LHPP Suppresses Proliferation And Metastasis And Promotes Apoptosis In Pancreatic Cancer

【Background】Pancreatic cancer(Pa Ca)is a common malignant tumor of the digestive system with poor prognosis and no ideal treatment for inoperable patients.It is recently reported that the protein histidine phosphatase LHPP is a tumor suppressor in hepatocellular carcinoma,cervical cancer,bladder cancer and melanoma.So far,there is no study on the expression level of LHPP in Pa Ca,and its mechanism of action on tumors is unclear.This article mainly expounds the role of LHPP in proliferation,migration,invasion and promoting apoptosis of pancreatic cancer cells,and preliminarily explores its mechanism,in order to provide a new strategy for clinical treatment of pancreatic cancer.【Method】1.Immunohistochemistry was used to investigate the LHPP expression levels in PaCa tissues and matched normal tumor tissue.Western blotting and q-PCR were used to detect the difference of LHPP expression between Pa Ca Bx PC-3,As PC-1 cell line and HPDE6-C7 normal pancreatic duct cell line.2.The lentiviral vector of LHPP gene was constructed in vitro and infected with pancreatic cancer cell lines Bx PC-3 and As PC-1.The effect of LHPP on the proliferation of Pa Ca cell was determined by CCK-8 reagent and clone formation assay,and the effect of LHPP on the migration and invasion of Pa Ca cells was analyzed by Transwell chamber.3.The protein expression levels of PTEN,phosphorylated AKT and total AKT in Vector,LHPP,NC and sh LHPP groups were detected by western blotting,and the protein expression levels of phosphorylated AKT and total AKT were further detected by stimulating LHPP group with AKT activator SC79.4.The relationship between apoptosis and LHPP in pancreatic cancer cells was detected by western blotting and flow cytometry,and the protein expression levels of cleaved-Caspase-3 and cleaved-PARP were further detected by stimulating LHPP group with AKT activator SC79.【Result】1.Expression of LHPP in pancreatic cancer tissues and cellsWe performed immunohistochemical analyses of Pa Ca tissues and matched normal tumor tissues from 36 patients,we found that LHPP expression was lower in cancer tissues than that in normal pancreatic tissue(P<0.05).Additionally,the protein and m RNA expression levels in Bx PC-3 and As PC-1 cells were significantly lower than those in HPDE6-C7 cells(P<0.05).2.Effect of LHPP on proliferation,migration and invasion of pancreatic Cancer Cell LineIn Bx PC-3 and As PC-1 cell lines,CCK-8 test showed that the proliferation ability of LHPP group was lower than that of the control group,and the OD value at 96 h was significantly lower than that of the control group(P<0.01),however,the proliferation ability of sh LHPP group was higher than that of NC group,and the OD value of 96 hours was significantly higher than that of NC group(P<0.01).Clone formation analysis showed that cell proliferation was significantly decreased in the LHPP group and increased in the sh LHPP group.Transwell analysis showed that migration and invasion of Bx PC-3 and As PC-1 cells were inhibited in the LHPP group,whereas the sh LHPP group presented the opposite results(P<0.05),These results suggested that LHPP suppressed the proliferation,migration,and invasion of Pa Ca cells.3.Study on the mechanism of LHPP on proliferation,migration,and invasion of pancreatic cancer cellsThe LHPP group showed significantly inhibited proliferation and transfer of Pa Ca cells,as evidenced by the reduced m RNA expression levels of snail,laminin-5,L1 CAM,N-cadherin,and vimentin(P<0.05);the sh LHPP group showed the opposite results.Western blotting showed that the LHPP group could promote PTEN expression and inhibit phosphorylated AKT expression,whereas the opposite was observed in the sh LHPP group(P<0.05).The addition of the drug SC79(an important AKT activator)to the LHPP group led to an increase in phosphorylated AKT(P<0.05).In this experiment,we found that LHPP significantly reduced AKT phosphorylation and enhanced the expression of PTEN protein.5.Effect of LHPP on apoptosis of Pancreatic Cancer cellsThe passaging results showed that the apoptosis of BxPC-3 and AsPC-1 cells was promoted in the LHPP group,whereas the apoptosis rate was lower in the sh LHPP group than that in the NC group(P<0.05),and we further found that the LHPP group showed promoted c IAP1 expression and inhibited levels of cleaved-PARP and cleaved-Casp3 in Bx PC-3 and As PC-1 cells;however,these results differed in the sh LHPP group(P<0.05),SC79 pretreatment in the LHPP group reduced the levels of cleaved-PARP and cleaved-Casp3 in Pa Ca cells(P<0.05).These results suggested that LHPP could inhibit the apoptosis of Pa Ca cells.【Conclusion】1.The expression of LHPP in pancreatic cancer tissues was significantly lower than that in paracancerous tissues,and the expression of LHPP in pancreatic cancer cells Bx PC-3 and As PC-1 was also significantly lower than that in normal pancreatic duct cells of HPDE6-C7.2.LHPP inhibits the proliferation,migration and invasion of pancreatic cancer cells by interfering with AKT signal pathway.3.LHPP promotes apoptosis of pancreatic cancer cells by inhibiting c IAP1 and promoting the expression of cleaved-Casp-3 and cleaved-PARP proteins.4.SC79 reduces the inhibitory effect of LHPP on phosphorylated AKT,and weakens the promoting effect of LHPP on cleaved-Casp3 and cleaved-PARP proteins.