METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Comparative transcriptome analysis of experimental cryptorchidism: of mice and cynomolgus monkeys.

In most mammalian species, the testis descends from the abdomen into the scrotum during fetal or neonatal life. The failure of testicular descent, a pathological condition known as cryptorchidism, has long been the subject of scientific interest in a wide range of fields, including medicine, developmental biology, and evolutionary biology. In this study, we analyzed global gene expression changes associated with experimental cryptorchidism in mice by using RNA-seq. A total of 453 differentially expressed genes were identified, of which 236 genes were upregulated, and 217 genes were downregulated. Gene ontology, pathway, and gene network analysis highlighted the activation of inflammatory response in experimental cryptorchidism. By examining the promoter regions of differentially expressed genes, we identified 12 causal transcription factors. In addition, we also induced experimental cryptorchidism in two cynomolgus monkeys and performed RNA-seq. A cross-species comparison was performed at the gene expression level. Our study provides a valuable resource for further understanding the molecular mechanisms of cryptorchidism in mammals.

miR-142-3p Suppresses Cell Growth by Targeting CDK4 in Colorectal Cancer.

BACKGROUND/AIMS: Deregulation of microRNAs (miRNAs) has been associated with a variety of cancers, including colorectal cancer (CRC). Here, we investigated anomalous miR-142-3p expression and its possible functional consequences in primary CRC samples. METHODS: The expression of miR-142-3p was measured by quantitative RT-PCR in 116 primary CRC tissues and adjacent non-tumor tissues. The effect of miR-142-3p up- or down-regulation in CRC-derived cells was evaluated in vitro by cell viability and colony formation assays and in vivo by growth assays in xenografted nude mice. RESULTS: Using quantitative RT-PCR, we found that miR-142-3p was down-regulated in 78.4 % (91/116) of the primary CRC tissues tested when compared to the adjacent non-tumor tissues. We also found that the miR-142-3p mimic reduced in vitro cell viability and colony formation by inducing cell cycle arrest in CRC-derived cells, and inhibited in vivo tumor cell growth in xenografted nude mice. Inversely, we found that the miR-142-3p inhibitor increased the viability and colony forming capacity of CRC-derived cells and tumor cell growth in xenografted nude mice. In addition, we identified CDK4 as a potential target of miR-142-3p by predictions and dual-luciferase reporter assays. Concordantly, we found that miR-142-3p mimics and inhibitors could decrease and increase CDK4 protein levels in CRC-derived cells, respectively. CONCLUSION: From our results we conclude that miR-142-3p may act as a tumor suppressor in CRC and may serve as a tool for miRNA-based CRC therapy.

[Study on rapid identification of medicinal plants of Paris polyphylla from different origin areas by NIR spectroscopy].

Based on near infrared spectroscopy, seventy samples of wild medicinal plants of paris polyphylla from Guizhou, Guangxi and Yunnan Provinces were collected to identify their geographical origins. Multiplication signal correction (MSC), standard normal variate (SNV), first derivative (FD), second derivative (SD), savitzky-Golay filter (SG), and Norris derivative filter (ND) were conducted to optimize the original spectra of fifty samples of training set. The results showed that the method MSC combined with SD and ND presented the best results of spectra pretreatment. According to spectrum standard deviation, spectrum range (7 450-4 050 cm(-1)) was chosen and principal component analysis-mahalanobis distance (PCA-MD) method was used to build the model. Its first three principal components, i. e. cumulative contribution, determination coefficient (R2), root-mean-square error of calibration (RMSEC) and root-mean-square error of prediction (RMSEP) were 89.44%, 97.58%, 0.179 6 and 0.266 4, respectively, and the prediction accuracy is 90%. Furthermore, according to variable importance plot (VIP), spectrum range (7 135.33-4 007.35 cm(-1)) was chosen and partial least square discrimination analysis (PLS-DA) was applied to establish the model. Its first three principal components cumulative contribution, R2, RMSEC and RMSEP were 89.28%, 95.88%, 0.234 8 and 0.348 2, respectively, and the prediction accuracy is 100%. Comparing the two methods, we found that spectrum range chosen by VIP and model built by PLS-DA could provide greater accuracy in identifying paris polyphylla from different origin areas. The method supplied foundation for authenticity and quality evaluation of traditional Chinese medicine.

