Progesterone Receptor Protein Expression Research Articles

Validation of a new Custom Polyclonal Progesterone Receptor Antibody for Immunohistochemistry in the Female Mouse Brain.

Immunohistochemical visualization of progesterone receptor (PR)-expressing cells in the brain is a powerful technique to investigate the role of progesterone in the neuroendocrine regulation of fertility. A major obstacle to the immunohistochemical visualization of progesterone-sensitive cells in the rodent brain has been the discontinuation of the commercially produced A0098 rabbit polyclonal PR antibody by DAKO. To address the unavailability of this widely used PR antibody, we optimized and evaluated 4 alternative commercial PR antibodies and found that each lacked the specificity and/or sensitivity to immunohistochemically label PR-expressing cells in paraformaldehyde-fixed female mouse brain sections. As a result, we developed and validated a new custom RC269 PR antibody, directed against the same 533-547 amino acid sequence of the human PR as the discontinued A0098 DAKO PR antibody. Immunohistochemical application of the RC269 PR antibody on paraformaldehyde-fixed mouse brain sections resulted in nuclear PR labeling that was highly distinguishable from background, specific to its antigen, highly regulated by estradiol, matched the known distribution of PR protein expression in the female mouse hypothalamus, and nearly identical to that of the discontinued A0098 DAKO PR antibody. In summary, the RC269 PR antibody is a specific and sensitive antibody to immunohistochemically visualize PR-expressing cells in the mouse brain.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Clinicopathological features and prognoses of very young patients (≤35 years) with breast cancer: a retrospective population-based study in China.

Clinicopathological features and prognoses of very young patients (≤35 years) with breast cancer: a retrospective population-based study in China.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization invitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Significance of differential expression of OLFM4 in the development of endometrial adenocarcinoma.

The incidence of endometrial adenocarcinoma (EA) has increased worldwide in recent years due to the widespread use of estrogen therapy and the overall increase in life expectancy. However, we know of no sensitive molecular index that can be used to predict the onset of EA, evaluate the therapeutic effects of treatment agents, or provide prognostic benefit in post-treatment follow-up. To explore the correlation between human olfactomedin 4 (OLFM4) and the clinicopathologic parameters of EA, and to determine the precise involvement of OLFM4 as a related factor in the occurrence and development of EA. We enrolled 61 gynecologic patients for a retrospective study at the Tai'an Central Hospital of Shandong Province from January 1, 2016, to June 30, 2022. We determined the expression levels of estrogen receptor α (ERα), progesterone receptor (PR), and OLFM4 proteins in endometrial tissue with the immunohistochemical S-P staining method, and analyzed the correlations among ERα, PR, and OLFM4 protein expression levels and with the pathologic stage, histologic grade, myometrial invasiveness, and lymphatic metastasis of EA. The expression levels of OLFM4 in EA were higher than in normal endometrium (P = .036). The expression level of OLFM4 protein in stage II-III patients was higher than that in stage I patients (P = .034), and the expression levels of ERα and PR proteins in EA were lower than those in normal endometrial tissue (P = .014 and P = .0005). While we observed no correlation in endometrial tissues of disparate pathologic types between OLFM4 and the expression levels of ERα and PR proteins, we noted a positive correlation between the expression levels of ERα and PR protein. The expression level of OLFM4 protein increased with the malignant degree of endometrial lesions and OLFM4 protein expression was related to the FIGO stage of EA. And OLFM4 protein can be used as 1 of the potential diagnostic factors for endometrial lesions, which is worthy of further study.

