MicroRNAs Cooperatively Suppress The Effect Of RORA On Proliferation In Oral Squamous Cell Carcinoma

Part I Deep sequencing reveals the regulatory network of microRNA-transcription factor in oral squamous cell carcinomaObjective We aim to explore the potential relationship between differentially expressed microRNAs(miRNAs)and mRNAs in oral squamous cell carcinoma(OSCC),and identify miRNA-mediated transcription factor(TF)regulatory network.Materials and Methods 1.MRNA-sequencing was used to detect differently expressed mRNAs in 4 pairs of OSCC-adjacent normal tissues,and Gene Ontology(GO)was performed to analyze the signaling pathway differently expressed mRNAs enriched with.2.MiRNA-sequencing was used to detect differently expressed miRNAs in 4 pairs of OSCC-adjacent normal tissues.3.Inverse correlations of differently expressed mRNAs and miRNAs were constructed.MiRanda and Target Scan were used to predict the potential target gene of differently expressed miRNA.Moreover,the set of differentially expressed target genes and their corresponding miRNAs significantly changing in an opposite way were identified(upregulated miRNA-downregulated mRNA/ downregulated miRNA-upregulated mRNA).4.To demonstrate the function relationship between differentially expressed mRNAs and miRNAs,GO analysis was performed with down-regulated mRNAs targeted by up-regulated miRNAs in 4 OSCC paired samples,as well as the up-regulated mRNAs targeted by down-regulated miRNAs.Results 1.MRNA-seq analysis showed that 21977-24584 mRNAs expressed in 8 OSCC or normal tissue samples.Among them,11788 genes significantly differentially expressed in at least one pair of samples between tumor tissues and normal tissues.And 3042-6776 differently expressed mRNAs were detected in every paired tissues.In addition,183 mRNAs were coordinately upregulated and 185 mRNAs were coordinately downregulated in all of the cancerous tissues compared to normal tissues.GO analysis revealed that,up-regulated mRNAs were enriched with functional pathways related to tumor growth and invasion,while down-regulated mRNAs were enriched in pathways controlling normal epithelial biological functions.2.MiRNA-seq analysis showed that 1034-2067 miRNAs expressed in 8 OSCC or normal tissue samples.Among them,458 miRNAs significantly differentially expressed in at least one pair of samples between tumor tissues and normal tissues.And 120-268 differently expressed miRNAs were detected in every paired tissues.In addition,17 miRNAs were coordinately upregulated and 1 miRNA was coordinately downregulated in all four cancerous tissues compared to the corresponding normal tissues.3.A total of 16968 genes were predicted as potential targets of 2617 miRNAs.Among those,we identified 8137 negative miRNA-mRNA interactions,involving 213 differently expressed miRNAs and 2172 differently expressed mRNAs.4.GO analysis revealed that,downregulated mRNAs targeted by upregulated miRNAs in 4 OSCC paired samples mainly enriched with transcription related signaling pathways,while upregulated mRNAs enriched with tumorigenesisrelated pathways and showed significant individual difference in every paired OSCC tissues.We selected the upregulated miRNAs and the downregulated TFs to construct a network of miRNAs(133)-transcription factors(167).Conclusion The differentially up-regulated miRNAs promote OSCC tumorigenesis dominantly by mediating the down-regulation of TFs and controlling transcription regulation pathways.The miRNAs-TFs network was constructed.Part II MicroRNAs cooperatively suppress the expression of circadian rhythm gene RORA in oral squamous cell carcinomaObjective We aim to explore the cooperative target effect of 5 miRNAs on the inhibition of retinoic acid receptor-related orphan receptor(RORA),among the microRNA(miRNA)-transcription factor(TF)regulatory network in oral squamous cell carcinoma(OSCC).Materials and Methods 1.Among the miRNA-RORA subnet of OSCC miRNA-TF network,miRNA-seq data of different human cell lines were used to screen the appropriate miRNAs predicted to target RORA from 25 miRNAs for further research.2.QPCR was performed to evaluate the mRNA level of RORA in 44 paired OSCCnormal tissues.And the expression levels of 5 miRNAs(miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p)were detected in 38 paired OSCCnormal tissues by q PCR.3.The c DNA of RORA 3’ UTR sequence(enriched with the binding sites of the 5 miRNAs)was amplified and inserted to the luciferase reporter vector p Si CheckTM-2.Luciferase assay was performed to test the target effect of 5 miRNAs on RORA.4.To testify the cooperative regulation effect of miRNAs on RORA expression,inhibitors of miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p were transfected into Hela cells,individually,in pairs or together,and miR-NC was served as the negative control.QPCR was performed to detect the mRNA level of RORA in each group.Results 1.Among the OSCC miRNA-TF network,circadian rhythm genes(RORA,RORB,RORC,and CLOCK)were simultaneously regulated by multiple miRNAs were of particular interest.RORA was reported to be a tumor suppressor and was decreased in many cancers.We chose miRNA-RORA subnet for further research.According to miRNA-seq data of different human cell lines,8 miRNAs might be related to the macrophage infiltration,and was removed.In the remain 17 miRNAs,5 tumor proliferation-related miRNAs(miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p)were chose for further research.2.RORA was significantly decreased in most OSCC samples(37/44,84%).And five miRNAs