The lncRNA-MALAT1/miR-330-3p Axis Promotes Growth and Metastasis of Gastric Cancer Through Regulation of Vascular Endothelial Growth Factor A: A Genetic and Cell Evaluation Study

SUMMARY: Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in various tumor tissues and cell lines was found to promote tumor cell proliferation, migration, and invasion. However, the role of MALAT1 in gastric cancer (GC) is still unclear. We aimed to investigate the correlation between long-chain non-coding RNAs (lncRNAs), MALAT1, MicroRNAs (miRNA) and vascular endothelial growth factor A (VEGFA) in gastric cancer and to disclose underlying mechanism. The correlation between MALAT1 levels and clinical features was analyzed by bioinformatics data and human samples. The expression of MALAT1 was down regulated in AGS cells to detect the cell proliferation, migration, and invasion characteristics, as well as the effects on signal pathways. Furthermore, we validated the role of MALAT1/miR-330-3p axis in GC by dual luciferase reporter gene assays. Expression of MALAT1 was higher in cancer tissues than in para-cancerous tissues. The high MALAT1 level predicted malignancy and worse prognosis. Down-regulation of MALAT1 expression in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA. By dual luciferase reporter gene assay and miR-330-3p inhibitor treatment, we demonstrate that MALAT1 sponged miR-330-3p in GC, leading to VEGFA upregulation and activation of the mTOR signaling pathway. The MALAT1/miR-330-3p axis regulates VEGFA through the mTOR signaling pathway and promotes the growth and metastasis of gastric cancer.

Se descubrió que la sobreexpresión del transcrito 1 de adenocarcinoma de pulmón asociado a metástasis (MALAT1) en varios tejidos tumorales y líneas celulares promueve la proliferación, migración e invasión de células tumorales. Sin embargo, el papel de MALAT1 en el cáncer gástrico (CG) aún no está claro. Nuestro objetivo fue investigar la correlación entre los ARN no codificantes de cadena larga (lncRNA), MALAT1, los microARN (miARN) y el factor de crecimiento endotelial vascular A (VEGFA) en el cáncer gástrico y revelar el mecanismo subyacente. La correlación entre los niveles de MALAT1 y las características clínicas se analizó mediante datos bioinformáticos y muestras humanas. La expresión de MALAT1 se reguló negativamente en las células AGS para detectar las características de proliferación, migración e invasión celular, así como los efectos sobre las vías de señales. Además, validamos el papel del eje MALAT1/miR- 330-3p en GC mediante ensayos de genes indicadores de luciferasa dual. La expresión de MALAT1 fue mayor en tejidos cancerosos que en tejidos paracancerosos. El alto nivel de MALAT1 predijo malignidad y peor pronóstico. La regulación negativa de la expresión de MALAT1 en células AGS inhibió la proliferación, migración e invasión celular al apuntar a VEGFA. Mediante un ensayo de gen indicador de luciferasa dual y un tratamiento con inhibidor de miR-330-3p, demostramos que MALAT1 esponjaba miR-330-3p en GC, lo que lleva a la regulación positiva de VEGFA y la activación de la vía de señalización mTOR. El eje MALAT1/miR-330-3p regula VEGFA a través de la vía de señalización mTOR y promueve el crecimiento y la metástasis del cáncer gástrico.

Reversal of multi-drug resistance by pSUPER-shRNA-mdr1 in vivo and in vitro.

AIM: To explore the possibility of reversing multi-drug resistance (MDR) to HepG2/mdr1 in vitro and in vivo with RNA interference (RNAi). METHODS: HepG2/mdr1 was obtained by cloning the whole gene mdr1 into HepG2 cells. shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence. pSUPER-shRNA/mdr1 was constructed using the enzyme-digested technique. HepG2/mdr1 cells were transfected with vectors of pSUPER-shRNA/mdr1 to measure their efficacy by real-time PCR for mdr1 mRNA, flow cytometry (FCM) for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdr1 function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdr1 in situ and into intra-abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemotherapy with adriamycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp. RESULTS: pSUPER-shRNA/mdr1 was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression (11.0% vs 98.2%, P<0.01). Real-time PCR showed that mRNA/mdr1 was lower in test groups than in control groups (18.73+/-1.33 vs 68.03+/-2.21, P<0.001). Compared with HepG2, the sensitivity of HepG2/mdr1 and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups (65.1% vs 94.1%, P<0.05). The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups (700.14+/-35.61 vs 1659.70+/-152.54, P<0.05). Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis. CONCLUSION: pSUPER-shRNA/mdr1 vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.

