Autoregulatory Loop Between TGF-β1/miR-411/SPRY4 And MAPK Pathway In Rhabdomyosarcoma Modulates Proliferation And Differentiation

Objective:To explore TGF-β1-repressed micro RNAs and MAPK signal pathways in modulating proliferation and differentiation in human rhabdomyosarcoma(RMS),and provide a new therapeutic targets for the RMS differentiation。Methods:(1) TGF-β1 repressed mi RNAs were identified by Real-time PCR assays in different RMS cell lines and tissue samples, screening out the most obviously different and widely mi RNAs as the research object.(2) MTT and 3H thymidine incorporation assay were conducted to determine cell proliferation in RMS cell lines transfected with mi R-411-5pM.(3) Constructed nude mouse tumor model, observe the impact on tumor growth in vivo injected with M or I; Ki-67 in subcutaneous RMS tissue was stained by immunohistochemistry.(4) Predict mi R-411-5p target genes by bioinformatics technology and network resources.(5) Real-time PCR assay, dual luciferase report system assay and western blotting analysis were conducted to validate target gene of mi R-411-5p.(6) Construct the Elk-1-Luc, c-Jun-Luc fluorescence report plasmid, SPRY4-si RNA or si RNA-ctrl transfected RMS cell lines treated with PKCa-expression plasmid, and then detect the fluorescent signal of Elk-1-Luc,c-Jun-Luc;PKCa-expression plasmid and SPRY4 si RNA/si RNA-ctrl com-transfected RMS cell lines, and then detect the level of P-p38 protein.(7) MKK6EE-expression plasmid and/or SPRY4-si RNA com-transfected RMS cell lines, detect the level of P-p38 protein by western blotting analysis, observe the change of apoptosis index(caspase-3) and muscle differentiation index(MHC, Myosin) by immunofluorescence staining.MKK6EE-expression plasmid and/or mi R-411-5p M, I com-transfected RMS cell lines, detect the level of P-p38 protein by western blotting analysis, observe the change of apoptosis index(caspase-3) and muscle differentiation index(MHC, Myosin) by immunofluorescence staining, detect the cell cycle changes by flow cytometry technology.Mi R-411-5p M or SPRY4-si RNA transfected RMS cells, 3H thymidine incorporation assay was to detect cell proliferation, and detect the Cleaved caspase-3 expression by western blotting analysis.(8) Real-time PCR analysis the correlation between TGF-β1 and SPRY4 m RNA in human RMS tissues.(9) TGF-β1、SPRY4 and P-p38 protein were stained by immunohistochemical technique, and then clinical pathologic correlation analysis was carried on.Results:(1) Mi R-411-5p was the most widely and obvious different in RMS cell lines treated with TGF- β 1 si RNA and RMS tissue samples.(2)Mi R- 411-5p can restrain the proliferation of the RMS cell lines.(3) the mi R-411-5p can inhibit the growth of nude mice subcutaneous RMS tumor and the expression of Ki-67. The second part.(4)Mi R-411-5p candidate target genes follow:calmodulin-like4(CALML4), the sprouty homolog 4(SPRY4), general transcription factor 21(GTF21), transcription factor(SP2), SET nuclear oncogene(SET), and glutamate receptor metabotropic 3(GRM3).(5) SPRY4 is the direct target genes of mi R-411-5p.(6) SPRY4 si RNA can enhance the fluorescence signal of Elk-1-Luc, c-Jun-Luc; P-p-ERK,P- JNK protein expression significantly increased in Si RNA crtl group, P-p38 protein expression increase is not obvious, however P-p-ERK,P- JNK protein expression increase is not obvious in SPRY4 si RNA group, P-p38 protein expression increase is significantly obvious.(7) P-p38 protein expression was significantly increased in MKK6 EE expression plasmid+ SPRY4-si RNA group, apoptosis index(caspase-3) and muscle differentiation index(MHC, Myosin) were increased.MKK6 EE + mi R-411-5p-M group p38 protein increased significantly, promote cell apoptosis index(caspase-3) and increase muscle differentiation index(MHC, Myosin) expression and the ratio of cell cycle at the G1 phase were increased. mi R-411-5p-M or SPRY4-si RNA transfected RMS cells, the level of 3H thymidine incorporation was significantly lower, and the protein expression of Cleaved caspase-3 was increased.(8) TGF-β1 protein expression and SPRY4 m RNA expression were negative correlation in human RMS tissues.(9) In the poorly differentiated RMS tissues, TGF-β1 and SPRY4 protein were higher expression, and P-p38 protein was lower expression; In a well-differentiated RMS tissues, TGF-β1 and SPRY4 protein were lower expression, and P-p38 was high expression.Conclusions 1. TGF-β1 negative regulates mi R-411-5p and the latter can inhibit proliferation of RMS. 2. SPRY4 is directly downstream target genes of mi R-411-5p. 3. SPRY4 inhibit PKCa-mediated MAPK signaling pathways, particularly p38 MAPK activation, which block the RMS differentiation. 4. In the RMS tissue samples, the expression of TGF-β1 was positive correlation with SPRY4, while negative correlation with mi R-411-5p m RNA level and P-p38 MAPK protein expression.