Effect of GREM[STHZ]1 on the proliferation and metastasis of gastric cancer cells / 临床检验杂志

Objective@#To detect the expression of GREM1 gene in gastric cancer cells, investigate its effects on the biological characteristics of gastric cancer cells and evaluate its application value in the diagnosis and prognosis of gastric cancer. @*Methods@#The expression difference of GREM1 in gastric cancer tissues and adjacent normal tissues was analyzed by the database, and the correlation of GREM1 expression levels with the prognosis of gastric cancer patients was evaluated. The expression levels of GREM1 protein in gastric cancer cell lines were detected by western blot. After GREM1 gene in AGS cells was silenced, its effects on the proliferation, migration, epithelial-mesenchymal transition (EMT) and Wnt/β-catenin pathway of AGS cells were detected by the colony formation assay, Transwell and Western blot, respectively. @*Results@#Kaplan-Meier analysis showed that the patients with high expression of GREM1 gene had low overall survival (OS) and progression-free survival (PFS). The expression level of GREM1 protein in AGS cells was the highest in all gastric cancer cell lines (1.967 ± 0.056). The analysis of colony formation assay, Transwell and Western blot showed that the silencing of GREM1 gene could decrease the proliferation and migration of gastric cancer cells (t=22.00; t=29.60; P＜0.01), increase the expression of E-cadherin (t=10.65, P＜0.01), and decrease the expressions of ZEB1 and MMP2 (t=10.74; t=13.67; P＜0.01) and the expressions of β-catenin, Cyclin D1, c-myc, p-GSK3β and PCNA in the Wnt/β-catenin pathway (t=12.65; t=16.21; t=8.74; t=7.75; t=8.42; P＜0.01). @*Conclusion@#GREM1 may induce EMT by activating the Wnt/β-catenin pathway, [JP2]and promote the metastasis and growth of tumors, which may be used as a new molecular diagnostic and prognostic marker for gastric cancer.

Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

The expression and function of miRNAs in human endometrial cells / 天津医药

Objective To explore the expression and function of miR-125b, miR-30b and miR-424 in endometrial cells. Methods Human endometrial samples were obtained in natural cycles and stimulating cycles. Endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) were isolated and confirmed by immunofluorescence. The expressions of miR-125b, miR-30b and miR-424 were detected by real-time PCR. Results The expression levels of miR-125b, miR-30b and miR-424 were higher in proliferative phase in ESCs than those in EECs. And in EECs, the expression levels of miR-125b, miR-30b and miR-424 were significantly up-regulated in secretory phase than in proliferative phase, while it was stable in ESCs. In addition, the expressions of miR-125b in EECs and miR-30b were increased in ESCs in women with elevated progesterone on the day of HCG administration than those of the control. The target genes of miR-125b, miR-30b and miR-424 mainly participated in cell migration and motion, cell-cell adherens junction and Wnt signaling pathway. Conclusion miR-125b, miR-30b and miR-424 were differently expressed in endometrial cells in different phases, and may participate in regulation of endometrial receptivity.

Research progress of sequencing method for animal mitochondrial genome / 天津医药

Mitochondria, the power house of cells, are important organelles in eukaryotic cells. Having their own unique and complete DNA (mtDNA) and genetic system, mitochondria play an essential role in cellular energy metabolism, intracel?lular signaling and apoptotic pathways, as well as many other biological functions, which are closely related with cellular met?abolic network. A disruption of mitochondrial genes can therefore result in mitochondrial dysfunction and human diseases, thus they have been widely used in molecular biology, development biology, genetics, forensic identification and clinical diag?nosis. Consequently, sequencing mitochondrial genome has shown great significance in mitochondrial structure and function research. In this review, research progress in mitochondrial genome sequencing method is summarized, mainly focusing on Sanger sequencing, long-PCR and next-generation sequencing. Also rolling circle amplification and indirect sequencing of mtDNA are reviewed. The ambiguities caused by numts in indirect sequencing are mentioned and resolved.

Study on expression profile of mRNA in brain of pronuclear transfer mice / 天津医药

Objective To investigate the expression profile of mRNAs in brain samples collected from pronuclear transfer (PNT) mice. Methods Female CD-1 mice were superovulated, and zygotes were collected after mating with adult male mice. Zygotes with two pronuclei were selected for pronuclear transfer manipulation, and then the reconstructed zygotes were transferred into the oviduct of pseudopregnant female mice. The infant mice obtained from pronuclear transfer were called PNT group, while the embryoes that were not performed pronuclear transfer was regarded as control group. Total RNA were extracted from brain samples of both PNT and control mice, and cDNA were labeled with fluorescent dye. Genes that were differentially expressed were identified using the Agilent mouse mRNA array. Gene ontology analysis and pathway analysis were also completed. Results Compared with control group, 392 mRNAs were expressed differentially, which showed more than 2.0 times variation and statistical significance, accounting for 1.7% of all mRNAs. Among those 366 mRNAs were up-regulated and 26 mRNAs were down-regulated. Eleven mRNAs came to 4.0 times variation in total. Gene ontology analysis indicated that differentially expressed genes were significantly enriched in alternative mRNA splicing, small GTPase mediated signal transduction, regulation of insulin receptor signaling pathway, hydrolase activity, transmembrane transporter activity and pyrophosphatase activity. Significant enriched pathway terms contained ion channel transport, fatty acid metabolism, butanoate metabolism, triacylglycerol and ketone body metabolism. Conclusion Pronuclear transfer might influence some key metabolism process in mouse brain.

Analysis of the deafness gene screening results from newborns in Shijiazhuang / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To build information repository of the carrying rate of neonatal deafness gene in Shijiazhuang.@*METHOD@#Blood samples were collected from the heel in 3-days neonates. Mutations of the deafness related genes were detected by the method of fluorescent PCR. Neonates received the detection of 6 mutation sites from 3 genes, including GJB2 (235delC, 299-300delAT), SLC26A4 (IVS7-2A> G, 2168A> G), mitochondrial DNA12S rRNA(1494C>T,1555A>G).@*RESULT@#There were 384 neonates who carried mutations among 421 subjects and the carrying rate was 4.08%, 158 (1.68%) newborns carried heterozygous mutations and 1 (0.01%) case carried homogeneous mutation of GJB2 (235 delC), 55 (0.58%) neonates carried heterozygous mutations of GJB2 (299-300delAT); 133 (1.41%) neonates carried heterozygous mutations and 1 (0.01%) homogeneous of SLC26A4(IVS7-2A>G),19 (0.20%) newborns carried heterozygous mutations of SLC26A4 (2168A>G). The numbers of neonates who carried homogeneous and heterogeneous mutation of mitochondrial 12S rRNA gene were 14 and 3 with carring rates of 0.15% and 0.03%. Two newborns were found to carry more than one mutation. One carried 235delC, IVS7-2A>G and 1555A>G and another carried 235delC and IVS7-2A>G.@*CONCLUSION@#The main mutational patterns were 235delC from GJB2 gene and IVS7-2A>G from SLC26A4 gene in Shijiazhuang newborns. The carrying rate information repository of neonatal deafness gene has been built preliminarily.