Many microRNAs (miRNAs) are downregulated in proliferative vascular disease. Thus, upregulation of these miRNAs has become a major focus of research activity. However, there is a critical barrier in gene therapy to upregulate some miRNAs such as miR-145 and miR-143 because of their significant downregulation by the unclear endogenous mechanisms under disease conditions. The purpose of this study was to determine the molecular mechanisms responsible for their downregulation and to overcome the therapeutic barrier.

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR-382 was down-regulated in alcohol-treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo, we identified that the dopamine receptor D1 (Drd1) is a direct target gene of miR-382. Via this target gene, miR-382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR-382 significantly attenuated alcohol-induced up-regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1-induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism.

Reactive oxygen species (ROS)-induced cardiac cell injury via expression changes of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate about 30% of the genes in a cell via degradation or translational inhibition of their target mRNAs. Currently, the effects of ROS on miRNA expression and the roles of miRNAs in ROS-mediated injury on cardiac myocytes are uncertain. Using quantitative real-time RT-PCR (qRT-PCR), we demonstrated that microRNA-21 (miR-21) was upregulated in cardiac myocytes after treatment with hydrogen peroxide (H(2)O(2)). To determine the potential roles of miRNAs in H(2)O(2)-mediated gene regulation and cellular injury, miR-21 expression was downregulated by miR-21 inhibitor and upregulated by pre-miR-21. H(2)O(2)-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. Programmed cell death 4 (PDCD4) that was regulated by miR-21 and was a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H(2)O(2)-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, Activator protein 1 (AP-1) was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H(2)O(2) stimulation. miR-21 participates in H(2)O(2)-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to ROS such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.

Smooth muscle 22 alpha (SM22 alpha) is not required for the development and basal homeostatic function of smooth muscle cells (SMCs). However, a recent study demonstrated that SM22 alpha plays a role in inhibiting the phenotypic modulation of vascular SMCs (VSMCs) from contractile to synthetic/proliferative cells. The present study investigated the mechanism underlying the SM22 alpha-mediated maintenance of the contractile phenotype of VSMCs.

Lysophosphatidic acid (LPA) has been implicated as causative in phenotypic modulation (PM) of cultured vascular smooth muscle cells (VSMC) in their transition to the dedifferentiated phenotype. We evaluated the contribution of the three major LPA receptors, LPA1 and LPA2 GPCR and PPARgamma, on PM of VSMC. Expression of differentiated VSMC-specific marker genes, including smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM-22alpha, and h-caldesmon, was measured by quantitative real-time PCR in VSMC cultures and aortic rings kept in serum-free chemically defined medium or serum- or LPA-containing medium using wild-type C57BL/6, LPA1, LPA2, and LPA1&2 receptor knockout mice. Within hours after cells were deprived of physiological cues, the expression of VSMC marker genes, regardless of genotype, rapidly decreased. This early PM was neither prevented by IGF-I, inhibitors of p38, ERK1/2, or PPARgamma nor significantly accelerated by LPA or serum. To elucidate the mechanism of PM in vivo, carotid artery ligation with/without replacement of blood with Krebs solution was used to evaluate contributions of blood flow and pressure. Early PM in the common carotid was induced by depressurization regardless of the presence/absence of blood, but eliminating blood flow while maintaining blood pressure or after sham surgery elicited no early PM. The present results indicate that LPA, serum, dissociation of VSMC, IGF-I, p38, ERK1/2, LPA1, and LPA2 are not causative factors of early PM of VSMC. Tensile stress generated by blood pressure may be the fundamental signal maintaining the fully differentiated phenotype of VSMC.

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate gene expression. Although miRNAs are highly expressed in the heart, their roles in heart diseases are currently unclear. Using microarray analysis designed to detect the majority of mammalian miRNAs identified thus far, we demonstrated that miRNAs are aberrantly expressed in hypertrophic mouse hearts. The time course of the aberrant miRNA expression was further identified in mouse hearts at 7, 14, and 21 days after aortic banding. Nineteen of the most significantly dysregulated miRNAs were further confirmed by Northern blot and/or real-time polymerase chain reaction, in which miR-21 was striking because of its more than fourfold increase when compared with the sham surgical group. Similar aberrant expression of the most up-regulated miRNA, miR-21, was also found in cultured neonatal hypertrophic cardiomyocytes stimulated by angiotensin II or phenylephrine. Modulating miR-21 expression via antisense-mediated depletion (knockdown) had a significant negative effect on cardiomyocyte hypertrophy. The results suggest that miRNAs are involved in cardiac hypertrophy formation. miRNAs might be a new therapeutic target for cardiovascular diseases involving cardiac hypertrophy such as hypertension, ischemic heart disease, valvular diseases, and endocrine disorders.

Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g., KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)-a component of the RNA-induced silencing complex (RISC)-physically interacts with RAS and promotes its downstream signaling. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC (LSL-KrasG12D/+;p53f/f;Cre) mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53 Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the Clara cell-specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation (pERK) in tumor cells, indicating impaired KRAS signaling. RNA sequencing (RNA-seq) of KPC nodules and nodule-derived organoids demonstrated impaired canonical KRAS signaling with Ago2 ablation. Strikingly, accumulation of pERK in KPC organoids depended on physical interaction of AGO2 and KRAS. Taken together, our data demonstrate a pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work may have future translational relevance.

Selective degradation of the cyclin-dependent kinases 12 and 13 (CDK12/13) presents a novel therapeutic opportunity for triple-negative breast cancer (TNBC), but there is still a lack of dual CDK12/13 degraders. Here, we report the discovery of the first series of highly potent and selective dual CDK12/13 degraders by employing the proteolysis-targeting chimera (PROTAC) technology. The optimal compound 7f effectively degraded CDK12 and CDK13 with DC50 values of 2.2 and 2.1 nM, respectively, in MDA-MB-231 breast cancer cells. Global proteomic profiling demonstrated the target selectivity of 7f. In vitro, 7f suppressed expression of core DNA damage response (DDR) genes in a time- and dose-dependent manner. Further, 7f markedly inhibited proliferation of multiple TNBC cell lines including MFM223, with an IC50 value of 47 nM. Importantly, 7f displayed a significantly improved antiproliferative activity compared to the structurally similar inhibitor 4, suggesting the potential advantage of a CDK12/13 degrader for TNBC targeted therapy.

MicroRNAs (miRNAs) are noncoding RNAs that impact almost every aspect of biology and disease. Until now, the cell-specific effects of miRNAs in cardiovascular system have not been established. In the current study, the cellular functions of miR-221 and miR-222 (miR-221/222) in vascular smooth muscle cells (VSMCs) and vascular endothelial cells (ECs) were compared. In cultured cells, we identified that the effects of miR-221/222 on proliferation, migration, and apoptosis are opposite between VSMCs and ECs. In VSMCs, miR-221/222 had effects of pro-proliferation, pro-migration, and anti-apoptosis. In contrast, miR-221/222 had effects of anti-proliferation, anti-migration, and pro-apoptosis in ECs. The different expression profiles of their target genes, p27(Kip1), p57(kip2), and c-kit between the two cell types might be related to the opposite effects. Finally, the opposite cellular effects of miR-221/222 were verified in vivo in balloon-injured rat carotid artery as demonstrated by different consequences in neointimal growth and re-endothelialization. The results suggest that the biological functions of miR-221/222 in vascular walls are cell-specific. The opposite cellular effects of miR-221/222 on VSMCs and ECs may have important therapeutic applications in many vascular diseases such as atherosclerosis and restenosis after angioplasty.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.