[Circular RNA expression profiles and circRNA-miRNA-mRNA crosstalk in pre-eclamptic placenta].

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.

Extraction of DNA from Sperm Cells in Mixed Stain by Nylon Membrane Bushing Separation Technique.

OBJECTIVES: To establish a novel method for the separation of sperm cells in mixed stain, and to evaluate its application value. METHODS: Totally 40 mixed stain samples were collected from sexual assault cases. Sperm cells were separated by the conventional differential lysis method and the nylon membrane bushing separation technique, respectively. The DNA of sperm cells was extracted with the silicon membrane kit （Forensic DNA Extraction Kit for Soft Tissues）. The PCR amplification was performed using AmpFℓSTR® Identifiler® Plus kit, and the products were electrophoresed by 3500xL genetic analyser. The results of two separation methods were then compared. RESULTS: Complete and single-source male STR genotypes could be obtained from all the 40 mixed stain samples except three samples with minimal residual of female DNA by the nylon membrane bushing separation technique. The STR genotypes of sperm cells could not be detected in 25 samples, which were obtained in 15 samples （seven were of incomplete male STR genotypes, six with residual of female DNA, two were complete and single-source STR genotypes of sperm cells）. CONCLUSIONS: The nylon membrane bushing separation technique developed in present study can be used in the separation of sperm cells in mixed stain, especially for the extraction of a small amount of sperm from a large quantity of female cells, which is inexpensive, rapid and simple.

A functional insertion/deletion polymorphism in the IL1A gene is associated with decreased risk of breast cancer.

The insertion/deletion polymorphism (rs3783553 TTCA/-) in the 3' untranslated region of interleukin-1A (IL1A) has been studied intensively and has been shown to affect tumor risk. We studied the frequency of the IL1A gene polymorphism rs3783553 and evaluated its relationship with breast cancer (BC). A hospital-based case-control study comprising 228 patients with histologically confirmed BC and 241 healthy subjects was conducted. Polymerase chain reaction was used to detect the IL1A rs3783553 polymorphism. The ins/ins (ttca/ttca) genotype was significantly associated with a decreased risk of BC compared with the del/del (-/-) genotype (OR = 0.48, 95% CI = 0.27-0.85). Moreover, the ins (ttca) allele distribution between cases and controls was significantly different from the del (-) allele distribution (OR = 0.74, 95% CI = 0.57-0.96). Thus, the rs3783553 polymorphism is associated with a decreased incidence of breast cancer.

MicroRNA variants and colorectal cancer risk: a meta-analysis.

Colorectal cancer (CRC) is a multi-factorial disease, and genetic background may contribute to its etiology. Single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) may be used as specific markers of predisposition for CRC diagnosis and prevention. In this review, we summarize and discuss recent publications evaluating the roles of miRNA SNPs in CRC. A meta-analysis was also carried out to assess the association between the five most frequently studied miRNA SNPs and CRC risk. No relationship was established between this disease and the three SNPs rs11614913, rs2910164, and rs3746444 in miR-196a-2, miR-146a, and miR-499, respectively. However, polymorphisms of miR-149 (rs2292832; CT vs TT: odds ratio [OR] = 0.816, 95% confidence interval [CI] = 0.691-0.963; CC+CT vs TT: OR = 0.834, 95%CI = 0.715-0.972) and pre-miR-27a (rs895819; GG vs AA: OR = 1.534, 95%CI = 1.148-2.049; GG+AG vs AA: OR = 1.324, 95%CI = 1.066-1.645) were found to be associated with CRC in our analysis. In conclusion, the SNPs rs2292832 in miR-149 and rs895819 in pre-miR-27a were associated with CRC susceptibility, whereas rs11614913, rs2910164, and rs3746444 in miR-196a-2, miR-146a, and miR-499, respectively, were not. Further studies should be carried out to validate these findings.

Association of polymorphism within the interleukin-28 receptor alpha gene, but not in interleukin-28B, with lower urinary tract symptoms (LUTS) in Chinese.

