Accelerated Cell Proliferation Research Articles

Nanomaterials and Research on the Repair of Basketball Sports Ligament Injury

Sports ligament injury refers to muscle or joint injury caused by excessive use of limbs or long‐term use of wrong training methods. This is a very common sports disease, and the body’s ability to heal itself is poor. At present, the clinical methods to treat this disease mainly include autologous structure transplantation, allogeneic structure transplantation, and artificial ligament reconstruction. Nanomaterials refer to materials made of them as basic units. Transplantation of homologous and allogeneic structures may cause immune rejection in the human body. Therefore, the use of nanomaterials to reconstruct artificial ligaments is the current research hotspot of sports injury repair. This article mainly studies the repair effect of nanomaterials on basketball sports ligament injuries. In this paper, by implanting nanofiber scaffolds in sports‐injured rabbits, observing and comparing the healing, growth, and proliferation of damaged cells after implantation, and the compatibility of nanofibers with cell tissues, so as to determine that nanomaterials have an impact of lion sports injury repair and the interaction between nanomaterials and cells. According to the experimental results, the implantation of nanofiber scaffolds can greatly accelerate cell proliferation and differentiation and promote the healing of sports‐damaged tissues; nanofiber scaffolds have good histocompatibility, so the use of nanomaterials can promote the repair of sports ligament injury in basketball sports.

Identification of mitochondrial RNA polymerase as a potential therapeutic target of osteosarcoma

POLRMT (RNA polymerase mitochondrial) is essential for transcription of mitochondrial genome encoding components of oxidative phosphorylation process. The current study tested POLRMT expression and its potential function in osteosarcoma (OS). The Cancer Genome Atlas (TCGA) cohorts and Gene Expression Profiling Interactive Analysis (GEPIA) database both show that POLRMT transcripts are elevated in OS tissues. In addition, POLRMT mRNA and protein levels were upregulated in local OS tissues as well as in established and primary human OS cells. In different OS cells, shRNA-induced stable knockdown of POLRMT decreased cell viability, proliferation, migration, and invasion, whiling inducing apoptosis activation. CRISPR/Cas9-induced POLRMT knockout induced potent anti-OS cell activity as well. Conversely, in primary OS cells ectopic POLRMT overexpression accelerated cell proliferation and migration. In vivo, intratumoral injection of adeno-associated virus-packed POLRMT shRNA potently inhibited U2OS xenograft growth in nude mice. Importantly, levels of mitochondrial DNA, mitochondrial transcripts and expression of respiratory chain complex subunits were significantly decreased in U2OS xenografts with POLRMT shRNA virus injection. Together, POLRMT is overexpressed in human OS, promoting cell growth in vitro and in vivo. POLRMT could be a novel therapeutic target for OS.

RETRACTED ARTICLE: FAM83H-AS1/miR-485-5p/MEF2D axis facilitates proliferation, migration and invasion of hepatocellular carcinoma cells

BackgroundAbundant evidence has manifested that long noncoding RNAs (lncRNAs) are closely implicated in human cancers, including hepatocellular carcinoma (HCC). Remarkably, lncRNA FAM83H antisense RNA 1 (FAM83H-AS1) has been reported to be a tumor-propeller in multiple cancers. However, its effect on HCC progression remains unknown.MethodsFAM83H-AS1 expression was analyzed by RT-qPCR. Colony formation, EdU, and flow cytometry as well as transwell assays were implemented to analyze the biological functions of FAM83H-AS1 on HCC progression. Luciferase reporter, RIP and RNA pull-down assays were implemented to detect the interaction among FAM83H-AS1, microRNA-485-5p (miR-485-5p), and myocyte enhancer factor 2D (MEF2D) in HCC cells.ResultsFAM83H-AS1 expression in HCC cells was markedly elevated. FAM83H-AS1 accelerated cell proliferation, migration and invasion whereas inhibiting cell apoptosis in HCC. Besides, we confirmed that FAM83H-AS1 acts as a miR-485-5p sponge in HCC cells. Additionally, MEF2D was verified to be a direct target of miR-485-5p. FAM83H-AS1 could upregulate MEF2D expression via sponging miR-485-5p. Further, rescue experiments testified that MEF2D upregulation or miR-485-5p downregulation offset the repressive effect of FAM83H-AS1 depletion on HCC cell progression.ConclusionsFAM83H-AS1 facilitates HCC malignant progression via targeting miR-485-5p/MEF2D axis, suggesting that FAM83H-AS1 may be a promising biomarker for HCC treatment in the future.

