Long transcripts minus touchdown qPCR (LTMT-qPCR): a simplified and convenient method for the screening and quantification of microRNA profiles.

Due to the short length and differences in abundance of microRNAs, microRNA profile screening and quantification is challenging. In this study, we found that size selection magnetic beads could be employed to easily and efficiently remove long RNA transcripts. After removing the long transcripts, the remaining small RNAs could be concentrated and then reverse-transcribed using universal stem-loop primers (USLP), with six randomized nucleotides at the 3' end region. The efficiency of reverse transcription decreased when the number of randomized nucleotides was reduced. In addition, we found that touchdown qPCR improved microRNA profile detection, with lower CT values and better detection efficiency than the regular qPCR protocol, especially for those low-abundance microRNAs. Finally, we incorporated these observations to create a new protocol we named long transcripts minus touchdown qPCR (LTMT-qPCR). We performed a side-by-side comparison of LTMT with USLP and traditional stem-loop primer (TSLP) protocols. We found that LTMT has higher detection efficiency than USLP, especially for the detection of low-abundance microRNAs. Although LTMT was equivalent to TSLP in terms of microRNA profile detection, LTMT is more convenient, user-friendly, and cost-effective. Taken together, the present data indicate that LTMT is a simple, rapid, and user-friendly approach that has higher precision, accuracy, and sensitivity than the previously described methods, making it more suitable for microRNA profile screening and quantification.

BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs.

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer.

Chromosome region 3p12-14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down-regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation-specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9-transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E-cadherin, VIM, SNAIL, VEGFA, NFκB-p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFß1/TßR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR- and TGFß1/TßR(I/II)-activated AKT signaling.

Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

Niclosamide Exhibits Potent Anticancer Activity and Synergizes with Sorafenib in Human Renal Cell Cancer Cells.

BACKGROUND/AIMS: As the most lethal urological cancers, renal cell carcinoma (RCC) comprises a heterogeneous group of cancer with diverse genetic and molecular alterations. There is an unmet clinical need to develop efficacious therapeutics for advanced, metastatic and/or relapsed RCC. Here, we investigate whether anthelmintic drug Niclosamide exhibits anticancer activity and synergizes with targeted therapy Sorafenib in suppressing RCC cell proliferation. METHODS: Cell proliferation and migration were assessed by Crystal violet staining, WST-1 assay, cell wounding and cell cycle analysis. Gene expression was assessed by qPCR. In vivo anticancer activity was assessed in xenograft tumor model. RESULTS: We find that Niclosamide effectively inhibits cell proliferation, cell migration and cell cycle progression, and induces apoptosis in human renal cancer cells. Mechanistically, Niclosamide inhibits the expression of C-MYC and E2F1 while inducing the expression of PTEN in RCC cells. Niclosamide is further shown to synergize with Sorafenib in suppressing RCC cell proliferation and survival. In the xenograft tumor model, Niclosamide is shown to effectively inhibit tumor growth and suppress RCC cell proliferation. CONCLUSIONS: Niclosamide may be repurposed as a potent anticancer agent, which can potentiate the anticancer activity of the other agents targeting different signaling pathways in the treatment of human RCC.

Downregulated miR-1247-5p associates with poor prognosis and facilitates tumor cell growth via DVL1/Wnt/ß-catenin signaling in breast cancer.

The abnormal expression of microRNAs is a key hallmark of breast cancer. Nevertheless, the biological roles of miR-1247-5p in breast cancer remain unknown. In this study, we revealed that miR-1247-5p expression was markedly decreased in breast cancer. It was a valuable diagnostic biomarker for breast cancer with the area under the curve of more than 0.80. Reduced miR-1247-5p expression was significantly correlated with patient age, tumor size, and poor prognosis in The Cancer Genome Atlas cohort including 839 breast cancer patients. Multivariate Cox regression analysis demonstrated that miR-1247-5p was an independent prognostic indicator for overall survival (hazard radio [HR] = 1.683, 95% confidence interval [CI] = 1.087-2.606, p = 0.020) and recurrence-free survival (HR = 2.496, 95% CI = 1.576-3.951, p < 0.001). Moreover, functional studies showed that overexpression of miR-1247-5p inhibited proliferation and induced apoptosis in breast cancer cells. Bioinformatics analysis and mechanistic investigations revealed that Dishevelled 1 (DVL1) was a direct target of miR-1247-5p. Inhibition of DVL1 by miR-1247-5p resulted in the suppression of Wnt/ß-catenin signaling, whereas overexpression of DVL1 abrogated the miR-1247-5p-mediated effect. These data reveal that miR-1247-5p, as an oncosuppressor in breast cancer, may be a promising prognostic biomarker and therapeutic target.