The aim of this study was to determine the relationship between polymorphisms in the IL-28B and IL-28R genes and lower urinary tract symptoms (LUTS) in Chinese patients. Genomic DNA was extracted from 553 whole blood samples from 233 patients with LUTS resulted from benign prostatic hyperplasia and 320 control subjects. The IL-28B rs12979860 and rs8099917, and IL-28Rα rs10903035 and rs11249006 polymorphisms were genotyped using a polymerase chain reaction-restriction fragment length polymorphism assay. For rs10903035, the frequencies of the "G" allele and the "AG/GG" genotypes in the LUTS group were significantly lower than those in the control group ("G" vs "A": OR = 0.655, 95%CI = 0.506-0.849; AG/GG vs "AA": OR = 0.538, 95%CI = 0.379-0.764, respectively). Combined effects analysis of rs12979860 and rs10903035 showed that the "CC+AG/GG" and "CT+AA" genotypes were significantly less frequent in the LUTS group ("CC+AG/GG" vs "CC+AA": OR = 0.553, 95%CI = 0.381-0.801; "CT+AG/GG" vs "CC+AA": OR = 0.429, 95%CI = 0.198- 0.927, respectively). In addition, the combined effects of the rs8099917 and rs10903035 "TT+AG/GG" and "GT+AG/GG" genotypes were also significantly lower in the LUTS group ("TT+AG/GG" vs "TT+AA": OR = 0.569, 95%CI = 0.395-0.821; "GT+AG/GG" vs "TT+AA": OR = 0.318, 95%CI = 0.128-0.788, respectively). Stratification analysis revealed that the frequencies of the rs11249006 "AG/GG" genotypes in the subgroups of size ≤4.11 and IPSS ≤ 28 were significantly higher than those in the subgroups of size >4.11 and IPSS > 28. Therefore, the IL-28Rαgene polymorphism might be involved in the development of LUTS.

Association of a let-7 KRAS rs712 polymorphism with the risk of breast cancer.

Breast cancer (BC) is a common malignancy affecting women, with increasing incidences of this disease in China every year. Recent studies have extensively investigated a single nucleotide polymorphism in the let-7 miRNA binding site of the 3'-untranslated region of KRAS mRNA. The aim of this study was to determine the genotype frequency of the KRAS rs712 polymorphism, and evaluate its effect on BC risk. This hospital-based case-control study comprised 228 patients with histologically confirmed BC and 251 healthy controls. The let-7a KRAS rs712 polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism. We observed no statistically significant association between BC risk and the let-7a KRAS rs712 polymorphism (GT vs GG, OR = 0.98, 95%CI = 0.66-1.46; TT vs GG, OR = 0.78, 95%CI = 0.28-2.21). However, the rs712 polymorphism was significantly associated with the N status of BC patients (GG vs GT/TT, OR = 0.52, 95%CI = 0.30- 0.92; G allele vs T allele, OR = 0.60, 95%CI = 0.37-0.97). We found no association between the let-7 rs712 polymorphism and BC risk. However, the let-7 rs712 G/T polymorphism was discovered to play a potential role in BC tumor metastasis; therefore, it may be employed as a new biomarker or therapy targeted towards resistant tumor metastasis.

[Biomechanics of wedge-osteotomy for kyphosis].

In 100 patients with kyphosis who received wedge-osteotomy, Cobb's degree greater than 90 degrees was noted in 44, of whom 44 patients showed radiologically aortic calcification. Follow-up for more than 1.5 years showed an average correction degree of 57.2 degrees and an average correction rate of 77.2%. Body height increased on the average by 16 cm. We analyzed biomechanics of this operation.

Apolipoprotein A5 polymorphisms and risk of coronary artery disease: a meta-analysis.

The relation has not been reported consistently between the polymorphisms in the gene of apolipoprotein A5 (APO A5) and coronary artery disease (CAD). To clarify the discrepancy, we conducted a comprehensive search of PubMed and EMBASE for all available casecontrol studies to explore the association between two APO A5 polymorphisms and CAD. Two reviewers independently selected studies. Statistical analyses were carried out using the STATA software package v 10.0. Thirteen studies investigated the association between the APO A5 -1131T>C polymorphism and risk of CAD were selected in this meta-analysis with 5,050 cases and 7,272 controls. For the S19W APO A5 gene polymorphism, 5 studies were included with 2,196 cases and 3,933 controls. We observed a significant statistical association between Apo A5 -1131T>C polymorphism and CAD (recessive genetic model: OR = 1.73, 95% CI = 1.37-2.19; dominant genetic model: OR = 1.42, 95% CI = 1.25-1.61; allelic contrast: OR = 1.31, 95% CI = 1.22-1.39, respectively). After restricting our analysis to Chinese individuals, we found that the association was stronger. We also observed strong association between the APO A5 S19>W polymorphism and risk of CAD under a recessive genetic model. This meta-analysis reveals that the minor allele of the -1131T>C polymorphism in the promoter of APO A5 gene significantly increases the susceptibility to CAD. This effect is more pronounced in Chinese subjects.