Knocking down Sterol regulatory element binding protein 2 (SREBF2) inhibits the Serine Protease 8 (PRSS8) /sodium channel epithelial 1alpha subunit (SCNN1A) axis to reduce the cell proliferation, migration and epithelial-mesenchymal transformation of ovarian cancer

ABSTRACT The pathogenesis of ovarian cancer (OC) is complex. Serine Protease 8 (PRSS8) is a potential biomarker for early detection of OC. Multiple databases were used to predict the expression of PRSS8, Sterol regulatory element binding protein (SREBP) and sodium channel epithelial 1alpha subunit (SCNN1A) in OC patients and to detect the relationship among the three. The expressions of PRSS8, SREBF2, SCNN1A and related factors of the pathway were detected by RT-qPCR and Western blot. The cell transfection was used to overexpress or inhibit the expression of PRSS8 and SREBF2, so as to explore its mechanism. MTT assay and Colony formation assay were used to detect cell proliferation. The Transwell and Wound Healing assays were utilized to measure cell invasion and migration. We have further confirmed cell-level studies in animals. We found that PRSS8 expression was up-regulated in OC patients and cell lines. Knocking down PRSS8 reduced the proliferation, migration and epithelial-mesenchymal transition (EMT) of OC cells, which was realized by SREBF2 transcriptional regulation. Knocking down SREBF2 reduced PRSS8 and then inhibited the expression of SCNN1A, thus affecting the proliferation, migration and EMT of OC cells. These results also applied to animals experiments. In conclusion, SREBF2 activates the PRSS8/SCNN1A axis to accelerate cell proliferation, migration and EMT of OC.

Abstract 12932: An Epigenetic Upregulation of Dynamin 2, a Regulator of Mitochondrial Fission, Promotes Human and Experimental Pulmonary Arterial Hypertension

Introduction: Increase in mitotic fission in pulmonary arterial smooth muscle cells (PASMC) contributes to the obstructive vasculopathy in pulmonary arterial hypertension (PAH). PAH’s fissogenic phenotype is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp1). Recent observation suggests that to complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). In this study we explore the role of DNM2 in the pathogenesis of PAH. Hypothesis: In PAH, increased expression of DNM2 increases mitochondrial fission and proliferation of PASMC. Methods: Immunoblot was used to quantify the expression of DNM2 in control (n=4) and PAH PASMC (n=5). Immunofluorescence was used to quantify the expression of DNM2 in the pulmonary arteries of monocrotaline (MCT) vs control rats. Flow cytometry was used to assess the effects of manipulating the expression of DNM2 on cell proliferation, cell cycle progression and rates of unstimulated apoptosis. Mitochondrial network structure was assessed using confocal imaging of cells infected with Adv-TOM20 mNeon green. Results: The expression of DNM2 was increased in human PAH PASMC vs control PASMC. Increase in the expression of DNM2 expression was also observed in the pulmonary arteries of MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission in PAH PASMC. Furthermore, silencing DNM2 inhibited proliferation in PAH PASMC by blocking the cell cycle at G1/G0. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation, recapitulating the PAH phenotype. In silico analysis identified miR-124-3p as a putative negative regulator of DNM2. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: DNM2 promotes mitochondrial fission and regulates cell proliferation in human PAH PASMC. Decreased miR-124-3p expression may contribute to upregulation of DNM2 in PAH. This work highlights the importance of DNM2 as partner with Drp1 in regulation of mitochondrial fission and shows that like Drp1, DNM2 is increased in human PAH. DNM2 may constitute new therapeutic target for PAH.

RGDSP-Decorated Hyaluronate Hydrogels Facilitate Rapid 3D Expansion of Amylase-Expressing Salivary Gland Progenitor Cells.

