Epigenetic editing of BRCA1 promoter increases cisplatin and olaparib sensitivity of ovarian cancer cells.

Drug resistance is the primary contributor to the high mortality rate of ovarian cancer (OC). The loss of BRCA1/2 function is linked to drug sensitivity in OC cells. The aim of this study is to enhance the drug sensitivity of OC cells by inducing BRCA1 dysfunction through promoter epigenetic editing. Epigenetic regulatory regions within the BRCA1 promoter, affecting gene expression, were initially discerned through analysis of clinical samples. Subsequently, we designed and rigorously validated epigenetic editing tools. Ultimately, we evaluated the cisplatin and olaparib sensitivity of the OC cells after editing. The BRCA1 promoter contains two CpG-rich regions, with methylation of the region covering the transcription start site (TSS) strongly correlating with transcription and influencing OC development, prognosis, and homologous recombination (HR) defects. Targeting this region in OC cells using our designed epigenetic editing tools led to substantial and persistent DNA methylation changes, accompanied by significant reductions in H3K27ac histone modifications. This resulted in a notable suppression of BRCA1 expression and a decrease in HR repair capacity. Consequently, edited OC cells exhibited heightened sensitivity to cisplatin and olaparib, leading to increased apoptosis rates. Epigenetic inactivation of the BRCA1 promoter can enhance cisplatin and olaparib sensitivity of OC cells through a reduction in HR repair capacity, indicating the potential utility of epigenetic editing technology in sensitization therapy for OC.

Prenatal exposure to per- and polyfluoroalkyl substances and DNA methylation in the placenta: A prospective cohort study.

Epidemiological studies regarding the relationship between per- and polyfluoroalkyl substances (PFAS) and DNA methylation were limited. We investigated the associations of maternal PFAS concentrations with placental DNA methylation and examined the mediating role of methylation changes between PFAS and infant development. We measured the concentrations of 11 PFAS in maternal plasma during early pregnancy and infant development at six months of age. We analyzed genome-wide DNA methylation in 16 placental samples using reduced representation bisulfite sequencing. Additionally, we measured DNA methylation levels using bisulfite amplicon sequencing in 345 mother-infant pairs for five candidate genes, including carbohydrate sulfotransferase 7 (CHST7), fibroblast growth factor 13 (FGF13), insulin receptor substrate 4 (IRS4), paired like homeobox 2Ap (PHOX2A), and plexin domain containing 1 (PLXDC1). We found that placental DNA methylation profiles related to PFOA mainly enriched in angiogenesis and neuronal signaling pathways. PFOA was associated with hypomethylation of IRS4 and PLXDC1, and PFNA was associated with PLXDC1 hypomethylation. There were positive associations of CHST7 methylation with PFTrDA and IRS4 methylation with PFDoA and PFTrDA. PLXDC1 hypomethylation mediated the association between PFOA and suspected developmental delay in infants. Future studies with larger sample sizes are warranted to confirm these findings.

Hysterectomy by Transvaginal Natural Orifice Transluminal Endoscopic Surgery versus Transumbilical Laparoscopic Single-Site Surgery: A Single-Center Experience from East China.

Objective: To compare hysterectomy by transvaginal natural orifice transluminal endoscopic surgery (VNOTES) versus transumbilical laparoscopic single-site surgery (LESS) as a minimal invasive technique. Materials and Method. The women undergoing hysterectomy for benign diseases by VNOTES and LESS from January 2020 to June 2021 in a tertiary hospital in Shanghai were retrospectively analyzed. Results: 361 women were included in our study, with 228 in the VNOTES groups, 129 in the LESS groups, and 4 conversions from VNOTES to LESS technique. The length of a VNOTES hysterectomy was shorter than that of LESS (80.76 min versus 112.09 min; MD -31.34 min; 95% CI -40.24 to -22.43 min; P < 0.001). VNOTES hysterectomy has a quicker gas passage by the anus (18.80 versus 36.49 hours, MD -17.68 hours, 95% CI -20.23 to -15.14 hours, P < 0.001) and associated with a shorter length of hospital stay (2.31 versus 3.77 days, MD -1.46 days, 95% CI -1.75 to -1.17 days, P < 0.001), while with no increase in blood loss during the operation (median 50 versus 50 ml, P = 0.25). Besides, the VAS pain score in the 24th hour after the operation was lower (median 0 versus 0.5, P < 0.001) in the VNOTES group. Four unique phases of the learning curve were identified using cumulative analysis: the mean operation time of phase I was 82.81 ± 31.45 min (the initial learning curve of 43 cases), phase II was 72.48 ± 23.66 min (the acquisition of command of 91 cases), phase III was 103.77 ± 45.69 min (the further learning of 26 cases), and phase IV was 73.18 ± 26.89 min (postlearning in 68 cases). Conclusions: VNOTES is noninferior to LESS as a new minimal invasive procedure for hysterectomy, which also allows patients a faster recovery from surgery and to suffer less pain, and its efficiency and feasibility in large uterine need further exploring.

