Objective:To develop a real-time fluorescent quantitative reverse transcription polymerase chain reaction(RFQ-RT-PCR) system for determination of the expression of guanylyl cyclase-C(GC-C) mRNA in the peripheral blood mononuclear cells(PBMC) in patients with colorectal cancer.Methods: The specific primers and TaqMan probe targeting the high conservative region of GC-C gene were designed for PCR amplification and the fluorescence was monitored in a real time manner.The expression levels of GC-C mRNA in clinical samples(including 30 healthy blood donors,11 patients with benign intestinal lesions and 37 with colorectal cancer) were calculated according to the standard curve.The mRNA levels of GC-C were presented as the ratios of GC-C mRNA to ?_2-microgluobulin mRNA.Results: The linear detection range of this RFQ-RT-PCR system was from 10~1 to 10~9 pg/ml and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 6.87% to 11.12% and from 8.86% to 15.19%,respectively.GC-C mRNA was expressed in 31/37 colorectal cancer patients,but not in healthy blood donors and patients with benign intestinal lesions.The mean level of GC-C mRNA was(0.88?)(0.06) in the above 31 colorectal cancer patients.GC-C mRNA levels were positively correlated to the Duke stage,lymph node metastasis and liver metastasis but not to patients' age,sex and tumor size.Conclusion: RFQ-RT-PCR has good sensitivity,specificity and reproducibility in determination of GC-C mRNA levels.Determination of GC-C mRNA levels in colorectal cancer patients may play a role in assessing Duke stage,lymph node metastasis,liver metastasis and approaching post-operation therapy.

Objective To investigate the role of the expression of a proliferation-inducing ligand (APRIL) and its receptors in the gastric carcinogenesis. Methods The real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) was used to detect expression of APRIL and its receptors. Based on the standard curves, the quantitative levels of target genes in tissue samples were determined by using software, and the results were presented as the ratios of mRNA levels of target genes to ?2-microgluobulin(?2M). Results The detection linear range of RFQ-PCR was 101-109 pg/ml and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged 6.52% - 12.02% and 8.76% - 14.16%, respectively.The expression levels of APRIL in the tissue of intestinal metaplasia , dysplasia and gastric cancer were significantly higher than those in normal tissue(P0.05 , respectively). Conclusions The present study indicated that RFQ-PCR had satisfied sensitivity and reproducibility in quantitative measurement of APRIL and its receptors. APRIL may play an important role in the development and progress of gastric cancer and could be established as a target molecule for early diagnosis and anti-cancer therapy. Besides, there maybe some unknown receptors of APRIL expressed on tumor tissue.

Objective To construct small hairpin RNA(shRNA) lentiviral vectors targeting human a proliferation-inducing ligand(APRIL) gene and detect the titer of virus and suitable multiplicity of infection (MOI) after 293T cells were infected by the lentival vectors. Methods Three RNA interference targeting sequences of APRIL gene were screened including shAPRIL1210, shAPRIL1754 and shAPRIL1604. Both sense and antisense Oligo DNA of the targeting sequences were synthesized and cloned into the pGCL-GFP vector, respectively. The resulting lentiviral vectors containing shAPRIL were named LV-shAPRIL1210, LV-shAPRIL1754, LV-shAPRILI604. Then they were confirmed by PCR and DNA sequencing. 293T cells were co-transfected with LV-shAPRIL, pHelper 1.0 and pHelper 2. 0 to product lentivirus, respectively. The titer of virus and suitable MOI were tested according to the expression level of GFP in the 293T cells. Results PCR analysis and DNA sequencing confirmed that three shAPRIL DNA were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5 × 107, 6 × 107 and 4 × 107(transduction units )TU/ml, respectively, and the suitable MOI was 5. Conclusions Three shRNA lentiviral vectors targeting human APRIL gene have been successfully constructed, which lays a foundation for future studying APRIL's gene silencing in related target cells.