In vitro engineering of salivary glands relies on the availability of synthetic matrices presenting essential cell-instructive signals to guide tissue growth. Here, we describe a biomimetic, hyaluronic acid (HA)-based hydrogel platform containing covalently immobilized bioactive peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP). The HA network was established by the thiol/acrylate reaction, and bioactive peptides were conjugated to the network with high efficiency without significantly altering the mechanical property of the matrix. When encapsulated as single cells in peptide-modified HA hydrogels, human salivary gland stem/progenitor cells (hS/PCs) spontaneously organized into multicellular spheroids with close cell-cell contacts. Conjugation of RGDSP and TWSKV signals in HA gels significantly accelerated cell proliferation, with the largest spheroids observed in RGDSP-tagged gels. Peptide conjugation did not significantly alter the expression of acinar (AMY1), ductal (TFCP2L1), and progenitor (KRT14) markers at the mRNA level. Characterization of three-dimensional (3D) cultures by immunocytochemistry showed positive staining for keratin-5 (K5), keratin-14 (K14), integrin-β1, and α-amylase under all culture conditions, confirming the maintenance of the secretory progenitor cell population. Two-dimensional (2D) adhesion studies revealed that integrin-β1 played a key role in facilitating cell-matrix interaction in gels with RGDSP, IKVAV, and TWSKV signals. Overall, conjugation of the RGDSP peptide to HA gels improved cell viability, accelerated the formation of epithelial spheroids, and promoted the expansion of the progenitor cell population in 3D. This work represents an essential first step toward the development of an engineered salivary gland.

Sprouty3, but Not Sprouty1, Expression Is Beneficial for the Malignant Potential of Osteosarcoma Cells.

Sprouty proteins are widely accepted modulators of receptor tyrosine kinase-associated pathways and fulfill diversified roles in cancerogenesis dependent on the originating cells. In this study we detected a high expression of Sprouty3 in osteosarcoma-derived cells and addressed the question of whether Sprouty3 and Sprouty1 influence the malignant phenotype of this bone tumor entity. By using adenoviruses, the Sprouty proteins were expressed in two different cell lines and their influence on cellular behavior was assessed. Growth curve analyses and Scratch assays revealed that Sprouty3 accelerates cell proliferation and migration. Additionally, more colonies were grown in Soft agar if the cells express Sprouty3. In parallel, Sprouty1 had no significant effect on the measured endpoints of the study in osteosarcoma-derived cells. The promotion of the tumorigenic capacities in the presence of Sprouty3 coincided with an increased activation of signaling as measured by evaluating the phosphorylation of extracellular signal-regulated kinases (ERKs). Ectopic expression of a mutated Sprouty3 protein, in which the tyrosine necessary for its activation was substituted, resulted in inhibited migration of the treated cells. Our findings identify Sprouty3 as a candidate for a tumor promoter in osteosarcoma.

LncRNA HLA Complex Group 18 (HCG18) Facilitated Cell Proliferation, Invasion, and Migration of Prostate Cancer Through Modulating miR-370-3p/DDX3X Axis.

Long non-coding RNAs (lncRNAs) have been reported to exert critical functions in the malignant development of many cancers. lncRNA HLA complex group 18 (HCG18) has been confirmed to have a promoting effect on various cancers. However, whether HCG18 functions in PC is still unclear. Therefore, the current study aimed at unveiling the role of HCG18 in PC progression and its regulatory mechanism on the biological behaviors of PC. Here, RT-qPCR was utilized to detect HCG18 expression, and then, functional experiments were conducted to verify the effects of HCG18 on PC cell proliferation, migration, invasion, and apoptosis. According to the results, HCG18 was significantly up-regulated in PC cells and it facilitated cell proliferation, migration, and invasion in PC. Furthermore, a series of mechanism experiments were carried out to verify the relationship among HCG18, miR-370-3p, and DEAD-box helicase 3 X-linked(DDX3X) in PC cells. Final rescue assays showed that DDX3X overexpression could reverse the inhibitory function of silencing HCG18 on PC progression. In summary, our study showed that lncRNA HCG18 accelerated cell proliferation, invasion, and migration of PC via up-regulating DDX3X through sponging miR-370-3p, providing a novel finding about PC-related regulatory mechanism.