OR16-2 Steroid Receptor Co-Activator Complexes Regulate Metabolic PFKFB Enzymes to Drive Therapy Resistant ER+ Breast Cancer

Abstract Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the survival and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in recurrent tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. We previously showed that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. PELP1 interacts with PFKFB proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB inhibitors in combination with ER targeted therapies blocked proliferation and tumorsphere formation in multiple models of advanced breast cancer, including paclitaxel (TaxR) and tamoxifen (TamR) resistant models and ER+ patient-derived organoids (PDxO). Chemotherapy and endocrine therapy resistant models contained increased levels of cytoplasmic PELP1, PELP1/SRC-3 complexes, and phenocopied PELP1/SRC-3/PFKFB biology and CSC behavior. To better understand CSC-mediated pathways in the context of therapy resistance, we performed RNA-seq on 3D-cultured TaxR tumorspheres to enrich for CSCs. Pathway analysis revealed that TaxR tumorspheres upregulate stem (Sox2-Oct4-Nanog), HIF, and progesterone mediated pathways. Progesterone receptor (PR) is a known contributor to CSC populations, and we observed increased PR mRNA and protein expression in TaxR cells grown as tumorspheres. Additionally, in vivo emergence of circulating tumor cell (CTC) populations was observed in ER+ TaxR mammary intraductal (MIND) xenografts. Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Our findings are applicable to therapy resistant models, indicating that SR co-activator complexes are a key mediator of resistance in ER+ breast cancer and may cooperate with PR to promote therapy resistant CSCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

Zearalenone Promotes Uterine Development of Weaned Gilts by Interfering with Serum Hormones and Up-Regulating Expression of Estrogen and Progesterone Receptors.

In this study, we aimed to assess the effect of diet ZEA on serum hormones, the location and expression of estrogen receptor ERα/β and progesterone receptor (PR) of the uterus in weaned piglets and to reveal the mechanism underneath. A total of 40 healthy weaned gilts were randomly allocated to basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0) and 1.5 (ZEA1.5) mg ZEA/kg and fed individually for 35 days. Meanwhile, the porcine endometrial epithelial cells (PECs) were incubated for 24 h with ZEA at 0 (Control), 5 (ZEA5), 20 (ZEA20) and 80 (ZEA80) μmol/L, respectively. The results showed that nutrient apparent digestibility (CP and GE), nutrient apparent availability (ME/GE, BV and NPU), the uterine immunoreactive integrated optic density (IOD), relative mRNA and protein expression of ER-α, ER-β and PR and the relative mRNA and protein expression of ER-α and ER-β in PECs all increased linearly (p < 0.05) with ZEA. Collectively, ZEA can interfere with the secretion of some reproductive hormones in the serum and promote the expression of estrogen/progesterone receptors in the uterus and PECs. All these indicate that ZEA may promote the development of the uterus in weaned gilts through estrogen receptor pathway.

Anti-inflammatory and hemostatic effects of total extract, saponins, and flavonoids of Clinopodium chinense in female rats with abnormal uterine bleeding and mechanism

This study aims to explore the anti-inflammatory and hemostatic effects of the total extract of Clinopodium chinense(TEC), total saponins of C. chinense(TSC), and total flavonoids of C. chinense(TFC) in female rats with abnormal uterine bleeding(AUB), and the possible mechanism. Mifepristone(i.g., 12.4 mg·kg~(-1)) and misoprostol(i.g., 130 μg·kg~(-1)) were used to induce AUB in SD female rats conceiving on the same day. Then the AUB rats were randomized into model group, TEC group, TSC group, TFC group, Yimucao Granules(LG) group, and estradiol valerate(EV) group, with 8 rats in each group. Another 8 non-pregnant female rats were selected as normal group. During the experiment, each group was given the corresponding drug by gavage once a day for 7 days. After the administration, blood and uterine tissue were collected. The uterine bleeding volume was measured by ultraviolet spectrophotometry and the pathological changes of endometrium were observed based on hematoxylin-eosin(HE) staining. In addition, the microvessel density of endometrium was determined by immunohistochemistry, and the content of thromboxane B2(TXB2), 6-keto-PGF_(1α), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in plasma and levels of lutenizing hormone(LH), follicle stimulating hormone(FSH), estradiol(E_2), and progesterone in serum were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression of estrogenreceptor α(ERα), progesterone receptor(PR), matrix metalloproteinase(MMP)-2, MMP-9, and vascular endothelial growth factor(VEGF) in uterine tissue was determined by Western blot. Compared with the model group, TEC, TSC, and TFC can reduce uterine bleeding volume, alleviate the pathological damage of endometrium, and increase the microvessel density in endometrium. Moreover, TEC and TSC can significantly raise plasma TXB2 level and ratio of TXB2 to 6-keto-PGF_(1α), and TEC and TFC can significantly reduce the levels of IL-6 and TNF-α. In addition, TEC significantly elevated serum progesterone level and TFC significantly increased serum levels of E_2, FSH, and LH. TSC can significantly raise serum progesterone and FSH levels. In addition, TEC can significantly down-regulate the protein expression of PR, MMP-2, and VEGF and TSC significantly reduced the expression of MMP-9. TFC significantly decreased the expression of PR, MMP-9, and VEGF, and up-regulated the expression of ERα. In conclusion, TEC, TSC, and TFC all show therapeutic effects on AUB, particularly TEC. TSC exerts the effects by enhancing the coagulation function and promoting endometrial repair, and TFC by regulating estrogen levels and reducing inflammatory response. This study reveals the mechanism of C. chinense against AUB and also explains the holistic characteristics of Chinese medicine.

