The transcription factor GATA6 accelerates vascular smooth muscle cell senescence-related arterial calcification by counteracting the role of anti-aging factor SIRT6 and impeding DNA damage repair.

Arterial calcification is a hallmark of vascular pathology in the elderly and in individuals with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs), after attaining a senescent phenotype, are implicated in the calcifying process. However, the underlying mechanism remains to be elucidated. Here, we reveal an aberrant upregulation of transcriptional factor GATA6 in the calcified aortas of humans, mice with CKD and mice subjected to vitamin D3 injection. Knockdown of GATA6, via recombinant adeno-associated virus carrying GATA6 shRNA, inhibited the development of arterial calcification in mice with CKD. Further gain- and loss-of function experiments in vitro verified the contribution of GATA6 in osteogenic differentiation of VSMCs. Samples of human aorta exhibited a positive relationship between age and GATA6 expression and GATA6 was also elevated in the aortas of old as compared to young mice. Calcified aortas displayed senescent features with VSMCs undergoing premature senescence, blunted by GATA6 downregulation. Notably, abnormal induction of GATA6 in senescent and calcified aortas was rescued in Sirtuin 6 (SIRT6)-transgenic mice, a well-established longevity mouse model. Suppression of GATA6 accounted for the favorable effect of SIRT6 on VSMCs senescence prevention. Mechanistically, SIRT6 inhibited the transcription of GATA6 by deacetylation and increased degradation of transcription factor Nkx2.5. Moreover, GATA6 was induced by DNA damage stress during arterial calcification and subsequently impeded the Ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair process, leading to accelerated VSMCs senescence and osteogenic differentiation. Thus, GATA6 is a novel regulator in VSMCs senescence. Our findings provide novel insight in arterial calcification and a potential new target for intervention.

C5a-C5aR1 induces endoplasmic reticulum stress to accelerate vascular calcification via PERK-eIF2α-ATF4-CREB3L1 pathway.

AIMS: Vascular calcification (VC) predicts the morbidity and mortality in cardiovascular diseases. Vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation is the crucial pathological basis for VC. To date, the molecular pathogenesis is still largely unclear. Notably, C5a-C5aR1 contributes to the development of cardiovascular diseases, and its closely related to physiological bone mineralization which is similar to VSMCs osteogenic transdifferentiation. However, the role and underlying mechanisms of C5a-C5aR1 in VC remain unexplored. METHODS AND RESULTS: A cross-sectional clinical study was utilized to examine the association between C5a and VC. Chronic kidney diseases mice and calcifying VSMCs models were established to investigate the effect of C5a-C5aR1 in VC, evaluated by changes in calcium deposition and osteogenic markers. The cross-sectional study identified that high level of C5a was associated with increased risk of VC. C5a dose-responsively accelerated VSMCs osteogenic transdifferentiation accompanying with increased the expression of C5aR1. Meanwhile, the antagonists of C5aR1, PMX 53, reduced calcium deposition, and osteogenic transdifferentiation both in vivo and in vitro. Mechanistically, C5a-C5aR1 induced endoplasmic reticulum (ER) stress and then activated PERK-eIF2α-ATF4 pathway to accelerated VSMCs osteogenic transdifferentiation. In addition, cAMP-response element-binding protein 3-like 1 (CREB3L1) was a key downstream mediator of PERK-eIF2α-ATF4 pathway which accelerated VSMCs osteogenic transdifferentiation by promoting the expression of COL1α1. CONCLUSIONS: High level of C5a was associated with increased risk of VC, and it accelerated VC by activating the receptor C5aR1. PERK-eIF2α-ATF4-CREB3L1 pathway of ER stress was activated by C5a-C5aR1, hence promoting VSMCs osteogenic transdifferentiation. Targeting C5 or C5aR1 may be an appealing therapeutic target for VC.

UBQLN1 deficiency mediates telomere shortening and IPF through interacting with RPA1.

Premature telomere shortening is a known factor correlated to idiopathic pulmonary fibrosis (IPF) occurrence, which is a chronic, progressive, age-related disease with high mortality. The etiology of IPF is still unknown. Here, we found that UBQLN1 plays a key role in telomere length maintenance and is potentially relevant to IPF. UBQLN1 involves in DNA replication by interacting with RPA1 and shuttling it off from the replication fork. The deficiency of UBQLN1 retains RPA1 at replication fork, hinders replication and thus causes cell cycle arrest and genome instability. Especially at telomere regions of the genome, where more endogenous replication stress exists because of G rich sequences, UBQLN1 depletion leads to rapid telomere shortening in HeLa cells. It revealed that UBQLN1 depletion also shortens telomere length at mouse lung and accelerates mouse lung fibrosis. In addition, the UBQLN1 expression level in IPF patients is downregulated and correlated to poor prognosis. Altogether, these results uncover a new role of UBQLN1 in ensuring DNA replication and maintaining telomere stability, which may shed light on IPF pathogenesis and prevention.

TERC suppresses PD-L1 expression by downregulating RNA binding protein HuR.

