ABSTRACT
Objective:To explore the regulatory role of miR-210 in hypoxia-induced epithelial-mesenchymal transition (EMT) of pancreatic cancer PANC1 cells.Methods:PANC1 cells cultured in normoxia and hypoxia were established in normoxia group and hypoxia group. Recombinant plasmid carrrying miR-210 mimics and miR-210 antagomirs were constructed. The recombinant plasmids were transfected with PANC1 cells cultured in normoxia and hypoxia by liposome method to establish cell lines of miR-210 overexpression (miR-210 mimics normoxia group) and miR-210 expression inhibition (miR-210 antagomirs hypoxia group). The blank plasmids were transfected to establish blank plasmid normoxia group and blank plasmid hypoxia group. Relative expression levels of miR-210 for PANC1 cells were determined by qRT-PCR in each group. Western blot was used to measure the expressions of HIF-1α, NF-κB and EMT related protein such as E-cadherin, β-catenin, vimentin and N-cadherin. Cell relative viability under gemcitabine and in vitro cell invasion ability were detected by CCK8 and Transwell chamber experiments, respectively. Results:The relative expressions of miR-210 in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group. The expression levels of HIF-1α, NF-κB and mesenchymal cell markers such as vimentin and N-cadherin in hypoxia group were significantly higher than those in normoxia group (0.74±0.06 vs 0.40±0.05, 1.58±0.16 vs 1.09±0.13, 0.46±0.04 vs 0.17±0.02, 1.27±0.07 vs 0.40±0.03) and the epithelial cell markers such as E-cadherin and β-catenin were significantly lower (0.40±0.07 vs 0.77±0.10, 0.35±0.02 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 mimics normoxia group were significantly higher than those in blank plasmid normoxia group (0.91±0.08 vs 0.40±0.06, 1.52±0.17 vs 1.05±0.14, 0.82±0.06 vs 0.66±0.07, 0.76±0.04 vs 0.46±0.03) and E-cadherin and β-catenin were significantly lower (0.38±0.07 vs 0.65±0.09, 0.50±0.03 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 antagomirs hypoxia group were significantly lower than those in blank plasmid hypoxia group (0.31±0.05 vs 0.55±0.06, 0.68±0.05 vs 1.11±0.13, 0.41±0.03 vs 0.74±0.07, 0.69±0.06 vs 0.78±0.05), while E-cadherin and β-catenin were significantly higher (0.72±0.13 vs 0.50±0.07, 0.71±0.04 vs 0.54±0.05). All the differences among the groups were statistically significant (all P value <0.05). Under gemcitabine, the relative viability of PANC1 cells in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group at 48 h (1.10±0.10 vs 0.76±0.05, 1.46±0.11 vs 1.12±0.09) and 72 h (1.12±0.13 vs 0.76±0.05, 1.54±0.13 vs 1.12±0.09) accordingly. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group at 48 and 72 h (0.75±0.09 vs 1.10±0.10, 1.19±0.12 vs 1.46±0.11). All the differences among the groups were statistically significant (all P value <0.05). The number of transmembrane cells in hypoxia group and miR-210 mimics normoxia group was significantly higher than those in normoxia group and blank plasmid normoxia group, respectively (417.50±81.22 vs 228.30±47.71, 371.30±72.81 vs 245.00±33.62 per high field), while those in miR-210 antagomirs hypoxia group was significantly lower than those in blank plasmid hypoxia group (228.30±54.01 vs 433.30±65.63 per high field). All the differences among the groups were statistically significant (all P value <0.05). Conclusions:miR-210 could weaken the sensitivity to gemcitabine and promote the invasion of PANC1 cells by regulating the occurrence of the hypoxia-induced epithelial-mesenchymal transition.