were generally up-regulated in the majority of samples: miR-503-5p in 52.6%(20/38);miR-450b-5p in 68.4%(26/38);miR-27a-3p in 52.6%(20/38);miR-181a-5p in 78.9%(30/38),and;miR-183-5p in 60.5%(23/38).3.Compared with the control group,the miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p mimic group resulted in approximately 49.4%,15.5%,49.8%,39.0%,and 46.3% luciferase activity reduction,respectively.While the inhibitor group increased nearly 34.7%,41.7%,87.2%,16.1%,and 60.4% luciferase activity,respectively.4.The mRNA expression of RORA was slightly increased about 1.2-fold,1.3-fold,1.1-fold,1.2-fold,and 1.1–fold in miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p inhibitor group,respectively.Additionally,when cells were transfected by miRNA inhibitors in pairs,there was an obviously increased RORA expression.And the five miRNA inhibitors mixing led to a much stronger up-regulation effect on RORA mRNA expression(about 4.9-fold)than the groups transfected with single miRNA inhibitor(p<0.001).Conclusion Five up-regulated miRNAs(miR-503-5p,miR-450b-5p,miR-27a-3p,miR-181a-5p and miR-183-5p)could suppress the expression of circadian rhythm gene RORA by direct target,and could exert more significant effect by cooperative regulation.Part III The effect of abnormal expression of RORA on the proliferation and prognosis of oral squamous cell carcinomaObjective We aim to explore the effect of retinoic acid receptor-related orphan receptor(RORA)on the prognosis and proliferation of oral squamous cell carcinoma(OSCC)and the underlying molecular mechanism.Materials and Methods 1.The mRNA-seq data of 340 OSCC and 32 normal epithelial patient samples were downloaded from TCGA to analyze the relationship between RORA mRNA level and clinicopathological characteristics.125 pairs of OSCC and normal epithelial patient samples were subjected to microarray and performed immunohistochemical staining to analyze the relationship between RORα protein level and clinicopathological characteristics.2.The lentiviral vector recombinant plasmid p CDH-RORA was constructed to overexpress RORA in Cal-27 cell line and UM-SCC-23 cell line(p CDH-puro served as control),while GV-248-RORA-sh1/2/3 for the knockdown of RORA in Cal-27 cell line(GV-248-con served as control).3.QPCR and western blot were performed to detect the expression level of RORA mRNA and protein in Cal-27/UM-SCC-23-p CDH-RORA and Cal-27-GV-248-RORA-sh1/2/3 to evaluate the over-expressing and knockdown efficiency of RORA.4.CCK-8 assay and mice xenograft assay were used to assess the effects of RORα on OSCC proliferation in vitro and in vivo.5.Western blot was used to detect the expression level of HIPK2 protein,p53 protein and p53 phosphorylation protein in HIPK2/p53 signaling pathway after suppressing or overexpressing RORA in Cal-27 cell line.6.The luciferase reporter vector p Si CheckTM-2-p53 including the sequence of p53 promoter enriched with the binding sites of RORA was constructed,as well as the corresponding mutant plasmid p Si CheckTM-2-p53-mut.Luciferase reporter assay was performed to test the target effect of RORA on p53 promoter.7.Immunoprecipitation was performed to detect the reciprocal protein binding effect of RORα and HIPK2 in Cal-27 cells.Results 1.RORA exhibited significantly lower expression in OSCC samples from TCGA,compared to normal samples(p < 0.001),and relatively low RORA mRNA abundance was associated with poor 3-year survival(p = 0.0456).In 125 OSCC tissues and paired adjacent epithelia microarray,the level of RORα protein in OSCC cells was significant lower than that in adjacent normal epithelial cells(p < 0.001).Attenuated nuclear RORα abundance was detected in tumors with poorlydifferentiated histology(p < 0.05),advanced clinical stage(p < 0.01)and unfavorable 5-year survival(p = 0.0265).2.Compared with control cell line,the relative expression of RORA mRNA in Cal-27/UM-SCC-23-p CDH-RORA cell lines was overexpressed about 903.6-fold and 156.2-fold(p<0.001)respectively,while the expression of RORα protein was significantly increased.The relative expression of RORA mRNA in Cal-27-GV-248-RORA-sh1/2/3 cell lines decreased about 3.13-,2.37-,2.40-folds respectively(p<0.001),while the expression of RORα protein was significantly decreased.3.In CCK-8 assay,Cal-27/UM-SCC-23 cells overexpressing RORα suppressed cell proliferation about 48% and 34% at 96 h respectively(p <0.01),while attenuated RORα in Cal-27 cells promoted cell proliferation about 1.51-fold(p<0.01).In mice xenograft assay,compared with the control group,the growth rate,final volumes and weights of Cal-27-p CDH-RORA xenografts were slower and slighter(p<0.05).4.The total and phosphorylated p53 protein and HIPK2 protein expression levels decreased after blocking RORα expression,while increased when enforcing RORα in Cal-27 cells.5.Compared with the control group,p Si CheckTM-2-p53 group resulted in approximately 2.34-fold luciferase activity increase(p<0.05),while the luciferase activity of p Si CheckTM-2-p53-mut was similar to control group.6.Immunoprecipitation assay showed that,in Cal-27 cells,HIPK2 protein was detected as binding protein by using RORA antibody,while RORα protein was detected as binding protein by using HIPK2 antibody to bind protein.Conclusion RORA showed lower expression in OSCC tissue compared to normal tissue,and the low RORA expression associated with poor prognosis.RORA acted as a tumor suppressor and could decrease the tumor proliferation,while the underlying mechanism might be related to p53,p53-Ser46 and HIPK2.