Hybrid Broussonetia papyrifera Fermented Feed Can Play a Role Through Flavonoid Extracts to Increase Milk Production and Milk Fatty Acid Synthesis in Dairy Goats.

In order to explore the effect of hybrid Broussonetia papyrifera fermented feed on milk production and milk quality of dairy goats, and to compare with alfalfa hay, three dairy goat diets were designed based on the principle of equal energy and equal protein. The goats in the control group were fed a basic TMR diet (CG group), and the other two groups were supplemented with 10% alfalfa hay (AH group) and 10% hybrid B. papyrifera fermented feed (BP group). The results showed that the dry matter intake and milk production of BP group increased significantly. The total amount of amino acids and the content of each amino acid in the milk of AH group and BP group were lower than those of CG group. The content of saturated fatty acids in the milk of BP group decreased while the content of unsaturated fatty acids increased. The contents of prolactin, estrogen and progesterone in BP goat serum were generally higher than those of AH goat and CG goat. Subsequently, this study separated and cultured mammary epithelial cells from breast tissue, and added flavone extracted from the leaves of hybrid B. papyrifera and alfalfa to their culture medium for comparison, which is one of their important bioactive components. The results showed that low-dose alfalfa flavone (AH) and hybrid B. papyrifera flavone (BP) can increase cell viability. They also can increase the accumulation of intracellular triglyceride and the formation of lipid droplets. Both AH flavone and BP flavone significantly up-regulated the expression of genes related to milk fat synthesis, including genes related to fatty acid de novo synthesis (ACACA, FASN, and SCD1), long-chain fatty acid activation and transport related genes (ACSL1), and genes related to transcription regulation (SREBP1). The three genes related to triglyceride synthesis (DGAT1, DGAT2, and GPAM) were all significantly increased by BP flavone. Both AH flavone and BP flavone significantly increased the protein expression of progesterone receptor and estrogen receptor in mammary epithelial cells but had no effect on prolactin receptor.

Effect of vitamin D3 on hyperplasia of mammary glands in experimental rats.

1,25(OH)2D3/vitamin D3 receptor (VD3/VDR) signaling pathway can inhibit the occurrence of breast cancer in many ways. We used vitamin D3 to interfere with Hyperplasia of mammary glands (HMG) model rats, and to explore the intervention effect of vitamin D3 on HMG. We divided 42 female rats into six groups randomly: blank control group, hyperplasia model group, negative control group, and vitamin D3 (VD3) groups of low-dose (LVD, 5 µg/kg), medium-dose (MVD, 10 µg/kg), and high-dose (HVD, 20 µg/kg). We established HMG model in all groups except for the blank control group, then different dose of VD3 was intraperitoneal injected for VD3 groups and normal saline (NS) was given to the negative control group. After the experiment, the body weights, heights and diameters of nipples, and the thickness of the mammary gland of rats were measured. The serum content of sex hormone and VD3 were detected by enzyme-linked immunosorbent assay (ELISA). The tissues of mammary glands were analyzed by hematoxylin and eosin (HE) stain, and the expression of estrogen receptor α (ERα), progesterone receptor (PR), and VDR were detected by immunohistochemical (IHC) stain. Similarly, the total protein expression of ERα, PR, and VDR were measured by western blot. Compared with the hyperplasia group, rats in the VD3 groups displayed a marked decrease of the thickness of the mammary glands and the height and diameter of the nipples. The serum estrogen (E2), testosterone (T), luteinizing hormone (LH), and VD3 was markedly decreased in all VD3 groups (P<0.05). The IHC results showed that ERα and PR was decreased in all three VD3 groups; however, VDR was increased. Western blot demonstrated that both ERα and PR were reduced in VD3 groups, while the VDR level was significantly enhanced. Overall, the findings suggested that VD3 could be used in HMG treatment. Supplementation of VD3 could markedly decrease the mammary gland thickness, nipple diameter, and nipple height of rats, accompanied by the decrease of serum E2, T, and LH. Intervention with VD3 can lead to decreased expression of ERa and PR, in conjunction with the increase of VDR.

