Correlation research of Runt-related transcription factor 2 with proliferation genes, tumor suppressor genes and angiogenesis molecules in colon cancer lesions

Objective: To investigate the correlation of Runt-related transcription factor 2 (RunX2) with proliferation genes, tumor suppressor genes and angiogenesis molecules in colon cancer lesions. Methods: A total of 90 patients with primary colon cancer were enrolled in colon cancer group, 68 patients with benign colon polyps were enrolled in colon polyps group, the differences in the expression levels of RunX2, proliferation genes, tumor suppressor genes and angiogenesis molecules in the two groups of lesions were compared, and Pearson test was further used to evaluate the correlation of RunX2 expression level with proliferation gene, tumor suppressor gene and angiogenesis molecule expression levels in colon cancer tissues. Results: RunX2 mRNA expression level in the lesions of colon cancer group was higher than that of colon polyps group. Proliferation genes GTPBP4, HOXB7, ZNF331, ADAM17 and HSP60 mRNA expression levels in the lesions of colon cancer group were higher than those of colon polyps group;tumor suppressor genes ATF3, FOXN3, OTUD1 and NDRG2 mRNA expression levels were lower than those of colon polyps group;angiogenesis molecules Musashi 1, NF-κB, RegⅣ and STAT3 mRNA expression levels were higher than those of colon polyps group. RunX2 mRNA expression level in the colon cancer lesions was directly correlated with the expression levels of the above proliferation genes, tumor suppressor genes and angiogenesis molecules. Conclusion: RunX2 expression is abnormally high in colon cancer lesions, the specific expression level is positively correlated with cancer cell proliferation activity and angiogenesis activity, and it is an important molecular target that can lead to the occurrence and development of colon cancer.

Correlation of Runt-related transcription factor gene 3 expression in osteosarcoma tissue with cell proliferation and angiogenesis

Objective:To study the correlation of Runt-related transcription factor gene 3 (RUNX3) expression in osteosarcoma tissue with cell proliferation and angiogenesis.Methods: A total of 80 patients with osteosarcoma who were treated in our hospital between February 2014 and February 2017 were collected, and the RUNX3 expression in osteosarcoma tissue and adjacent tissue were detected. According to the RUNX3 expression in tumor tissue, the patients were further divided into high RUNX3 expression group and low RUNX3 expression group, and the proliferation gene and angiogenesis gene expression were compared.Results:RUNX3, KISS-1 and RanBP9 mRNA expression in osteosarcoma tissue were significantly lower than those in adjacent tissue while VCP, Six1, S100A6, IF-1α, MMP-14, bFGF and Ang-2 mRNA expression were significantly higher than those in adjacent tissue;KISS-1 and RanBP9 mRNA expression in osteosarcoma tissue of high RUNX3 expression group were significantly higher than those of low RUNX3 expression group while VCP, Six1, S100A6, IF-1 , MMP-14, bFGF and Ang-2 mRNA expression were significantly lower than those of low RUNX3 expression group.Conclusions:The desease of RUNX3 expression in osteosarcoma tissue is one of the direct causes of increased tumor proliferation activity and strong angiogenesis.

Novel Mutation of Cleidocranial Dysplasia-related Frameshift Runt-related Transcription Factor 2 in a Sporadic Chinese Case

Background： Cleidocranial dysplasia （CCD） is an autosomal dominant disease that affects the skeletal system. Common symptoms of CCD include hypoplasia or aplasia of the clavicles, delayed or even absent closure of the fontanels, midface hypoplasia, short stature, and delayed eruption of permanent and supernumerary teeth. Previous studies reported a connection between CCD and the haploinsufficiency of runt-related transcription factor 2 （RUNX2）. Here, we report a sporadic Chinese case presenting typical symptoms of CCD. Methods： We made genetic testing on this sporadic Chinese case and identified a novel RUNX2 ffameshift mutation： c.1111 dupT. In situ immunofluorescence microscopy and osteocalcin promoter luciferase assay were performed to compare the functions of the RUNX2 mutation with those of wild-type RUNX2. Results： RUNX2 mutation was observed in the perinuclear region, cytoplasm, and nuclei. In contrast, wild-type RUNX2 was confined in the nuclei, which indicated that the subcellular compartmentalization of RUNX2 mutation was partially perturbed. The transactivation function on osteocalcin promoter of the RUNX2 mutation was obviously abrogated. Conclusions： We identified a sporadic CCD patient carrying a novel insertion/frameshift mutation of RUNX2. This finding expanded our understanding of CCD-related phenotypes.

Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice

Background Runt-related transcription factor 1 （Runxl） plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runxl will induce leukemia and how the change of Runxl expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runxl in BaF3 cells alone would induce teukemogenesis. And we also wanted to know if abnormal expression of Runxl in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runxl in BaF3 cells would induce leukemogenesis. Methods Plasmids containing full-length Runxl cDNA were transduced into BaF3 cells and BaF3-P185wt cells （BCR-ABL transformed BaF3 cells） by electroporation. Plasmids containing a short hairpin RNA of Runxl were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runxl expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runxl on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes. Results In vitro analysis revealed that overexpression of Runxl in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runxl could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runxl cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runxl group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. Conclusions Overexpression or knock-down of Runxl gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runxl expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemoaenesis in vivo.

Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts

Bone is a dynamic tissue that is constantly renewed by the coordinated action of two cell types, i.e., the bone-resorbing osteoclasts and the bone-forming osteoblasts. However, in some circumstances, bone regeneration exceeds bone self repair capacities. This is notably often the case after bone fractures, osteolytic bone tumor surgery, or osteonecrosis. In this regard,bone tissue engineering with autologous or allogenic mesenchymal stem cells(MSCs) is been widely developed. MSCs can be isolated from bone marrow or other tissues such as adipose tissue or umbilical cord, and can be implanted in bone defects with or without prior amplification and stimulation. However, the outcome of most pre-clinical studies remains relatively disappointing. A better understanding of the successive steps and molecular mechanisms involved in MSC-osteoblastic differentiation appears to be crucial to optimize MSC-bone therapy. In this review, we first present the important growth factors that stimulate osteoblastogenesis. Then we review the main transcription factors that modulate osteoblast differentiation, and the microRNAs(miRs)that inhibit their expression. Finally, we also discuss articles dealing with the use of these factors and miRs in the development of new bone MSC therapy strategies. We particularly focus on the studies using human MSCs, since significant differences exist between osteoblast differentiation mechanisms in humans and mice for instance.

Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids

AIM:To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.METHODS:DNA was extracted from undiluted pancreatic and biliary fluids.As a surrogate for a genomewide hypomethylation assay,levels of long interspersed nuclear element-1(LINE-1) methylation were analyzed using bisulfite pyrosequencing.CpG island hypermethylation of 10 tumor-associated genes,aryl-hydrocarbon receptor repressor,adenomatous polyposis coli,calcium channel,voltage dependent,T type α1G subunit,insulin-like growth factor 2,O-6-methyl-guanine-DNA methyltransferase,neurogenin 1,CDKN2A,runt-related transcription factor 3(RUNX3),secreted frizzled-related protein 1,and ubiquitin carboxyl-terminal esterase L1(UCHL1),was analyzed using MethyLight.To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1,human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h.After the treatment,UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction.RESULTS:Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease(58.7% ± 4.3% vs 61.7% ± 2.2%,P = 0.027;53.8% ± 6.6% vs 57.5% ± 1.7%,P = 0.007);however,LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids(45.4% ± 5.5% vs 58.7% ± 4.3%,P < 0.001).CpG island hypermethylation of tumor-associated genes was detected at various frequencies,but it was not correlated with LINE-1 hypomethylation.Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic(67%) or biliary(70%) fluids from patients with pancreatobiliary cancer.As a single marker,hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers,respectively(100% specificity).Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic(87% sensitivity and 100% specificity) and pancreatobiliary(97% sensitivity and 100% specificity) cancers.Treatment with a demethylating agent,5-AZA-2'-deoxycytidine,restored UCHL1 expression in pancreatobiliary cancer cell lines.CONCLUSION:Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.