Profiling the DNA methylation patterns of imprinted genes in abnormal semen samples by next-generation bisulfite sequencing.

PURPOSE: Changes in DNA methylation modifications have been associated with male infertility. With the development of assisted reproductive technologies (ARTs), abnormal DNA methylation in sperm, especially in imprinted genes, may impact the health of offspring and requires an in-depth study. METHODS: In this study, we collected abnormal human semen samples, including asthenospermic, oligospermic, oligoasthenospermic and deformed sperm, and investigated the methylation of imprinted genes by reduced representation bisulfite sequencing (RRBS) and bisulfite amplicon sequencing on the Illumina platform. RESULTS: The differentially methylated regions (DMRs) of imprinted genes, including H19, GNAS, MEG8 and SNRPN, were different in the abnormal semen groups. MEG8 DMR methylation in the asthenospermic group was significantly increased. Furthermore, higher methylation levels of MEG8, GNAS and SNRPN DMR in the oligospermic and oligoasthenospermic groups and a decrease in the H19 DMR methylation level in the oligospermic group were observed. However, the methylation levels of these regions varied greatly among the different semen samples and among individual sperm within the same semen sample. The SNP rs2525883 genotype in the H19 DMR affected DNA methylation. Moreover, DNA methylation levels differed in the abnormal semen groups in the non-imprinted genomic regions, including repetitive sequence DNA transposons and long/short interspersed nuclear elements (LINEs and SINEs). CONCLUSION: Our study established that imprinted gene DMRs, such as H19, GNAS, SNRPN and MEG8, were differentially methylated in the abnormal semen groups with obvious inter- and intra-sample heterogeneities. These results suggest that special attention needs to be paid to possible epigenetic risks during reproduction.

Hypermethylation of the PTTG1IP promoter leads to low expression in early-stage non-small cell lung cancer.

Despite the clinical requirement for early diagnosis, the early events in lung cancer and their mechanisms are not fully understood. Pituitary tumor transforming gene 1 binding factor (PTTG1IP) is a tumor-associated gene; however, to the best of our knowledge, its association with lung cancer has not been reported. The present study analyzed PTTG1IP expression in early-stage non-small cell lung cancer (NSCLC) samples and investigated its epigenetic regulatory mechanisms. The results revealed that the mRNA level of PTTG1IP in NSCLC tissues was significantly downregulated by 43% compared with that in adjacent tissues. In addition, overexpression of this gene significantly inhibited cell proliferation. According to data from The Cancer Genome Atlas, a significant negative correlation was identified between the PTTG1IP gene methylation level and expression level in lung adenocarcinoma and lung squamous cell carcinoma cases. Reduced representation bisulfite sequencing (RRBS) analysis of six paired early-stage NSCLC tissue samples indicated that the CpG island shore of the PTTG1IP promoter is hypermethylated in lung cancer tissues, which was further validated in 12 paired early-stage NSCLC samples via bisulfite amplicon sequencing. Following treatment with 5-aza-2'-deoxycytidine to reduce DNA methylation in the promoter region, the PTTG1IP mRNA level increased, indicating that the PTTG1IP promoter DNA methylation level negatively regulates PTTG1IP transcription. In conclusion, in early-stage NSCLC, the PTTG1IP gene is regulated by DNA methylation in its promoter region, which may participate in the development and progression of lung cancer.

siRNA-based breast cancer therapy by suppressing 17ß-hydroxysteroid dehydrogenase type 1 in an optimized xenograft cell and molecular biology model in vivo.