Pregnancy after liver transplantation: four-year follow-up of the first case in mainland China.

The safety and feasibility of pregnancy following liver transplantation (LT) have been accredited in a series of LT center. The first case in mainland China is reported. The follow-up data of a 22-year-old pregnant patient with end-stage liver disease undergone orthotopic liver transplantation were analyzed retrospectively. After surgery, the patient was uneventfully recovered and became pregnant 33 mo after LT. The patient was closely monitored and treated with a standard and individualized triple-drug immunosuppressive therapy throughout her pregnancy. Caesarean section was performed in March 18, 2004, and a health live-born infant was delivered. After the delivery, a 4-year follow-up period indicated that the patient was satisfactory with her condition and her baby was healthy. Our case shows that a successful pregnancy following LT is possible and safe in women with end-stage liver diseases under close monitoring. Three factors including mother, baby, and transplanted liver function must be considered for the safety of high-risk pregnancies.

Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein.

AIM: To investigate the effects of interferon-alpha (IFN-alpha) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-alpha. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-alpha-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-alpha-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-alpha-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-alpha. CONCLUSION: IFN-alpha can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-alpha therapy of HCC.

A successful pregnancy following liver transplantation.

BACKGROUND: The safety and possibility of pregnancy following liver transplantation has been the hot topic in transplant. A case is reported with a review of the literature. METHOD: The data of a 22-year-old pregnant patient with end-stage liver disease who had undergone orthotopic liver transplantation in September 28, 2000 were analyzed retrospectively. RESULTS: After surgery, the patient was uneventfully recovered and was pregnant at the time of the 33rd month postoperation. The patient experienced a rejection on the 8th week of pregnancy and was successfully treated at this hospital. The patient was closely monitored throughout her pregnancy, and received routine antenatal care with respect to sonographic screening. Caesarean section was performed in March 18, 2004, and a health live-born infant weighing 2000 g was delivered at full-term. After the delivery, the patient was satisfactory with her health and the baby was healthy. CONCLUSION: Under close monitoring, successful pregnancy following liver transplantation is possible and safe in women with end-stage liver diseases.

Problems in adult living donor liver transplantation using the right hepatic lobe.

BACKGROUND: Adult living donor liver transplantation (LDLT) is now widely applied to patients, children or adults, and the graft extends from the left hepatic lobe to the right hepatic lobe. Harvesting the right hepatic lobe would mean putting the donor at high risk. The congestion of a graft may cause small-for-size syndrome. The safety of the donor and its evaluation, which are related to the outcome for the recipient, play an important role in LDLT. How to decrease the congestion of the graft is another challenge to transplant experts. DATA SOURCES: A literature search from MEDLINE about adult LDLT in recent years was made to analyze the safety of the living donor and the innovation of surgical techniques for preventing small-for-size syndrome. RESULTS: The top priority for adult LDLT is donor safety. Preoperative donor evaluation consists of three stages: phase 1 for general evaluation, phase 2 for laboratory tests, and phase 3 for radiological evaluation of graft volume and vessel anatomy. The potential pathogenic mechanisms of small-for-size syndrome seem to be related to persistent portal hypertension and portal overperfusion. Improved surgical techniques for decreasing portal hypertension and preventing congestion of a graft may reduce the incidence of small-for-size syndrome. The improved techniques include reconstruction of the tributaries of the middle hepatic vein, end-to-side portocaval shunting, ligation of the splenic artery, dual-graft transplantation, and modified reconstruction of hepatic veins. CONCLUSION: With the careful preoperative assessment and the safety of the living donor, as well as improved surgical techniques, adult LDLT using the right lobe is safe.

