Super enhancer-associated circRNA-circLrch3 regulates hypoxia-induced pulmonary arterial smooth muscle cells pyroptosis by formation of R-loop with host gene.

BACKGROUND: Pulmonary hypertension (PH) is a complex vascular disorder, characterized by pulmonary vessel remodeling and perivascular inflammation. Pulmonary arterial smooth muscle cells (PASMCs) pyroptosis is a novel pathological mechanism implicated of pulmonary vessel remodeling. However, the involvement of circRNAs in the process of pyroptosis and the underlying regulatory mechanisms remain inadequately understood. METHODS: Western blotting, PI staining and LDH release were used to explore the role of circLrch3 in PASMCs pyroptosis. Moreover, S9.6 dot blot and DRIP-PCR were used to assess the formation of R-loop between circLrch3 and its host gene Lrch3. Chip-qPCR were used to evaluate the mechanism of super enhancer-associated circLrh3, which is transcriptionally activated by the transcription factor Tbx2. RESULTS: CircLrch3 was markedly upregulated in hypoxic PASMCs. CircLrch3 knockdown inhibited hypoxia induced PASMCs pyroptosis in vivo and in vitro. Mechanistically, circLrch3 can form R-loop with host gene to upregulate the protein and mRNA expression of Lrch3. Furthermore, super enhancer interacted with the Tbx2 at the Lrch3 promoter locus, mediating the augmented transcription of circLrch3. CONCLUSION: Our findings clarify the role of a super enhancer-associated circLrch3 in the formation of R-loop with the host gene Lrch3 to modulate pyroptosis in PASMCs, ultimately promoting the development of PH.

Presenilin 1 Is a Therapeutic Target in Pulmonary Hypertension and Promotes Vascular Remodeling.

Small muscular pulmonary artery remodeling is a dominant feature of pulmonary arterial hypertension (PAH). PSEN1 affects angiogenesis, cancer, and Alzheimer's disease. We aimed to determine the role of PSEN1 in the pathogenesis of vascular remodeling in pulmonary hypertension (PH). Hemodynamics and vascular remodeling in the Psen1-knockin and smooth muscle-specific Psen1-knockout mice were assessed. The functional partners of PSEN1 were predicted by bioinformatics analysis and biochemical experiments. The therapeutic effect of PH was evaluated by administration of the PSEN1-specific inhibitor ELN318463. We discovered that both the mRNA and protein levels of PSEN1 were increased over time in hypoxic rats, monocrotaline rats, and Su5416/hypoxia mice. Psen1 transgenic mice were highly susceptible to PH, whereas smooth muscle-specific Psen1-knockout mice were resistant to hypoxic PH. STRING analysis showed that Notch1/2/3, ß-catenin, Cadherin-1, DNER (delta/notch-like epidermal growth factor-related receptor), TMP10, and ERBB4 appeared to be highly correlated with PSEN1. Immunoprecipitation confirmed that PSEN1 interacts with ß-catenin and DNER, and these interactions were suppressed by the catalytic PSEN1 mutations D257A, D385A, and C410Y. PSEN1 was found to mediate the nuclear translocation of the Notch1 intracellular domains and activated RBP-Jκ. Octaarginine-coated liposome-mediated pharmacological inhibition of PSEN1 significantly prevented and reversed the pathological process in hypoxic and monocrotaline-induced PH. PSEN1 essentially drives the pathogenesis of PAH and interacted with the noncanonical Notch ligand DNER. PSEN1 can be used as a promising molecular target for treating PAH. PSEN1 inhibitor ELN318463 can prevent and reverse the progression of PH and can be developed as a potential anti-PAH drug.

Orthogroup and phylotranscriptomic analyses identify transcription factors involved in the plant cold response: A case study of Arabidopsis BBX29.

