[Correlation between differential expression of microRNA and quality of embryos].

OBJECTIVE: To explore the correlation between microRNA (miRNA) differential expression and quality of embryo. METHODS: The miRNA expression profiles of 8 blastocysts were detected by a TaqMan microRNA array, and miRNAs with a stable expression were selected. Additional blastocysts were selected, and the candidate miRNA was detected by real-time PCR. Meanwhile, chromosomal abnormalities of the embryos were detected by using next-generation sequencing, and the results were compared. RESULTS: The expression of mir-720, mir-372, mir-886-3p and mir-512-3p was higher than that of miR-145, which suggested that mir-720, mir-372, mir-886-3p and mir-512-3p are related to early embryo development. The expression of miR-145 and mir-886-3p were significantly lower in the normal chromosome group. With the threshold values of above 9 and 3 for the relative expression of miR-145 and mir-886-3p, respectively, there was no embryo without a chromosomal abnormality. CONCLUSION: There is a correlation between the expression level of specific miRNA and chromosomal abnormalities of embryos, which may be used as a novel biomarker for embryo selection.

[Data analysis of non-invasive prenatal testing based on special loci in cell-free fetal DNA].

OBJECTIVE To analyze the data of non-invasive prenatal testing based on specific loci of circulating cell-free fetal DNA (cffDNA). METHODS Selected loci of target chromosomes were analyzed by sequence capture and sequencing. Meanwhile, 600 loci were selected from other chromosomes for determining the concentration of cffDNA. RESULTS A total of 768 specific loci were captured on chromosomes 21 and 18, and used to determine whether the two were abnormal. When the minimum concentration of detected cffDNA was set at 3% and the threshold of Z score was set to [-6,6], the specificity of the analysis was 99.37% and the sensitivity was 100%. CONCLUSION A reliable, convenient and low-cost analytical method has been developed. The method requires less sequencing data for non-invasive prenatal testing, and can accurately detect abnormalities of fetal chromosomes 21 and 18, and simultaneously determine the concentration of cffDNA.

The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection.

With the development and clinical application of genomics, more and more concern is focused on single-cell sequencing. In the process of single-cell sequencing, whole genome amplification is a key step to enrich sample DNA. Previous studies have compared the performance of different whole genome amplification (WGA) strategies on Illumina sequencing platforms, but there is no related research aimed at Ion Proton platform, which is also a popular next-generation sequencing platform. Here by amplifying cells from six cell lines with different karyotypes, we estimated the data features of four common commercial WGA kits (PicoPLEX WGA Kit, GenomePlex Single Cell Whole Genome Amplification Kit, MALBAC Single Cell Whole Genome Amplification Kit, and REPLI-g Single Cell Kit), including median absolute pairwise difference, uniformity, reproducibility, and fidelity, and examined their performance of copy number variation detection. The results showed that both MALBAC and PicoPLEX could yield high-quality data and had high reproducibility and fidelity; and as for uniformity, PicoPLEX was slightly superior to MALBAC.

MicroRNA-581 promotes hepatitis B virus surface antigen expression by targeting Dicer and EDEM1.

Hepatitis B virus surface antigen (HBsAg) is an important risk factor for hepatocellular carcinoma (HCC) and is downregulated during hepatocarcinogenesis. MicroRNAs (miRNAs) are frequently deregulated in HCC tissues. However, whether the deregulation of certain miRNAs in HCC has an impact on HBsAg expression remains unclear. We found here that microRNA-581 (miR-581), which is deregulated during hepatocarcinogenesis, promoted HBsAg expression. Additionally, miR-581 targeted Dicer and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1) and repressed their expression. Although Dicer cannot process HBV transcripts, Dicer knockdown led to increased HBsAg secretion, most likely due to a reduction in the levels of Dicer-processed 7SL RNA fragments. Moreover, Dicer-processed 7SL RNA fragments partially inhibited the ability of miR-581 to stimulate HBsAg expression. Furthermore, we found that forced EDEM1 expression inhibited miR-581-mediated induction of HBsAg. Finally, transfection of miR-581 into HepG2.2.15 cells promoted cell proliferation and led to upregulation of genes involved in development, cell proliferation and protein secretion. Altogether, we conclude that miR-581 promotes HBsAg expression by targeting Dicer and EDEM1. Our findings suggest that downregulation of miR-581 during hepatocarcinogenesis may lead to a reduction in HBsAg expression and impede HCC development.

Hepatitis B surface antigen inhibits MICA and MICB expression via induction of cellular miRNAs in hepatocellular carcinoma cells.

Hepatitis B surface antigen (HBsAg) seropositivity is an important risk factor for hepatocellular carcinoma (HCC), and HBsAg-transgenic mice have been reported to spontaneously develop HCC. The major histocompatibility complex class I-related molecules A and B (MICA and MICB) are NKG2D ligands that play important roles in tumor immune surveillance. In the present study, we found that HBsAg overexpression in HepG2 cells led to upregulation of 133 and downregulation of 9 microRNAs (miRNAs). Interestingly, several HBsAg-induced miRNAs repressed the expression of MICA and MICB via targeting their 3'-untranslated regions. In addition, the expression of MICA and MICB was significantly reduced upon HBsAg overexpression, which was partially restored by inhibiting the activities of HBsAg-induced miRNAs. Moreover, HBsAg-overexpressing HCC cells exhibited reduced sensitivity to natural killer cell-mediated cytolysis. Taken together, our data suggest that HBsAg supresses the expression of MICA and MICB via induction of cellular miRNAs, thereby preventing NKG2D-mediated elimination of HCC cells.