Effect of peroxisome proliferator-activated receptor-gamma ligand on inflammation of human gallbladder epithelial cells.

AIM: To investigate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) and its ligand, ciglitazone, on inflammatory regulation of human gallbladder epithelial cells (HGBECs) and to assess the effect of human epithelial growth factor (hEGF) on growth of HGBECs. METHODS: HGBECs were cultured in media containing hEGF or hEGF-free media. HGBECs were divided into normal control group, inflammatory control group and ciglitazone group (test group). Inflammatory control group and ciglitazone group were treated with 5 microg/L of human interleukin-1beta (hIL-1beta) to make inflammatory model of HGBECs. The ciglitazone group was treated with various concentrations of ciglitazone, a potent ligand of PPAR-gamma. Subsequently, interleukin-8 (IL-8), IL-6, and tumor necrosis factor-alpha (TNF-alpha) concentrations in all groups were measured. The data were analyzed statistically. RESULTS: HGBECs were cultured in medium successfully. The longevity of HGBECs in groups containing hEGF was longer than that in hEGF-free groups. So was the number of HGBECs. The longest survival time of HGBEC was 25 d. The inflammatory model of HGBECs was obtained by treating with hIL-1beta. The concentrations of IL-6 and IL-8 in ciglitazone group were lower than those in inflammatory control group (P<0.05). The secretion of IL-6 in inflammatory control group was higher (350.31+/-37.05 microg/L) than that in normal control group (50.0+/-0.00 microg/L, P<0.001). Compared to normal control group, IL-8 concentration in inflammatory control was higher (P<0.05). CONCLUSION: hEGF improves the growth of HGBECs in vitro. Ciglitazone inhibits the inflammation of HGBECs in vitro and has potential therapeutic effect on cholecystitis in vivo.

Liver transplantation for patients with hepatolithiasis.

BACKGROUND: Because of its complicated pathological features such as repeated cholangitis and multiple operations, hepatolithiasis is difficult to treat and often lead to portal hypertension, and liver failure. The aim of this study was to investigate the indications of orthotopic liver transplantation (OLT)for patients with hepatolithiasis and the improvement of operative techniques and the treatment of postoperative complications. METHODS: The data of 4 patients with hepatolithiasis who had undergone OLT at our department in recent years were reviewed retrospectively. RESULTS: The 4 patients with hepatolithiasis complicated with secondary biliary cirrhosis in end-stage liver disease included 1 woman and 3 men. One patient underwent OLT using veno-venous bypass prior to the mobilization of the liver. Two patients were subjected to liver transplantation with improved piggyback technique without bypass. One patient received OLT without bypass. All patients were operated on successfully and recovered uneventfully. Patient 1 had bleeding from the digestive tract on the postoperative day 6. Patient 2 had hemorrhage from the digestive tract and a leakage of end-to-side intestinal anastomosis on the postoperative day 44. Patient 4 was reoperated on because of hemorrhage from the anastomotic stoma of the hepatic artery on the postoperative days 8 and 10. In the 4 patients the mean operative time was 7.9 hours and blood loss was 910 ml. Postoperative pathological analysis revealed biliary liver cirrhosis. Follow-up of all patients showed good conditions. CONCLUSIONS: Hepatolithiasis with secondary biliary liver cirrhosis in patients with end-stage liver disease is indicated for liver transplantation. Veno-venous bypass prior to the mobilization of the liver and improved piggyback technique can lower the incidence of intraoperative bleeding, the duration of surgery and postoperative complications.

[An experimental study on the gastroantrium-cholecystostomy using pylorus sphincter to replace Oddi sphincter for preventing reflux].

