[The Effect of TALENs-mediated Downregulation Expression of Nanog on Malignant Behavior of Cervical Cancer HeLa Cells].

OBJECTIVE: To study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells. METHODS: Gene editing tool TALENs was employed to induce downregulation expression of Nanog, and Nanog mutation was evaluated by sequencing. RT-PCR and Western blot was used to detect the mRNA and protein expression level, respectively. Colony-formation assay, Transwell invasion assay, and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation, invasion, and chemoresistance, respectively. RESULTS: TALENs successfully induced Nanog mutation and downregulated Nanog expression. Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3 times compared to thoses of wild-type HeLa cells (P < 0.05). Additionally, significant weakened abilities of colony-formation, invasion, and chemoresistance in monoclonal HeLa cells were observed when compared to those of wild-type HeLa cells (P < 0.05). CONCLUSION: Nanog mutation attenuates the malignant behavior of HeLa cells. Importantly, downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.

Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

BACKGROUND AIMS: Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. METHODS: In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. RESULTS: The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. CONCLUSIONS: Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins.

Circular RNA CircCCNB1 sponges micro RNA-449a to inhibit cellular senescence by targeting CCNE2.

Circular RNAs (CircRNAs) are a novel subset of non-coding RNA widely present in eukaryotes that play a central role in physiological and pathological conditions. Accumulating evidence has indicated that CircRNAs participated in modulating tumorigenesis by acting as a competing endogenous RNA (CeRNA). However, the roles and functions of CircRNAs in cellular senescence and aging of organisms remain largely obscure. We performed whole transcriptome sequencing to compare the expression patterns of circular RNAs in young and prematurely senescent human diploid fibroblast 2BS cells, and identified senescence-associated circRNAs (SAC-RNAs). Among these SAC-RNAs, we observed the significantly downregulated expression of CircRNAs originating from exons 6 and 7 circularization of the cyclin B1 gene (CCNB1), termed CircCCNB1. Reduced CircCCNB1 expression triggered senescence in young 2BS cells, as measured by increased senescence associated-beta-galactosidase (SA-ß-gal) activity, enhanced expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/P21 and tumor protein 53 (TP53) expression, and reduced cell proliferation. Mechanistically, reduced CircCCNB1 level inhibited cyclin E2 (CCNE2) expression by modulating micro RNA (miR)-449a activity, which repressed cellular proliferation. Our data suggested that CircCCNB1may serve as a sponge against miR-449a to delay cellular senescence by targeting CCNE2. Targeting CircCCNB1 may represent a promising strategy for aging and age-related disease interventions. Furthermore, we also identified and characterized several kinds of the CircCCNB1-binding proteins (CBPs), which may contribute to the degradation of CircCCNB1.

3-(4-Fluoro-phen-yl)-2-(4-methoxy-phen-oxy)-4-oxo-5-phenyl-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile.

There are two crystallographically independent mol-ecules in the asymmetric unit of the title compound, C(26)H(17)FN(4)O(3), which differ in the dihedral angles between the aromatic rings (fluorophenyl, phenyl) and the pyrrolopyrimidine rings [0.6 (3)/76.3° and 73.7 (3)/64.6°, respectively]. The crystal structure is mainly stabilized by C-H⋯O and C-H⋯F inter-actions.

TALENs-mediated homozygous CCR5Δ32 mutations endow CD4+ U87 cells with resistance against HIV­1 infection.

Since evidence suggests that transplantation of bone marrow stem cells with the C­C chemokine receptor type 5 (CCR5)Δ32/Δ32 genotype may cure patients infected with human immunodeficiency virus (HIV)­1, the present study aimed to reproduce the CCR5Δ32 mutation in cluster of differentiation (CD)4+ U87 cells using genome engineering methods. A modified transcription activator­like effector nucleases (TALENs) technique, combined with homologous recombination for site­specific, size­controlled and homozygous DNA deletions, was used to reproduce the homozygous CCR5Δ32 mutation in CD4+ U87 cells. The results indicated that the frequency of the TALENs­targeted mutation reached 50.4% without any selection, whereas homologous recombination from CCR5 to CCR5Δ32 occurred in 8.8% of targeted cells. Notably, a HIV­1 challenge test demonstrated that CCR5Δ32/Δ32 CD4+ U87 cells were resistant to HIV infection. In conclusion, engineered CCR5Δ32/Δ32 mutations endowed CD4+ U87 cells with resistance against HIV­1 infection; this site­specific, size­controlled and homozygous DNA deletion technique was able to induce precise genomic editing, i.e., the deletion or insertion of a predetermined length of DNA sequence at a specific locus throughout the genome.

Autophagy regulates the stemness of cervical cancer stem cells.

Cancer stem cells (CSCs) are a rare population of multipotent cells with the capacity to self-renew. It has been reported that there are CSCs in cervical cancer cells. Pluripotency-associated (PA) transcription factors such as Oct4, Sox2, Nanog and CD44 have been used to isolate CSCs subpopulations. In this study, we showed that autophagy plays an important role in the biological behavior of cervical cancer cells. The expression of the autophagy protein Beclin 1 and LC3B was higher in tumorspheres established from human cervical cancers cell lines (and CaSki) than in the parental adherent cells. It was also observed that the basal and starvation-induced autophagy flux was higher in tumorspheres than in the bulk population. Autophagy could regulate the expression level of PA proteins in cervical CSCs. In addition, CRISPR/Cas 9-mediated Beclin 1 knockout enhanced the malignancy of HeLa cells, leading to accumulation of PA proteins and promoted tumorsphere formation. Our findings suggest that autophagy modulates homeostasis of PA proteins, and Beclin 1 is critical for CSC maintenance and tumor development in nude mice. This demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance.

TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.

Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

[Constructing the recombinant of pET28a-TAT-LacZ].

A synthesized double-stranded oligomeric nucleotide encoding 11-amino acid TAT protein transduction domain3 was inserted into pET28a vector after 6 histidine coding sequence, LacZ gene from pcDNA4/Myc-His/LacZ was digested by EcoR I and Hind III, then cloned into pET28a-TAT. The highly expressed TAT-beta-galactosidase was purified by affinity chromatography. TAT-beta-Gal fusion protein can across vascular smooth muscle cells in vitro. This result open new possibility for direct delivery of protein into tissues for therapy.