Studies On The Role Of MiR-155-5p Regulates The Polarization Of Tumor Associated Macrophage In Lung Cancer And The Correlation With C/EBP-?

Background and Objective:Lung cancer have became the leading cause of cancer-related death in both men and women.In recent years,so many researches have been done on the mechanism and treatment of lung cancer.But,the long-term survival rate is still low due to the high rate OF recurrence and metastasis.Macrophages are immune cells with phagocytosis,which are involved in a variety of immune responses,and also widely existed in tumor microenvironment.These macrophages,are also known as tumor associated macrophage(TAM).Currently,accumulating studies have shown TAM is involved in regulating biological behaviors such as tumor proliferation,invasion and metastasis,as well as radiochemotherapy resistance.TAM may become a new target for tumor immunotherapy.According to different functions and phenotypes,TAM is divided into two types: M1 type(Classic active macrophage),which mainly secretes inflammatory promotion factors to stimulate the occurrence of inflammatory response.M2 type,which is replaced by activated macrophage,mainly secretes anti-inflammatory factors and has the effect of inhibiting immune response.M2 TAM also secretes some proteins that alter the extra cellular environment.Studies have shown that M2 TAM not only participates in a variety of malignant tumor biological behaviors,but also related to the poor prognosis of the patients.Therefore,the molecular mechanism regulating TAM's M1/M2 polarization will be a new idea for targeted tumor immunotherapy.The polarization of M1-M2 is regulated by multiple signaling pathways.The CCAAT/ enhancer binding protein(C/EBP)family plays an important role in regulating the differentiation of the activation of macrophages.Studies have shown that C/EBP is an important transcription factor that regulates the polarization of M2 TAM and is highly expressed in M2.C/EBP can induce TAM to M2 polarized by regulating the related genes of M2 in the downstream.Therefore,C/EBP is an important factor regulating M2 polarization of macrophages.If the endogenous molecules of inhibit C/EBP expression can be found,it will be significance for M2 TAM polarization.miRNA plays an important role in regulating tumor biological behavior.The expression of miR-155-5p in M1 TAM was significantly higher than that of M2 TAM.Previous studies have shown its expression in gastric cancer,colorectal cancer,liver cancer and other malignant tumor tissues were abnormal.However,the regulation effect of miR-155-5p on TAM phenotypic transformation has not been known yet.Our previous prediction suggested that miR-155-5p might have the effect of inhibiting the M2 TAM polarization of lung cancer,and it also indicated that miR-155-5p might have the effect of targeting inhibition of C/EBP,which was closely related to the mediating TAM phenotype transformation.Therefore,the study on the molecular mechanism affecting TAM endogenous regulation of miR-155-5p is helpful to find new targets to regulate TAM polarization.Methods:We collected the patients from September 2004 to September 2011,with stage I lung adenocarcinoma underwent radical surgery in the first hospital of China medical university.182 patients in our study,including 84 males and 98 females,with an average age of 66.7.Further classified into Lepidic(LPD,N=104),Acinar(ACI,N=39),Papillary(PAP,N=14),Solid(SOL,N=21)and Mucinous(MUC,N=4).The diameter of tumors were 6 mm-20 mm,with 116 cases of T1 a and 66 cases of T1 b.All the patients were treated with lobectomy combined with systematic lymph node dissection,and all the patients were not treated by adjuvant therapy such as radiotherapy and chemotherapy before operation.After the pathological tissues were constructed into tissue microarrays and stained with CD204 antibody,the image analysis software identified the percentage of CD204 positive macrophages and divided them into high expression and low expression due to the average expression intensity.Based on the clinical data of related patients,the relationship between CD204+TAM and clinical features and prognosis of patients was analyzed.Experimental cells: human lung adenocarcinoma A549 cell line,thp-1 cell line.First,tumor conditioned medium was collected for further macrophage culture.The cultured macrophages were polarized,and the polarized M1-type and M2-type macrophages were obtained.The growth morphology