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

Stable and biocompatible fibrillar hydrogels based on the self-crosslinking between collagen and oxidized chondroitin sulfate

Stable and biocompatible collagen-based hydrogels based on natural extracellular matrix (ECM) with a homogeneous microstructure are ideal scaffolds for tissue engineering; however, lack of structural stability and mechanical property have hindered their application. In this work, Chondroitin Sulfate (CS) was periodate-oxidized to prepare dialdehyde ECM-derived biopolymer with the weight average molecular weight of 3.7 × 104 g/moL to construct the stable collagen-based fibrillar hydrogels. The results of Fourier Transform Infrared (FTIR) analysis and hydroxylamine hydrochloride titration assay confirmed the presence of aldehyde groups in oxidized CS (CSox) with an oxidization degree of 30.67%. Gelation time test and scanning electron microscopy (SEM) observation showed that gel formation and fibrillogenesis process were accelerated by adding CSox, and all hydrogels exhibited a tight fibrillar structure. With an increase in CSox concentration from 0.125 to 2.000 mg/mL, the crosslinking degree increased from 24.95% to 35.05% due to the formation of covalent bonds between the ε-amino groups of collagen and aldehyde of CSox. The mechanical strength of collagen/CSox hydrogels reached a maximum value of 11.79 KPa, which was more than triple that of pure collagen hydrogel (3.58 KPa) at 0.125 mg/mL CSox. The swelling ratio, enzymatic resistance, and thermal stability were also improved with the increasing concentration of CSox. When the CSox concentration reached 0.500 mg/mL, the collagen-based hydrogels exhibited the highest thermal denaturation temperature, which was approximately 7.5 °C higher than that of the pure collagen hydrogel. Moreover, the results of the CCK-8 assay and LIVE/DEAD staining demonstrated that all the collagen/CSox hydrogels accelerated cell proliferation and showed acceptable biocompatibility. This work broadens the avenue for the design of natural collagen-based hydrogels for tissue engineering.

LncRNA DLGAP1-AS1 accelerates glioblastoma cell proliferation through targeting miR-515-5p/ROCK1/NFE2L1 axis and activating Wnt signaling pathway.

IntroductionGlioblastoma (GBM), the primary malignant tumor in the central nervous system, features high aggressiveness and mortality. Long noncoding RNAs (lncRNAs) can exert the crucial function in regulating various human diseases, including GBM. However, the function and mechanism of lncRNA DLGAP1 antisense RNA 1 (DLGAP1‐AS1) in GBM remain still unknown.MethodsDLGAP1‐AS1 expression in GBM cells was detected by RT‐qPCR. Functional assays were conducted to determine GBM cell proliferation and apoptosis. RIP, RNA pull down, and luciferase reporter assay were applied for measuring the interplay of DLGAP1‐AS1 with other RNAs.ResultsDLGAP1‐AS1 was distinctly upregulated in GBM cells. DLGAP1‐AS1 depletion inhibited cell proliferation, but induced apoptosis. MiR‐515‐5p could be sponged by DLGAP1‐AS1 in GBM cells and to repress cell proliferation in GBM. Further, Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) and Nuclear factor erythroid‐2 like 1 (NFE2L1) were confirmed as the target gene of miR‐515‐5p. Wnt signaling pathway could be activated by DLGAP1‐AS1 via regulating ROCK1 and NFE2L1 expression. Rescue assays proved that overexpression of both ROCK1 and NFE2L1 could totally reverse the inhibitory effect of silencing DLGAP1‐AS1 on GBM cell proliferation.ConclusionLncRNA DLGAP1‐AS1 accelerated cell proliferation in GBM via targeting miR‐515‐5p/ROCK1/NFE2L1 axis and activating Wnt signaling pathway.

Abstracts from the World Federation for Laser Dentistry—Asia Pacific Division Virtual Conference July 16–17, 2021

Abstracts from the World Federation for Laser Dentistry—Asia Pacific Division Virtual Conference July 16–17, 2021

SKA3, negatively regulated by miR-128-3p, promotes the progression of non-small-cell lung cancer.

