| Objective:In solid tumors,multiple cytokines in the tumor microenvironment recruit monocytes from blood circulation system to the tumor site and induce them to activate and differentiate into tumor-associated macrophages(TAMs),which play an anti-tumor role in the early stage and a pro-tumor role in the later stage.Due to the plasticity of macrophages,long noncoding RNA(lnc RNA)may potentially regulate the phenotypic changes of macrophages,thereby promoting its anti-tumor effect and inhibiting its anti-tumor effect.This study preliminarily discusses the regulation of linc RNA-p21 in the phenotypic transformation of tumor-associated macrophages,and researches its potential mechanism to provide a new target for the diagnosis and treatment of cancer.Method:(1)The breast tissues of mice which spontaneous breast cancer were separated and the infiltration of macrophages in breast tumor tissues was detected by immunohistochemistry.Cultured mouse macrophage cell line RAW264.7 and culture supernatant of 4T1 mouse breast cancer cells was collected for stimulated normal macrophages,and then collected them for lnc RNA expression chip detection and subsequent advanced analysis(Shanghai oyi medical biotechnology).Lnc RNA with significant differences were screened and validated by real time fluorescence quantitative PCR(RT-q PCR).The high expression level and stable expression lnc RNA were selected as the research objects.In addition,the expression of linc RNA-p21 was detected by RT-q PCR in macrophages stimulated by the culture supernatant of mouse tumor cells CT26 and Lewis.Design mice linc RNA-p21 specific small interference RNA(small interfering RNA,si RNA)(si-linc RNA-p21),and used RT-q PCR and si-linc RNA-p21 with fluorescent labeling verify its interference effect and efficiency.(2)To determine the influence of the inhibition of linc RNA-p21 in macrophages which stimulated by the supernatant from 4T1 tumor cells.CCK-8 kit was used to detect the proliferation of macrophages.Annexin V/PI kit was used to detect the apoptosis of macrophages.The secretion of IL-4,IL-6,IL-10,IL-12 and TNF-? was detected by ELISA.Using flow cytometry(FCM)to detect the expression of CD80,CD86,CD206 and MHC? in macrophage.The expression of argininase-1(Arg-1)and nitric oxide synthase(i NOS)in macrophages was detected by western-blot.The phagocytic function of macrophages was detected by standard strain of Escherichia coli.(3)To study the effect of tumor cells which co-culture with macrophages suppression the expression of linc RNA-p21.The knockdown of linc RNA-p21 in macrophages was co-cultured with tumor cells,and CCK-8 kit was used to detect the proliferation of tumor cells.Annexin V/PI kit was used to detect apoptosis.Transwell assay was used to detect the migration ability of tumor cells.The migration ability of tumor cells was detected by scratch test.FCM was used to detect the Fas L expression in macrophages and Fas expression in tumor cells.(4)Mouse spleen mononuclear cell isolation kit was used to isolate mouse spleen mononuclear cells,and flow cytometry was used to select macrophages in tumor tissues of tumor-bearing mice,and the expression of linc RNA-p21 was detected by RT-q PCR.BALB/c mice were injected subcutaneously with si-linc RNA-p21 knockdown macrophages mixed with the same amount of 4T1 tumor cells,and macrophages knockdown linc RNA-p21 were injected at the same location every 7days to continuously monitor the growth and survival of subcutaneous tumors in mice.The tumor quality of mice was measured after the mice were sacrificed.To investigate the potential mechanism of linc RNA-p21 regulates macrophage phenotypic transformation,the expression of Bax,Bcl-2,p53 and MDM2 were determined by western-blot.FISH probe was used to detect the localization of linc RNA-p21 in macrophages and its interaction with p53 and MDM2.Western-blot analysis of the phosphorylation levels of STAT3 and p65 in macrophages with linc RNA-p21 knockdown and stimulated by 4T1 tumor supernatant.Results:(1)Compared with normal macrophage RAW264.7,the expression of lincRNA-p21 in RAW264.7 was up-regulated after 4T1 tumor supernatant stimulation(p<0.05).The expression of linc RNA-p21 was also up-regulated in RAW264.7 after stimulation by CT26 and Lewis tumor supernatant(p<0.05).(2)Compared with the NC group,CCK-8 detected the proliferation of linc RNA-p21 knockdown macrophages showed increased(p<0.05)and apoptosis was decreased.The secretion of proinflammatory cytokines IL-6,IL-12 and TNF-? increased(p<0.05),and the anti-inflammatory cytokines IL-4 decreased(p<0.05).FCM results showed that compare with the control group,the expression of CD206 in macrophage which inhibit linc RNA-p21 were reduced,the expression of CD86 in macrophages were increased,and the expression of CD80 and MHC ? in macrophages were no significant influence.In addition,compared with the NC group,the linc RNA-p21 knockdown group expressed less Arg-1 and more i NOS.Meanwhile,inhibition of linc RNA-p21 has no effect on macrophage phagocytosis.(3)After co-culture linc RNA-p21 knockdown macrophages with 4T1 tumor cells,CCK-8 detection showed that the proliferation of 4T1 tumor cells slowed down(p<0.05)and apoptosis increased.Compared with the NC group,the migration ability of 4T1 tumor cells was decreased after co-culture with macrophages knockdown linc RNA-p21(p<0.05).FCM results showed that the expression of Fas L in macrophages and the expression of Fas in 4T1 tumor cells have no significant change.(4)Primary monocytes and tumor-associated macrophages were isolated,and the expression of linc RNA-p21 detected by RT-q PCR was consistent with the results of cell lines,and the expression of linc RNA-p21 in tumor tissues was up-regulated(p<0.05).Compared with the control group,the mice injected with si-linc RNA-p21 showed lower tumor growth rate(p<0.05),lower tumor quality(p<0.05),and longer survival time(p<0.05).Western-blot results showed that linc RNA-p21 knockdown could promote the expression of Bcl-2 in macrophages and inhibit the expression of Bax,but had no significant effect on p53 and MDM2.Moreover,the phosphorylation level of STAT3 and p65 was significantly up-regulated when 4T1 tumor supernatant stimulated the macrophages which knockdown linc RNA-p21 at 24 hours.The results of FISH probe showed that linc RNA-p21 was mainly located in the cytoplasm of macrophages,and the binding of linc RNA-p21 to p53 was significantly enhanced after the supernatant stimulation of 4T1 tumor cells,while the binding of linc RNA-p21 to MDM2 was weak.Conclusion:The expression of linc RNA-p21 in tumor-associated macrophages was up-regulated,and the inhibition of linc RNA-p21 expression could promote the anti-tumor immunity of macrophages. |
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