OBJECTIVE: This experimental study of gastroantrium-cholecystostomy was designed to identify the possible use of pylorus sphincter as a substitute for Oddi sphincter. METHODS: Fifteen prairie dogs were randomized to three groups. The dogs in the control group underwent sham operation. The dogs in the first experiment group were subjected to gastroantrium-cholecystostomy and gastrojejunostomy. Meanwhile, two thin tubes were placed in stomach antrium and duodenum separately for post-operative photography. One week after operation, 20% cyctografin (10 ml) was injected into the duodenum through the relevant tube so as to check whether any backflow came out from duodenum to the bile duct. Then, 20% cyctografin (5 ml) was injected into the stomach antrium so as to demonstrate how it went from stomach antrium into duodenum. The samples of liver, stomach, stomach antrium, gallbladder and extra hepatic duct were collected for pathological examinations. The dogs of the second experiment group underwent the same operations, but the samples for pathological examination were collected four weeks after operation. RESULTS: Roentgenography showed that there was no contrast medium backflowing into the stomach antrium, and the contrast medium could flow from stomach antrium into duodenum smoothly. The results of histo-pathological examination showed that there were no obvious changes in the liver, stomach antrium and bile duct. Under light microscope, a few inflammatory cells could be seen in the gallbladder wall, and the mucosa of stomach was slightly atrophic. CONCLUSION: The gastroantrium-cholecystostomy which uses pylorus sphincter to take over the functions of Oddi sphincter can effectively prevent intestinal juice from flowing back into the bile duct.

Liver transplantation for patients with hepatic alveolar echinococcosis in late stage.

BACKGROUND: Failed surgical treatment and multiple operations often lead to liver failure and make it difficult to be treated by traditional methods. Liver transplantation may be the ideal and the last choice. In this study we tried to explore the indication of liver transplantation for hepatic alveolar echinococcosis (HAE) and the improvement of intraoperative treatment. METHODS: Five patients who had received liver transplantation of hepatic alveolar echinococcosis in our hospital from 1999 through 2003 were analyzed retrospectively. RESULTS: All the patients (3 were male and 2 female) were in late stage of hepatic alveolar echinococcosis. During orthotopic liver transplantation(OLT), 4 patients underwent veno-venous bypass, 3 were subjected to veno-venous bypass prior to the mobilization of the liver, and 2 received placement of T tube in the bile duct. In all the patients treated successfully by OLT, 4 recovered, and 1 died of severe infection and acute rejection after operation. T tube fell off in one patient. Postoperatively, pathological diagnosis verified hepatic alveolar echinococcosis. Four patients were followed up to the present, showing a good life quality and work capacity. CONCLUSIONS: Hepatic alveolar echinococcosis in late stage can be considered as one of the indications of liver transplantation. Technique of veno-venous bypass prior to the mobilization of the liver could decrease operative time and bleeding. Early diagnosis and treatment of the disease ensures a better prognosis.

[A study on inflammatory regulation of human gallbladder epithelial cells by PPAR-gamma ligand].

OBJECTIVE: To investigate the inflammatory regulation of human gallbladder epithelial cells (HGBEC) by peroxisome proliferator activated receptor gamma (PPAR-gamma) ligand ciglitazone. METHODS: HGBEC were cultured in medium containing human epidermal growth factor (hEGF). HIL-1beta were added into the ciglitazone groups and inflammatory control groups to make inflammatory model . IL-6 and TNF-alpha concentration in ciglitazone groups and all control groups were measured. RESULTS: HGBEC were cultured in medium successfully. The inflammatory model was made. The longest duration is 25 d. In inflammatory control groups, cells were edema with unclear cellular membrane and plasmid. In ciglitazone groups, the inflammatory edema of cells were less evident than that in inflammatory control groups, especially in 50 micromol/ml group. The IL-6 concentration in ciglitazone groups is lower than that in control group (P<0.01). The relation between the inhibitory effect and the concentration of ciglitazone is positive correlation. CONCLUSION: Ciglitazone that can inhibit the inflammation of HGBEC maybe an effective treatment for acute and chronic cholecystitis.