Aim: To investigate the effects of SKA3on cell proliferation and metastasis in non-small-cell lung cancer (NSCLC) and its underlying mechanism. Methods: Immunohistochemistry was employed to analyze the expression of SKA3 in NSCLC. CCK-8 assay, EdU assay, Transwell assay and flow cytometry analysis were employed to assess cell proliferation, metastatic potentialand apoptosis in vitro, respectively. A lung metastasis model was used to evaluate metastasis of NSCLC cells in vivo.A luciferase reporter gene assay was conducted to verify the targeting relationship. Results: SKA3 exhibited high expression in NSCLC tissues and cells. Overexpression of SKA3 remarkably accelerated cell proliferation and metastasis and suppressed apoptosis of NSCLC cells and promoted lung metastasis in a mousemodel. miR-128-3p repressed SKA3 expression by targeting it. Conclusion:miR-128-3p inhibited the progression of NSCLC through targeting SKA3.

Zinc Signaling in the Mammary Gland: For Better and for Worse.

Zinc (Zn2+) plays an essential role in epithelial physiology. Among its many effects, most prominent is its action to accelerate cell proliferation, thereby modulating wound healing. It also mediates affects in the gastrointestinal system, in the testes, and in secretory organs, including the pancreas, salivary, and prostate glands. On the cellular level, Zn2+ is involved in protein folding, DNA, and RNA synthesis, and in the function of numerous enzymes. In the mammary gland, Zn2+ accumulation in maternal milk is essential for supporting infant growth during the neonatal period. Importantly, Zn2+ signaling also has direct roles in controlling mammary gland development or, alternatively, involution. During breast cancer progression, accumulation or redistribution of Zn2+ occurs in the mammary gland, with aberrant Zn2+ signaling observed in the malignant cells. Here, we review the current understanding of the role of in Zn2+ the mammary gland, and the proteins controlling cellular Zn2+ homeostasis and signaling, including Zn2+ transporters and the Gq-coupled Zn2+ sensing receptor, ZnR/GPR39. Significant advances in our understanding of Zn2+ signaling in the normal mammary gland as well as in the context of breast cancer provides new avenues for identification of specific targets for breast cancer therapy.

Effects of compound stimulation of fluid shear stress plus ultrasound on stem cell proliferation and osteogenesis.

Bone tissue with strong adaptability is often in a complex dynamical microenvironment in vivo, which is associated with the pathogenesis and treatment of orthopedic diseases. Therefore, it is of great significance to investigate the effects of corresponding compound stimulation on cell behaviors. Herein, a fluid shear stress (FSS) plus ultrasound stimulation platform suitable for cell studies based on a microfluidic chip was constructed and bone marrow mesenchymal stem cell (BMSC) was chosen as a model cell. The proliferation and osteogenesis of BMSCs under the compound stimulation of FSS plus ultrasound in growth medium without any soluble induction factors were firstly investigated. Single FSS stimulation and static culture conditions were also examined. Results illustrated that suitable single FSS stimulation (about 0.06 dyn/cm2) could significantly enhance cell proliferation and osteogenesis simultaneously when compared to the static control, while greater FSS mitigated or even restricted these enhancing effects. Interestingly, ultrasound stimulation combined with this suitable FSS stimulation further accelerated cell proliferation as the intensity of ultrasound increasing. As for the osteogenesis under compound stimulation, it was relatively restricted under lower ultrasound intensity (about 0.075 W/cm2), while promoted when the intensity became higher (about 1.75 W/cm2). This study suggests that both the cell proliferation and osteogenesis are very responsive to the magnitudes of FSS and ultrasound stimulations and can be both significantly enhanced by proper combination strategies. Moreover, these findings will provide valuable references for the construction of effective cell bioreactors and also the treatment of orthopedic diseases.

KIF15 knockdown suppresses gallbladder cancer development.

Gallbladder cancer (GBC) is commonly regarded as one of the most lethal malignant tumor types with poor prognosis. Kinesin family member 15 (KIF15) is reported to be tightly related with progression of multiple cancer types which, however, has not been clarified in GBC so far. KIF15 was significantly up-regulated in clinical GBC tissues compared with that in para-carcinoma tissues and the expression level was also correlated with tumor malignancies. In addition to tissues, GBC cells also exhibited a high expression abundance of KIF15. After down-regulating KIF15 via lentiviral transfection, GBC cell proliferation and migration were both inhibited, while cell apoptosis was promoted markedly. Likewise, silencing KIF15 significantly interfered the growth of nude mouse xenografts. Our experiments in GBC cell lines also demonstrated that KIF15 overexpression accelerated cell proliferation but lessened cell apoptosis in both GBC-SD and SGC-996 cells. Further investigation of the mechanism occurring in GBC inhibition mediated by KIF15 knockdown revealed that KIF15 deficiency led to decreased activity of several signaling pathways (TNF, PI3K/AKT and MAPK), a reduction of CDK6 expression regulated by enhanced p21, and HSP60 absence. Following the treatment of shCtrl- and shKIF15-transfected cells with AKT activator, we found that anti-tumor effects resulting from KIF15 deficiency could be relieved by AKT activator in both experimental cells. Overall, for the first time, we demonstrated that KIF15 was overexpressed in GBC and displayed a close relationship between KIF15 levels and GBC clinical stages. Furthermore, low expression of KIF15 resulted in obvious anti-tumor effects.