Runx3 might participate in regulating dendriti cell function in patients with irritable bowel syndrome

Objective:To evaluate the expression levels and correlations among the expressions of transforming growth factor-β1（TGF-β1）,Kunx3 and CD83 in colonic mucosal specimens from IBS patients.Methods:A total of 40 patients were selected,who were confirmed as IBS by Rome III standard and 40 healthy volunteers served as control.Colonic mucosal specimens of each subject were collected from colon sigmoideum with biopsy forceps.Runx3,TGF-β1?and CD83(the marker for immunecompetent mature dendritic cells（DCs）mRNA in the sigmoid colon tissue were measured by real-time fluorescence quantitative PCR.Results:Compared with the control group,CD83 mRNA expressions were higher in patients with IBS than in healthy controls（P【0.05）and were associated with runt-related transcription factor 3（Runx3）mRNA levels（r=-0.361,P【0.05）.Meanwhile,Runx3 mRNA levels were associated with TGF-β1,mRNA expressions in irritable bowel syndrome（IBS）patients（r=0.402,P【0.05）.However,there was no correlation between the mRNA expressions of TGF-β1 and CD83（P】0.05）.Conclusions:The increase of abnormal dendritic cells might influence the occurrence and development of IBS.TGF-β1 signal pathway might not be involved in Runx 3-regulated maturation of dendritic cells in IBS.

Dysregulated RUNX1 Predicts Poor Prognosis by Mediating Epithelialmesenchymal Transition in Cervical Cancer

Objective Runt-related transcription factor 1(RUNX1)has been proven to be over-expressed and vital in many malignancies.However,its role in cervical cancer is still unclear.Methods Some online databases(Oncomine,GEPIA,UALCAN,LinkedOmics,and others)were used to explore the expression level,prognostic significance,and gene mutation characteristics of RUNX1 in cervical cancer.The protein levels of RUNX1 in cervical cancer were measured by immunohistochemistry(IHC).The functional changes of cervical cancer cells were measured in vitro after decreasing RUNX1.Results Bioinformatic results revealed that RUNX1 was upregulated in cervical cancer compared to normal tissues.Moreover,over-expression of RUNX1 was significantly correlated with cervical cancer patients’clinical parameters(e.g.,individual cancer stages,patients’age,nodal metastasis status,and others).Meanwhile,functional enrichment analysis of RUNX1-related genes indicated that RUNX1 was mainly involved in the epithelial-mesenchymal transition(EMT)process in cervical cancer.Furthermore,RUNX1 may be upregulated by hsamiR-616-5p and hsa-miR-766 identified by miRDB,TargetScan,and miRWalk.Finally,RUNX1 was upregulated in cervical cancer compared to normal tissues by IHC in collected cervical cancer samples.The invasion and migration abilities of cervical cancer cells were significantly reduced by repressing EMT after knocking down RUNX1 in vitro.Conclusion RUNX1 was highly expressed in cervical cancer,and upregulated RUNX1 could significantly promote the invasive abilities of cervical cancer cells by inducing EMT.Therefore,RUNX1 may be a potential biomarker for early diagnosis and targeted therapy of cervical cancer.

Prohibitins, novel vitamin K2 target factors in osteoblast

Vitamin K2 (VK2, menaquinone) is a drug for osteoporosis. VK2 acts as a cofactor for γ-glutamyl carboxylase, which catalyzes the carboxylation of specific glutamic acid residues (γ-carboxylation) of substrate proteins. Here we demonstrate that VK2 also regulate osteoblastgenic marker gene expression. Using VK2-immobilzed nanobeads new target proteins were purified and identified from osteoblastic cell line. They are prohibitin 1 and 2 (PHB1 & 2), respectively. To confirm the PHBs function on VK2-dependent transcription, PHB1 & 2 were knock-down and osteocalcin gene 2 transcriptions were analyzed, indicating that PHBs regulate VK2-dependent transcription. Taken together PHBs are VK2 target proteins for osteoblastgenic transcription.

Rhizoma Drynariae-derived nanovesicles reverse osteoporosis by potentiating osteogenic differentiation of human bone marrow mesenchymal stem cells via targeting ERα signaling