C-repeat binding factors (CBFs) are well-known transcription factors (TFs) that regulate plant cold acclimation. RNA sequencing (RNA-seq) data from diverse plant species provide opportunities to identify other TFs involved in the cold response. However, this task is challenging because gene gain and loss has led to an intertwined community of co-orthologs and in-paralogs between and within species. Using orthogroup (closely related homologs) analysis, we identified 10,549 orthogroups in five representative eudicots. A phylotranscriptomic analysis of cold-treated seedlings from eudicots identified 35 high-confidence conserved cold-responsive transcription factor orthogroups (CoCoFos). These 35 CoCoFos included the well-known cold-responsive regulators CBFs, HSFC1, ZAT6/10, and CZF1 among others. We used Arabidopsis BBX29 for experimental validation. Expression and genetic analyses showed that cold-induction of BBX29 is CBF- and abscisic acid-independent, and BBX29 is a negative regulator of cold tolerance. Integrative RNA-seq and Cleavage Under Targets and Tagmentation followed by sequencing analyses revealed that BBX29 represses a set of cold-induced TFs (ZAT12, PRR9, RVE1, MYB96, etc.). Altogether, our analysis yielded a library of eudicot CoCoFos and demonstrated that BBX29 is a negative regulator of cold tolerance in Arabidopsis.

A phylotranscriptomic dataset of angiosperm species under cold stress.

Angiosperms are one of the most diverse and abundant plant groups that are widely distributed on Earth, from tropical to temperate and polar zones. The wide distribution of angiosperms may be attributed to the evolution of sophisticated mechanisms of environmental adaptability, including cold tolerance. Since the development of high-throughput sequencing, transcriptome has been widely utilized to gain insights into the molecular mechanisms of plants in response to cold stress. However, previous studies generally focused on single or two species, and comparative transcriptome analyses for multispecies responding to cold stress were limited. In this study, we selected 11 representative angiosperm species, performed phylotranscriptome experiments at four time points before and after cold stress, and presented a profile of cold-induced transcriptome changes in angiosperms. Our multispecies cold-responsive RNA-seq datasets provide valuable references for exploring conserved and evolutionary mechanisms of angiosperms in adaptation to cold stress.

Transcriptome analysis provides StMYBA1 gene that regulates potato anthocyanin biosynthesis by activating structural genes.

Anthocyanin biosynthesis is affected by light, temperature, and other environmental factors. The regulation mode of light on anthocyanin synthesis in apple, pear, tomato and other species has been reported, while not clear in potato. In this study, potato RM-210 tubers whose peel will turn purple gradually after exposure to light were selected. Transcriptome analysis was performed on RM-210 tubers during anthocyanin accumulation. The expression of StMYBA1 gene continued to increase during the anthocyanin accumulation in RM-210 tubers. Moreover, co-expression cluster analysis of differentially expressed genes showed that the expression patterns of StMYBA1 gene were highly correlated with structural genes CHS and CHI. The promoter activity of StMYBA1 was significantly higher in light conditions, and StMYBA1 could activate the promoter activity of structural genes StCHS, StCHI, and StF3H. Further gene function analysis found that overexpression of StMYBA1 gene could promote anthocyanin accumulation and structural gene expression in potato leaves. These results demonstrated that StMYBA1 gene promoted potato anthocyanin biosynthesis by activating the expression of structural genes under light conditions. These findings provide a theoretical basis and genetic resources for the regulatory mechanism of potato anthocyanin synthesis.

QTL analysis of tuber shape in a diploid potato population.

Tuber shape is one of the most important traits for potato breeding. Since poor or irregular shape increases the difficulty of handling and processing, researching the inheritance of potato tuber shape for potato breeding is highly important. To efficiently identify QTL for tuber shape, a diploid potato population (PM7) was generated by self-pollinated M6 (S. chacoense). A QTL TScha6 for tuber shape was identified by the QTL-seq approach at 50.91-59.93 Mb on chromosome 6 in the potato DM reference genome. To confirm TScha6, four SSR and twenty CAPS markers around the QTL were developed and the TScha6 was narrowed down to an interval of ~ 1.85 Mb. The CAPS marker C6-58.27_665 linked to TScha6 was then used to screen 86 potato cultivars and advanced breeding lines. The tuber length/width (LW) ratio was significantly correlated with the presence/absence of C6-58.27_665, and the correlation coefficient was r = 0.55 (p < 0.01). These results showed that C6-58.27_665 could be applied in marker-assisted selection (MAS) for tuber shape breeding in the future. Our research sets the important stage for the future cloning of the tuber shape gene and utilities of the marker in the breeding program.