Expression of nuclear progesterone receptor, progesterone receptor membrane components 1 and 2 and prostaglandin-endoperoxide synthase 2 in the endometrium and oviduct of spontaneously ovulating cats

Although ovulations not followed by pregnancy occur regularly in cats, differences in endometrial function between cats in the luteal and non-luteal phase have not been studied so far. Progesterone exerts its effects through a nuclear progesterone receptor (PGR) and via cell-membrane bound receptors referred to as progesterone receptor membrane component (PGRMC) 1 and 2. Progesterone receptor expression is regulated by gonadal steroid hormones and therefore may change throughout the oestrous cycle. Protein expression of PGR, PGRMC-1 and 2 and prostaglandin-endoperoxide synthase 2 (PTGS2) was analysed in the endometrium and oviduct of non-pregnant female cats in the follicular (n = 8) and luteal phase (n = 9). We hypothesized that the presence of corpora lutea (CL) is associated with downregulation of progesterone receptors and PTGS2. Cells of the luminal endometrial epithelium, endometrial stroma and oviductal epithelium were assessed by immunohistochemistry. The PGR protein expression was more pronounced in the endometrial epithelium than stroma (p < 0.001) and less pronounced in cats with a CL than without CL (p < 0.001) but did not differ between groups in the oviduct. The PTGS2 was localized only in the endometrial and oviductal epithelium and its expression was reduced in cats with CL (p = 0.001). In the endometrial epithelium, PGRMC-1 expression was reduced in cats with CL (p < 0.05). Expression of PGRMC-2 was highest in the endometrial epithelium and lowest in the endometrial stroma (p = 0.01) but did not differ between cats with and without CL. In conclusion, progesterone receptor and PTGS2 downregulation in the female cat closely resembles findings in other spontaneously ovulating domestic animal species.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10mg/kg) was injected subcutaneously 5days before ovariohysterectomy in the treatment group (n=6); bitches without treatment served as a control group (n=6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p<.05) and ERα mRNA increased (p<.01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p<.05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p<.01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Hydrolyzed seawater pearl tablet modulates the immunity via attenuating Th1/Th2 imbalance in an immunosuppressed mouse model.