Pancreatic agenesis and altered m6A methylation in the pancreas of PDX1-mutant cynomolgus macaques.

As an essential transcriptional activator, PDX1 plays a crucial role in pancreatic development and ß-cell function. Mutations in the PDX1 gene may lead to type 4 maturity-onset diabetes of the young (MODY4) and neonatal diabetes mellitus. However, the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models. In this study, three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology, all of which succumbed shortly postpartum, exhibiting pancreatic agenesis. Notably, one tri-allelic PDX1-mutant cynomolgus macaque (designated as M4) developed a pancreas, whereas the two mono-allelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation. RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions, indicating developmental delay and PDX1 haploinsufficiency. A marked change in m6A methylation was identified in the M4 pancreas, confirmed through cultured PDX1-mutant islet organoids. Notably, overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids. This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.

[Utilization of Werner syndrome mouse model in studying premature aging and tumor].

Werner syndrome (WS) is a rare autosomal recessive genetic disease in human. It is considered as a good model disease in studying human premature syndrome. Werner protein (WRN) is a nuclear protein mutated in WS. Recent biochemical and genetic studies indicated that WRN plays important roles in DNA replication, DNA repair, and telomere maintenance. Here, we reviewed the molecular genetics of WS and the importance of telomere and WRN in the development of WS. Knocking out both telomerase and Wrn genes in mouse faithfully manifests human WS. The mouse model provides a unique genetic platform to explore the crosstalk of premature aging and tumor.

Sp1 contributes to overexpression of stanniocalcin 2 through regulation of promoter activity in colon adenocarcinoma.

BACKGROUND: Aberrant expression of stanniocalcin 2 (STC2) is implicated in colon adenocarcinoma (COAD). A previous study identified that STC2 functions as a tumor promoter to drive development of some cancers, but the role of its overexpression in the development of COAD remains unclear. AIM: To evaluate the regulation mechanism of STC2 overexpression in COAD. METHODS: The expression of STC2 in COAD was assessed by TCGA COAD database and GEO (GSE50760). Methylation level of the STC2 promoter was evaluated with beta value in UALCAN platform, and the correlation between STC2 expression and survival rate was investigated with TCGA COAD. Transcription binding site prediction was conducted by TRANSFAC and LASAGNA, and a luciferase reporter system was used to identify STC2 promoter activity in several cell lines, including HEK293T, NCM460, HT29, SW480, and HCT116. Western blotting was performed to evaluate the role of Sp1 on the expression of STC2. RESULTS: The central finding of this work is that STC2 is overexpressed in COAD tissues and positively correlated with poor prognosis. Importantly, the binding site of the transcription factor Sp1 is widely located in the promoter region of STC2. A luciferase reporter system was successfully constructed to analyze the transcription activity of STC2, and knocking down the expression of Sp1 significantly inhibited the transcription activity of STC2. Furthermore, inhibition of Sp1 remarkably decreased protein levels of STC2. CONCLUSION: Our data provide evidence that the transcription factor Sp1 is essential for the overexpression of STC2 in COAD through activation of promoter activity. Taken together, our finding provides new insights into the mechanism of oncogenic function of COAD by STC2.

Knockdown of CHPF suppresses cell progression of non-small-cell lung cancer.

Purpose: The aim of the present study was to explore the role of CHPF in non-small-cell lung cancer (NSCLC) and to develop an shRNA vector-based therapy to repress the expression of CHPF gene in NSCLC cell lines. Methods: In this study, we used immunohistochemical staining to verify the expression of CHPF in NSCLC tissue. Then, we determined the expression of CHPF gene in different NSCLC cell lines with RT-PCR and Western blotting. Specific CHPF shRNA was used to knockdown the expression of CHPF. Celigo image cytometry, cell cycle analysis, and flow cytometry assay were performed. Results: The results showed that expression level of CHPF was higher in NSCLC tissues than normal lung tissues. Further, we established that CHPF expression knockdown in NSCLC cells could substantially restrain the cell proliferation, apoptosis, and cell cycle in vitro. Conclusion: On the basis of these results, we concluded that CHPF expression has an important role in the progression of human NSCLC cells. Therefore, its interference could possibly be used as a potential therapeutic target against NSCLC.

Prevalence and risk factors for human papillomavirus infection among Chinese ethnic women in southern of Yunnan, China.