ADAMTS9 is Silenced by Epigenetic Disruption in Colorectal Cancer and Inhibits Cell Growth and Metastasis by Regulating Akt/p53 Signaling.

BACKGROUND/AIMS: ADAMTS (disintegrin-like and metalloproteinase with thrombospondin motifs) proteins are extracellular zinc metalloproteinases that play an important role in extracellular matrix assembly and degradation, connective tissue structuring, angiogenesis, and cell migration. Multiple studies suggest that ADAMTS proteins (e.g. ADAMTS9) can act as tumor suppressors. In gastric, esophageal, and nasopharyngeal carcinomas ADAMTS9 is frequently down-regulated by promoter methylation. Whether ADAMTS9 can function as a tumor suppressor gene (TSG) in colorectal cancer is still unclear. METHODS: We performed immunohistochemistry, RT-PCR, and qRT-PCR, to examine the expression of ADAMTS9 in colorectal cancer cell lines and primary colorectal cancer tissues. Methylation-specific PCR was also carried out to investigate the promoter methylation status of ADAMTS9. We also explored the functions of ADAMTS9 in colorectal cancer cell lines through in vitro experiments. RESULTS: ADAMTS9 expression was down-requlated or silenced in 83.3% (5/6) of colorectal cancer cell lines, and frequently repressed in 65.6% (21/32) of colorectal cancer tissues. Down-regulation of ADAMTS9 was partially due to promoter methylation. Exogenous expression of ADAMTS9 in colorectal cancer cell lines inhibited cell proliferation and migration through the regulation of cell cycle and apoptosis. In addition, ADAMTS9 prevented the activation of Akt, and its downstream targets in colorectal cancer cell lines. CONCLUSION: Our findings suggest ADAMTS9 is a TSG in colorectal cancer.

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering ß-catenin signalling and GADD45G expression.

Recent studies suggest that paired box 5 (PAX5) is down-regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non-small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down-regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non-cancerous tissues (P = 0.0201). PAX5 down-regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down-regulation of ß-catenin and up-regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down-regulating the ß-catenin pathway and up-regulating GADD45G expression.

A Quasi-relativistic Density Functional Theory Study of the Actinyl(VI, V) (An = U, Np, Pu) Complexes with a Six-Membered Macrocycle Containing Pyrrole, Pyridine, and Furan Subunits.

Actinyl(VI, V) (An = U, Np and Pu) complexes of the recently reported hybrid macrocycle, cyclo[1]furan[1]pyridine[4]pyrrole (denoted as H4L), have been studied using density functional theory in combination with the small-core scalar-relativistic effective core potentials and corresponding (14s13p10d8f6g)/[ 10s9p5d4f3g] basis sets in the segmented contraction scheme. On the basis of our calculations, the pyrrole nitrogen atoms that possess the shortest An-L bonds and strongest basicity are the main donor atoms that contribute to the formation of actinyl(VI, V) complexes. The natural population analysis (NPA) suggests higher ligand-to-actinyl charge transfer in the actinyl(VI) complexes than in their actinyl(V) analogues, which account for the higher decomposition energies of the former. A significant actinide-to-ligand spin density delocalization in the uranyl(V) and neptunyl(V) complexes was observed owing to the redistribution of spin density caused by complexation. A thermodynamic analysis indicates that the formation of the actinyl(VI, V) complexes are exothermic reactions in CH2Cl2 solvent, where the uranyl cations show the highest selectivity. In aqueous solution containing chloride ions, for complexing with macrocycle H4L, the plutonyl(VI) and uranyl(V) cations possess the highest selectivity among actinyl(VI) and (V) cations, respectively. This work can shed light on the design of macrocycle complexes for actinide recognition and extraction in the future.

Rh(II)/Brønsted acid cocatalyzed intramolecular trapping of ammonium ylides with enones: diastereoselective synthesis of 2,2,3-trisubstituted indolines.

Highly diastereoselective intramolecular trapping of ammonium ylides with enones has been developed through a Rh(II)/Brønsted acid cocatalytic strategy. This process allows rapid and efficient construction of N-unprotected polyfunctional 2,2,3-trisubstituted indolines in moderate to good yields with excellent diastereoselectivity.

Alleviating binary toxicity of polystyrene nanoplastics and atrazine to Chlorella vulgaris through humic acid interaction: Long-term toxicity using environmentally relevant concentrations.