To investigate whether Hydrolyzed Seawater Pearl tablet (HSPT) could modulate the Th1/Th2 imbalance in an immunosuppressed mouse model with Th1 to Th2 shift induced by Cyclosporine A (CsA) which can be used in the clinical treatment of Th2 to Th1 shift diseases, and explore the possible mechanism for the adjuvant therapeutic efficacy of HSPT on recurrent respiratory infections (RRI) and acquired immune deficiency syndrome (AIDS). The mice were randomly divided into six groups of five animals each, namely normal group, model group, lentinan polysaccharide tablet (LPT) group and three HPST treated groups. HPST treated groups were administered with HPST (0.51, 1.02, 2.04 g/kg) via intragastric gavage (i.g) for 30 consecutive days. LPT used as reference drug for positive control, LPT group was administered with LPT (8.2 mg/kg) for 30 consecutive days. Normal group and model group were received distilled water. The animals in model group, LPT group and HPST treated groups were injected intraperitoneally with CsA (50 mg/kg) to establish the immunosuppressed mice model with Th1 to Th2 shift on the 20th, 22nd and 24th day, one hour after the administration of the respective treatment. Animals were sacrificed one hour after the last administration to collect blood and splenic tissue. The proportion of T cells including CD8+ and CD4+ T cells, Th1 and Th2 in peripheral blood of experimental mice were measured by flow cytometric. The protein level in serum and mRNA level in splenic tissue of experimental mice for interleukin (IL)-2, IL-12, interferon-γ (IFN-γ), IL-4, IL-6, IL-10 and IL-13 were measured by enzyme linked immunosorbent assay and fluorescence quantitative polymerase chain reaction respectively. HSPT elevated the proportion of T cells including both CD8+ and CD4+ T cells, in which the proportion of Th1 and Th2 cells increased, while the ratio of Th1/Th2 cells decreased in peripheral blood of the immunosuppressed mouse model with Th1 to Th2 shift induced by CsA. Furthermore, HSPT elevated both protein and mRNA level of Th1-type cytokines IL-2 and IFN-γ, while had no significant effect on protein and mRNA level of Th1-type cytokine IL-12 and Th2-type cytokines IL-4, IL-6, IL-10, IL- 13 in mouse model. Our findings suggest that HSPT can increase proportion of T cells including both CD8+ and CD4+ T cells and induce Th2 to Th1 shift in both cells and cytokines, which probably was the mechanism to account for the adjuvant therapeutic efficacy of HSPT on RRI and AIDS.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Effect of combined administration of electroacupuncture and mesenchymal stem cells on expression of endometrium estrogen receptor and progesterone receptor in thin endometrium rats

To investigate the effect of joint administration of electroacupuncture (EA) and bone marrow mesenchymal stem cells (BMMSC) on the expression of estrogen receptor (ER) and progesterone receptor (PR) in thin endometrium rats, so as to explore its mechanisms underlying improvement of conception rate. Forty female SD rats were divided into control, model, BMMSC and EA+BMMSC groups. The thin endometrium model was established by intrauterine infusion of 95% ethanol and saline. EA (1 mA, 2 Hz/15 Hz) was applied to "Guanyuan"(CV4), unilateral "Zigong" (EX-CA1) and unila-teral "Sanyinjiao" (SP6) for 15 min, once daily for 10 d. Rats of the BMMSC and EA+BMMSC groups received injection of BMMSC suspension through caudal vein on the day of modeling, the 3rd day after surgery, and the 2nd and the 3rd estrus phases, respectively. Changes of body weight and estrus phase were continuously recorded. After three estrus cycles, uterine specimens were taken and the uterine coefficient was calculated. The immunoactivity of KI67(an antigen for cell proliferation) was detected by immunohistochemistry, and the expression of ER and PR was detected by Western blot. Compared with the control group, the uterine coefficient, and expression of endometrium ER and PR proteins were significantly decreased in the model group (P<0.001, P<0.01). In Comparison with the model group, the uterine coefficient, immunoactivity of KI67, and expression of ER and PR proteins were significantly increased in both BMMSC and EA+BMMSC groups (P<0.000 1,P<0.001, P<0.01). The expression level of PR in the EA+BMMSC group was notably higher than that in the BMMSC group (P<0.05), but no significant difference was found between the BMMSC and EA+BMMSC groups in the levels of uterine coefficient and ER expression (P>0.05). EA combined with BMMSC injection has a positive effect in promoting the proliferation of endometrium cells in rats with thin endometrium, which may be related to its effect in up-regulating the expression of ER and PR proteins.

