Study On The Effect Of Heparanase Gene Silencing By Mirna Interference On The Biological Behaviors Of Gallbladder Carcinoma Cells In Vitro

Gallbladder cancer (GBC), a rare and highly lethal disease is the most commonmalignant neoplasm of the biliary tract. Although steady progress has been achievedin clinical treatment, the prognosis of the GBC is very poor, primarily due to lack oftypical, specific symptoms and low clinical suspicion. Because the symptoms of GBCare usually nonspecific, some patients are diagnosed at the time of cholecystectomyfor biliary colic and cholelithiasis. GBC is one of the most commonly diagnosedmalignancies in bile duct system, indeed, GBC is the fifth most commongastrointestinal cancer. The goal for treating GBC is an operative resection which isthe most efficacious. No drug therapy has proven efficacious for GBC. Traditionaladjuvant chemotherapeutic, adjuvant radiotherapy and other adjuvant therapy wereprover to be ineffective. The majority of cases were diagnosed at a late stage, mostpatients already have occult or overt metastasis.Heparanase (HPA), a mammalian endo-beta-D-glucuronidase, is capable ofdegrading the heparan sulfate proteoglycans (HSPGs) in the ECM and BM. HPA isthe only enzyme that degrades heparan sulfate chains of heparan sulfate proteoglycansand is proposed to have a promoting role in cancer invasion, angiogenesis andmetastasis. Overexpression of HPA has been detected in many malignant tumors,showing a correlation with the metastatic potential, with positive association withprimary tumor local invasion and distant metastasis. HPA expression is inverselycorrelated with surgical resection survival. Thus HPA may represent an importantprognostic mark. Recently, it was found that in malignant tumors, including those ofmalignant parotid gland tumors, blood cancer and pancreatic cancer, HPA is overexpression, indicating that HPA may represent an ideal prognostic mark. Wethought that the reduced invasiveness of the cell lines through Matrigel might due tothe down-regulated expression of HPA by miRNA.In this study, we constructed HPA miRNA and transfected it into culturedGBC-SD cells by lipofectamineTM2000. The subclonal cells were generated forstable knockdown of HPA, to investigate the effects of HPA gene silencing bymiRNA interference on biological behaviors of human gallbladder cancer GBC-SDcells. We found that miRNA decreased HPA mRNA and protein expression inGallbladder cancer cells. Stable knockdown of HPA expression decreased the invitro migration, invasiveness and metastasis of Gallbladder cancer cells. Our resultsshowed that HPA is highly expressed in clear cell GBC-SD and suggested that HPAplays a role in invasion and metastasis of clear cell GBC-SD. These data suggestedthat HPA might be a novel target for treating the invasion and metastasis ofgallbladder cancer, and RNAi is might be a novel therapeutic agent for humanneoplasms.Part oneStudy of HPA Expression in Human Gallbladder CarcinomaGBC-SD CellsObjective: To detect the expression of HPA mRNA and protein in human gallbladdercarcinoma GBC-SD cells.Methods: Human gallbladder carcinoma GBC-SD cells in Logarithmic growth phasewere harvested, RT-PCR was performed to detect HPA mRNA expression levels.Western blot analysis and immunohistochemical staining were used to detect the HPAprotein expression in human gallbladder carcinoma GBC-SD cells.Results: RT-PCR results showed that the size of HPA mRNA fragment and β-actinmRNA fragment is585bp and218bp. Western-blot results showed that HPA proteinmolecular is65KDa and β-actin is43KDa. They are consistent with the theoretical value. RT-PCR results showed that HPA mRNA expression in GBC-SD cells was1.00±0.04. Western blot and Immunohistochemical staining results showed thatHPA protein expression was0.76±0.06and6.80±0.42. Immunohistochemicalstaining results showed positive significantly.Conclusions: Overexpression of HPA was detected in human gallbladder carcinomaGBC-SD cells. Part twoEffects of HPA Gene Silencing by miRNA Interference on HPAmRNA and Protein Expression in Human Gallbladder CancerGBC-SD CellsObjective: To construct specific miRNA expression vectors targeting human HPAgene sequence and detect the suppression efficiency of the constructed vectors inhuman gallbladder cancer GBC-SD cells.Methods: According to provide the sequence of Gene ID：10855, four pairs ofsingle-stranded recombinant plasmid miRNA oligo-DNA targeting human HPA genesequence were designed by making use of Invitrogen’s RNAi Designer on line. Thesesequences which correspond with miRNA were BLAST analyzed to make sure thatthey were not homology with genome data base. The artificial miRNA, which