| The macrophage is known as a multifunctional antigen presenting cells and playing a central role in the hematopoitic system.Due to its multiple functions,the macrophage is involved in the normal organ development,internal balance,tissue repair and immune response to pathogens.The macrophages have two well-established polarized phenotypes,the classically activated macrophages(M1)and alternatively activated macrophages(M2).Macrophages are the most common immune cells in the tumor tissue,known as tumor-associated macrophages,whose function and phenotype are M2-like,and affected by multiple signaling pathways.Long non-coding RNAs(lncRNAs)are a specific class of RNA that transcribes more than 200 bases in length without coding any proteins.Increasing evidences suggest that lncRNAs are participated in many aspects of biochemical actions,such as epigenetic regulation,transcriptional regulation,cellular and tissue differentiation.However,its function in the M2 macrophage polarization remains exclusive.Our study is to identify lncRNA(s)which may play a vital role in the macrophage M2 polarization and to investigate the underlying mechanism.Based on this finding,series of compounds were screened and 4-HPR was found to a promising small molecular compound that could inhibit the expression of lncRNA-RIK.Furthermore,4-HPR could impact the polarization of M2 macrophage via the inhibition of the phosphorylated STAT6.In vivo,the inhibition of M2 macrophage polarization is involved in 4-HPR-mediated chemopreventive effect on the colon cancer.Our study suggest that targeting the M2 macrophage may be a potential target for tumorigenesis.Section 1 The role of lncRNA-RIK in regulating macrophage M2 polarization and its underlying mechanismObjective:The aim of this section is not only to identify the lncRNA(s)that play a central role in the polarization of the M2 macrophage but also to investigate the mechanism and biological function of the lncRNA(s).Our study is trying to provide a new target to inhibit the polarization of M2 macrophage.Methods:Mouse macrophage cell line RAW264.7 and bone marrow derived macrophage(BMDM)were used in this study.(1)Flow cytometry was applied to analyze the surface markers CD206 and CD209 of M2 polarized macrophages.(2)Cells were collected from different stimuli and different time point for the IncRNAs microarray assay.(3)Small-interfer RNAs were applied to knockdown the specific lncRNA to assess its role in the M2 polarization of macrophage.(4)Transwell assay was conducted to evaluate the migration of different cancer cell lines after treatament of different macrophage conditioned medium.(5)Tube formation assasy was applied to investigate the effect of conditioned medium on tumor angiogenesis.(6)Western blot assay was employed to detect proteins expression level of related pathways after the knockdown of IncRNAs.Results:(1)We established the M2 macrophage polarization model in vitro by using the classical stimuli IL4 and IL13.(2)LncRNA microarray assay was further performed to identify the lncRNAs which might regulate the polarization of M2 macrophage and IncRNA-RIK was found to be up-regualted in the process of M2 polarization.(3)In vitro,we used different stimuli LPS,IFN-?,IL4 and IL13 for treating the RAW264.7 cells or BMDMs.The results of Real-time PCR suggest that IncRNA-RIK was specifically up-regulated during M2 polarization(compared to the control group,the expression of IncRNA-RIK in the IL4 or IL13 treated group increased respectively to 8.39 ± 2.94 and 8.17 ±3.77,p value is 0.0486 and 0.0301,as evaluated using Student's T-test).(4)Knockdown of IncRNA-RIK significantly attenuated the M2 polarization rate of macrophage.(5)There is no effect on the migration of tumor cells after knockdown IncRNA-RIK expressing in macrophages.(5)Knockdown of IncRNA-RIK dramatically blocked the tube formation of HUVEC cells.