Objective To investigate the inhibitive effect of shRNA (short hairpin RNA) targeting APRIL gene on the pancreatic cancer cells in vitro and in vivo, in order to explore the feasibility of gene therapy for pancreatic cancer. Methods The LV-shAPRIL targeting APRIL gene had been constructed before, and was used to infect the CFPAC-1 cells. Cell proliferation and apoptosis were examined by MTT and flow cytometry. Then CFPAC-1 cells were used to construct the model of transplantation tumor into the nude mice, the tumor growth was assessed after LV-shAPRIL treatment. Results 96 hours after the LV-shAPRIL infection into CFPAC-1 cells, the cell proliferation was significantly inhibited when compared with control group and lentivirus infection group (P＜0.05 ). Flow cytometry showed the apoptosis ratio of the CFPAC-1 cells was (17.35±0.96)% in LV-shAPRIL group, which was higher than that in control group and lentivirus infection group (P＜0.05 ). After LV-shAPRIL injection into the model of nude mice, the tumor growth was slower than that in the two control groups. The tumor's volume of the LV-shAPRIL group was(821.8±123.3) mm3 and the mass was (2.16±0.18)g at 27 day, and were obviously depressed, when compared with two control groups (P±0.05). Conclusions LV-shAPRIL targeting APRIL gene can inhibit the growth of the CFPAC-1 cells in vitro and vivo. This may provide a new gene therapy approach for pancreatic cancer.

Objective To construct of shRNA lentiviral expression vector targeting APRIL (aproliferation-inducing ligand) gene in CFPAC-1 cell of human pancreatic cancer. Methods We used gene engineering to screen RNA interference targeting sequence of APRIL gene. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCL-GFP vector. The resulting lentiviral vector containing shAPRIL were named LV-shAPRIL. Then it was conformed by PCR and DNA sequencing identification. 293T cells were eotransfected with LV-shAPRIL,pHelper 1.0 and pHelper 2.0 to product ientivirus. The titer of virus was tested according to the expression level of GFP in the 293T cells. After recombinant lentivirus infection into CFPAC-1 cells, we used real-time RT-PCR and Western blotting to examine APRIL mRNA and protein expression at different cell culture period.Results PCR analysis and DNA sequencing conformed that shAPRIL DNA was successfully inserted into the lentiviral vector. The titer of concentrated virus were 5 × 107 TU/ml. APRIL expression in CFPAC-1 cells were inhibited significantly at both mRNA and protein level. APRIL mRNA expression were decreased 73%, 70%and 71% , respectively, after the infection of 4 days, 4 weeks and 8 weeks by LV-shAPRIL. APRIL protein expression were decreased 66%, 63% and 62%, respectively , after the infection of 4 days , 4 weeks and 8weeks by LV-shAPRIL. Conclusions ShRNA lentiviral expression vector targeting APRIL gene has been successully constructed, and it can effectively inhibit the expression of APRIL gene in CFPAC-1 ceils. This study lays a foundatin for in vivo research APRIL gene scilence in pancreatic cancer cell using the model of nude mice.

Objectives To establish real-time fluorenscence quantitative polymerase chain reaction ( RFQ-PCR ) for measurement of the expression level of guanylyl cyclase-C(GC-C) in the Peripheral blood mononuclear cell(PBMC)in 30 blood donors, 10 cases colorectal cancer tissue and 1 case T84 human colon cancer cell line. Methods Specific primers and TaqMan probe have been designed,and fluorenscence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression level of GC-C in clinical samples has been determined using software, and the results were presented as the ratios of GC-C mRNA to?2-microgluobulin(?2M)mRNA. Results The detection range of the assay was from 101 pg/ml to109pg/ml,the coefficient of variation values of both intra-experimental and inter- experimental reproducibility were 6. 87% to 11. 12% and 8. 86% to 15. 19% . None of 30 blood donors and 11 benign intestinal patients expressed GC-C mRNA,it was expressed in 31/37 colorectal cancer patients. The expression level of GC-C mRNA in colorectal cancer patients was 0. 88?0.06,and the expression level of its in colorectal carcinoma tissue and T84 cells were 0. 86?0.07/ug tissue and 0.0082/per cell. Conclusions This assay had high sensitivity,specificity and reproducibility.