Although various anti-osteoporosis drugs are available,the limitations of these therapies,including drug resistance and collateral responses,require the development of novel anti-osteoporosis agents.Rhizoma Drynariae displays a promising anti-osteoporosis effect,while the effective component and mechanism remain unclear.Here,we revealed the therapeutic potential of Rhizoma Drynariae-derived nanovesicles(RDNVs)for postmenopausal osteoporosis and demonstrated that RDNVs potentiated osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)by targeting estrogen receptor-alpha(ERα).RDNVs,a natural product isolated from fresh Rhizoma Drynariae root juice by differential ultracentrifugation,exhibited potent bone tissue-targeting activity and anti-osteoporosis efficacy in an ovariectomized mouse model.RDNVs,effectively internalized by hBMSCs,enhanced proliferation and ERαexpression levels of hBMSC,and promoted osteogenic differentiation and bone formation.Mechanistically,via the ERαsignaling pathway,RDNVs facilitated mRNA and protein expression of bone morphogenetic protein 2 and runt-related transcription factor 2 in hBMSCs,which are involved in regulating osteogenic differentiation.Further analysis revealed that naringin,existing in RDNVs,was the active component targeting ERαin the osteogenic effect.Taken together,our study identified that naringin in RDNVs displays exciting bone tissue-targeting activity to reverse osteoporosis by promoting hBMSCs proliferation and osteogenic differentiation through estrogen-like effects.

Ziyin Huatan Recipe, a Chinese herbal compound, inhibits migration and invasion of gastric cancer by upregulating RUNX3 expression

Objectives: Ziyin Huatan Recipe(ZYHT), a traditional Chinese medicine comprised of Lilii Bulbus, Pinelliae Rhizoma, and Hedyotis Diffusa, has shown promise in treating gastric cancer(GC). However, its potential mechanism has not yet been clearly addressed. This study aimed to predict targets and molecular mechanisms of ZYHT in treating GC by network pharmacology analysis and to explore the role of ZYHT in GC both in vitro and in vivo.Methods: Targets and molecular mechanisms of ZYHT were predicted via network pharmacology analysis. The effects of ZYHT on the expression of metastasis-associated targets were further validated by Western blot and quantitative real-time polymerase chain reaction. To explore the specific molecular mechanisms of the effects of ZYHT on migration and invasion, the runt-related transcription factor 3(RUNX3) gene was knocked out by clustered regularly interspaced short palindromic repeats/Cas9, and lentiviral vectors were transfected into SGC-7901 cells. Then lung metastasis model of GC in nude mice was established to explore the anti-metastasis effect of ZYHT. Western blot and immunohistochemistry were used to explore the impact of ZYHT on the expression of metastasis-related proteins with or without RUNX3 gene.Results: The network pharmacology analysis showed that ZYHT might inhibit focal adhesion, migration,invasion and metastasis of GC. ZYHT inhibited the proliferation, migration and invasion of GC cells in vitro via regulating the expression of metastasis-associated targets. Knocking out RUNX3 almost completely reversed the cell phenotypes(migration and invasion) and protein expression levels elicited by ZYHT.In vivo studies showed that ZYHT inhibited the metastasis of GC cells to the lung and prolonged the survival time of the nude mice. Knocking out RUNX3 partly reversed the metastasis of GC cells to the lung and the protein expression levels elicited by ZYHT.Conclusion: ZYHT can effectively inhibit the invasion and migration of GC in vitro and in vivo, and its molecular mechanism may relate to the upregulation of RUNX3 expression.

A Systems Biology Approach for Studying Heterotopic Ossification： Proteomic Analysis of Clinical Serum and Tissue Samples

Heterotopic ossification （HO） refers to the abnormal formation of bone in soft tissue. Although some of the underlying processes of HO have been described, there are currently no clinical tests using validated biomarkers for predicting HO formation. As such, the diagnosis is made radiographically after HO has formed. To identify potential and novel biomarkers for HO, we used isobaric tags for relative and absolute quantitation （iTRAQ） and high-throughput antibody arrays to produce a semi-quantitative proteomics survey of serum and tissue from subjects with （HO ＋） and without （HO-） heterotopic ossification. The resulting data were then analyzed using a systems biology approach. We found that serum samples from subjects experiencing traumatic injuries with resulting HO have a different proteomic expression controls. Subsequent quantitative ELISA identified profile compared to those from the matched five blood serum proteins that were differentially regulated between the HO-- and HO- groups. Compared to HO- samples, the amount of insulin-like growth factor I （IGF1） was up-regulated in HO＋ samples, whereas a lower amount of osteopontin （OPN）, myeloperoxidase （MPO）, runt-related transcription factor 2 （RUNX2）,and growth differentiation factor 2 or bone morphogenetic protein 9 （BMP-9） was found in HO ＋ samples （Welch two sample t-test; P 〈 0.05）. These proteins, in combination with potential serum biomarkers previously reported, are key candidates for a serum diagnostic panel that may enable early detection of HO prior to radiographic and clinical manifestations.