In this study, microalgae Chlorella vulgaris (C. vulgaris) were simultaneously exposed to environmental concentrations of amino-functionalized polystyrene nanoplastics (PS-NH2; 0.05, 0.1, 0.2, 0.3 and 0.4 mg/L) and the world's second most used pesticide, the herbicide atrazine (ATZ; 10 µg/L), in the absence and presence of humic acid (HA; 1 mg/L) for 21 days. Due to the low concentrations of PS-NH2, the majority of them could not cause a significant difference in the end-points of biomass, chlorophylls a and b, total antioxidant, total protein, and superoxide dismutase and malondialdehyde compared to the control group (p > 0.05). On the other hand, by adding ATZ to the PS-NH2, all the mentioned end-point values showed a considerable difference from the control (p < 0.05). The exposure of PS-NH2+ATZ treatments to the HA could remarkably reduce their toxicity, additionally, HA was able to decrease the changes in the expression of genes related to oxidative stress (e.g., superoxide dismutase, glutathione reductase, and catalase) in the C. vulgaris in the most toxic treatment group (e.g., PS-NH2+ATZ). The synergistic toxicity of the PS-NH2+ATZ group could be due to their enhanced bioavailability for algal cells. Nevertheless, the toxicity alleviation in the PS-NH2+ATZ treatment group after the addition of HA could be due to the eco-corona formation, and changes in their zeta potential from positive to negative value, which would increase their electrostatic repulsion with the C. vulgaris cells, in such a way that HA also caused a decrease in the formation of C. vulgaris-NPs hetero-aggregates. This research underscores the complex interplay between PS-NH2, ATZ, and HA in aquatic environments and their collective impact on microalgal communities.

Hemiprotonic ph-ph+ with two targets inhibits metastatic breast cancer and concurrent candidiasis.

Concurrent infection in breast cancer patients is the direct cause of the high mortality rate of the disease. However, there is no available method to increase the survival rate until now. To address the problem, we propose one drug with two target strategy to treat the refractory disease. A small chemical, ph-ph+, was attempted to be used in the study to explore the feasibility of the approach in anticancer and antifungus at the same time. The results showed that ph-ph+ could prevent the proliferation and metastasis of breast cancer cells, and kill C. albicans simultaneously. The molecular mechanism was associated with the activation of an evolutionarily conserved protease CLpP in the cancer and C. albicans cells. Also, the signaling pathway mediated by PLAGL2 that highly expressed in cancer cells participated in preventing cell metastasis and inducing apoptosis of ph-ph+. The one drug with dual targets inhibited the growth and metastasis of the cancer cells, and meanwhile eliminated C. albicans in tissues in the experimental animals. The results suggested that ph-ph+ with dual targets of CLpP and PLAGL2 would be a feasible approach to prolong the survival rate in patients with metastatic breast cancer and pathogenic infection.

Presence of humic acid in the environment holds promise as a potential mitigating factor for the joint toxicity of polystyrene nanoplastics and herbicide atrazine to Chlorella vulgaris: 96-H acute toxicity.

Increasing amounts of amino-functionalized polystyrene nanoplastics (PS-NH2) are entering aquatic ecosystems, raising concerns. Hence, this study investigated 96-h acute toxicity of PS-NH2 and its combination with the pesticide atrazine (ATZ) in the absence/presence of humic acid (HA) on the microalgae Chlorella vulgaris (C. vulgaris). Results showed that both PS-NH2 and PS-NH2+ATZ reduced algal growth, photosynthetic pigments, protein content, and antioxidant capacity, while increasing enzymatic activities. Gene expression related to oxidative stress was altered in C. vulgaris exposed to these treatments. Morphological and intracellular changes were also observed. The combined toxicity of PS-NH2+ATZ demonstrated a synergistic effect, but the addition of environmentally relevant concentration of HA significantly alleviated its toxicity to C. vulgaris, indicating an antagonistic effect due to the emergence of an eco-corona, and entrapment and sedimentation of PS-NH2+ATZ particles by HA. This study firstly highlights the role of HA in mitigating the toxicity of PS-NH2 when combined with other harmful compounds, enhancing our understanding of HA's presence in the environment.

Treatment of colorectal cancer by anticancer and antibacterial effects of hemiprotonic phenanthroline-phenanthroline+ with nanomicelle delivery.

