Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.

BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.

Identification of miRNAs associated with dark-induced senescence in Arabidopsis.

BACKGROUND: microRNAs (miRNAs) are endogenous small (~21 nucleotide) single-stranded non-coding RNAs that typically function by guiding cleavage of target genes. To find the miRNAs that may be involved in dark-induced leaf senescence, we identified miRNAs by microarray platform using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs). RESULTS: We found that the expressions of 137 miRNAs (P < 0.01, signal intensity >0) were significantly changed both in DP and IDL leaves. Among them, the expression levels of 44 miRNAs were relative higher than others (P < 0.01, signal intensity > 500). Of these differentially expressed miRNAs, 6 miRNAs (miR319a, 319c, miR159, miR164a, miR164c and miR390a) have been previously reported to be involved in dark-induced leaf senescence, and the remaining 38 miRNAs have not been implicated in leaf senescence before. Target genes of all 44 miRNAs were predicted, and some of them, such as NAC1, At3g28690, At2g17640 and At2g45160, were found in the Leaf Senescence Database (LSD). GO and KEGG analysis of 137 miRNAs showed that the predicted target genes were significantly enriched in transcription regulation, development-related biological processes and metabolic pathways. Expression levels of some of the corresponding miRNA targets (At1g73440, At2g03220 and At5g54810) were analysed and found to be significantly different in DP/IDL than that in WT. CONCLUSIONS: A microarray analysis about dark-induced miRNAs involved in leaf senescence are present here. Further expression analysis revealed that some new founding miRNAs maybe regulate leaf senescence in Arabidopsis, and the findings highlight the important role of miRNAs in dark-induced leaf senescence.

lncRNA Gm15290 promotes cell proliferation and invasion in lung cancer through directly interacting with and suppressing the tumor suppressor miR-615-5p.

Long non-coding RNAs (lncRNAs) have been involved in occurrence and progression of multiple cancers. In the present study, we investigated the role of lncRNA Gm15290 in the proliferation and invasion of non-small cell lung cancer (NSCLC) cells. First, we found that lncRNA Gm15290 was markedly up-regulated in tumor tissues from NSCLC patients and NSCLC cell lines, compared with adjacent normal tissues and normal lung cell line HBE respectively. Then, different concentrations of pcDNA-Gm15290 expression vector and Gm15290 siRNA were respectively transfected into A549 NSCLC cells. Our results showed that overexpression of Gm15290 significantly increased the proliferation and invasion of A549 cells and suppressed cell apoptosis. Knockdown of Gm15290 suppressed A549 cell proliferation and invasion and promoted cell apoptosis. Subsequently, we explored the underlying mechanism through which Gm15290 promoted cell proliferation and invasion. The output of RNA hybrid bioinformatic tool revealed that Gm15290 potentially interacted with tumor suppressor miR-615-5p which displayed an opposite expression pattern in the cell lines and a strong negative correlation with the levels of Gm15290 in NSCLC patients (r2 = 0.9677, P<0.0001). The results of RNA pull-down assays confirmed that Gm15290 directly bound with miR-615-5p Gm15290 negatively regulated the expression of miR-615-5p and increased the protein levels of miR-615-5p target genes, including IGF2, AKT2, and SHMT2 Moreover, miR-615-5p mimic could antagonize the promoting effect of Gm15290 on cell proliferation and invasion.

A review of S100 protein family in lung cancer.

S100 protein family, representing 25 relatively small calcium binding proteins, has been reported to be involved in multiple stages of tumorigenesis and progression. These proteins are considered having potential value to be adopted as novel biomarkers in the detection and accurate prediction of many kinds of tumors, including lung cancer. As the one having the highest morbidity and mortality among all cancers, lung carcinoma is still occult for detection, especially at early stage. S100 proteins take participation in the lung neoplasia through playing intracellular and/or extracellular functions, therefore getting involved in a variety of biological processes such as differentiation, proliferation, and migration. A few members have also been testified to modulate TGF-ß/Smad-3 mediated transcriptional activity of target genes involved in tumor promotion. In addition to that, a number of proteins in this family have already been reported to experience an abnormal trend in lung cancer at cell, serum and tissue levels. Thus, S100 proteins may serve as effective biomarkers for suspected or already diagnosed lung cancer patients. In future, S100 protein family might be applied as therapeutic targets in clinical treatment of lung cancer. In this review, we firstly summed up the biological and clinical evidence connecting S100 proteins and lung cancer, which has not been summarized before.

LncRNA Gm15290 Promotes Cell Proliferation and Invasion in Non-Small Cell Lung Cancer Through Directly Interacting With and Suppressing the Tumor Suppressor miR-615-5p.

Long non-coding RNAs (lncRNAs) have been involved in occurrence and progression of multiple cancers. In this study, weinvestigated the role of lncRNA Gm15290 in the proliferation and invasion of non-small cell lung cancer (NSCLC) cells. First, we found that lncRNA Gm15290 was markedly upregulated in tumor tissues from NSCLC patients and NSCLC cell lines, compared with adjacent normal tissues and normal lung cell line HBE respectively. Then, different concentrations of pcDNA-Gm15290 expression vector and Gm15290 siRNA were respectively transfected into A549 NSCLC cells. Our results showed that overexpression of Gm15290 significantly increased the proliferation and invasion of A549 cells, and suppressed cell apoptosis. Knockdown of Gm15290 suppressed A549 cell proliferation and invasion, and promoted cell apoptosis. Subsequently, we explored the underlying mechanism through which Gm15290 promoted cell proliferation and invasion. The output of RNAhybrid bioinformatic tool revealed that Gm15290 potentially interacted with tumor suppressor miR-615-5p which displayed an opposite expression pattern in the cell lines and a strong negative correlation with the levels of Gm15290 in NSCLC patients (r2 = 0.9677, P < 0.0001). The results of RNA pull-down assays confirmed that Gm15290 directly bound with miR-615-5p. Gm15290 negatively regulated the expression of miR-615-5p and increased the protein levels of miR-615-5p target genes, including IGF2, AKT2 and SHMT2. Moreover, miR-615-5p mimic could antagonize the promoting effect of Gm15290 on cell proliferation and invasion.