BACKGROUND: Dai is a major Chinese ethnic minority group residing in rural areas of the southern part of Yunnan. However, no data exist on the Human Papillomavirus (HPV) prevalence and genotype distribution among Dai women. METHOD: A total of 793 participants (Dai=324, Han=251, other ethnic=218) were included in this study. PCR was performed to detect the HPV-positive samples, and genotyping was performed with an HPV Geno-Array. RESULT: The overall HPV prevalence was very low among Dai women compared to the others. The prevalence of high-risk-HPV infections was significantly higher (p=0.001) among other ethnic women (22.0%) than that among Han (13.1%) and Dai women (7.1%). The overall HPV, high-risk-HPV, single and multiple infection prevalence among rural women were 12.9%, 12.1%, 12.3%, and 0.5%, respectively. HPV-16 (5.5%) was shown to be the most prevalent genotype, followed by HPV-52 (2.6%) and HPV-58 (2.4%). Urban women had relatively higher rates of overall HPV (16.0%), high-risk-HPV (14.1%), single genotype (11.9%), and multiple genotype (4.1%) infections. In urban women, HPV-52 (3.6%) was the most prevalent genotype, followed by HPV-39 (2.7%) and HPV-16 (1.2%). In the urban area, HPV prevalence was highest in women aged <29 years, but declined with increasing age. However, in rural women, the highest HPV prevalence was observed among women at older age (>50 years). Education and smoking habit were significantly associated with HPV infection among both rural and urban area women (p<0.001). CONCLUSION: The prevalence and genotype distribution of HPV varied among ethnic women in urban and rural area of Yunnan Province.

Complete chloroplast genome sequence of Nicotiana tabacum TN90 (Solanaceae).

This study reported the complete nucleotide sequence of the Nicotiana tabacum TN90 chloroplast (cp) genome. The cpDNA was 155 992 bp in length and contained 133 individual genes (79 protein encoding genes, 30 tRNA genes and four rRNA genes). Maximum-likelihood (ML) phylogenetic tree for 17 species with Arabidopsis thaliana, Oryza sativa, and Anomochloa marantoidea as an outgroup resulted in a single tree with - lnL =542 222.71, where the Nicotiana tabacum TN90 plastid was clustered with three previous reported Nicotiana species: N. tomentosiformis, N. undulata and N. tabacum. The TN90 variety of tobacco cp genome sequence reported in this study will accelerate tobacco improvement in the future.

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

[cDNA cloning and sequence analysis of pluripotency genes in tree shrews (Tupaia belangeri)].

In this paper, partial sequences of the tree shrew (Tupaia belangeri) Klf4, Sox2, and c-Myc genes were cloned and sequenced, which were 382, 612, and 485 bp in length and encoded 127, 204, and 161 amino acids, respectively. Whereas, their cDNA sequence identities with those of human were 89%, 98%, and 89%, respectively. Their phylogenetic tree results indicated different topologies and suggested individual evolutional pathways. These results can facilitate further functional studies.

[Research on MicroRNAs in pluripotent stem cells].

MicroRNAs (miRNAs) are a newly identified class of small regulatory non-coding endogenous RNAs that take part in a series of important processes by regulating gene expression. Recent studies have provided evidence that miRNAs may be involved in nearly all biological and metabolic processes, especially influencing self-renewal and differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In this review, we briefly summarize the biological characteristics of miRNAs, the detection technologies, and the role of miRNAs regulation in ESCs and iPSCs to frame a discussion on the future prospects of miRNA research.

[Morphological characteristics and cryodamage of Chinese tree shrew (Tupaia belangeri chinensis) sperm].

The tree shrew (Tupaia belangeri chinensis) is a small non-rodent mammal, which is a relatively new experimental animal in medicine due to its close evolutionary relationship to primates and its rapid propagation. Sperm characteristics and cryopreservation in the tree shrew were the main contents of our spermatological research. Epididymal sperm were surgically harvested from male tree shrews captured from the Kunming area. The rate of testis weight to body weight was (1.05±0.07)%, volume of both testis was (1.12 ± 0.10) mL, total sperm from epididymis and vas deferens were 2.2-8.8×10(7), and sperm motility and acrosome integrity were (68.8 ± 3.9)% and (90.0 ± 2.1)%, respectively. Sperm ultrastructure of the tree shrew was examined by scanning electron microscopy and transmission electron microscopy. Tree shrew sperm had a round or oval shaped head of approximately 6.65×5.82 µm, and midpiece, principal piece, tail, and total sperm lengths were 13.39, 52.35, 65.74, and 73.05 µm, respectively. The mitochondria in the midpiece consisted of approximately 48 gyres and had a 9+9+2 axonemal pattern. After freezing and thawing, sperm showed partly intact acrosomes and plasma membrane defects, and sperm breakages, twists, and swellings were found. The tree shrew had similar ultrastructure with other mammalians except for the mitochondria number and the sperm size. Ultrastructural alteration is still the main cause resulting in poor sperm after cryopreservation.

