Study On The Mechanism Of MiRNA154 In Regulating Osteoclast Differentiation In Bone Metastasis Of Lung Cancer

Tumor bone metastasis is regulated by multiple factors,through the strict regulation of tumor cells and bone microenvironment of the cell gene expression interaction to achieve.In this case,miRNAs can be used as a major regulatory factor in gene expression,involved in controlling multiple aspects of bone metastasis,including cancer cells that escape from the primary tumor site,the spread of cancer cells to bone marrow,and bone marrow invasion,and Secondary growth and tumor interstitial cell interactions.In clinical practice,some specific mi RNAs have been identified in bone-forming cells,thereby increasing the likelihood that mi RNAs can be used as biomarkers for tumor bone metastases.The regulatory activity of mi RNAs in the bone microenvironment also suggests that miRNAs may be promising therapeutic targets.The research objectives are:(1)Co-expression Analysis Reveals Key Gene Modules of Bone Metastases(2)Down regulation of lysosomal and further gene expression characterization in lung cancer patients with bone metastasis(3)Screening miRNAs differentially expressed in bone metastases of lung cancer;(4)Identify mi RNAs that have an important regulatory effect on osteoclast function during bone metastases;(5)to clarify the signal pathway and molecular mechanism of miRNA regulation of osteoclast differentiation,growth and maturation.Methods(1)Here,we analyzed the coexpression modules with the help of WGCNA and investigated the function enrichment of coexpression genes form important modules.Firstly,the coexpression modules were conducted for 6,922 genes in the 20 bone metastases samples recently.Then,the interaction relationships of hub-genes between pairwise modules were analyzed using WGCNA algorithm.Following that,the genes in the bone metastases coexpression modules were subjected to do functional annotation analysis.(2)In this study,gene expression of bone marrow negative and bone marrow positive samples were analyzed.Besides,the protein-protein interaction(PPI)network was constructed to figure out the regulation relationship among these differentially expressed genes(DEGs).Functional enrichment biological process and pathway were analyzed by gene ontology(GO)and pathway enrichment analysis.Furthermore,core proteins were screened out from the constructed protein-protein interaction(PPI)network.(3)Three patients with bone metastases were collected from the clinical cohort and 3 patients with spinal injury were also collected.The specimens of lung cancer were collected from the clinical operation.MiRNA microarray and qRT-PCR were used to analyze the expression of miRNAs in different groups and the candidate miRNAs were verified.In this study,LNATM miRNA chips(version 11.0)produced by EXIQON,Denmark,each containing 1807 specific probes and 435 specific probes were used.To ensure the reliability of the results,each of these probes is repeated four times in the chip,that is,each chip is repeated 4 times for the same sample.The data were analyzed with Genepix 6.0 software.We defined the expression level of the tumor group relative to the control group greater than 2 or less than 0.5 for differential expression.(4)Two sgRNAs with different GC% were designed for hsa-miR-30 dgene,hsa-miR-154 gene,hsa-miR-34 b gene,and then constructed into sgRNA expression vector and Cas9 protein / sgRNA co-expression vector.The co-expression vector of Cas9 protein / sgRNA can express both sgRNA and Cas9 protein at the same time,so the sgRNA and Cas9 protein can co-transfer together.The constructed expression vector was transfected into mammalian cells by Lipofectamine 2000,and the shear efficiency was detected by T7E1 assay and DNA sequencing.The results showed that the CRISPR / Cas system was able to work to targetthe hsa-miR-30 d,hsa-miR-154,hsa-miR-34 b,and results revealed that the CRISPR / Cas system had different shear efficiency.In order to knock out hsa-miR-30 d,hsa-miR-154 and hsa-miR-34 b gene completely,we transfected the cells with the CRISPR / Cas system vector and hsa-miR-30 d,Hsa-miR-154,hsa-mi R-34 b targeted vector together,and cells were obtained by the method of puromycin resistance screening,and the effect of hsa-miR-30 d,hsa-miR-154,hsa-miR-34 b knockout cells were analysised.The expression of hsa-miR-30 d,hsa-miR-154 and hsa-miR-34 b were detected by Real-Time PCR,and the Differences of hsa-miR-30 d,hsa-mi R-154 and hsa-miR-34 b expression were detected between knockout cells and wild-type cells.(5)According to our online bioinformatics analysis,it was found that mir-154 was involved in the regulation of Wnt/?-catenin signaling pathway.DKK2 protein was highly specific for regulate Wnt/?-catenin signaling pathway.The Wnt/?-catenin signaling pathway has a specific DKK2 target sequence.Mir-154 recognizes the 3 'untranslated region(3'-UTR)of DKK2 by the way of base complementary.The expression of DKK2 in these transfected cells was detected by Western blot analysis.The expression of DKK2 was detected in Raw264.7 cells treated with mir-154 mimetic or mir-154 inhibitor by Western blot.A 3'-UTR sequence of mutant was prepared by point-mutation technique.The mutant 3'-UTR sequence was prepared by bioinformatics prediction of DKK2 and wild type 3'-UTR sequence of mir-154 was prepared.Then mutant 3'-UTR and 3'-UTR of wild type-UTR was cloned into the psiCHECK-2 plasmid.The psiCHECK-2 plasmid containing mutant 3'-UTR and wild type 3'-UTR was transfected into HEK-293 T cells with mir-154,and the fluorescence intensity was detected by double fluorescence analysis.