Research of Fluridone's Effects on Growth and Pigment Accumulation of Haematococcus pluvialis Based on Transcriptome Sequencing.

Haematococcus pluvialis has high economic value because of its high astaxanthin-producing ability. The mutation breeding of Haematococcus pluvialis is an important method to improve the yield of astaxanthin. Fluoridone, an inhibitor of phytoene dehydrogenase, can be used as a screening reagent for mutation breeding of Haematococcus pluvialis. This study describes the effect of fluridone on the biomass, chlorophyll, and astaxanthin content of Haematococcus pluvialis at different growth stages. Five fluridone concentrations (0.00 mg/L, 0.25 mg/L, 0.50 mg/L, 1.00 mg/L, and 2.00 mg/L) were set to treat Haematococcus pluvialis. It was found that fluridone significantly inhibited the growth and accumulation of astaxanthin in the red dormant stage. In addition, transcriptome sequencing was used to analyze the expression of genes related to four metabolic pathways in photosynthesis, carotenoid synthesis, fatty acid metabolism, and cellular antioxidant in algae after fluridone treatment. The results showed that six genes related to photosynthesis were downregulated. FPPS, lcyB genes related to carotenoid synthesis are downregulated, but carotenoid ß-cyclic hydroxylase gene (LUT5), which plays a role in the conversion of carotenoid to abscisic acid (ABA), was upregulated, while the expression of phytoene dehydrogenase gene did not change. Two genes related to cell antioxidant capacity were upregulated. In the fatty acid metabolism pathway, the acetyl-CoA carboxylase gene (ACACA) was downregulated in the green stage, but upregulated in the red stage, and the stearoyl-CoA desaturase gene (SAD) was upregulated. According to the transcriptome results, fluridone can affect the astaxanthin accumulation and growth of Haematococcus pluvialis by regulating the synthesis of carotenoids, chlorophyll, fatty acids, and so on. It is expected to be used as a screening agent for the breeding of Haematococcus pluvialis. This research also provides an experimental basis for research on the mechanism of astaxanthin metabolism in Haematococcus pluvialis.

Characterization of deoxyribonucleic methylation and transcript abundance of sex-related genes during tempera ture-dependent sex determination in Mauremys reevesii†.

A number of genes relevant for sex determination have been found in species with temperature-dependent sex determination. Epigenetics play a key role in sex determination, but characterization of deoxyribonucleic acid methylation of sex-related genes on temperature-dependent sex determination remains unclear. Mauremys reevesii is a typical species with temperature-dependent sex determination. In this study, we analyzed the Cytosine Guanine (CpG) methylation status of the proximal promoters, the messenger ribonucleic acid expression patterns and the correlation between methylation and expression levels of Aromatase, Forkhead box protein L2, Doublesex and mab3-related transcription factor 1, sex-determining region on Y chromosome-box 9, and anti-Müllerian hormone, which are key genes in sex determination in other species. We also analyzed the expression level of genes that encode enzymes involved in methylation and demethylation. The expression levels of Aromatase and Forkhead box protein L2 at the female producing temperature were higher than those at the male producing temperature; the expression levels of Doublesex and mab3-related transcription factor 1, sex-determining region on Y chromosome-box 9, and anti-Müllerian hormone were higher at MPT. The expression of some genes involved in methylation and demethylation is significantly different between male producing temperature and female producing temperature. The expression of messenger ribonucleic acid of genes involved in deoxyribonucleic acid methylation and demethylation affected by temperature, together with other factors, may change the methylation level of the regulatory regions of sex-related genes, which may further lead to temperature-specific expression of sex-related genes, and eventually affect the differentiation of the gonads.

Transcriptome sequencing and comparative analysis of adult ovary and testis identify potential gonadal maintenance-related genes in Mauremys reevesii with temperature-dependent sex determination.