isbased on the murine miR-155, were constructed to produce silencing of HPA. Fourpairs of recombinant plasmid miRNA targeting human HPA gene sequence and apairs of negative control were transfected into GBC-SD cells by lipofectamineTM2000,respectively. Experimental design was divided into six groups: four interferencegroups, negative and blank. The transfection efficiency was monitored by theenhanced green fluorescent protein (EGFP) reporter within these vectors,48h after the Genesilencer-mediated transfection, EGFP was expressed within the cytoplasm ofGBC-SD cells. EGFP was observed under inverted fluorescence microscop. ThemRNA and protein expression of HPA were examined by RT-PCR and Western blotand adopt to select the recombinant plasmid which showed the best silencing effect.Results: Four pairs of HPA targeted microRNA (miRNA)-1,-2,-3and-4weresuccessfully inserted into the plasmid vector pcDNA6.2-GW/EmGFPmiR，and thecoding sequences of the obtained miRNA were consistent with the designedfragments．The recombinant plasmids(pcDNA6.2-GW/EmGFPmiR-HPA-1(HPA1)、pcDNA6.2-GW/EmGFPmiR-HPA-2(HPA2)、pcDNA6.2-GW/EmGFPmiR-HPA-3(HPA3)、pcDNA6.2-GW/EmGFPmiR-HPA-4(HPA4)and negative control were transfectedinto GBC-SD cells by lipofectamineTM2000, respectively. After transfecting48hours, RT-PCR results showed that the relative HPA mRNA expression levels inGBC-SD cells transfected with HPA1, HPA2, HPA3and HPA4were0.30±0.04,0.24±0.05,1.06±0.06and、0.36±0.09. GBC-SD cells transfected with HPA1, HPA2and HPA4showed significantly(p<0.05) decreased HPA mRNA expression levelswhen compared with the negative control and blank (0.90±0.07,0.96±0.08). Thewestern blotting analysis results indicated that the relative HPA protein expressionlevels in GBC-SD cells transfected with HPA1, HPA2, HPA3and HPA4were0.39±0.01,0.34±0.02,0.80±0.03, and0.42±0.01. GBC-SD cells transfected withHPA1, HPA2and HPA4showed significantly (p<0.05) decreased HPA proteinexpression levels when compared with the negative control and blank (0.76±0.02,0.79±0.03). RT-PCR and western blot were used to detected the HPA mRNA andprotein expression levels, the results indicated that recombinant plasmid HPA1,HPA2and HPA4could effectively knockdown the HPA gene expression in humangallbladder cancer cells, and select the recombinant plasmid HPA2which showed themost best silencing effect. Recombinant plasmid HPA2was as interference groupfor further experiments.Conclusions: Four pairs of recombinant plasmid pcDNA6.2-GW/EmGFPmiR expression vectors targerting HPA gene sequence were constructed successfully.Recombinant plasmid HPA2showed the best silencing effect was as interferencegroup for further experiments. Part threeEffect of HPA Gene Silencing by miRNA Interference on BiologicalBehaviors of Human Gallbladder Cancer CellsObjective: To investigate the effect of HPA gene silencing by miRNA interferenceon biological behaviors of human gallbladder cancer GBC-SD cells.Methods: Recombinant plasmid HPA2which showed the highest inhibited rate wasas interference group for further experiments. Experimental design was divided intothree groups: interference group HPA2, negative control and blank. The cellularshape of GBC-SD cell was observed through microscopy and Confocal LaserScanning Fluorescence Microscope (LSFM）, Cell proliferation was analyzed by MTTassay, Cell Cycle and cell apoptosis was detected by flowcytometry, Cell migrationand invasion was detected by matrigel invasion assays. Wound closrue assays wereperformed to examine the migration and invasive ability of the GBC-SD cells.Results: We observed through microscopy and LSFM that the cellular shape was notaffected obviously before and after transfection. MTT assay results indicated thatcells proliferation ability was ineffective when compared with the negative controland blank. However, stable knockdown of HPA expression did not affect theproliferation and cell cycle of GBC-SD cells obviously. HPA gene silencing bymiRNA interference of GBC-SD cells showed significantly (P<0.05) increased onearly apoptosis rate when compared with the negative control and blank. GBC-SDcells transfected with HPA2showed significantly (p<0.05) decreased invasiive ability when compared with the negative control and blank. GBC-SD cells transfected withrecombinant plasmid HPA2showed the significant shorter(p<0.05) migration distanceat0h,12h,24h when compared with the negative control and blank.Conclusions：HPA Gene Silencing by miRNA Interference can effectively inhibit cellularmigration and invasiveness and affect apoptosis of GBC-SD cells. Howere, Stableknockdown of HPA expression did not affect the proliferation and cell cycle ofGBC-SD cells in vitro．...