(6)The key proteins of Wnt/?-catenin signaling pathway in Raw264.7 cells were stained with tartrate-resistant acid phosphatase,and Wesrtern Blot was used to detect tartrate-resistant acid phosphatase and cathepsin K Osteoblast differentiation and maturation,and to analyze the relationship between the key protein of Wnt/?-catenin signaling pathway and the differentiation and maturation of Raw264.7 cells.Results(1)The functional enrichment for the top five modules exhibited great differences,with the module two enriched in focal adhesion,ECM-receptor interaction and cell adhesion molecules.We then inferred genes of the module two were the important genes which were associated with bone metastases.(2)This study was to screen out differentially expressed genes(DEGs)and functional proteins in bone metastases.Our results suggested CTSS,CTSD,MX1,NKX2-1 might play a decisive role in bone metastasis.(3)We detected the bone tissue microarray inall the 3 cases of lung cancer patients with bone metastases and 3 cases of non-lung cancer patients,cluster analysis showed a total of 82 species down regulation and a total of 112 were raised.The results showed that hsa-miR-30 d,hsa-miR-154 and hsa-miR-34 b were low expression in bone tissue of lung cancer withbone metastases by bioinformatics predictive analysis.Then we used real-time quantitative PCR to verify the above-mentioned miRNAs in bone tissues of lung cancer with bone metastases.The results showed that the expression of miRNAs such as hsa-mi R-30 d,hsa-miR-154 and hsa-miR-34 b were significantly decreased in the bone tissue of lung cancer compared with the control group(P <0.01),among which hsa-miR-154 was the most obvious decreased,which may be regulated lung cancer bone metastasis of a fairly conservative small RNA,and may affect the occurrence and development of tumors.(4)T7E1 digestion experiment confirmed that hsa-miR-30 d gene fragment was able to observe 250 bp and 360 bp small fragments after digestion,hsa-miR-154 gene fragment was digested to produce 240 bp and 480 bp fragments,hsa-miR-34 b gene fragment was cleaved into fragments of 220 bp and 520 bp.The CRISPR/Cas system designed for hsa-miR-30 d,hsa-miR-154 and hsa-mi R-34 b genes can recognize and knock down the target gene.The sequences of hsa-miR-30 d,hsa-miR-154 and hsa-miR-34 b were sequenced and found in the CRISPR/Cas system,and the sequences at the cleavage site became very disorderly,indicating that the CRISPR / Cas system designed to effectively target the target site knockout.(5)Western blot Results showed that Raw264.7 cells transfected with mir-154 mimetic showed a lower level of DKK2 protein.In contrast,cells transfected with the MIR-154 inhibitor expressed a higher level of DKK2 protein.The mutant 3'-UTR sequence did not bind to mir-154,and the wild-type 3'-UTR sequence was bound to MIR-154 after mutant 3'-UTR sequence transfection was prepared by point mutation technique.The mutant 3'-UTR and wild-type 3'-UTR were cloned into the psiCHECK-2 plasmid.The psiCHECK-2 plasmid containing mutant 3'-UTR and wild type 3'-UTR was transfected into HEK-293 T cells with mir-154,and the fluorescence intensity was detected by double fluorescence analysis.The results showed that the fluorescence intensity of HEK-293 T cells transfected with wild-type plasmids and mir-154 was significantly lower than that of the control group.The fluorescence intensity of HEK-293 T cells transfected with mutant plasmids and mir-154 was not significantly different from that of the control group.(6)Anti-tartaric acid phosphatase staining and Wesrtern Blot detection of these two proteins.TRAP-positive multinucleated cells were observed in Ad-?-catenin+RANKL group.In the blank group,a small number of TRAP-positive multinucleated cells were observed in the osteoblastic differentiation of both osteotrophic acid phosphatase and cathepsin K.The TRAP-positive multinucleated cells in the RANKL group and Ad-GFP+RANKL with the induction factor were significantly increased.Western Blot results showed that a color band was detected in all groups,but the color band of Ad-?-catenin+RANKL group was lighter than that of the other three groups.These results confirmed that the Wnt/?-catenin signaling pathway is directly involved in the differentiation and maturation of osteoclasts and showed an inhibitory effect.Conclusion(1)Together,our findings provided the framework of coexpression gene modules of bone metastases and furthered the understanding of these modules at functional aspect.(2)these results demonstrated that bone metastasis from lung cancer would lead to changes in lysosome function,which may affect the decomposition and elimination of old bone matrix,thus affecting bone turnover.In addition,our findings provided new insights into the prediction and treatment of bone metastases.(3)We screened and identified the differentially expressed miRNA profiles in the bone tissue of lung cancer after bone metastasis,and screened and demonstrated the low expression of hsa-miR-30d?hsa-miR-154?hsa-miR-34 b in bone tissue of lung cancer with bone metastasis(4)The miRNA154 plays an important role in the differentiation and maturation in Raw264.7 cells through CRISPR / Cas system.(5)Results demonstrated that mir-154 can up-regulate the expression of ?-catenin by directly inhibiting the translation and expression of DKK2,thereby activating the classical Wnt / ?-catenin signaling pathway.(6)Results confirmed the Wnt / ?-catenin signaling pathway directly osteoclast differentiation process,and is the inhibitory effect...