Decreased dicer expression enhances SRP-mediated protein targeting.

We have shown that Dicer processes 7SL RNA into different fragments ranging from ∼20 to more than 200 nucleotides. Here we addressed the molecular functions of these 7SL RNA fragments and found that some of them functioned as dominant-negative regulators of the full-length 7SL RNA, interfering with signal recognition particle (SRP) complex formation. Transfection of these 7SL RNA fragments inhibited the expression of cell surface glycoproteins, the targeting of a reporter protein to the endoplasmic reticulum, and the secretion of secreted alkaline phosphatase. These results suggest that some Dicer-processed 7SL RNA fragments interfered with SRP-mediated protein targeting. Moreover, we showed that Dicer knockdown enhanced SRP-mediated protein targeting and that transfection of a mixture of the 7SL RNA fragments partially restored this effect. Our data indicate that Dicer can fine-tune the efficiency of SRP-mediated protein targeting via processing a proportion of 7SL RNA into fragments of different lengths.

Dicer-dependent biogenesis of small RNAs derived from 7SL RNA.

It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting. In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.

MicroRNA-34a inhibits migration and invasion of colon cancer cells via targeting to Fra-1.

MicroRNA-34a (miR-34a), a transcriptional target of p53, is a well-known tumor suppressor gene. Here, we identified Fra-1 as a new target of miR-34a and demonstrated that miR-34a inhibits Fra-1 expression at both protein and messenger RNA levels. In addition, we found that p53 indirectly regulates Fra-1 expression via a miR-34a-dependant manner in colon cancer cells. Overexpression of miR-34a strongly inhibited colon cancer cell migration and invasion, which can be partially rescued by forced expression of the Fra-1 transcript lacking the 3'-untranslated region. The expression of matrix metalloproteinase (MMP)-1 and MMP-9, two enzymes involved in cell migration and invasion, was decreased in miR-34a-transfected cells, and this can be rescued by Fra-1 overexpression. Moreover, we found that miR-34a was downregulated in 25 of 40 (62.5%) colon cancer tissues, as compared with the adjacent normal colon tissues and that the expression of miR-34a was correlated with the DNA-binding activity of p53. Unexpectedly, the DNA-binding activity of p53 was not inversely correlated with Fra-1 expression, and a significant statistical inverse correlation between miR-34a and Fra-1 expression was only observed in 14 of 40 (35%) colon cancer tissues. Taken together, our in vitro data suggest that p53 regulates Fra-1 expression, and eventually cell migration/invasion, via a miR-34a-dependent manner. However, in vivo data indicate that the p53-miR-34a pathway is not the major regulator of Fra-1 expression in human colon cancer tissues.

[Dynamic research of density on Platycodon grandiflorum dry material accumulation and platycodin D content in Platycodon grandiflorum].

OBJECTIVE: To study the dynamic changes of dry material accumulation and platycodin D content in Platycodon grandiflorum in different planting densities. METHOD: Five different planting densities M1 (4 cm x 25 cm), M2 (6 cm x 25 cm), M3 (8 cm x 25 cm), M4 (10 cm x 25 cm) and M5 (12 cm x 25 cm) were designed in the plot experiment. The individual and colony biomass accumulation, dry material distribution, root yield and platycodin D content were measured in different stage. RESULT: In a certain density range the individual biomass in P. grandiflorum obviously declined with increasing density with the exception of biomass M2 > biomass M3. On the contrary, the colony biomass increased with the increasing density. Dry material accumulation in each organ in P. grandiflorum in different planting densities showed significance (P<0.05). The dry material distribution in organs in the different planting densities showed significance (P<0.05), and the dry material distribution in flower and fruit reached the minimal level in M2, in the same planting density the distribution in root reached the maximal; The dry material in stem, flower and fruit obviously declined with the increasing density, while the dry material in leaf increased. The individual root output increased with the increasing density, and it reached the highest in M2. The colony root yield increased with the increasing density. The platycodin D content in P. grandiflorum reached the highest in M2. CONCLUSION: The result showed that a suitable planting density is very important to P. grandiflorum dry material accumulation and distribution, root yield, platycodin D content and colony yield.

[Relationship between photosynthetic characteristics and environmental factors in leaves of Pueraria lobata].

OBJECTIVE: To study the relationship between photosynthetic characteristics and environmental factors in leaves of P. lobata. METHOD: Photosynthetic characteristics and environmental factors were measured by using CIRAS-2 portable photosynthesis system. RESULT: The apparent quantum yield in leaves was 0.0173 micromol CO2 x micromol(-1) photon. The dark respiration rate was 2.9333 micromol x m(-2) x s(-1). The light compensation point of photosynthesis was 180 micromol x m(-2) x s(-1). The light saturation point was 1600 micromol x m(-2) x s(-1). The carboxylation efficiency was 0.0338 micromol x m(-2) x s(-1). The light respiration rate was 2.5 micromol x m(-2) x s(-1). The CO2 compensation point was 100 micromol x mol(-1), The CO2 saturation point was 1 600 micromol x mol(-1). CONCLUSION: Photo flux density and air temperature are major environmental factors influencing diumal changes of net photosynthetic rate.