The Study On The Mechanism Of SALL4 Promoting Glycolysis In HCC And Inducing M2-type Polarization Of TAMs

BackgroundChina is a country disturbed by liver cancer seriously.More than half of the world’s liver cancer patients are in China,in which hepatocellular carcinoma(HCC)is the most common form,accounting for about 75%-85%of the total number of cases.And its high morbidity and mortality seriously threaten human life and health.With the continuous advancement of medical technology,the combination of surgery,local chemoradiotherapy and immunotherapy is gradually adopted clinically.Immune checkpoint inhibitor or CAR-T therapy is mostly used in tumor immunotherapy at present,which has problems such as small audience,high cost,and low overall effective rate.Safer and more effective treatments of HCC are still being explored.In addition to immunotherapy,targeted metabolic therapy for tumors is also gradually emerging.For example,glycolysis inhibitors such as 2-DG and 3-PO have entered the stage of clinical research.In the early twentieth century,Otto Warburg proposed that even in an aerobic environment,tumor cells are more inclined to use the glycolysis pathway to provide the necessary ATP for their own life activities,that is,the"Warburg effect".More evidences show that targeting key molecules of glycolysis can effectively inhibit the occurrence and development of cancer,and the aerobic glycolysis pathway is a potential target for cancer therapy.SALL4(sal-like protein 4)is a transcription factor necessary for embryonic stem cell self-renewal and maintenance of pluripotency.It is often silenced in the adult liver but reactivated in liver cancer.SALL4 regulates biological behaviors of tumor such as proliferation,apoptosis,migration/invasion,drug resistance,and sternness by targeting multiple genes,and is associated with poor patient prognosis.In recent years,studies have shown that SALL4 can upregulate the expression of GLUT1 and HK2 directly or indirectly,and induce glycolysis in tumor cells to promote tumor growth.However,there are few studies in the field of tumor metabolism,and whether the HCC immune microenvironment can be altered by targeting SALL4-mediated glycolysis is unclear.Therefore,we constructed a short hairpin RNA(shRNA)targeting SALL4,observed the effect of knocking down SALL4 on the proliferation,migration,apoptosis and stemness of HCC cells,and explored the molecular mechanism of SALL4 regulating HCC glycolysis.We also clarified the impact of SALL4-induced glucose metabolism on the tumor immune microenvironment,especially the polarization and metabolic changes of tumor-associated macrophages(TAM),in order to provide new insights for the targeted therapy of clinical HCC strategy.Purpose1.To clarify the effect of targeting and interfering with SALL4 on the biological behavior of HCC tumors;2.To clarify the molecular mechanism of SALL4 promoting glycolysis in HCC;3.To explore the effect and underlying mechanism of SALL4 on TAM polarization in HCC.Methods(1)Extract the SALL4 expression data of HCC patients in the TCGA database and conduct integrated analysis,and analyze the correlation between SALL4 and key glycolysis genes in HCC.(2)Construct a lentiviral vector targeting human SALL4,carry out lentiviral packaging by calcium phosphate transfection method,prepare a virus-containing medium for knocking down SALL4,and infect Huh7 and Hep3B human HCC cell lines.(3)After infection with lentivirus targeting knocking down of SALL4,the viability of HCC cells was detected by CCK8 method;the migration ability of HCC cells was evaluated by scratch assay;the apoptosis level of HCC cells was detected by Annexin-V/PI method;the difference of HCC cell stem was detected by clone formation test;the cell cycle arrest of HCC was observed by PI method.(4)The expression of SALL4 in the above cell lines was detected by qPCR and Western blot,and the expression changes of key molecules such as HCC migration,apoptosis and glycolysis were detected after knocking down SALL4.(5)After lentivirus infection targeting knocking down of SALL4,the color difference of HCC culture supernatant was observed,the glucose and lactate concentrations were detected to calculate the glucose uptake and lactate production rate indirectly,and the cell glycolytic capacity was evaluated by Seahorse energy metabolism analyzer(6)Use PMA(TPA)to induce THP-1-associated macrophages(THP-1-M),and observe the cell morphology under a microscope.(7)Collect the culture supernatant of BAY-876-treated HCC and THP-1-M cells,detect the glucose concentration,calculate the glucose uptake inhibition rate of BAY-876 on the above cells,and select the optimal concentration.(8)Flow cytometry was used to detect the contents of M1/M2 macrophage polarization marker molecules NOS2 and CD 163,and qPCR was used to detect the expression levels of key molecules of glycolysis and oxidative phosphorylation in HCC-TAMs;(9)Incubate THP-1-M with HCC culture supernatant,and continue the culture after replacing with fresh medium.Collect the supernatant and incubate the HCC cell line for counter-incubation.The changes in cell proliferation and migration ability are detected by CCK8 and scratch assays,and the expression changes of proliferation and migration-related molecules were detected by qPCR.