FAM126A Activates PI3K/AKT Pathway Through Regulating ENO1 Promotes The Proliferation,Invasion And Metastasis Of Pancreatic Cancer

Objective:Studies have confirmed that some members of family with sequence similarity(FAMs)play a promoting role in tumor progression,and a variety of malignant tumors including pancreatic cancer can be regulated by members of the FAMs family.In the early stage,the research group sieved out differentially expressed FAMs family members in pancreatic cancer tissues and paracancerous tissues,then analyzed the expression and clinical significance of FAM126 A in pancreatic cancer tissues and cell lines.In the course of further experiments,we investigated the effect of FAM126 A on the proliferation,invasion and metastasis of pancreatic cancer cells.Besides,we explored the molecular mechanism of FAM126 A promoting the progression of pancreatic cancer.Methods:The members of FAMs family were screened by GEO,GEPIA2 and LOGpc database,subsequently the differentially expressed FAM126 A was identified.Real-time fluorescence quantitative PCR(q RT-PCR),Western Blot assay and tissue microarray worked for detecting the expression of FAM126 A in 49 pairs of human pancreatic cancer tissues and paracancerous tissues,6 pancreatic cancer cell lines and normal pancreatic ductal epithelial cells(HPDE).After silencing FAM126 A by small molecular interference RNA,CCK-8 test,Ed U test,Transwell test and cell scratch test were reacted on examining the effect of FAM126 A on the proliferation,invasion and metastasis of pancreatic cancer cells.Construct a negative control,over-expressed and down-regulated stable transgenic strain of lentivirus,and verify its infection efficiency.CCK-8 cell,Ed U,clone plate,flow cytometry,Transwell and scratch test were also used to detect the effect of FAM126 A on the proliferation,invasion and metastasis of pancreatic cancer cells in vitro.Western blot experiment worked for detecting the changes of PI3K/AKT,cycle and EMT-related proteins.The effects of FAM126 A on the proliferation,invasion and metastasis of pancreatic cancer cells in vivo were proved by subcutaneous tumorigenesis and spleen capsule liver metastasis in nude mice.The tumor obtained by subcutaneous tumorigenesis were subjected to Ki67 and PCNA immunohistochemical staining to observe their changes of expression.The liver tissue taken from the spleen capsule liver metastasis model was stained with HE to observe the liver metastasis.Co-immunoprecipitation assay goes for analyzing protein spectrum,the interaction between ENO1 and FAM126 A was screened out by pre-experiment and literature review,which may be acted as a potential functional target.The bioinformatics website GEPIA2 was used to analyze the expression of ENO1 in pancreatic cancer and its effect on the prognosis of patients with pancreatic cancer,then found its downstream pathway by pre-experiment and literature review.Construct a ENO1 down-regulated stable transgenic strain of lentivirus and a co-treat stable transgenic strain of lentivirus with up-regulation of FAM126 A and down-regulation of ENO1,verifying its infection efficiency.Western blot,CCK-8,Ed U,clone plate,flow cytometry,Transwell and scratch test were conducted in vitro recovery experiment,the subcutaneous tumorigenesis,spleen capsule liver metastasis model and immunohistochemical experiment were used in vivo recovery experiment to further verify that ENO1 is a potential target for FAM126 A.FAM126A up-regulation group,FAM126 A up-regulation and ENO1down-regulation co-treatment group,FAM126 A up-regulation group and PI3K/AKT pathway inhibitor(LY294002)co-treatment group were detected by Western blot to further verify the role of ENO1 in the process of FAM126 A activating PI3K/AKT.Results:The GEO,GEPIA2 and LOGpc databases showed that the expression of FAM126 A in pancreatic cancer tissues was higher than that in paracancer tissues and had a negative effect on the prognosis of patients.The q RT-PCR,Western Blot assay and tissue microarray revealed that the expression of FAM126 A in pancreatic cancer tissues and cells was apparently higher than that in normal tissues and cells.CCK-8test,Ed U test,Transwell test and cell scratch test confirmed that silencing FAM126 A can inhibit the proliferation,invasion and metastasis of pancreatic cancer cells.Compared with the control arm,the expression of lentivirus was dramatically increased after overexpression of FAM126 A and decreased after down-regulation of FAM126 A,which revealed the successful construction of lentivirus stable transgenic strain.After overexpression of FAM126 A,the cloning ability,cell viability,subcutaneous tumorigenesis ability,invasion,metastasis and the number of liver metastasis of pancreatic cancer cells were substantially higher than those of control arm,after down-regulating FAM126 A,the above-mentioned in vivo and in vitro experiments showed opposite results.After overexpression of FAM126 A,the expression of Cyclin D1,Cyclin E1,CDK2,CDK4,N-cadherin,Vimentin,Snail,p-PI3 K and p-AKT increased,E-cadherin expression decreased,the results showed that FAM126 A accelerated cell cycle G1/S phase transition and induced the occurrence of EMT,activated PI3K/AKT signal pathway,and cell proliferation,invasion and metastasis increased;After down-regulating of FAM126 A,the opposite results were found in vivo and in vitro experiments above.After down-regulating FAM126 A,the staining of Ki67 and PCNA became lighter,inhibited cell proliferation,the number and size of liver metastasis were obviously less than those in the control arm.A total of 29 proteins that may interact with FAM126 A were screened by co-immunoprecipitation and proteome analysis,and ENO1 was determined to be the target gene of FAM126 A through pre-experiments and literature review.After consulting the bioinformatics website GEPIA2 we analyzed that compared with the paracancerous tissues,ENO1 in pancreatic cancer tissues is remarkably up-regulated,meanwhile ENO1 may have a negative impact on the prognosis of pancreatic cancer patients.Construct an ENO1 down-regulated stable transgenic strain of lentivirus and a co-treat stable transgenic strain of lentivirus with up-regulation of FAM126 A and down-regulation of ENO1,verifying that compared with the overexpression of FAM126 A,the co-treatment group of FAM126 A up-regulating and ENO1 down-regulating had a reduced expression of FAM126 A and ENO1,while there was further declined in the ENO1 down-regulating group.In vitro experiments(CCK-8,Ed U,clone plate,flow cytometry,Transwell and scratch test)and in vivo experiments(subcutaneous tumorigenesis,spleen capsule liver metastasis model and immunohistochemical experiment)were performed as recovery experiments to display that the proliferation,invasion,metastasis and EMT process of pancreatic cancer cells were apparently decreased in the co-treatment group with up-regulation of FAM126 A and down-regulation of ENO1 compared with the overexpression of FAM126 A,while the ENO1 down-regulation group was further weakened.The effect of ENO1 on PI3K/AKT pathway was detected by Western Blot,it was found that down-regulation of ENO1 could significantly reduce the phosphorylation level of PI3 K and AKT upregulated by FAM126 A,concurrently the effect of pathway inhibitor LY294002 on p-PI3 K and p-AKT of pancreatic cancer cells overexpressing FAM126 A was similar to that of down-regulation of ENO1.Conclusion:FAM126A can activate PI3K/AKT signal pathway and induce EMT process by changing the expression level of ENO1,and finally promote the progression of pancreatic cancer.