TERC is the RNA component of telomerase, and provides a template for TERT to synthesize telomere repeats at chromosome ends. Increasing evidence has revealed that TERC is involved in other biological processes beyond telomerase. Here, we found that the expression level of TERC is negatively correlated with PD-L1 and that ectopic expression of TERC but not TERT in ALT cells significantly inhibits PD-L1, suggesting that TERC suppresses PD-L1 expression in a telomerase-independent manner. Mechanistically, instead of regulating PD-L1 mRNA directly, TERC accelerates PD-L1 mRNA degradation by inhibiting the expression of HuR, which binds to the 3'UTR of PD-L1 mRNA and maintains its stability. We also found that the small molecule AS1842856, a FoxO1 inhibitor, promotes TERC expression and reverses the PD-L1 upregulation caused by chemotherapy, providing a potential combination cancer therapy that avoids cancer immune escape during chemotherapy.

Increased local miR-21 expressions are linked with clinical severity in lumbar disc herniation patients with sciatic pain.

BACKGROUND: The miR-21 has been implicated in the process of neuroinflammation as well as neuropathic pain. OBJECTIVES: To explore the relationship between the plasma and local expression of miR-21 with disease severity of lumbar disc herniation (LDH) patients with sciatic pain. MATERIAL AND METHODS: Ninety-two LDH patients with sciatic pain and 25 scoliosis patients as painless controls were enrolled in the current study. Samples from nucleus pulposus (NP), annulus fibrosus (AF) and soft tissues around nerve root (STANR) were obtained. The plasma and local expressions of miR-21 were detected with quantitative reverse transcription polymerase chain reaction (qRT-PCR). The visual analogue scale (VAS) for lumbar pain and leg pain, and Japanese Orthopedic Association (JOA) score were selected to evaluate the clinical severity. The degree of disc compression on nerve was evaluated using the Pfirrmann grade based on the magnetic resonance imaging (MRI) findings. For the convenience of analysis, LDH patients with sciatic pain were classified into a severe pain (SP) group (VAS ≥ 6) and a mild-moderate pain (MP) group (VAS < 6). Receiver operating characteristic (ROC) curve analysis was performed to detect the potential diagnostic power of miR-21 with regard to the Pfirrmann grade. RESULTS: There were no significant differences in serum miR-21 expressions among SP LDH patients, MP LDH patients and scoliosis painless controls. Local expressions of miR-21 in STANR, AF and NP were all drastically upregulated in the SP group in comparison with the MP group and scoliosis painless group. Local NP and STANR miR-21 expressions were positively associated with the Pfirrmann grade. Local miR-21 expressions in STANR and AF were positively associated with VAS score and negatively related to JOA score. The ROC curve analysis indicated that both STANR and AF miR-21 expressions may serve as significant diagnostic factors for the Pfirrmann grade. CONCLUSIONS: Increased local miR-21 expressions are linked with clinical severity of LDH in patients with sciatic pain.

LIN28/let-7/PD-L1 Pathway as a Target for Cancer Immunotherapy.

The immunocheckpoint protein PD-1/PD-L1 is considered a promising target for cancer immunotherapeutics. However, the objective response rate using antibodies that block the interaction between PD-1 and PD-L1 was less than 40%, and the mechanism underlying regulation of PD-1/PD-L1 expression is poorly understood. In this study, we identified the miRNA let-7 that posttranscriptionally suppresses PD-L1 expression. LIN28, an RNA binding protein upregulated in most cancer cells, inhibits the biogenesis of let-7, thus promoting PD-L1 expression. Therefore, inhibition of LIN28 may be a strategy to prevent immune evasion of cancer cells. We found that treatment with a LIN28 inhibitor, the small compound C1632, increases let-7 and suppresses PD-L1 expression, leading to reactivation of antitumor immunity in vitro and in vivo In addition, C1632 also displayed the capacity to inhibit cancer cell proliferation and tumor growth in mice. Altogether, these findings identified LIN28/let-7 as a target for PD-L1-mediated immunotherapeutics and reveal the potential of C1632 and its derivatives as promising oncotherapeutic agents.

Author Correction: HuR regulates telomerase activity through TERC methylation.

In the original version of this Article, the affiliation details for Fan Yang were incorrectly given as 'Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, 510632, China' and 'Leibniz Institute for Age Research - Fritz Lipmann Institute, Friedrich-Schiller University of Jena, Jena, 07745, Germany'. This has now been corrected in both the PDF and HTML versions of the Article.

HuR regulates telomerase activity through TERC methylation.

Telomerase consists of the catalytic protein TERT and the RNA TERC. Mutations in TERC are linked to human diseases, but the underlying mechanisms are poorly understood. Here we report that the RNA-binding protein HuR associates with TERC and promotes the assembly of the TERC/TERT complex by facilitating TERC C106 methylation. Dyskeratosis congenita (DC)-related TERC U100A mutation impair the association of HuR with TERC, thereby reducing C106 methylation. Two other TERC mutations linked to aplastic anemia and autosomal dominant DC, G107U, and GC107/108AG, likewise disrupt methylation at C106. Loss-of-HuR binding and hence lower TERC methylation leads to decreased telomerase activity and telomere shortening. Furthermore, HuR deficiency or mutation of mTERC HuR binding or methylation sites impair the renewal of mouse hematopoietic stem cells, recapitulating the bone marrow failure seen in DC. Collectively, our findings reveal a novel function of HuR, linking HuR to telomerase function and TERC-associated DC.