Methylation level of potato gene OMT30376 regulates tuber anthocyanin transformations.

After anthocyanin synthesis, a variety of anthocyanin compounds are produced through further methylation, glycosylation, and acylation. However, the effect of the potato methylase gene on anthocyanin biosynthesis has not been reported. Red and purple mutation types appear in tubers of the potato cultivar 'Purple Viking' with chimeric skin phenotypes. In this study, transcriptome and anthocyanin metabolome analyses were performed on skin of Purple Viking tubers and associated mutants. According to the metabolome analysis, the transformation of delphinidin into malvidin-3-O-glucoside and petunidin 3-O-glucoside and that of cyanidin into rosinidin O-hexoside and peonidin-3-O-glucoside were hindered in red tubers. Expression of methyltransferase gene OMT30376 was significantly lower in red tubers than in purple ones, whereas the methylation level of OMT30376 was significantly higher in red tubers. In addition, red skin appeared in tubers from purple tuber plants treated with S-adenosylmethionine (SAM), indicating the difference between purple and red was caused by the methylation degree of the gene OMT30376. Thus, the results of the study suggest that the OMT30376 gene is involved in the transformation of anthocyanins in potato tubers. The results also provide an important reference to reveal the regulatory mechanisms of anthocyanin biosynthesis and transformation.

RPS4XL encoded by lnc-Rps4l inhibits hypoxia-induced pyroptosis by binding HSC70 glycosylation site.

Pyroptosis is involved in pulmonary hypertension (PH); however, whether this process is regulated by long non-coding RNAs (lncRNAs) is unclear. Some lncRNAs encode peptides; therefore, whether the regulation of pyroptosis in PH depends on lncRNAs themselves or their encoded peptides needs to be explored. We aimed to characterize the role of the peptide RPS4XL encoded by lnc-Rps4l and its regulatory mechanisms during pyroptosis in PH. Transgenic mice overexpression of lnc-Rps4l was established to rescue the inhibition of hypoxia-induced pyroptosis in pulmonary artery smooth muscle cells (PASMCs). An adeno-associated virus 9 construct with a mutation in the open reading frame of lnc-Rps4l was used to verify that it could inhibit hypoxia-induced PASMCs pyroptosis through its encoded peptide RPS4XL. Glutathione S-transferase (GST) pull-down assays revealed that RPS4XL bound to HSC70, and microscale thermophoresis (MST) was performed to determine the HSC70 domain that interacted with RPS4XL. Through glycosylation site mutation, we confirmed that RPS4XL inhibited hypoxia-induced PASMCs pyroptosis by regulating HSC70 glycosylation. Our results showed that RPS4XL inhibits pyroptosis in a PH mouse model and hypoxic PASMCs by regulating HSC70 glycosylation. These results further clarify the important mechanism of vascular remodeling in PH pathology.

Innovation and Emerging Roles of Populus trichocarpa TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR Transcription Factors in Abiotic Stresses by Whole-Genome Duplication.

The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family proteins are plant-specific transcription factors that have been well-acknowledged for designing the architectures of plant branch, shoot, and inflorescence. However, evidence for their innovation and emerging role in abiotic stress has been lacking. In this study, we identified a total of 36 TCP genes in Populus trichocarpa, 50% more than that in Arabidopsis (i.e., 24). Comparative intra-genomes showed that such significant innovation was mainly due to the most recent whole genome duplication (rWGD) in Populus lineage around Cretaceous-Paleogene (K-Pg) boundary after the divergence from Arabidopsis. Transcriptome analysis showed that the expressions of PtrTCP genes varied among leaf, stem, and root, and they could also be elaborately regulated by abiotic stresses (e.g., cold and salt). Moreover, co-expression network identified a cold-associated regulatory module including PtrTCP31, PtrTCP10, and PtrTCP36. Of them, PtrTCP10 was rWGD-duplicated from PtrTCP31 and evolved a strong capability of cold induction, which might suggest a neofunctionalization of PtrTCP genes and contribute to the adaptation of Populus lineage during the Cenozoic global cooling. Evidentially, overexpression of PtrTCP10 into Arabidopsis increased freezing tolerance and salt susceptibility. Integrating co-expression network and cis-regulatory element analysis confirmed that PtrTCP10 can regulate the well-known cold- and salt-relevant genes (e.g., ZAT10, GolS2, and SOS1), proving that PtrTCP10 is an evolutionary innovation in P. trichocarpa response to environmental changes. Altogether, our results provide evidence of the rWGD in P. trichocarpa responsible for the innovation of PtrTCP genes and their emerging roles in environmental stresses.

