusion A liver metastasis model of human gallbladder cancer has been established, a reliable and efficient tool for study on the metastasis of gallbladder cancer to liver in vivo. Isolated from hepatic metastasis, the line of GBC-SD/M is a subpopulation with high metastatic potential, retaining the histological properties and identification of genetic background of its parent cell line GBC-SD.

Ke-qiang Li

Papers

Establishment of liver metastasis model of human gallbladder cancer and isolation of the subpopulation with high metastatic potential