Quantification of distinct let-7 microRNA family members by a modified stem-loop RT-qPCR.

Lethal-7 (let-7) microRNA (miRNA) serves a pivotal role in a number of physiological processes and is associated with the occurrence and development of multiple disorders such as cancer. The present study aimed to use a newly developed stem­loop strategy for reverse transcription­quantitative polymerase chain reaction (RT­qPCR) to distinguish let­7 miRNA family members that differ by as little as a single nucleotide. For the miRNAs comprising 16 identical nucleotides at the 5'­end, different stem­loop RT primers were designed and used in RT­qPCR to assess the expression profiles of a panel of let­7 family member miRNAs in human glioblastoma U87 cells. Amplification efficiency was evaluated through correlation analysis between total RNA input and the quantification threshold values. Melting curve profiles were measured to estimate the amplification specificity of the improved stem­loop RT­qPCR compared with those of the poly(A)­tailing method. In addition, the discrimination ability of the modified stem­loop method was examined. Compared with poly(A) tailing, the modified stem­loop RT method was able to specifically reverse transcribe the diverse let­7 miRNA family members followed by accurate quantification, with a theoretical amplification efficiency of ~100%. This modified stem­loop method was able to distinguish miRNAs with a single base difference. This innovative method may be used in the clinical detection of let­7 expression levels in a variety of tumour samples, and may provide valuable data for disease diagnosis and prognostic evaluation. In addition, this method may offer a new avenue for developing particular stem­loop approaches in measuring other miRNAs with little discrepancy.

SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5'- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.

Preferential extension of short telomeres induced by low extracellular pH.

The majority of tumor cells overcome proliferative limit by expressing telomerase. Whether or not telomerase preferentially extends the shortest telomeres is still under debate. When human cancer cells are cultured at neutral pH, telomerase extends telomeres in telomere length-independent manner. However, the microenvironment of tumor is slightly acidic, and it is not yet known how this influences telomerase action. Here, we examine telomere length homeostasis in tumor cells cultured at pHe 6.8. The results indicate that telomerase preferentially extends short telomeres, such that telomere length distribution narrows and telomeres become nearly uniform in size. After growth at pHe 6.8, the expression of telomerase, TRF1, TRF2 and TIN2 decreases, and the abundance of Cajal bodies decreases. Therefore, telomerase are insufficient for extending every telomere and shorter telomeres bearing less shelterin proteins are more accessible for telomerase recruitment. The findings support the 'protein-counting mechanism' in which extended and unextended state of telomere is determined by the number of associated shelterin proteins and the abundance of telomerase. Decreased expression of telomerase and preferential extension of short telomeres have important implications for tumor cell viability, and generate a strong rationale for research on telomerase-targeted anti-cancer therapeutics.

Energy-Efficient Control with Harvesting Predictions for Solar-Powered Wireless Sensor Networks.

Wireless sensor networks equipped with rechargeable batteries are useful for outdoor environmental monitoring. However, the severe energy constraints of the sensor nodes present major challenges for long-term applications. To achieve sustainability, solar cells can be used to acquire energy from the environment. Unfortunately, the energy supplied by the harvesting system is generally intermittent and considerably influenced by the weather. To improve the energy efficiency and extend the lifetime of the networks, we propose algorithms for harvested energy prediction using environmental shadow detection. Thus, the sensor nodes can adjust their scheduling plans accordingly to best suit their energy production and residual battery levels. Furthermore, we introduce clustering and routing selection methods to optimize the data transmission, and a Bayesian network is used for warning notifications of bottlenecks along the path. The entire system is implemented on a real-time Texas Instruments CC2530 embedded platform, and the experimental results indicate that these mechanisms sustain the networks' activities in an uninterrupted and efficient manner.

Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance.

The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating that telomerase assembly occurs in the absence of Cajal bodies. Moreover, dispersed hTR aggregates and forms foci specifically during S and G2 phase in coilin-KO cells. Colocalization of these hTR foci with telomeres implies proper telomerase trafficking, independent of Cajal bodies. Therefore, telomerase adds similar numbers of TTAGGG repeats to telomeres in coilin-KO and controls cells. Overexpression of TPP1-OB-fold blocks cell cycle-dependent formation of hTR foci and inhibits telomere extension. These findings suggest that telomerase assembly, trafficking and extension occur with normal efficiency in Cajal bodies deficient human cells. Thus, Cajal bodies, as such, are not essential in these processes, although it remains possible that non-coilin components of Cajal bodies and/or telomere binding proteins (e.g. TPP1) do play roles in telomerase biogenesis and telomere homeostasis.