The Research Of Activation For Fibroblast By PDGF-BB

【Objective】During tumor progression,the interaction between tumor cells and stromal cells has attracted much attention.Compared to normal fibroblasts,the fibroblasts in the tumor stroma,called cancer-associated fibroblasts,are in an activated state,but the activation mechanism is unknown yet.Therefore,we have been studying for it and found that oral cancer cells can induce fibroblast activation and are closely related to platelet derived growth factor PDGF-BB and receptor PDGFR-beta.In this study,the co-culture models of oral cancer cells and fibroblasts were established to explore the mechanisms in the activation of fibroblasts by PDGF-BB/PDGFR-beta axis.【Methods】(1)The human TSCC cell line Cal-27(ATCC,USA),hOMF(Normal human Oral Mucosa(P2)500K Keratinocytes cells,Singapore Cell Research Corp)and hOMK(Normal human Oral Mucosa(P3)500K fibroblast cells,Singapore Cell Research Corp)cells was cultured in high-glucose DMEM with 10%FBS,at 37℃and 5%C02.(2)PDGF-BB in the difference expression of hOMK and Cal-27 cells were detected by RayBio QAH-CAA-1000.(3)Preparation of conditioned medium.After hOMK and Cal-27 cells inoculated to 150 ml culture bottle,and growed convergence to 70-80%,replaced with fresh complete medium,developed continuesly for 24h,the supernatant was collected and centrifuged in 15000 g for 3 minutes at 4℃ to get rid of cellular debris,and filtered in 0.22μm microporous membrane to bacteria,and stored in-20℃.(4)The activation of fibroblast.(5)The microtubules and dense patches in activated fibroblasts was observed by transmission electron microscopy.(6)Immunofluorescence was used to detect PDGFR-beta in normal oral mucosa fibroblasts and activated fibroblasts under fluorescence microscopy.(7)PDGF-BB factor detected between the normal oral mucosa epithelial cells and oral squamous cancer cells secrete differently by QAH-CAA-1000 array.Human Chemokine Array C1 was used to screen CCL25 chemotactic factor differently from fibroblasts and AF.(8)Human Cancer Focus microRNA&Target mRNA PCR Array was used to detect microRNA-26a expressing differently from normal oral mucosa fibroblasts and activated fibroblasts.(9)CCL25,TAB3 mRNA and microRNA-26a were detected by qPCR.(10)The expressions of TAB3、NF-κB and a-SMA were analyzed by Western blot.(11)Overexpression and knockdown microRNA-26a cells were constructed by gene transfection and gene knockdown in AF and hOMF cell.(12)The data obtained is expressed as mean ± SD.T test was used to analyz compared with the two groups.ANOVA analysis between groups showed that p<0.05 was statistically significant.The data was processed and analyzed using Graphpad Prism 5.0(Graphpad software.San Diego,SA).【Results】(1)The secretory supernatant of Cal-27 cell induced fibroblast to activate.Compared with the control group,the fibroblasts were stimulated by the secretory supernatant of Cal-27 cells,the growth of fibroblasts was accelerated and the contact inhibition disappeared by HE staining and optical microscopy;the intracellular microtubules and the dense plaques were increased by the transmission electron microscope;the expression of activated marker protein a-SMA was increased Significance by Western blot(p<0.05);the expression of PDGFR-β increased by immunofluorescence.(2)Oral tongue squamous cell carcinoma cell highly secreted PDGF-BB factor.QAH-CAA-1000 chip revealed that Cal-27 cell secreted PDGF-BB is higher than hOMK.Compared with normal oral mucosa epithelial hOMK cell supernatant,the PDGF-BB of oral tongue squamous carcinoma Cal-27 cell supernatant is 5.7 times than normal oral mucosa epithelial cells hOMK supernatant.PDGF-BB came from Cal-27 cell is higher than hOMK cell by ELISA,the difference was statistically significant(p<0.01),the result is similar to the QAH-CAA-1000 chip results.(3)PDGF-BB induced fibroblasts to enhance chemokine CCL25 secretion.Human Chemokine Array(RayBiotech)detected that PDGF-BB can promote hOMF cell secrete chemotactic factor CCL25 compared with control group;after stimulated by PDGF-BB,the concentration of CCL25 was 1.8 times than the control group,the difference was statistically significant(p<0.05);The result of detection by ELISA was similar to that of the chip hybridization,and the difference was statistically obviously(p<0.01).(4)The expression of miR-26a in hOMF cell was reduced after stimulated by PDGF-BB.The Human Cancer Focus microRNA&Target mRNA PCR Array chip(Arraystar)showed that the expression of hsa-mir-26a-5p in hOMF cells was reduced by PDGF-BB(p<0.05).qPCR detected the same results,and the difference was statistically significant(p<0.05).(5)OTSC induced high expression of TAB3 and NF-κB phosphorylation in fibroblasts.Western blotting found that the relative expression quantity of the α-SMA,TAB3,and NF-kappa B(p-p65)was significantly upregulated compared with the control group after the stimulated via the Cal-27 cells supernatant,and the difference was statistically significant(p<0.05).The protein expression of a-SMA,TAB3,and and NF-kappa B(p-p65)was downregulated compared with the Cal-27 cells supernatant group after blocked PDGF-BB or PDGFR-beta by anti-PDGF-BB antibody or anti-PDGFR-βantibody,and the difference was statistically significant(p<0.05).(6)The TAB3 of activated hOMF by PDGF-BB was upregulated by miR-26a-5p decreasing.Bioinformatics analysis and biluciferase detection revealed that hOMF cells was activated by PDGF-BB through dergulating the inhibition of TAB3 and activating NF-kappa B by miR-26a-5p.Bioinformatics analysis,dual luciferase detection showed that the fluorescence value of TAB3-WT-hsa-miR-26 a group was lower than those of TAB3-WT-mimics NC group,the results are similar with the PC-mimics NC,PC-mimics NC-hsa-miR-26 a positive control group(PC-mimics NC,PC-mimics NC-hsa-miR-26 a),the difference was statistically significant(p<0.05);and the results were contrary to the negative control group(TAB3-mut-mimics NC,TAB3-mut-hsa-miR-26a),and the differences were statistically significant(p<0.05)in 293T cells and AF.(7)miR-26a-5p dysfunction promoted the activation of fibroblast.The expression of a-SMA,TAB3,and NF-kappa B of hOMF-hsa-miR-26a-5p inhibitor group was significantly upregulated compared with hOMF-ZsGreen-Puro group,and the difference was statistically significant(p<0.05).After the PDGF-BB stimulation,a-SMA,TAB3 and NF-kappa B of hOMF-hsa-miR-26a-5p inhibitor group was significantly higher than that of hOMF-ZsGreen-Puro group,and the difference was statistically significant(p<0.01).(8)The activation of fibroblast was inhibited or reversed by miR-26a-5p.The expression of a-SMA,TAB3,and NF-kappa B protein of AF-hsa-miR-26a-5p group was obviously reduced compared with the AF-ZsGreen-Puro group,and the difference was statistically significant(p<0.05).After PDGF-BB stimulated,the expression of a-SMA,TAB3,and NF-kappa B of AF-hsa-miR-26a-5p and AF-ZsGreen-Puro group was significantly increased compared with the control group,and the difference was statistically significant(p<0.01).[Conclusion]The oral cancer cells have induced fibroblast activation by PDGF-BB and PDGF-BB/PDGFR-beta axis through reduced inhibition of TAB3 and activated NF-kappa B by miR-26a-5p.