PURPOSE: Hormone-dependent breast cancer is the most common form of breast cancer, and inhibiting 17ß-HSD1 can play an attractive role in decreasing estrogen and cancer cell proliferation. However, the majority of existing inhibitors have been developed from estrogens and inevitably possess residual estrogenicity. siRNA knockdown provides a highly specific way to block a targeted enzyme, being especially useful to avoid estrogenicity. Application of 17ß-HSD1-siRNA in vivo is limited by the establishment of an animal model, as well as the potential nuclease activity in vivo. We tried to reveal the in vivo potential of 17ß-HSD1-siRNA-based breast cancer therapy. MATERIALS AND METHODS: To establish a competent animal model, daily subcutaneous injection of an estrone micellar aqueous solution was adopted to provide the substrate for estradiol biosynthesis. The effects of three different doses of estrone (0.1, 0.5, and 2.5 µg/kg/day) on tumor growth in T47D-17ß-HSD1-inoculated group were investigated and compared with the animals inoculated with wild type T47D cells. To solve in vivo delivery problem of siRNA, "17ß-HSD1-siRNA/LPD", a PEGylated and modified liposome-polycation-DNA nanoparticle containing 17ß-HSD1-siRNA was prepared by the thin film hydration method and postinsertion technology. Finally, "17ß-HSD1-siRNA/LPD" was tested in the optimized model. Tumor growth and 17ß-HSD1 expression were assessed. RESULTS: Comparison with the untreated group revealed significant suppression of tumor growth in "17ß-HSD1-siRNA/LPD"-treated group when HSD17B1 gene expression was knocked down. CONCLUSION: These findings showed promising in vivo assessments of 17ß-HSD1-siRNA candidates. This is the first report of an in vivo application of siRNA for steroid-converting enzymes in a nude mouse model.

Current knowledge of the multifunctional 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1).

At the late 1940s, 17ß-HSD1 was discovered as the first member of the 17ß-HSD family with its gene cloned. The three-dimensional structure of human 17ß-HSD1 is the first example of any human steroid converting enzyme. The human enzyme's structure and biological function have thus been studied extensively in the last two decades. In humans, the enzyme is expressed in placenta, ovary, endometrium and breast. The high activity of estrogen activation provides the basis of 17ß-HSD1's implication in estrogen-dependent diseases, such as breast cancer, endometriosis and non-small cell lung carcinomas. Its dual function in estrogen activation and androgen inactivation has been revealed in molecular and breast cancer cell levels, significantly stimulating the proliferation of such cells. The enzyme's overexpression in breast cancer was demonstrated by clinical samples. Inhibition of human 17ß-HSD1 led to xenograft tumor shrinkage. Unfortunately, through decades of studies, there is still no drug using the enzyme's inhibitors available. This is due to the difficulty to get rid of the estrogenic activity of its inhibitors, which are mostly estrogen analogues. New non-steroid inhibitors for the enzyme provide new hope for non-estrogenic inhibitors of the enzyme.

Age-related changes in the anatomical characteristics of Peyer's patches in small intestine of Bactrian camels (Camelus bactrianus).

The distribution, size, and appearance of Peyer's patches vary according to species. In order to determine the anatomical characteristics of Peyer's patches in small intestine of Bactrian camel, and age-related changes in the number of Peyer's patches, 40 Bactrian camels of the following four age groups were studied: young (0.5-2 years), pubertal (3-5 years), middle-aged (6-16 years), and old (17-20 years). The exact number of Peyer's patches was recorded, and the appearance of Peyer's patches was described in detail. The results indicated that Peyer's patches of Bactrian camels not only have a particular anatomical location and distinct appearance but also change with age. They were distributed in the whole small intestine and there were four distinct types of Peyer's patches: nodular, faviform, cup-shaped, and cystic form Peyer's patches. However, the nodular and cystic form Peyer's patches are specific to Bactrian camel, which have not been found in other animals including Dromedary camel. In addition, the distribution density of Peyer's patches in ileum was the maximum, then was jejunum and duodenum. Further statistical analysis showed that the number of Peyer's patches was altered with age. The number peaked in 5-year-old camels and declined subsequently with age. However, there was little change in the size of Peyer's patches in different age groups; no age-related macroscopic variations in the shape or size of the Peyer's patches were found. Results obtained from this study provide the basic information to further study on the gastrointestinal mucosal immunity of Bactrian camel.