Objective To study the expression of long non-coding RNA (LncRNA) stomach cancer-associated transcript 16 (STCAT16) in gastric cancer tissues and its effects on the proliferation, migration and invasion of gastric cancer cells.Methods The different expression of STCAT16 in 32 cases of gastric carcinoma and corresponding adjacent tissues was detected by real-time fluorescence quantitative polymerase chain reaction (PCR).The STCAT16 overexpression plasmid and empty vector was separately transfected gastric cancer cell line AGS with low expression of STCAT16.The cell proliferation of empty vector group, non-transfection group and STCAT16 analogue transfection group was evaluated by cell counting kit-8 (CCK-8) assay at 0, 24, 48, 72 and 96 hours after transfection.The colony forming ability was tested by colony formation assay.The cell invasion ability was measured by Transwell chamber assay and migration ability was tested by scratch-wound assay.The effects of STCAT16 on tumorigenicity in vivo were verified by tumorigenicity experiments in nude mice.T-test and one-way analysis of variance were performed for data analysis.Repeated measures analysis of variance was used to compare the repeated measured data among groups.Chi square test and Fisher exact probability method were used for comparison of counting data.Results The expression of STCAT16 in gastric cancer tissues was low (0.87±0.19), while it was high in corresponding adjacent tissues (2.32±0.37), and the difference was statistically significant (t=-20.859, P<0.05).The expression of STCAT16 of STCAT16 analogue transfection group was higher than that of empty vector group (3.43±0.25 vs 1.00±0.06), and the difference was statistically significant (t=-16.795,P<0.05).Compared to empty vector group and non-transfection group, the cell proliferation decreased in STCAT16 analogue transfection group at 72 and 96 hours after transfection (1.41±0.07, 1.42±0.08, 1.03±0.09, and 1.72±0.11, 1.78±0.14, 1.24±0.08, respectively), and the differences were statistically significant (t=15.043,5.358, 12.193 and 8.109, all P<0.05).The results of colony formation assay indicated that the colony forming ability of gastric cells in STCAT16 analogue transfection group was lower than that in empty vector group (97.3±9.1 vs 185.0±20.1) and non-transfection group (97.3±9.1 vs 138.0±11.1), and the differences were statistically significant (t=11.634 and 4.417,both P<0.05).The results of Transwell assay showed that the number of AGS cells passing through the membrane of STCAT16 analogue transfection group was significantly less than those of empty vector group and non-transfection group (151.0±28.1 vs 228.0±38.2 and 151.0±28.1 vs 199.3±17.9), and the differences were statistically significant (t=4.823 and 4.747,both P<0.05).After transfection for 48 hours and 72 hours, the scratch-wound repair rate of STCAT16 analogue transfection group decreased, compared with those of empty vector group and non-transfection group ((52.67±6.11)%, (53.33±5.51)%, (42.67±4.72)%, and (90.67±2.51)%, (90.60±5.41)%, (69.67±1.52)%, respectively), and the differences were statistically significant (t=5.773, 5.955, 21.000 and 5.881, all P<0.05).The results of tumorigenicity in nude mice showed that compared with those of empty vector group, the tumor size of STCAT16 analogue transfection group was smaller at one-, two-, three-and four-week ((0.42±0.10) cm3 vs (0.16±0.05) cm3, (0.66±0.13) cm3 vs (0.34±0.05) cm3, (1.25±0.22) cm3 vs (0.54±0.13) cm3, (2.54±0.46) cm3 vs (0.78±0.41) cm3)), and the differences were statistically significant (t=3.175, 4.190, 7.996 and 9.705, all P<0.05).Conclusions STCAT16 is lowly expressed in gastric cancer tissues.The proliferation, migration, invasion ability and tumorigenicity in nude mice of gastric cancer cell is inhibited after upregulating the expression of STCAT16.