Functional Characterization of a Flavone Synthase That Participates in a Kumquat Flavone Metabolon.

Flavones predominantly accumulate as O- and C-glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for C-glycosylation) and another involving the direct catalysis of flavanones to flavones for O-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O-glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein-protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an O-glycosyl flavone metabolon that channels flavanones for O-glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits.

Role of lncRNAs in cis- and trans-regulatory responses to salt in Populus trichocarpa.

Long non-coding RNAs (lncRNAs) are emerging as versatile regulators in diverse biological processes. However, little is known about their cis- and trans-regulatory contributions in gene expression under salt stress. Using 27 RNA-seq data sets from Populus trichocarpa leaves, stems and roots, we identified 2988 high-confidence lncRNAs, including 1183 salt-induced differentially expressed lncRNAs. Among them, 301 lncRNAs have potential for positively affecting their neighboring genes, predominantly in a cis-regulatory manner rather than by co-transcription. Additionally, a co-expression network identified six striking salt-associated modules with a total of 5639 genes, including 426 lncRNAs, and in these lncRNA sequences, the DNA/RNA binding motifs are enriched. This suggests that lncRNAs might contribute to distant gene expression of the salt-associated modules in a trans-regulatory manner. Moreover, we found 30 lncRNAs that have potential to simultaneously cis- and trans-regulate salt-responsive homologous genes, and Ptlinc-NAC72, significantly induced under long-term salt stress, was selected for validating its regulation of the expression and functional roles of the homologs PtNAC72.A and PtNAC72.B (PtNAC72.A/B). The transient transformation of Ptlinc-NAC72 and a dual-luciferase assay of Ptlinc-NAC72 and PtNAC72.A/B promoters confirmed that Ptlinc-NAC72 can directly upregulate PtNAC72.A/B expression, and a presence/absence assay was further conducted to show that the regulation is probably mediated by Ptlinc-NAC72 recognizing the tandem elements (GAAAAA) in the PtNAC72.A/B 5' untranslated region (5'-UTR). Finally, the overexpression of Ptlinc-NAC72 produces a hypersensitive phenotype under salt stress. Altogether, our results shed light on the cis- and trans-regulation of gene expression by lncRNAs in Populus and provides an example of long-term salt-induced Ptlinc-NAC72 that could be used to mitigate growth costs by conferring plant resilience to salt stress.

The SR Splicing Factors: Providing Perspectives on Their Evolution, Expression, Alternative Splicing, and Function in Populus trichocarpa.

Serine/arginine-rich (SR) proteins are important splicing factors in plant development and abiotic/hormone-related stresses. However, evidence that SR proteins contribute to the process in woody plants has been lacking. Using phylogenetics, gene synteny, transgenic experiments, and RNA-seq analysis, we identified 24 PtSR genes and explored their evolution, expression, and function in Popolus trichocarpa. The PtSR genes were divided into six subfamilies, generated by at least two events of genome triplication and duplication. Notably, they were constitutively expressed in roots, stems, and leaves, demonstrating their fundamental role in P. trichocarpa. Additionally, most PtSR genes (~83%) responded to at least one stress (cold, drought, salt, SA, MeJA, or ABA), and, especially, cold stress induced a dramatic perturbation in the expression and/or alternative splicing (AS) of 18 PtSR genes (~75%). Evidentially, the overexpression of PtSCL30 in Arabidopsis decreased freezing tolerance, which probably resulted from AS changes of the genes (e.g., ICE2 and COR15A) critical for cold tolerance. Moreover, the transgenic plants were salt-hypersensitive at the germination stage. These indicate that PtSCL30 may act as a negative regulator under cold and salt stress. Altogether, this study sheds light on the evolution, expression, and AS of PtSR genes, and the functional mechanisms of PtSCL30 in woody plants.