Colorectal cancer (CRC) is a common digestive tract tumor worldwide. Specific microorganisms, including Fusobacterium nucleatum (F. nucleatum) and Escherichia coli (E. coli), are abundant in colonic mucosa and can promote the cancer progression and malignancy. Therefore, a therapeutic strategy is proposed to deliver effective drugs to colorectum for both anticancer and antibacteria. Here we used thin-film dispersion method to encapsulate hemiprotonic phenanthroline-phenanthroline+ (ph-ph+) into nanomicelle. The results showed that the drug-loading nanomicelle had good dispersion, and the particle size was about 28 nm. In vitro assay indicated that the nanomicelle was active against CRC-related obligate and facultative anaerobes. In human CRC cells, the nanomicelle could effectively inhibit cell proliferation and induce apoptosis. In vivo distribution showed that the nanomicelle could release ph-ph+ mainly in the colorectum. In CRC model mice, the nanomicelle significantly reduced tumor number and volume, and decreased the bacteria load and colorectal inflammation. Together, the study identifies that the ph-ph+nanomicelle has the potential to apply in treating CRC, and also suggests that anticancer combined with antimicrobial therapy would be a feasible way for CRC therapy.

NTF4 plays a dual role in breast cancer in mammary tumorigenesis and metastatic progression.

Breast cancer metastasis can happen even when the primary tumor is relatively small. But the mechanism for such early metastasis is poorly understood. Herein, we report that neurotrophin 4 (NTF4) plays a dual role in breast cancer proliferation and metastasis. Clinical data showed high levels of NTF4, especially in the early stage, to be associated with poor clinical outcomes, supporting the notion that metastasis, rather than primary cancer, was the major determinant of breast cancer mortality for patients. NTF4 promoted epithelial-mesenchymal transition (EMT), cell motility, and invasiveness of breast cancer cells in vitro and in vivo. Interestingly, NTF4 inhibited cell proliferation while promoting cellular apoptosis in vitro and inhibited xenograft tumorigenicity in vivo. Mechanistically, NTF4 elicited its pro-metastatic effects by activating PRKDC/AKT and ANXA1/NF-κB pathways to stabilize SNAIL protein, therefore decreasing the level of E-cadherin. Conversely, NTF4 increased ANXA1 phosphorylation and sumoylation and the interaction with importin ß, leading to nuclear import and retention of ANXA1, which in turn activates the caspase-3 apoptosis cascade. Our findings identified an unexpected dual role for NTF4 in breast cancer which contributes to early metastasis of the disease. Therefore, NTF4 may serve as a prognostic marker and a potential therapeutic target for breast cancer.

Corrigendum to "Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)" [Genes & Diseases 5 (2018) 137-149].

[This corrects the article DOI: 10.1016/j.gendis.2018.04.003.].

Corrigendum to "The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs" [Genes & Diseases 5 (2018) 62-74].

[This corrects the article DOI: 10.1016/j.gendis.2018.02.001.].

Corrigendum to "Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference" [Genes & Diseases 5(2018):172-184].

[This corrects the article DOI: 10.1016/j.gendis.2018.04.006.].

Thermodynamic study on the complexation of Am(III) and Eu(III) with tetradentate nitrogen ligands: a probe of complex species and reactions in aqueous solution.

A thermodynamic investigation has been performed to study the complexation of trivalent metal (M) ions (M = Am(III), Eu(III)) with tetradentate ligands (L), 6,6'-bis(5,6-dialkyl-1,2,4-triazin-3-yl)-2,2'-bipyridines (BTBPs), by using relativistic quantum mechanical calculations. The structures and stabilities of the inner-sphere BTBPs complexes were explored in the presence of various counterions such as NO(3)(-), Cl(-), and ClO(4)(-). According to our calculations, Am(III) and Eu(III) can chelate eight or nine water molecules at most, whereas more stable species like M(NO(3))(3)(H(2)O)(4) tend to be formed in the presence of nitrate ions. The inner sphere of the BTBPs complexes can accommodate four water molecules or three nitrate ions based on our calculations, forming species such as [ML(H(2)O)(4)](3+) and ML(NO(3))(3). Compared with Eu(III) complexes, the Am(III) counterparts have obviously lower binding energies in both the gas phase and solution. In addition, the solvent effect significantly decreases the binding energies of the BTBPs complexes. It has been found that the complexing reactions, in which products and reactants possess the same or close number of nitrate ions, are more favorable for formation of the BTBPs complexes. In short, the reactions of M(NO(3))(3)(H(2)O)(4) → ML(NO(3))(3) and [M(NO(3))(H(2)O)(7)](2+) → [ML(2)(NO(3))](2+) are probably the dominant ones in the Am(III)/Eu(III) separation process.

Long non-coding RNA (LncRNA) HOTAIR regulates BMP9-induced osteogenic differentiation by targeting the proliferation of mesenchymal stem cells (MSCs).

Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.