Abstract GS4-09: Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study

Abstract Background: Several biomarkers such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and Ki67 have been implicated in the pathogenesis and/or as prognostic biomarkers of breast cancer, and are utilized to determine treatment. Given the heterogeneity of response to endocrine therapy, however, predictive biomarkers are critical to better individualize patient care. Previous results from the Trans-aTTom study demonstrated that the Breast Cancer Index HOXB13/IL17BR [BCI (H/I)] biomarker significantly predicts extended endocrine benefit from 10 vs 5y of tamoxifen. In this correlative study, the predictive activity of BCI (H/I) was compared with ER, PR, AR and Ki67 protein expression in node positive patients treated in the aTTom trial. Methods: Patients with available tumor tissue and biomarker analyses were included. ER, PR, AR and Ki67 were centrally assessed by immunohistochemistry (IHC) utilizing tissue microarrays. BCI gene expression analysis by RT-PCR was performed blinded to clinical outcome. Multivariate Cox models adjusting for age, tumor size, tumor grade and HER2 status were used to assess the significance of the interaction between treatment and each biomarker as continuous variables. 17-year risk of recurrence, as a function of each continuous biomarker, was estimated from Cox models in each of the 2 treatment arms. Results: Analysis of 789 HR+, N+ patients showed a weak negative correlation between BCI (H/I) and ER, PR, and AR expression whereas Ki67 and the AR/ER ratio showed no correlation (ER, cor=−0.18; PR, cor=−0.25; AR, cor=−0.14; Ki67, cor=0.04; AR/ER ratio, cor=0.02). The interaction between BCI (H/I) and extended tamoxifen treatment was significant (p=0.014). In addition, analysis of risk of recurrence as a function of continuous BCI (H/I) demonstrated that the magnitude in the reduction in recurrence risk with extended tamoxifen correlated with increasing H/I levels. In contrast, interaction P values were nonsignificant (ER, p=0.829; PR, p=0.659; AR, p=0.783; Ki67, p=0.865; AR/ER ratio, p=0.835) and the magnitude of endocrine benefit did not correlate with expression levels of any of other biomarkers. Conclusion: Results from this post-hoc analysis of the Trans-aTTom study demonstrated that whereas BCI(H/I) is a significant predictive biomarker of endocrine response, analysis of ER, PR, AR, Ki67 and AR/ER expression showed no interaction with treatment, and lacked the ability to predict benefit of extended tamoxifen in HR+ early stage breast cancer. These results add to the growing body of evidence that BCI (H/I) is distinct in its ability to predict benefit from therapy and interrogates distinct tumor biology that is not captured by other traditional biomarkers. Citation Format: Dennis C Sgroi, Kai Treuner, Yi Zhang, Tammy Piper, Ranelle Salunga, Ikhlaaq Ahmed, Lucy Doos, Sarah Thornber, Karen J Taylor, Elena F Brachtel, Sarah Pirrie, Catherine A Schnabel, Daniel W Rea, John MS Bartlett. Correlative studies of the breast cancer index (HOXB13/IL17BR) and ER, PR, AR, AR/ER ratio and Ki67 for prediction of extended endocrine benefit: A trans-aTTom study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-09.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.

Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

MicroRNA Dysregulation and Steroid Hormone Receptor Expression in Uterine Tissues of Rats with Endometriosis during the Implantation Window.

Background:Estrogen receptor (ER) and progesterone receptor (PR) are involved in endometriosis, but the involvement of microRNAs (miRNAs) is unknown. The aim of the study was to explore the correlation between miRNA and ER/PR in uterine tissues of rats with endometriosis during the implantation window.Methods:Twenty female Sprague-Dawley rats were randomized in three groups: endometriosis (n = 7), fat tissue control (n = 6), and normal (n = 7) groups. The female rats were mated and sacrificed on day 5 (implantation). Uterine tissues were obtained for hematoxylin-eosin staining, immunohistochemistry, and miRNA expression. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the expression of rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p.Results:The 475 miRNAs were found to differentially express between the endometriosis and normal control groups, with 127 being upregulated and 348 being downregulated. Expression of five miRNAs (rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p) were validated by RT-PCR and found to be differentially expressed among the three groups. Expression of ER and PR proteins (immunohistochemistry) in the glandular epithelium and endometrial stroma was significantly different among the three groups (all P < 0.05). Five miRNAs were involved in pathways probably taking part in implantation and fertility.Conclusions:The results suggested that miRNAs, ER, and PR could play important roles in the embryo implantation period of rats with endometriosis. These miRNAs might play a role in endometrial receptivity in endometriosis.