HMGB1 Promotes Invasion And Migration Of Human Pancreatic Cancer Cell Through Lipid Synthesis

Objective:The aim of this study was to investigate the pathway of the release of high mobility group box-1 protein(HMGB1)in human pancreatic cancer cells under hypoxic conditions by simulating tumor microenvironment,and further study on its role in promotion of invasion and migration of human pancreatic cancer cells and its potential molecular mechanism.This provides a new experimental basis and theoretical basis for molecular targeted therapy of pancreatic cancer,and provides a new direction for guiding the clinical treatment of pancreatic cancer.Methods:1.The clinical significance of HMGB1 in pancreatic cancer was analyzed by using bioinformatics methods.2.Western blot was used to detect the expression of HMGB1 in different pancreatic cancer cells(Bx PC3,As PC1,Pa Tu8988,SW1990,Panc1).3.Western blot and ELISA were used to detect the expression of HMGB1 in supernatant of human pancreatic cancer cell line Pa Tu8988 after treatment with different hypoxia time.4.IF was used to detect the nuclear translocation process of HMGB1 in human pancreatic cancer cell line Pa Tu8988 after hypoxic treatment.5.Western blot,TEM and IF were used to detect the correlation between HMGB1 released from human pancreatic cancer cell line Pa Tu8988 and exosomes under hypoxic conditions.6.The efficiency of interference with HMGB1 in human pancreatic cancer cell line Pa Tu8988 was detected by ZEISS fluorescence microscope,Western blot and q PCR.7.Western blot was used to detect epithelial mesenchymal transition-related proteins(E-cadherin,N-cadherin,Snail,MMP-9)of human pancreatic cancer cell line Pa Tu8988 when co-cultured with rh HMGB1 and hypoxic supernatant(Sup-sh-EGFP,Sup-sh-HMGB1).8.Transwell experiments and scratch test were used to detect the invasion and migration of human pancreatic cancer cell line Pa Tu8988 when co-cultured with rh HMGB1 and hypoxia supernatant(Sup-sh-EGFP,Sup-sh-HMGB1).9.Western blot was used to detect lipid synthesis related proteins(Pro-SREBP1?M-SREBP1?FAS?SCD1?ACLY)of human pancreatic cancer cell line Pa Tu8988 when co-cultured with rh HMGB1 and hypoxic supernatant(Sup-sh-EGFP,Sup-sh-HMGB1).10.The efficiency of interference with SREBP1 in human pancreatic cancer cell line Pa Tu8988 was detected by ZEISS fluorescence microscope,Western blot and q PCR.11.Western blot was used to detect epithelial mesenchymal transition-related proteins(E-cadherin,N-cadherin,Snail,MMP-9)and lipid synthesis related proteins(Pro-SREBP1?M-SREBP1?ACC?FAS?ACLY)of human pancreatic cancer cell line Pa Tu8988-sh-SREBP1 when co-cultured with rh HMGB1.Results:1.Database results showed that HMGB1 was highly expressed in pancreatic cancer tissues compared with normal pancreatic tissue,and the expression level was negatively correlated with survival rate,and the expression of HMGB1 was not correlated with the stage of pancreatic cancer.2.Western blot analysis showed that HMGB1 has the highest expression in human pancreatic cancer cell line Pa Tu8988.3.The results of Western blot and ELISA showed that the expression of HMGB1 in the supernatant of human pancreatic cancer cell line Pa Tu8988 was the highest after 48 h hypoxic treatment.4.IF showed the translocation of HMGB1 from the nucleus to the cytosol after hypoxic treatment of human pancreatic cancer cell line Pa Tu8988.5.TEM showed that the cell culture supernatant had microvesicle structure;The expression of exosome membrane-associated marker proteins(CD9,CD63,CD81,HSP70)and the expression of HMGB1 in exosomes were detected by Western blot;The colocalization of HMGB1 and CD63 increased under hypoxic conditions.6.ZEISS fluorescence microscopy,Western blot and q PCR showed that the interference plasmid of sh-HMGB1 was successfully transferred into human pancreatic cancer cell line Pa Tu8988 with high interference efficiency.The pancreatic cancer cell line Pa Tu8988-sh-HMGB1 was successfully constructed.7.Western blot showed that the expression of E-cadherin was decreased and N-cadherin,Snail,MMP-9 expression were increased in rh HMGB1 and Sup-sh-EGFP groups compared with PBS and Sup-sh-HMGB1 groups.8.Transwell experiments and scratch test showed that the invasion and migration ability of rh HMGB1 and Sup-sh-EGFP groups were enhanced compared with PBS and Sup-sh-HMGB1.9.Western blot showed that the expression of Pro-SREBP1,M-SREBP1,FAS and SCD1 were up-regulated in rh HMGB1 and Sup-sh-EGFP groups compared with PBS and Sup-sh-HMGB1 groups.10.ZEISS fluorescence microscopy,Western blot and q PCR showed that the interference plasmid of sh-SREBP1 was successfully transferred into human pancreatic cancer cell line Pa Tu8988 with high interference efficiency.The pancreatic cancer cell line Pa Tu8988-sh-SREBP1 was successfully constructed.11.Western blot showed that the expression of E-cadherin was up-regulated and the expression of N-cadherin,Snail,MMP-9,Pro-SREBP1,M-SREBP1,FAS,ACLY and ACC were down-regulated in the rh HMGB1 group compared with control groups.Conclusions:1.Hypoxia promoted the release of HMGB1 from human pancreatic cancer cells and can be released by the exosomal pathway.2.HMGB1 could promote invasion and migration of human pancreatic cancer cells.3.HMGB1 could enhance the invasion and migration ability of human pancreatic cancer cells by promoting lipid synthesis of human pancreatic cancer cells.