[Research advance and application in the gene therapy of gene editing technologies].

Gene editing technologies are used to specifically edit the target sequence. With the development of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), regular clustering of short palindrome repeats (CRISPR) and single base editing (BE) techniques, gene editing technologies not only provide powerful tools for gene functional studies, but also offer new therapeutic strategies in biomedical research. Gene editing has demonstrated broad application prospects in the gene therapy field, as well as in the construction of animal and cell models, drug target screening and gene functional research. In this review, we summarize several typical gene editing technologies, their characteristics and applications in gene therapy and discusses their opportunities and challenges in gene therapy, thereby providing critical insights and references on the clinical application of gene editing technologies.

[Synergistic regulation of the erythroid differentiation of K562 cells by KLF1 and KLF9].

Krüppel-like factors (KLFs) regulate diverse physiological processes such as the differentiation and development of red blood cells. However, it remains unclear whether KLFs exhibit synergistic regulatory effects. Transcriptomic data from our previous study showed that KLF1 and KLF9 expression was significantly higher in differentiated red blood cells than in hematopoietic stem cells. In the present study, we manipulated KLF1 and KLP9 gene expression by overexpressing or knocking down KLF1 and KLF9 in K562 cells and revealed a positive correlation between the expression of KLF1 and KLF9; their co-expression can significantly promote erythroid differentiation and specifically enhance ß-globin gene expression. Further, we analyzed the transcriptome data of K562 cells with altered KLF1/KLF9 levels and found that KLF1 and KLF9 synergistically regulated erythroid differentiation through the PI3K-Akt and FoxO signaling pathways. KLF1 and KLF9 may exert this synergistic effect through FOS, TF, and IL8 in K562 cells. We have provided evidence that KLF1 and KLF9 play a synergistic role in regulating erythroid differentiation.

[Study on the level changes of obesity-associated factors and puberty-associated hormones and their relationship between obese and normal 13 years old children].

OBJECTIVE: To investigate the level changes of obesity-associated factors and puberty-associated hormones and their relationship between obese and normal Children. METHODS: The concentrations of orexins, leptin, insulin, testosterone (T), estradiol (E), luteotrophic hormone (LH), as well as follicle stimulating hormone (FSH) in blood were measured respectively in 78 cases of 13 years old obesity children and 84 normal children as a control group by the means of RIA methods. The bone ages were evaluated with "bone age standard of hundred percent scale". RESULTS: The leptin and insulin concentrations were significantly higher in obesity children as compared with the control group (P < 0.01). The concentrations of plasma orexinA were significantly lower in obesity children as compared with the control group (P < 0.01). The concentrations of T were significantly lower in obesity boys as compared with the control group (P < 0.01). The concentrations of E2 and LH were significantly higher in obesity girls as compared with the control group (P < 0.01). The bone age of the obese children was significantly older than that of the control group. Along with the bone age of children became older, the concentration of leptin and LH in blood were significantly increased. There was a positive correlation between LH and FSH in two groups of either boys and girls and so did between leptin and insulin, C-peptide, E2 (P < 0.01). In the group of normal boys, the positive correlations between LH, FSH and E2 were shown (r = 0.373, 0.314, P <0.05), and a negative correlations between leptin and T were shown as( r = -0.423, P < 0.01), a positive correlation between leptin and FSH was also shown as (r = 0.308, P < 0.05). In the control group of girls, there were positive correlations between leptin, LH and E2 (r = 0.585, 0.647, P < 0.01). Any correlations between LH, FSH T and E2 were not shown in the two obese groups of boys and girls. CONCLUSION: The leptin, orexinA, insulin, T, E2, and LH concentrations had been changed significantly in blood among those obesity children, and so as their interactions. The high level of leptin in obesity children may change the concentrations of T, E2 and LH and affect the development of puberty.

[Blood leptin, orexins and NPY levels and their relations in obese children].

OBJECTIVE: To investigate the concentration levels of leptin, orexins and neuropeptide Y (NPY) in the blood of obese children, and to analysed the relationship between these substances. METHODS: RIA methods were used to measure the concentrations of leptin, orexinA, orexinB, and NPY in the blood of 98 obese children [BMI: male (29.24 +/- 1.87) kg/mZ, female (28.12 +/- 2.30) kg/m2] and in 104 normal children [BMI: male (20.49 +/- 1.95) kg/m2, female (19.59 +/- 1.51) kg/m2] as the control group. RESULTS: The leptin concentrations in obese children [male (26.00 +/- 14.66) ng/mL; female (33.59 +/- 14.63) ng/mL] were higher than those in the control group [male (6.65 +/- 44.49) ng/mL; female [10.48 +/- 5.52) ng/mL P < 0.013]. The concentrations of plasma orexinA in obes children [male (3.23 +/- 1.86) pg/mL; female (3.38 +/- 1.80) pg/mL] were lower than those in the control group [male (4.52 +/- 1.52) pg/mL; female (4.71 +/- 1.53) pg/mL P < 0.05]; Negative correlations between leptin and NPY were noted in the obesity group (r = -0.302) and the control group (r = -0.310, P < 0.01), while the slopes in the two groups were different (control group -2.969; obese group -0.809). A positive correlation between NPY and orexinA was noted (r = 0.207, P < 0.05). The fluctuation range of orexinA in obese children was markedly narrowed when compared with that in the control group. CONCLUSION: The concentration level of peripheral orexinA and leptin in the obese and non-obese children change inversely. The obesity in children correlates with the concentrations of orexinA, leptin, NPY as well as with their interactions.

Upregulation of spondin-2 predicts poor survival of colorectal carcinoma patients.

Colorectal cancer (CRC) is the third and second most common cancer in males and females worldwide, respectively. Spondin-2 is a conserved secreted extracellular matrix protein and a candidate cancer biomarker. Here we found that Spondin-2 mRNA was upregulated in CRC tissues using quantitative RT-PCR and data-mining of public Oncomine microarray datasets. Spondin-2 protein was increased in CRC tissues, as revealed by immunohistochemistry analyses of two tissue microarrays containing 180 cases. Spondin-2 gene expression was significantly associated with CRC stage, T stage, M stage and Dukes stage, while its protein was associated with age and M stage. Kaplan-Meier analysis revealed that the upregulated Spondin-2 mRNA and protein predicted poor prognosis of CRC patients. Univariate and multivariate Cox regression analyses indicated that grade, recurrence, N stage and high Spondin-2 were independent predictors of overall survival of CRC patients. ELISA revealed that plasma Spondin-2 was upregulated in CRC and dropped after surgery. Receiver operating characteristic curve analysis demonstrated that plasma Spondin-2 has superior predictive performance for CRC with an area under the curve of 0.959 and the best sensitivity/specificity of 100%/90%. Furthermore, ectopic expression of Spondin-2 enhanced colon cancer cell proliferation. Spondin-2 could be an independent diagnostic and prognostic biomarker of colon cancer.

Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.