MiR-1273g-3p Promotes Malignant Progression and has Prognostic Implications in Prostate Cancer.

Prostate cancer (PCa) is the most popular cancer of mankind. Our study aimed to provide the expression and the predictive significance of miR-1273g-3p in PCa. Moreover, the effects on cell biological activities were also investigated. The relative expression of miR-1273g-3p in PCa tissues and cell lines was validated by quantitative real-time PCR. Kaplan-Meier curve and Cox regression analyses were performed to indicate the prognostic value. The implications of miR-1273g-3p on cell proliferation, migration, and invasion were validated using the CCK-8 and Transwell assay. Our results provided that the expression of miR-1273g-3p was increased in PCa tissues and cell lines. The levels of miR-1273g-3p were associated with Gleason score, TNM stage, clinical stage, and lymph node metastasis. Overexpression of miR-1273g-3p indicated a promising overall survival rate. Cox regression results indicated miR-1273g-3p might be an independent marker for PCa patients. Silenced miR-1273g-3p inhibited PCa cell proliferation, migration, and invasion. In total, miR-1273g-3p was increased in PCa and identified as a therapeutic target and a prognostic factor for PCa patients. Overexpression of miR-1273g-3p might be an oncogene via accelerating cell proliferation, migration, and invasion.

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System.

The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.

Effects of spermine on the proliferation and migration of porcine intestinal epithelial cells

Whether spermine promotes the repair of porcine intestinal epithelium damage through Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase C-γ1 (PLC-γ1) signaling remains unclear. The current study investigated the effects of spermine addition on the proliferation and migration of IPEC-J2 cells and the effects of Rac1/PLC-γ1 signaling on cell migration. We showed that the inhibitors of Rac1 (NSC-23766) and PLC-γ1 (U73122) reduced cell migration and decreased the protein levels of Rac1 and PLC-γ1 in the cells. Moreover, spermine promoted the proliferation and migration of the IPEC-J2 cells, that is, 1 µM spermine exhibited the best effect, and spermine treatment increased the protein levels of Rac1 and PLC-γ1. Further experiments showed that spermine treatment increased cell migration and enhanced Rac1 and PLC-γ1 protein levels, compared with NSC-23766 and U73122 treatments with spermine. In conclusion, spermine treatment promoted the repair of damaged porcine intestinal epithelium by accelerating cell proliferation and migration mediated by Rac1/PLC-γ1 signaling.

The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter.

Cyanide is traditionally viewed as a cytotoxic agent, with its primary mode of action being the inhibition of mitochondrial Complex IV (cytochrome c oxidase). However, recent studies demonstrate that the effect of cyanide on Complex IV in various mammalian cells is biphasic: in lower concentrations (nanomolar to low micromolar) cyanide stimulates Complex IV activity, increases ATP production and accelerates cell proliferation, while at higher concentrations (high micromolar to low millimolar) it produces the previously known (‘classic’) toxic effects. The first part of the article describes the cytotoxic actions of cyanide in the context of environmental toxicology, and highlights pathophysiological conditions (e.g., cystic fibrosis with Pseudomonas colonization) where bacterially produced cyanide exerts deleterious effects to the host. The second part of the article summarizes the mammalian sources of cyanide production and overviews the emerging concept that mammalian cells may produce cyanide, in low concentrations, to serve biological regulatory roles. Cyanide fulfills many of the general criteria as a ‘classical’ mammalian gasotransmitter and shares some common features with the current members of this class: nitric oxide, carbon monoxide, and hydrogen sulfide.