Mauremys reevesii is a classical organism with temperature-dependent sex determination (TSD). Gonad development in early life has recently received considerable attention but gonadal maintenance after sex differentiation in turtles with TSD remains a mystery. In this study, we sequenced the transcriptomes for the adult testis and ovary using RNA-seq, and 36,221 transcripts were identified. In total, 1,594 differentially expressed genes (DEGs) were identified where 756 DEGs were upregulated in the testis and 838 DEGs were upregulated in the ovary. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the TGF-beta signaling pathway and Hedgehog signaling pathway have important roles in testis maintenance and spermatogenesis, whereas the Hippo signaling pathway and Wnt signaling pathway are likely to participate in ovary maintenance. We determined the existence of antagonistic networks containing significant specific-expressed genes and pathways related to gonadal maintenance and gametogenesis in the adult gonads of M. reevesii. The candidate gene Fibronectin type 3 and ankyrin repeat domains 1 (FANK1) might be involved with the regulation of testis spermatogenesis.

[miR-593 inhibits proliferation of colon cancer cells in vitro by down-regulating PLK1].

OBJECTIVE: To explore the role of miR-593 in regulating the proliferation of colon cancer cells and the molecular mechanism. METHODS: Bioinformatics analysis identified PLK1 as the possible target gene of miR-593. Luciferase assay was employed to verify the binding between miR-593 and PLK1, and qRT-PCR and Western blotting were used to verify that PLK1 was the direct target gene of miR-593. CCK-8 assay was performed to test the hypothesis that miR-593 inhibited the proliferation of colon cancer cells by targeting PLK1. RESULTS: Luciferase assay identified the specific site of miR-593 binding with PLK1. Western blotting showed a significantly decreased expression of PLK1 in the colon cancer cells transfected with miR-593 mimics and an increased PLK1 expression in the cells transfected with the miR-593 inhibitor as compared with the control cells (P < 0.05). The results of qRT-PCR showed no significant differences in the expression levels of PLK1 among the cells with different treatments (P > 0.05). The cell proliferation assay showed opposite effects of miR-593 and PLK1 on the proliferation of colon cancer cells, and the effect of co-transfection with miR-593 mimic and a PLK1-overexpressing plasmid on the cell proliferation was between those in PLK1 over-expressing group and miR-593 mimic group. CONCLUSIONS: miR-593 inhibits the proliferation of colon cancer cells by down-regulating PLK1 and plays the role as a tumor suppressor in colon cancer.

Detecting coevolution of positively selected in turtles sperm-egg fusion proteins.

Physically interacting sperm-egg proteins have been identified using gene-modified animals in some mammal species. Three proteins are essential for sperm-egg binding: Izumo1 on the sperm surface, and JUNO and CD9 on the egg surface. Most proteins linked to reproductive function evolve rapidly among species by positive selection, and have correlated evolutionary rates to compensate for changes on both the sperm and egg. Up to now, interactions between sperm and egg proteins have not been identified in non-mammalian vertebrates, such as turtles that have interspecific hybrids that can produce surviving F1 generations. To explore the potential physical interactions of sperm-egg proteins in turtle species, the coding region of Izumo1, JUNO, and CD9 homologous genes (named Tu-Izumo1, Tu-JUNO, and Tu-CD9) in six turtle species (Mauremys reevesii, M. mutica, M. sinensis, Cistoclemmys flavomarginata, Platysternon megacephalum and Chrysemys picta bellii) were identified, amplified, and sequenced, and tissue-specific expression was analyzed in M. reevesii. We constructed phylogenetic trees and analyzed the signatures of coevolution between sperm-egg protein pairs using MirrorTree Server and linear regression methods. The results showed that Tu-Izumo1, Tu-JUNO, and Tu-CD9 proteins have correlated evolutionary rates, and that the area where Tu-Izumo1 interacts with Tu-JUNO has only one positive selection site in some turtle species. These results suggest there is a potential interaction between Tu-Izumo1 and Tu-JUNO among turtles that can interbreed, and that a significantly lower positive selection in the interaction region may be one of the reasons why turtle hybrids are so common. Further studies are required to uncover Tu-Izumo1, Tu-JUNO and Tu-CD9 protein biological functions during gamete fusion.

miR-21-5p induces cell proliferation by targeting TGFBI in non-small cell lung cancer cells.