[Effects of some extenders and monoamines on sperm cryopreservation in tree shrews (Tupaia belangeri)].

The tree shrew may be an important experimental animal for disease models in humans. The effects of some extenders and momamines on sperm cryopreservation will provide helpful data for experimentation of strains and conservation of genetic resources in tree shrews. Epididymal sperm were surgically harvested from male tree shrews captured around Kunming, China and sperm motility, acrosome integrity and fertility were assessed during cryopreservation. In Experiment 1 eight extenders (TTE, TCG, TCF, TTG, BWW, BTS, DM, and SR) supplemented with 0.4 mol/L DMSO were used to dilute the sperm: only TTE, DM and SR showed no differences in motility and acrosome integrity compared to fresh controls after equilibration. After freezing and thawing, sperm in any extender showed lower motility than fresh control and sperm in DM showed higher motility than other groups. However, BWW produced the lowest motility. For acrosome integrity, TTE and DM showed higher than BWW, BTS and SR after equilibration. The parameter in DM was higher than other groups (except TTE) after thawing. In Experiment 2 four penetrating cryoprotectant agents (CPA) [dimethyl-formamide (DF), formamide (F), dimethylacetamide (DA), and acetamide (A)] at 0.2 mol/L, 0.4 mol/L, 0.8 mol/L, and 1.2 mol/L, respectively were added to the DM extender. Motility showed no difference among CPA groups and non-CPA group (control) after equilibration, but all thawed sperm showed lower values in motility and acrosome integrity than pre-freezing groups. However, sperm in 0.8 mol/L DF and 0.4 mol/L DMSO showed higher values in both parameters than that in other CPA groups (P>0.05). In Experiment 3 the fertilization rate of oocytes inseminated with 0.4mol/L DMSO (50%) were higher than that with 0.8mol/L DF (16%). In conclusion, non-ion extenders supplemented with egg yolk may be better for sperm cryopreservation in tree shrews and cryoprotectant effects of monoamines agents should be further studied in this species.

Prevalence and risk factors for human papillomavirus infection among Chinese ethnic women in southern of Yunnan, China

ABSTRACT Background: Dai is a major Chinese ethnic minority group residing in rural areas of the southern part of Yunnan. However, no data exist on the Human Papillomavirus (HPV) prevalence and genotype distribution among Dai women. Method: A total of 793 participants (Dai = 324, Han = 251, other ethnic = 218) were included in this study. PCR was performed to detect the HPV-positive samples, and genotyping was performed with an HPV Geno-Array. Result: The overall HPV prevalence was very low among Dai women compared to the others. The prevalence of high-risk-HPV infections was significantly higher (p = 0.001) among other ethnic women (22.0%) than that among Han (13.1%) and Dai women (7.1%). The overall HPV, high-risk-HPV, single and multiple infection prevalence among rural women were 12.9%, 12.1%, 12.3%, and 0.5%, respectively. HPV-16 (5.5%) was shown to be the most prevalent genotype, followed by HPV-52 (2.6%) and HPV-58 (2.4%). Urban women had relatively higher rates of overall HPV (16.0%), high-risk-HPV (14.1%), single genotype (11.9%), and multiple genotype (4.1%) infections. In urban women, HPV-52 (3.6%) was the most prevalent genotype, followed by HPV-39 (2.7%) and HPV-16 (1.2%). In the urban area, HPV prevalence was highest in women aged <29 years, but declined with increasing age. However, in rural women, the highest HPV prevalence was observed among women at older age (>50 years). Education and smoking habit were significantly associated with HPV infection among both rural and urban area women (p < 0.001). Conclusion: The prevalence and genotype distribution of HPV varied among ethnic women in urban and rural area of Yunnan Province.