of M?,M1,M2 and TAM was observed by inverted phase microscope.The phenotypic expression of macrophages was identified by flow cytometry.Real time PCR was used to detect the expression of cytokine il-1 beta,TNF-?,CCL22 and TGF-?.The second part of research application of Realtime PCR detection M ?,M1,M2,TAM,four groups of cells expression levels of miR-155-5p.TAM flow detection,TAM + miR-NC,TAM + miR-155-5p,CD86 and CD204 expression of three groups of cells.Real-time PCR was used to detect il-1 expression,TNF-?,CCL22 and TGF-? expression in three groups.The expression levels of il-1 expression,TNF-?,CCL22 and TGF-? expression were detected by Western blot.In the third part,real-time PCR was used to detect the expression level of C/EBP in the three groups,and Western blot was used to detect the expression level of C/EBP in the three groups.Next,luciferase reporter plasmids that specifically bind to miR-155-5p were constructed: wild-type plasmids and mutant plasmids.The constructed plasmids were transfected into TAM cells,and then transfected into miR-155-5p mimic synchronously.At last,luciferase activity was determined by double luciferase detection box.Result:The expression of CD204 was not associated with gender(P=0.981),age(P=0.745),tumor location(P=0.769),smoking history(P=0.494),but associated with histological subtype(P=0.008),clinical stage of tumor(P=0.008),lymph node metastasis(P<0.001),and recurrence or not(P<0.001).The 5-year RFS of the CD204 high-expression group was significantly lower than that of the CD204 low-expression group(69.8% vs.98.3%,p<0.001),but there was no significant difference in OS between the two groups(84.1% vs.92.4%,p=0.052).TAMs cells were successfully cultured and polarized.M?,M1,M2 and TAM all expressed CD86,CD204,and M1 TAM mainly expressed CD86,while CD204 showed low expression.M2 TAM mainly express CD204,while CD86 expression is low.The surface molecules of TAM cells are similar to M2 TAM,and CD204 expression is increased while CD86 expression is low.Realtime PCR was used to detect the mRNA expression levels of il-1?,TNF-?,CCL22 and TGF-?,and the results showed that the mRNA expression levels of il-1?,TNF-?,CCL22 and TGF-?were all detected by the macrophages of the 4 groups.The mRNA expression in the M1 TAM group was significantly higher than that in the M2 TAM group(1.30± 0.35)(1.25± 0.42)and TAM group(2.02±0.17);(p<0.001).miRNA expression of CCL22 and TGF-scaffold in both the M2 macrophage group and the TAM group were significantly higher than that in the m1-macrophage group,and the difference was statistically significant(p<0.001).M2-macrophage group and TAM group showed no statistically significant differences in the mRNA expression levels of il-1 apoptosis,TNF-1,CCL22 and TGF-foot transcription(p>0.05).The expression of mir-155-5p-related genes in the M1 TAM group was significantly higher than that in the M2 TAM and TAM group.miT-155-5p in M1 group(1.89±0.28),mir-155-5p in M2 group(0.97±0.45),miR-155-5p in TAM group(1.25±0.29),and miR-155-5p expression in M1 and M2 were statistically significant(p<0.01).The expressions of miR-155-5p in the M1 and the TAM were also statistically significant(p<0.01).Comparison of miR-155-5p expression between M2 and TAM showed no significant difference(p>0.05).It suggested that the expression of miR-155-5p was significantly different in macrophages with different polarization,which might be related to cell polarization,while TAM expression was similar to M2 macrophages,which might be related to the low expression of miR-155-5p.TAM,TAM+ miR-nc,TAM+ miR-155-5p three groups of cells all expressed CD86,CD204,and reverse TAM with high expression of miR-155-5p showed the transition of surface molecular expression,the positive rate of CD86 expression increased compared with TAM,and CD204 expression decreased compared with TAM,suggesting that TAM changed from an M2-like phenotype to a M1-like phenotype.The expression of four major cytokines was detected by real-time PCR to verify the effect of mir-155-5p expression on cell polarization.The results showed that il-1,TNF-?,CCL22 and TGF-? receptor mRNA expression were detected in all three groups.The mRNA expression in TAM+ mir-155-5p group was significantly higher than that in TAM+ miR-155-5p group(1.94± 0.24),TNF-?(1.77 ± 0.09)was significantly higher than that in TAM group(1.03±0.08),TNF-?(1.04±0.0.1)and TAM+ mir-nc group(0.97±0.18),TGF-?