HSPs(+) Macrophages Promote Tumor Cells Proliferation And Metastasis In Hepatocellular Carcinoma

Objective: To explore the effects of macrophages with high expression of heat shock proteins(HSPs)on the occurrence and development of hepatocellular carcinoma(HCC)and its related mechanisms.Methods:(1).The HCC and matched adjacent normal tissues were obtained from patients after surgery,and the tumor-associated macrophages with high expression of HSPs were localized in the tissues by immunofluorescence co-staining.(2).The expression levels of HSPs in macrophages under inflammatory conditions(LPS/IFN-γ)were detected by qPCR.(3).The effects of HSPs+ macrophages conditioned medium on tumor cells proliferation and metastasis were detected by CCK-8 assay and scratch assay,respectively.(4).Bioinformatics analysis were performed to predict the possible signaling pathways responsible for the regulation of HSPs in macrophages,and the related enriched pathways were then validated by qPCR and western blot.(5).P38α in HSPs+ macrophages was inhibited by siRNA,and the effects of conditioned medium of macrophages on the proliferation and metastasis of HCC cells were detected by CCK-8 and scratch assays,respectively.Results:(1).The results of immunofluorescence co-staining showed that HSPs+ macrophages were significantly increased in tumor tissues as compared to adjacent normal tissues(*p<0.05).(2).The results of qPCR showed that LPS/IFN-γ could up-regulate HSPA1 B,DNAJB1,HSP90AA1,HSPH1 in macrophages in a time-dependent manner(*p<0.05).In addition,the mRNA expression levels of cytokines such as TNF-α,IL-1β,IL-10 and TGF-β1 were up-regulated(**p<0.01).(3).The results of CCK-8 assay showed that HSPs+ macrophages conditioned medium could promote the proliferation of HepG2 and Hep3 B cells after LPS/IFN-γ stimulation(*p<0.05).Scratch assay results showed that HSPs+ macrophages conditioned medium could promote the metastasis of HepG2 and Hep3 B cells after LPS/IFN-γ stimulation(*p<0.05).(4).The results of qPCR showed that LPS/IFN-γ induced the up-regulation of HSPs in macrophages by activating the p38α pathway(**p<0.01).Detected with qPCR,the transcription levels of HSPs were significantly reduced after inhibition of p38α with siRNA in HSPs+ macrophages(*p<0.05).(5).After blocking the MAPK signaling pathway of HSPs+macrophages by p38α siRNA,the results of CCK-8 assay and scratch assay showed that the ability of HSPs+ macrophages to promote tumor proliferation and metastasis was weakened(*p<0.05).Conclusion: Persistent chronic inflammation promotes the proliferation and metastasis of HCC cells by activating the P38α-mediated MAPK signaling pathway in HSPs+ macrophages which secrete pro-tumor cytokines.