StWRKY13 promotes anthocyanin biosynthesis in potato (Solanum tuberosum) tubers.

Although the role of WRKY transcription factors (TFs) in colour formation has been reported in several species, their function in potato (Solanum tuberosum L.) anthocyanin biosynthesis remains unclear. In this study, the potato WRKY gene StWRKY13 was isolated and characterised. Expression analysis revealed a significantly higher StWRKY13 expression in chromatic tubers than in yellow ones. Transient activation assays showed that StWRKY13 could enhance the role of StAN2 in promoting anthocyanin biosynthesis in tobacco (Nicotiana tabacum L.). Over-expressing the StWRKY13 gene promoted anthocyanin biosynthesis in potato tubers. Further investigations indicated that StWRKY13 could interact with the StCHS, StF3H, StDFR, and StANS gene promoters and significantly enhance their activities. Our findings showed that StWRKY13 could promote anthocyanin biosynthesis by activating StCHS, StF3H, StDFR, and StANS transcription in potato tubers, thereby supporting the theoretical basis for anthocyanins formation in coloured potato tubers.

The underlying molecular conservation and diversification of dioecious flower and leaf buds provide insights into the development, dormancy breaking, flowering, and sex association of willows.

As harbingers of bursting growth, flower buds and leaf buds generally show similar surface morphologies but different structural and functional changes. Dioecious plants further generate four types of Female/Male Flower/Leaf Buds (FFB, FLB, MFB, and MLB), showing a complex regulation. However, little is known about their underlying molecular mechanisms. Here, we exemplify the woody dioecious Salix linearistipularis to investigate their morphological characteristics and potential molecular mechanisms by combining cytological, physiological, phenological, and transcriptomic datasets. First, FFB and MFB have simultaneous development dynamics and so do FLB and MLB. Interestingly, FLB and MLB show very similar expression profiles preparing for photosynthesis and stress-tolerance, whereas FFB and MFB show great similarities but also striking sexual differences. Comparing flower buds and leaf buds after their revival from dormancy shows different cold- and vernalization-responsive genes (e.g. SliVRN1, SliAGL19, and SliAGL24), implying different programming processes for dormancy breaking between the buds. Moreover, except SliAP3, the expression of ABCDE model genes is consistent with their roles in the buds, suggesting a conserved mechanism of flower development between dioecious Salix and hermaphrodite Arabidopsis. Finally, considering sex-associated genes (e.g. SliCLE25, SliTPS21, and SliARR9) on Salix chromosomes and other reports, we hypothesize a dynamic model of sex determination on chromosomes 15 and 19 in the last ancestor of Salix and Populus but evolutionarily on 15 in Salix after their divergence. Together, our study provides new insights into the molecular mechanisms of dioecious four-type buds by showing the genes involved in their development, dormancy breaking, flowering, and sexual association.

Circular RNA hsa_circ_101555 promotes hepatocellular carcinoma cell proliferation and migration by sponging miR-145-5p and regulating CDCA3 expression.

Circular RNAs have been reported to play significant roles in regulating pathophysiological processes while also guiding clinical diagnosis and treatment of hepatocellular carcinoma (HCC). However, only a few circRNAs have been identified thus far. Herein, we investigated the role of a specific closed-loop structure of hsa_circ_101555 that was generated by back-splicing of the host gene casein kinase 1 gamma 1 (CSNK1G1) in the development and proliferation of HCC. We investigated the expression of Hsa_circ_101555 in HCC and normal tissues using bioinformatics. The expression level of hsa_circ_101555 was further detected by fluorescence in situ hybridization and qRT-PCR in ten HCC patients. Transwell, migration, WST-1 assays, and colony formation assays were used to evaluate the role of hsa_circ_101555 in HCC development and proliferation. The regulatory mechanisms of hsa_circ_101555 in miR-145-5p and CDCA3 were determined by dual luciferase reporter assay. A mouse xenograft model was also used to determine the effect of hsa_circ_101555 on HCC growth in vivo. hsa_circ_101555 showed greater stability than the linear RNA; while in vitro and in vivo results demonstrated that hsa_circ_101555 silencing significantly suppressed cell proliferation, migration, and invasion of HCC cells. Rescue experiments further demonstrated that suppression of miR-145-5p significantly attenuated the biological effects of hsa_circ_101555 knockdown in HCC cells. We also identified a putative oncogene CDCA3 as a potential miR-145-5p target. Thus, our results demonstrated that hsa_circ_101555 might function as a competing endogenous RNA of miR-145-5p to upregulate CDCA3 expression in HCC. These findings suggest that hsa_circ_101555 may be a potential therapeutic target for patients with HCC.

Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis.

[Figure: see text].

lnc-Rps4l-encoded peptide RPS4XL regulates RPS6 phosphorylation and inhibits the proliferation of PASMCs caused by hypoxia.

Pulmonary artery smooth muscle cells (PASMCs) proliferation caused by hypoxia is an important pathological process of pulmonary hypertension (PH). Prevention of PASMCs proliferation can effectively reduce PH mortality. Long non-coding RNAs (lncRNAs) are involved in the proliferation process. Recent evidence has demonstrated that functional peptides encoded by lncRNAs play important roles in cell pathophysiological process. Our previous study has demonstrated that lnc-Rps4l with high coding ability mediates the PASMCs proliferation under hypoxic conditions. We hypothesize in this study that a lnc-Rps4l-encoded peptide is involved in hypoxic-induced PASMCs proliferation. The presence of peptide 40S ribosomal protein S4 X isoform-like (RPS4XL) encoded by lnc-Rps4l in PASMCs under hypoxic conditions was confirmed by bioinformatics, immunofluorescence, and immunohistochemistry. Inhibition of proliferation by the peptide RPS4XL was demonstrated in hypoxic PASMCs by MTT, bromodeoxyuridine (BrdU) incorporation, and immunofluorescence assays. By using the bioinformatics, coimmunoprecipitation (coIP), and mass spectrometry, RPS6 was identified to interact with RPS4XL. Furthermore, lnc-Rps4l-encoded peptide RPS4XL inhibited the RPS6 process via binding to RPS6 and inhibiting RPS6 phosphorylation at p-RPS6 (Ser240+Ser244) phosphorylation site. These results systematically elucidate the role and regulatory network of Rps4l-encoded peptide RPS4XL in PASMCs proliferation. These discoveries provide potential targets for early diagnosis and a leading compound for treatment of hypoxic PH.

circRNA CDR1as Promotes Pulmonary Artery Smooth Muscle Cell Calcification by Upregulating CAMK2D and CNN3 via Sponging miR-7-5p.

Emerging evidence has suggested that circular RNAs (circRNAs) are involved in multiple physiological processes and participate in a variety of human diseases. However, the underlying biological function of circRNAs in pulmonary hypertension (PH) is still ambiguous. Herein, we investigated the implication and regulatory effect of a typical circRNA, CDR1as, in the pathological process of vascular calcification in PH. Human pulmonary artery smooth muscle cell (HPASMC) calcification was analyzed by western blotting, immunofluorescence, alizarin red S staining, alkaline phosphatase activity analysis, and calcium deposition quantification. CDR1as targets were identified by bioinformatics analysis and validated by dual-luciferase reporter and RNA antisense purification assays. We identified that CDR1as was upregulated in hypoxic conditions and promoted a phenotypic switch of HPASMCs from a contractile to an osteogenic phenotype. Moreover, microRNA (miR)-7-5p was shown to be a target of CDR1as, and calcium/calmodulin-dependent kinase II-delta (CAMK2D) and calponin 3 (CNN3) were suggested to be the putative target genes and regulated by CDR1as/miR-7-5p. The results showed that the CDR1as/miR-7-5p/CNN3 and CAMK2D regulatory axis mediates HPASMC osteoblastic differentiation and calcification induced by hypoxia. This evidence reveals an approach to the treatment of PH.