(10)BALB/C nude mice were subcutaneously loaded with Huh7 and Huh7-TAMs cells,and the effect of knocking down SALL4 on tumor growth and glycolysis level was detected.Flow cytometry was used to evaluate the effect of knocking down SALL4 on the tumor-promoting effect of TAMs.Results1.SALL4 is highly expressed in liver cancerThe SALL4 expression data of HCC patients in the GEPIA2 and cBioportal database were extracted and integrated analysis was performed.It was found that HCC patients showed significantly higher SALL4 expression compared with healthy people,which was verified in the Western Blotting experiments.2.Construction of SALL4-knocking down lentiviral vector and evaluation of knocking down effectPCR and sequencing results showed that the SALL4-knocking down lentiviral vector was successfully constructed,and after stable infection of Huh7 and Hep3B cells,the expression of SALL4 was significantly decreased at both mRNA and protein levels comparing with the empty-vector group,indicating that the vector was effective.3.SALL4 regulates the tumor biological behaviorsof HCC cellsAfter knocking down S ALL4,the proliferation,migration,and stemness of HCC cells were significantly inhibited,and the level of apoptosis was significantly upregulated comparing with the empty-vector group,indicating that SALL4 is important for the growth of HCC cells.4.Knockdown of SALL4 arrests HCC cell cycle in G0/G1 phaseAfter knocking down SALL4,the proportion of HCC cells in G0/G1 phase was significantly increased comparing with the empty-vector group,while the proportion in S phase was decreased,indicating that knocking down SALL4 could arrest HCC cell cycle in G0/G1 phase.5.Knockdown of SALL4 inhibits glycolysis and down-regulates the expression of key glycolysis enzymes in HCC cellsAfter knocking down SALL4,the glucose uptake rate and extracellular acid production rate(ECAR)of HCC were significantly decreased comparing with the empty vector group,and bioinformatics analysis indicated that SALL4 was significantly correlated with the expression of key glycolysis molecules GLUT1 and HK2,and the results showed that SALL4 could promote glycolysis in HCC cells.6.SALL4-knocking down HCC culture supernatant inhibits the M2-type polarization of THP-1-MWhen THP-1-M was incubated with SALL4-knocking down HCC culture supernatant,it was found that M1-type marker molecules were regulated to varying degrees,while M2-type marker molecules were down-regulated.In addition,HCC cells were counter-incubated with HCC-TAMs culture supernatant and found that their growth was significantly inhibited,indicating that SALL4-knocking down HCC culture supernatant could inhibit the polarization of THP-1-M to M2 type.7.Knockdown of SALL4 affects THP-1-M polarization by reducing glucose competitiveness in HCCKnockdown of SALL4 significantly inhibited the expression of GLUT1,indicating that SALL4 can regulate the glucose uptake capacity of HCC.After inhibiting glucose uptake in HCC-TAMs,M2-type marker molecules were found to be significantly up-regulated.Further blocking the glucose uptake of HCC,and using its culture supernatant to incubate THP-1-M,the M1-type marker molecules were upregulated,and the M2-type marker molecules were slightly decreased.It is suggested that knocking down SALL4 can make HCC lose glucose competitiveness by downregulating the expression of GLUT1,and inhibit the polarization of THP-1-M towards M2 type.8.SALL4-knocking down HCC culture supernatant induces altered metabolism of HCC-TAMsBy detecting the glycolysis and OXPHOS marker molecules of HCC-TAMs,it was found that knockdown of SALL4 could simultaneously down-regulate the levels of glycolysis and oxidative phosphorylation of HCC-TAMs,inhibiting their M2-type polarization.Blocking the glucose uptake of HCC-TAMs or exogenous glucose supplementation can recover.9.Knockdown of SALL4 inhibits the proliferation of HCC in vivo and attenuates the tumor-promoting effect of TAMsCompared with the empty vector group,knockdown of SALL4 significantly inhibited tumor growth in tumor-bearing mice,with down-regulating the expressions of key glycolysis enzymes GLUT1,HK2,PKM2,PGK1 and LDHA at the mRNA level,and attenuated the expression of HCC-TAMs at the same time.10.Knockdown of SALL4 inhibits the polarization of HCC tumor-infiltrating TAMs towards M2 typeKnockdown of SALL4 can significantly up-regulate the expression of the Ml-type marker molecule iNOS in tumor-infiltrating macrophages,and down-regulate the M2type marker molecule CD 163,indicating that knockdown of SALL4 can inhibit the polarization degree of HCC-TAMs to M2-type in vivo.Conclusion and significance1.This study confirmed that SALL4 significantly promoted the proliferation,migration,and sternness but inhibited the apoptosis of HCC,indicating that targeting SALL4 may be an effective way to clinically treat HCC.2.This study proved that SALL4 promotes glycolysis in HCC cells to ensure the energy source and metabolic needs of tumors.3.This study provides a novel mechanism for SALL4 promoting HCC development.SALL4 increased the glucose competitiveness of HCC by up-regulating the expression of the glucose transporter GLUT1,which promoted the polarization of HCC-TAMs to the M2 type that enhancing the tumor biological characteristics of HCC.4.This study sugested SALL4 might be a target for HCC treatment and drug discovery and development.