Study On The Anti-apoptosis Role Of LIMS1 In Pancreatic Cancer

BackgroundLIMS1 also known as PINCH1(particularly new cysteine-histidine rich protein)was first discovered in 1994 in the human aged blood cell cDNA library by Rearden.LIMS1 consists of 5 tandem LIM1 domains as well as a short C tail that forms.LIMS1 is involved in the development and maturation of embryos,and plays an important role in the maintenance of some organs such as heart,lung,pancreas and so on.After our previous experimental results indicated that: compared with normal pancreatic tissue and pancreatic duct cells,the pancreatic cancer cell lines and pancreatic ductal adenocarcinoma in patients with LIMS1 expression have a clear upward trend,we design the experiment from the relationship,and explore the prognostic significance of the expression of LIMS1 in survival pancreatic cancer and various clinical parameters of the function,the underlying regulatory mechanisms,to explore the function and significance of LIMS1 in pancreatic ductal adenocarcinoma cells from clinical level,function level,the level of molecular biology and animal experiments Method 1 Application of pancreatic ductal adenocarcinoma tissue paraffin,serial sections of LIMS1 and HIF1 a immunohistochemical staining,analyzed the expression level of correlation between LIMS1 and HIF1 a patients were collected;prognosis,the relationship between the expression level of LIMS1 and clinical pathological parameters.2.Western blot,RT-PCR measures were used to probe the expression levels of LIMS1 and HIF1 a in five pancreatic cancer cell lines and one nomal pancreatic duct cells,and we analysis the relationship between LIMS1 and HIF1 a.detection the location of LIMS1 in pancreatic carcinoma cells by immunofluorescence technique 3.We construct The LIMS1 and HIF1a-overexpressing and-shRNA plasmid.And we successfully establish stably overexpressing or knockdown expressing LIMS1 and HIF1 a cell lines.WB,RT-PCR measures was used to verify the LIMS1 and HIF1 a expression levels in those 4.Effects of LIMS1 on the survival of pancreatic cancer cells in the presence of low glucose and hypoxia microenvironment.5.The application of Western blot,RT-PCR and glucose uptaking kit to detect the molecular mechanism of LIMS1 involved in the apoptosis-resisting of tumor cells 6.Subcutaneous pancreatic tumor model in nake mouse was used to verify the role of LIMS1 in facilitatingi pancreatic cancer apoptosis-resisting cell in vivo.The animal PET-CT was used to verify its mechanism in vitro 7.Constructing the subcutaneous tumor model of pancreatic cancer in NOD-SCID mice,and to observe the effect of LIMS1 on pancreatic cancer knockdown by si-LIMS1 through tail? The results 1.Expression and significance of 1 LIMS1 in pancreatic cancer tissues and adjacent normal tissues.Based on 115 cases of pancreatic cancer tissue samples by immunohistochemical staining,and from which 8 randomly selected specimens by Western RT-PCR found that,compared with adjacent normal tissues,the cancer tissue showed high expression of LIMS1 in pancreatic cancer,which was negatively correlated with LIMS1 expression and prognosis of pancreatic cancer survival(p<0.05*).TCGA(The Cancer Genome Atlas)data give the same answer.The LIMS1 and clinicopathological parameters of pancreatic cancer and LIMS1 analysis indicated that tumor size,T stage,N stage,TNM stage and vascular invasion have relationship with LIMS1(p<0.05*),TCGA database data also suggest that the expression of LIMS1 relate with T stage and tumor size,N stage,TNM stage,vascular invasion and tumor differentiation.The above information suggests that LIMS1 affects the size of pancreatic cancer.2.Effect of LIMS1 on anti hypoxia and low glucose in pancreatic cancer.Successfully constructed 2 pancreatic cancer cell line that overexpress LIMS1(CFPAC-and Bx PC-3)and two knowdown cell lines(MIA PaCa-2 and PANC-1).We put experimental group and control group in the conventional cell culture environment after treatment to detect the apoptosis of each group but no significant differences we have found,when we simulate the vivo culture conditions of tumor(hypoxia and low glucose)compared with the control group,the overexpression group have high activity with and less apoptosis,while compared with the knockdown group have high activity with and less apoptosis.The tumor volume was larger and the tumor apoptosis was less in the.subcutaneous tumor model of pancreatic cancer.3.LIMS1 plays a role in anti-apoptosis by participating in glucose metabolism of tumor cells.We have found that LIMS1 might play important role in the glucose uptaking under hypoxia and low glucose microenvironment.In situ mice and clinical patient data also confirm the phenomenon.According to the literature,AKT pathway can be activated by LIMS1,and the AKT pathway also affects the expression and distribution of glucose uptake protein(GLUT1).We found that LIMS1 did affect the ability of tumor cells to resist apoptosis through AKT pathway4.HIF1a can transcript LIMS1.The expression of LIMS1 in pancreatic cancer cell lines was significantly increased under hypoxia.HIF1 a plays a key role in the hypoxic environment of pancreatic cancer,and the expression of LIMS1 in HIF1 a is consistent with that of HIF1 a.HIF1a can bind to the promoter region of LIMS1 and start the translation of LIMS1.5.LIMS1 can regulate the translation of HIF1 a.We found that the expression of HIF1 in LIMS1 steady cell lines was positively correlated with LIMS1 expression.By blocking experiments,we found that LIMS1 could regulate HIF1 a translation by AKT/mTOR/4EBP1.6.LIMS1 and HIF1 a show a high correlation.It was found that LIMS1 and HIF1 a showed a positive correlation between HIF1 a and LIMS1 in patients with pancreatic cancer surgery.7.LIMS1 plays an important role in the anti-apoptotic effect of HIF1 a.Bottle-neck experiments were performed in vitro to further identical LIMS1 could play an important role of HIF1 a in pancreatic cancer cells against hypoxia and low sugar microenvironment.Conclusions1.LIMS1 is positively expressed in pancreatic cancer tissues and cell lines.LIMS1 high expression levels associated with poor prognosis.2.LIMS1 can activate the AKT pathway to regulate the expression and distribution of GLTU1 in pancreatic cancer cells,so that play an important role in the resistance to hypoxia and low glucose microenvironment.3.HIF1a can initiate the transcription of LIMS1,and LIMS1 can regulate the translation of HIF1 a.LIMS1 could play an important role of HIF1 a in pancreatic cancer cells against hypoxia and low sugar microenvironment.