(6)The phosphorylation of STAT6 was decreased after knockdown IncRNA-RIK in macrophage,which is the classical transcription factor for M2 macrophage polarization.Conclusions:The IncRNA-RIK plays a central role in the M2 polarization of macrophage.Knockdown the IncRNA-RIK could impact the angiogenesis-promoting ability of M2 macrophage.The study confirms that IncRNA-RIK is a key regulator of macrophage M2 polarization.Section 2 The involvement of M2 macrophage polarization inhibition in 4-HPR-mediated chemopreventive effects on the colon cancerObjective:The aim of this section is to identify small molecular compounds that could impact the expression of lncRNA-RIK.4-HPR was found and futher investigated to be a promising inhibitor in the M2 polarization of macrophages.Our study will clarify how 4-HPR regulates M2 polarization and evaluate the role of M2 macrophage in 4-HPR-mediated chemoprevention,thus providing the possibility for the clinical application of 4-HPR.Methods:Mouse macrophage cell line RAW264.7,bone marrow derived macrophages(BMDMs)and colon cancer cell lines were used in this study.(1)Real-time PCR was performed to evaluate the inhibiton rate of 4-HPR on the expression of IncRNA-RIK.(2)RAW264.7 and BMDMs were treated with IL4 or IL13(10 ng/mL)with or without 4-HPR.Flow cytometry was used to evaluate the inhibitory effect of 4-HPR on the M2 polarization.(3)RAW264.7 and BMDMs were treated with IL4 or IL13(10 ng/mL)with or without 4-HPR.Real-time PCR was conducted to evaluate the inhibitory effect of 4-HPR on the M2 polarization.(4)Tube formation assasy was applied to investigate the effect of conditioned medium on tumor angiogenesis.(5)Western Blotting assay was performed to detect the activation of different pathways involved in the M2 polarization with or without 4-HPR.(6)A total of 18 APCmin/+ transgenetic mice were randomly assigned to either 4-HPR(20 mg/kg)or placebo for 10 weeks.Treatment effects were investigated using histological and immunohistochemistry assay.(7)Immunohistochemistry was performed to detect the M2 macrophage in the tumor region.(8)Immunohistochemistry was performed to detect the angiogenesis in the tumor region.Results:(1)4-HPR was found to suppress the expression the lncRNA-RIK(compared to the IL4 group,the expression of IncRNA-RIK decreased from 6.09±1.83 to 0.12±0.03 in the presense of 4-HPR,p =0.0051.compared to the IL13 group,the expression of lncRNA-RIK decreased from 8.18±3.08 to 0.18±0.09 in the presense of 4-HPR,p=0.0020,as evaluated using Student' T-test).(2)4-HPR could significantly suppress IL4 or IL13 induced M2-like polarization of macrophages,as illustrated by reducing expression of M2 surface marker,down-regulation of M2 marker genes.(3)4-HPR could inhibit the angiogenesis promoted by M2 macrophages.(4)Mechanistically,to investigate the relationship between the inhibition of M2 polarization and ROS,we applied the ROS scavengers N-acetylcysteine(NAC)and glutathione(GSH)to remove intracellular ROS generated in response to 4-HPR treatment.The data suggested that the generation of oxidative stress,is not involved in the 4-HPR-driven inhibition of M2 polarization.(5)The phosphorylation status of STAT6 was increased after treating cells with IL4 or IL13 for 30 min.Meanwhile the increase of phosphorylated STAT6 was dramatically inhibited by co-treatment with 4-HPR,which suggests that STAT6 might participate in 4-HPR-mediated inhibition of TAMs M2 polarization,which was consistence with the result that 4-HPR could down-regulate the expression of lncRNA-RIK.(6)By applying APCmin/+ transgenetic mice,we demonstrated that the tumorigenesis was dramatically decreased by 4-HPR treatment accompanied with less M2-like macrophage in the tumor tissues.(7)We also investigated whether 4-HPR effected onthe tumor growth and apoptosis and found that 4-HPR did not induce colon tumor cell death nor inhibit the cancer cell growth in vivo.Conclusion:4-HPR not only can impact the expression of lncRNA-RIK,but also inhibit the M2 polarization.The inhibition of macrophage M2 polarization is involved in the 4-HPR-mediated chemoprevention.This study suggests that 4-HPR to be a promising compound for chemoprevention. |
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