ABSTRACT
Objective:To explore the effects of kallikrein 12 (KLK12) on the proliferation, invasion and migration ability of pancreatic cancer cells.Methods:Pancreatic cancer tissues and para-cancer tissues were collected from 95 patients who underwent radical surgical resection, and pathologically diagnosed as pancreatic cancer at the Department of Biliary and Pancreatic Surgery of the First Affiliated Hospital of USTC between February 2014 and October 2018. Expression of KLK12 in pancreatic cancer tissues was investigated using immunohistochemistry, and the correlation between KLK12 expression and clinicopathological characteristics of pancreatic cancer was analyzed. Western blot and qPCR methods were used to detect the expression of KLK12 protein and mRNA in pancreatic cancer cell line SW1990, PANC1 and normal pancreatic gland cells HPDE6-C7. Pancreatic cancer cell lines with up-regulated and down-regulated KLK12 expression were constructed by plasmid transfection. Non-transfected pancreatic cancer cells and transfected pancreatic cancer cells carrying negative control plasmids were used as blank control group and negative control group. CCK8 and Transwell chamber experiments were used to study the changes in cell proliferation, invasion and migration.Results:The positive rate of KLK12 in pancreatic cancer tissues was significantly higher than that in para-cancer tissues (70.5% vs 29.5%, P<0.001), and was significantly related to low tumor differentiation, late TNM stage and lymph node metastasis (all P value <0.05). The expression of KLK12 protein and mRNA in SW1990 and PANC1 cell lines was higher than that in HPDE6-C7 (0.34±0.01, 0.28±0.01 vs 0.21±0.01; 3.31±0.10, 2.91±0.09 vs 1.41±0.20; all P value <0.01). In cells with down-regulated KLK12 expression and then cultured for 72 h, the A450 value of SW1990 cell proliferation (0.94±0.02 vs 1.16±0.05), the number of invading membrane penetrating cells (373.7±14.8 vs 726.0±11.8 per high magnification field) and the number of migrating penetrating cells (696.0±13.1 vs 841.3±15.4 per high magnification field) were significantly decreased. The A450 value of PANC1 cell proliferation (0.96±0.03 vs 1.21±0.03), the number of invading membrane penetrating cells (556.3±13.6 vs 646.0±15.1 per high magnification field) and the number of migrating penetrating cells (449.0±16.5 vs 595.7±8.6 per high magnification field) were also significantly decreased. When the expression of KLKL12 was up-regulated in cells and cultured for 72 h, the A450 value of SW1990 cell proliferation (1.32±0.03 vs 1.11±0.03), the number of invading membrane penetrating cells (556.3±22.2 vs 402.7±10.5 per high magnification field) and the number of migrating penetrating cells (639.3±16.5 vs 433.0±11.8 per high magnification field) were significantly increased. The A450 value of PANC1 cell proliferation (1.26±0.04 vs 1.08±0.03), the number of invading membrane penetrating cells (571.0±17.4 vs 426.7±23.3 per high magnification field) and the number of migrating penetrating cells (740.3±13.0 vs 442.7±10.3 per high magnification field) were also significantly increased (all P value <0.05). Conclusions:KLK12 is highly expressed in pancreatic cancer, and up-regulated KLK12 expression can promote the proliferation, invasion and migration ability of pancreatic cancer cells.
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Objective:To explore the role of decoy receptor 3(DcR3) and its ligand tumor necrosis factor ligand-related molecule 1A(TL1A) in the treatment of pancreatic cancer implanted tumors in nude mice with CD 4 and CD 8 double negative T cells (DNT cells). Methods:DNT cells derived from peripheral blood of healthy volunteers were cultured in vitro by antibody adsorption method. A nude mouse model of pancreatic cancer implantation was established and randomly divided into DNT cell treatment group (tail vein injection of 1×10 8/ml DNT cell suspension), gemcitabine treatment group (tail vein injection of 50 mg/kg gemcitabine) and control group (no treatment). The tumor volume and weight in each group were measured after 50 days. Western blotting and qRT-PCR were used to detect the protein and mRNA expression of DcR3 and TL1A in nude mice implanted tumor tissues in each group, and TUNEL method was used to detect the apoptosis rate of nude mice implanted tumors in each group. Results:The tumor volume of the DNT cell treatment group, gemcitabine treatment group, and control group was (670.28±124.54), (604.60±179.16), (1738.80±391.39)mm 3, and the tumor weight was (225.60±8.12), (222.69±8.73), (265.07±10.76)mg, and the volume and weight of implanted tumors in the DNT cell treatment group and gemcitabine treatment group were significantly lower than those in the control group, and the differences were statistically significant (all P values <0.001). The expression levels of DcR3 protein and mRNA in the DNT cell treatment group and gemcitabine treatment group (0.56±0.02, 3.74±0.19; 0.57±0.03, 3.40±0.39) were significantly higher than those in the control group (0.39±0.04, 0.92±0.05), while the expression levels of TL1A protein and mRNA (0.41±0.03, 0.83±0.11; 0.40±0.05, 0.79±0.08) were significantly lower than those of the control group (0.81±0.05, 1.70±0.36), and the differences were statistically significant (all P values <0.001). The apoptotic rate of implanted tumors in the DNT cell treatment group was (53.2±11.2)%, and that in the gemcitabine treatment group was (56.2±8.6)%, which were significantly higher than the control group (10.3±3.2)%, and the differences were statistically significant ( all P values <0.001). Conclusions:DNT cells had a significant inhibitory effect on the growth of pancreatic cancer implanted tumors in nude mice. DcR3-TL1A may be involved in the anti-tumor mechanism of DNT cells.
ABSTRACT
Double negative T cell is a rare type of cells that regulates immune responses in a way that is unique.In the process of adoptive immunotherapy,it gradually becomes a hot spot,because of its obvious anti-tumor effect.However,the mechanism by which DNT cell kills tumor cell is not clear.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and natural-killer group 2,member D (NKG2D) pathway is a more important part of its killing mechanism,and NKG2D receptor can also regulate the production of soluble TRAIL,making the two channels to establish a certain link.In this paper,the effect of Double negative T cell and the mechanism of TRAIL and NKG2D pathway in DNT cell's anticancer were described.