Objective To investigate the expressions of guanylyl cyclase-c(GC-C) and caudal-type homeobox transcription factor 2 (CDX2) in human gastric tissues and precursor lesions and its significance. Methods The cancerous and paracancerous (5 cm from cancer lesion )samples from 30 cases of gastric cancer and 32 samples including 23 intestinal metaplasia and 9 dysplasia were collected. The mRNA expressions of GC-C and CDX-2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and proteins of GC-C and CDX-2 were measured by using Western blot and immunofluorescence methods. Results The mRNA expressions of GC-C and CDX-2 were absent in paracancerous tissues, but were 66.7% and 63.3% in cancerous tissues, respectively(P=0. 000). The Western blot indicated that expressions of GC-C and CDX-2 were 19/30 and 17/30 in cancerous tissues, but absent in paracancerous tissues(P=0. 000). The immunofluorescence examination revealed that GC-C and CDX-2 expressions were 39.1% and 39.1% in intestinal metaplasia, 55.6% and 55.6% in dysplasia, and 56.7% and 60.0% in cancerous tissues, respectively, but absent in paracancerous tissues. Moreover, expressions of GC-C and CDX-2 showed a statistical difference between intestinal-type and diffuse-type of gastric cancer (P＜ 0.05) ,but had no correlation with age, sex, size of the lesion, clinical stage and lymphnode metastasis. The positive correlation was found in expressions of GC-C and CDX2 between intestinal metaplasia and cancerous tissues(r=0. 4524 and 0. 3845, P= 0. 037 and 0. 0408, respectively). Conclusions The over expressions of GC-C and CDX2 in human gastric cancer is associated with precursor lesions and may play an important role in the carcinogenesis of gastric cancer. The examination of GC-C and CDX2 expressions will be helpful in diagnosing gastric cancer and precursor lesions.

Background:Recurrent acute pancreatitis(RAP)is a special type of acute pancreatitis(AP). Finding the cause is the key to avoid the recurrence of RAP. Aims:To investigate the risk factors of RAP. Methods:The clinical data of 43 patients with RAP and 130 patients with only one time AP(control group)were retrospectively analyzed. Risk factors of recurrence of RAP were analyzed. Results:Univariate analysis showed that no significant differences in etiology,severity, serum levels of cholesterol,calcium,white blood cell count,amylase,ALT,AST,total bilirubin,direct bilirubin and C-reactive protein were found between RAP group and control group(P > 0. 05). Serum triglyceride level,blood glucose level and CT score in RAP group were significantly higher than those in control group(t = 3. 260,P < 0. 05;t = 2. 720, P < 0. 05;t = 2. 162,P < 0. 05). Logistic regression analysis demonstrated that serum triglyceride level,blood glucose level and CT score were risk factors of RAP(oR = 1. 86,95% CI:1. 05-3. 68,P = 0. 03;oR = 1. 23,95% CI:1. 01-1. 50,P = 0. 04;oR = 2. 46,95% CI:1. 00-6. 03,P = 0. 04). Conclusions:The recurrence of RAP is related with serum triglyceride level,blood glucose level and CT score.

Objective To explore the effect of microRNA-32 (miRNA-32)on the biological behaviors of gastric cancer cell and its mechanism.Methods Gastric cancer cell line SGC-7901 cells were transiently transfected with miRNA-32 analogue,miRNA-32 inhibitor and empty plasmid vectors by lipofectamine and divided into analogue transfection group,inhibitor transfection group,empty plasmid transfection group and non-transfection group.The expression of green fluorescent protein was observed under fluorescent microscopy.The expression of miRNA-32 at mRNA level was detected by quantificational real-time polymerase chain reaction.The cell proliferation was evaluated by CCK-8 assay.The cell migration ability was measured by scratch test and Transwell chamber assays.The data were analyzed by one-way ANOVA.Results Compared with empty plasmid transfection group and non-transfection group,the expression of miRNA-32 mRNA of miRNA-32 analogue transfection group (relative quantitative value:2.327) was significantly up-regulated and that of miRNA-32 inhibitor transfection group (relative quantitative value:0.402) was significantly down regulated (F=11.238,P＜0.05).The width of scratch of miRNA-32 analogue transfection group was (61.39± 2.21) μm at 24 hours; miRNA-32 inhibitor transfection group was (29.97±0.66) μm.The migration distance of inhibitor transfection group was far than that of analogue transfection group (F=9.371,P＜0.05).After transfection for 48 hours,the cell number of migrated cells of analogue transfection group was significantly less than that of non transfection group,which was 16.93±4.63 and 93.93± 7.09,respectively (F=6.853,P＜0.05).After transfection for 48 hours and 72 hours,the cell growth inhibiting rate of miRNA 32 analogue transfection group was (43.474 ± 18.636)％ and (45.050±23.764)％,respectively,the cell growth was significantly inhibited (F=7.986 and 8.635,P=0.028 and 0.032).Conclusion The cell growth and migration ability of human gastric cancer cell line SGC-7901 are obviously inhibited through upregulating the expression of miRNA-32.