(1.03±0.06).The pairwise differences were statistically significant(p<0.001).In TAM+miR-155-5p group,the mRNA expression of CCL22(0.74± 0.08),TGF-?(1.00±0.07)were significantly lower than that of TAM group(1.54 ± 0.16),TGF-?(1.95±0.12)and TAM+ miR-nc group(1.69±0.14),and TGF-?(1.84±0.16).The pairwise differences were statistically significant(p<0.001).It can be seen that TAM with reverse artificial high expression of mir-155-5p is similar to m1-type macrophages,suggesting that the expression of mir-155-5p may be closely related to cell polarization.Protein expressions of the four cytokines were detected by western blot to further verify the effect of miR-155-5p expression on cell polarization.The results showed that the expression of il-1 proteins,TNF-?,CCL22 and TGF-? scaffold in all three groups were detected.In TAM+ mir-155-5p group,the protein expression of TNF-? was significantly increased,which was stronger than il-1 beta in TAM+ miR-nc group and TNF-?,and the pairwise comparison was statistically significant(p<0.001).In TAM+ miR-155-5p group,the protein expression of CCL22 and TGF-?was significantly decreased,which was lower than that of TAM+ miR-nc group,CCL22 and TGF-?.The pairwise difference was statistically significant(p<0.001).These results confirmed the conclusion that miR-155-5p regulates macrophage polarization,which was confirmed again from the level of protein expression.The expression level of C/EBP-?was detected by real-time PCR,and the results showed that the mRNA expression of C/EBP-? could be detected in all three groups of cells.The C/EBP-?of TAM+ miR-155-5p group(0.69±0.05)was significantly lower than that of TAM+ miR-nc group(1.42± 0.12)and TAM+ miR-nc group(1.56 ±0.09).The difference was statistically significant(p<0.01).The results suggested that TAM with high miR-155-5p expression in the reverse direction had a negative regulation effect on C/EBP-?.The expression of higher miR-155-5p and the lower C/EBP-?was suggesting that the negative regulation of miR-155-5p by C/EBP-? was closely related to cell polarization.The expression of C/EBP-? protein was detected by Western blot in three groups of cells.The C/EBP-? expression in TAM+ miR-155-5p group was significantly reduced,which was lower than that of TAM+ miRnc group C/EBP-?,and the difference was statistically significant(p<0.01).Furthermore,at the protein level,TAM with high reverse expression of miR-155-5p was negatively regulated by C/EBP-? protein expression.The higher the expression of mir-155-5p was,the more inhibited the expression of C/EBP-?.The results showed that the luciferase activity of the wild-type plasmid 3 'utr-wt + mir-155-5p was significantly lower than that of the wild-type plasmid 3' utr-wt + mir-nc,and the difference was statistically significant(p<0.001).The luciferase activity of the wildtype plasmid 3 'utr-wt + mir-155-5p was also significantly lower than that of the mutant plasmid 3' utr-mut + mir-155-5p,and the difference was statistically significant(p<0.001).There was no significant difference in luciferase activity between mutant 3 'utr-mut + mir-nc group and 3' utr-mut + mir-155-5p group and wild-type plasmid 3 'utr-wt + mir-nc(P > 0.05).It was suggested that the mir-155-5p mimic can bind with the wild-type plasmid C/EBP-? and down-regulate the expression of C/EBP-? miRNA,inhibit the luciferase activity of firefly,and thus reduce the luciferase activity.In contrast,the miR-155-5p mimic did not bind to the mutant plasmid C/EBP-?,and could not inhibit the expression of C/EBP-? miRNA,thus the luciferase activity was not affected.The results confirmed that miR-155-5p can directly bind to C/EBP-? and thereby inhibit C/EBP-? expression in lung adenocarcinoma cells by targeting at the further transcription level.It was further proved that miR-155-5p may regulate TAM phenotypic transformation by intervening C/EBP-?.Conclusion:1.The high expression of CD204+ TAM is related to the poor prognosis of patients;The expression of il-1 and TNF-? in M1 were significantly higher than that in M2 and TAMs.The expression of CCL22 and TGF-? was significantly higher than that in M1.The biological behavior of TAMs is more similar to the M2 TAM.2.The expression of miR-155-5p in TAM was inhibited,and it also can be seen in M2.Reverse the miR-155-5p expression can cause the repolarization of TAM.miR-155-5p may affect TAMs phenotypic transformation by regulating C/EBP-?.3.miR-155-5p regulates the expression of C/EBP-? and affects the repolarization of macrophages.