The mortality rate of non-small cell lung cancer (NSCLC) remains high worldwide. miR-21-5p plays an important part in many cancer types, including NSCLC. However, the effect of miR-21-5p in NSCLC tumorigenesis remains poorly understood. The present study investigated whether miR-21-5p promoted NSCLC cell proliferation in vitro. In order to study the molecular mechanism by which miR-21-5p contributes to NSCLC progression, three bioinformatics algorithms were used to predict the genes which miR-21-5p targeted. TGFBI was identfieid as a putative direct target in NSCLC cells via the luciferase reporter assay. Furthermore, miR-21-5p downregulated TGFBI protein expression by a post-transcriptional mechanism via western blotting and a reverse transcription-quantitative polymerase chain reaction analysis. Finally, TGFBI exhibited opposing effects to those of miR-21-5p on NSCLC cells, suggesting that miR-21-5p may promote cell proliferation by negative regulation of TGFBI. These results suggest miR-21-5p promote the proliferation of NSCLC cells via inhibiting TGFBI expression.

Single-molecule long-read transcriptome profiling of Platysternon megacephalum mitochondrial genome with gene rearrangement and control region duplication.

Platysternon megacephalum is the sole living representative of the poorly studied turtle lineage Platysternidae. Their mitochondrial genome has been subject to gene rearrangement and control region duplication, resulting in a unique mitochondrial gene order in vertebrates. In this study, we sequenced the first full-length turtle (P. megacephalum) liver transcriptome using single-molecule real-time sequencing to study the transcriptional mechanisms of its mitochondrial genome. ND5 and ND6 anti-sense (ND6AS) forms a single transcript with the same expression in the human mitochondrial genome, but here we demonstrated differential expression of the rearranged ND5 and ND6AS genes in P. megacephalum. And some polycistronic transcripts were also reported in this study. Notably, we detected some novel long non-coding RNAs with alternative polyadenylation from the duplicated control region, and a novel ND6AS transcript composed of a long non-coding sequence, ND6AS, and tRNA-GluAS. These results provide the first description of a mtDNA transcriptome with gene rearrangement and control region duplication. These findings further our understanding of the fundamental concepts of mitochondrial gene transcription and RNA processing, and provide a new insight into the mechanism of transcription regulation of the mitochondrial genome.

miR-24-3p promotes cell migration and proliferation in lung cancer by targeting SOX7.

Lung cancer (LC) is one of the leading causes of cancer-related death in the world. miR-24-3p plays critical roles in many cancer types, including LC. In this study, we first investigated whether miR-24-3p promoted LC cell migration and proliferation in vitro. We used three bioinformatics algorithms to predict the miR-24-3p target gene to study the molecular mechanism by which miR-24-3p contributes to LC progression. Then, we used the luciferase reporter assay to identify whether SOX7 was a direct target of miR-24-3p. Moreover, Western blotting and a quantitative real time-polymerase chain reaction analysis showed that miR-24-3p downregulated SOX7 protein expression by a post-transcriptional mechanism. Finally, we determined that SOX7 had opposing effects to those of miR-24-3p on LC cell proliferation and migration, suggesting that miR-24-3p promotes cell proliferation and migration by directly targeting SOX7. Furthermore, miR-24-3p accelerated tumor growth in xenograft mice by targeting SOX7. These results provide the first clue that miR-24-3p could play a role as an oncomiR in LC by regulating SOX7.

Complete mitochondrial genome of the Cyclemys pulchristriata (Chelonia: Geoemydidae).

In this study, we obtained complete mitochondrial genome sequence of Cyclemys pulchristriata. The mitochondrial genome reaches a length of 16,527 bp, containing 13 protein-coding genes (PCGs), 22tRNA genes, 2 rRNA genes and 1 control region. All protein-coding genes initiate with ATG as start codon, except for CO1 started with GTG. Most protein-coding genes ended by TAA as stop codon. Interestingly, there is an extra nucleotide A insertion in ND3 gene in C. pulchristriata. This study provides information on the genetic resources of C. pulchristriata that will contribute to protect this species.