TGF-β1Mediated Micrornas Regulate Myogenic Differentiation Of Rhabdomyosarcoma (RMS)

Objective: To explore correlation between TGF-β1pathway and TGF-β1mediatedmicroRNAs (miRNAs) in regulating myogenic differentiation of RMS.Methods:(1) RMS cell lines: RD, SMS-CTR and RH28cell were treated withTGF-β1-siRNA, and miRNA microassay detected the expression of TGF-β1mediatedmiRNAs in RMS cells. The expression of correlated miRNAs in RMS tissues ofTGF-β1-low and-high expression were determined by immunohistochemistry and RT-PCR,moreover, Western blot, Northern blot�'�RT-PCR assays were performed to confirm theTGF-β1regulated miRNA.(2) we transiently transfected mimics (M) and inhibitors (I) ofthe miRNA (miR-450b-5p) into RD (embroynal RMS) cell and RH28(alveolar RMS) cellin vitro, and expression of transfected miRNAs was confirmed by RT-PCR assays.3Hthymidine (3H-TdR) incorporation assay and MTT were conducted in transfected RD andRH28cells to determine differences of cell proliferation. Caspase-3and TUNEL wereexamined to confirm consequence of cell apoptosis by immunofluorescence.(3) After RDcell was inoculated in nude mice, tumors were injected with M or I, then tumorigenicity ofRD cells was initially confirmed by volume measurement of tumor. Ki67, myogenin,myosin and desmin in subcutaneous RMS tissue were stained by immunohistochemistryand expressions were confirmed by RT-PCR following.(4) we searched for predictedmiR-450b-5p targets with Target Scan6.1, Diana microT4.0, and MICRORNA.ORGindependently. TGF-β1-siRNA knockdown in RMS (ERMS: RD, RH36, SMS-CTR;ARMS: RH30, RH28, RH3) cells forced miR-450b-5p overexpression, and efficient targetswere determined by RT-PCR and Western blot. RD cell was co-transfected by wild-type ormutant3′-UTRs of targets (Wt.UTR/Mut.UTR) and M/I, and targets were conducted byLuciferase binding assays. Otherwise the clinical association between TGF-β1,miR-450b-5p and targets were explored by RT-PCR. Targets-siRNAs knocked down RDcells after M transfection. RT-PCR was performed to detected expression of mRNAs, andMHC and TUNEL were examined to confirm consequence of cell differentiation ane cell apoptosis by immunofluorescence.(5) Expression of pre-miR-450b-5p andmature-miR-450b-5p in TGF-β1-siRNA treated RD cells and RH28cells were examinedby RT-PCR. TGF-β1-siRNA, Smad4-siRNA, Smad2-siRNA or Smad3-siRNA combiningwith exogenous TGF-β1knocked down RD cell, and expressions of miR-450b-5p wereperformed by RT-PCR. RD cell was co-transfected by TGF-β1-siRNA, Smad4-siRNA,Smad2-siRNA, Smad3-siRNA and Wt.UTR/Mut.UTR, and Luciferase binding assaysdetected the signal.Results:(1) miRNA microassay displayed that the5miRNAs with overexpressionlevels in TGF-β1knock down RMS cell lines were miR-4275, miRNA-4298, miR-411,miR-493*and miR-450b-5p. Only miR-2113was significantly down-regulated expressedin TGF-β1knock down RMS cell lines. miR-450b-5p showed the most significant increasein expression in the TGF-β1-low expression RMS tissues by immunohistochemistry andRT-PCR, demonstrated by Western blot, Northern blot�'�RT-PCR.(2) M could effectivelyup-regulate the expression of miR-450b-5p and I effectively down-regulated miR-450b-5pexpression by RT-PCR. RMS cell proliferation was reduced after M treatment in atime-dependent manner by3H-TdR incorporation and MTT. The numbers of Caspase-3-and TUNEL-positive cells increased significantly after M transfection byimmunofluorescence, even the apoptosis was promoted by M and TGF-β1siRNAco-treatment.(3) After measurement, volumes of M transfected tumors notably decreasedin a time-dependent manner3weeks later. M induced an increase in myogenin and myosinafter immunohistochemistry staining and RT-PCR.(4) Among these candidates, onlyENOX2and PAX9were repressed after knockdown in RMS cells. M could down-regulatethe expression of ENOX2and PAX9, however I up-regulated. The Wt.UTR expression ofboth ENOX2and PAX9were decreased by co-transfection of M and increased byco-transfection of I in RD cells by Luciferase binding assays. High ENOX2and PAX9expression were associated with low miR-450b-5p expression and with high TGF-β1expression by RT-PCR. The numbers of Caspase-3and MHC–positive cells increasedsignificantly after ENOX2-siRNA or PAX9-siRNA co-transfected with M in RD cell byimmunofluorescence.(5) The expressions of pre-miR-450b-5p and mature-miR-450b-5pincreased in TGF-β1-siRNA treated RD cells and RH28cells, and mature-miR-450b-5pexpression increased effectively. miR-450b-5p expression was induced in TGF-β1-siRNA,Smad4-siRNA, and Smad3-siRNA-knock down RD cells but not Smad2-siRNA, and the exogenous TGF-β1antagonized miR-450b-5p level of TGF-β1-siRNA, Smad4-siRNA,and Smad3-siRNA-knock down RD, but not Smad2-siRNA. The signal of ENOX2andPAX9decreased in TGF-β1-siRNA, Smad4-siRNA, and Smad3-siRNA-knock down RDcells, but not Smad2.Conclusion:(1) miR-450b-5p was suggested as an anti-tumor regulater, and wassuccessfully selected for further study by miRNA microassay, immunohistochemistry,RT-PCR,Western blot�'�Northern blot.(2) M efficiently increased miR-450b-5pexpression, inhibited activity of RMS cell, and promoted cell apoptosis. Otherwise theco-transfection of M and TGF-β1-siRNA intensively promoted RMS cell apoptosis.(3) Minhibited tumorigenicity of RMS cell, but inhibited by TGF-β1pathway.(4) ENOX2andPAX9were functional targets of miR-450b-5p that regulated differentiation. miR-450b-5pcan directly bind to the3′-UTR sequences of ENOX2and PAX9. High ENOX2and PAX9expression were associated with low miR-450b-5p expression and with high TGF-β1expression.(5) The repression of miR-450b-5p by TGF-β1occurred at post-transcriptionstep. Smad3and Smad4, not Smad2, play an important role in control processing ofmiR-450b-5p expression.