Downregulation of HNRNPA1 induced neoantigen generation via regulating alternative splicing.

BACKGROUND: Immunotherapies effectively treat human malignancies, but the low response and resistance are major obstacles. Neoantigen is an emerging target for tumor immunotherapy that can enhance anti-tumor immunity and improve immunotherapy. Aberrant alternative splicing is an important source of neoantigens. HNRNPA1, an RNA splicing factor, was found to be upregulated in the majority of tumors and play an important role in the tumor immunosuppressive microenvironment. METHODS: Whole transcriptome sequencing was performed on shHNRNPA1 SKOV3 cells and transcriptomic data of shHNRNPA1 HepG2, MCF-7M, K562, and B-LL cells were downloaded from the GEO database. Enrichment analysis was performed to elucidate the mechanisms underlying the activation of anti-tumor immunity induced by HNRNPA1 knockdown. mRNA alternative splicing was analyzed and neoantigens were predicted by JCAST v.0.3.5 and Immune epitope database. The immunogenicity of candidate neoantigens was calculated by Class I pMHC Immunogenicity and validated by the IFN-γ ELISpot assay. The effect of shHNRNPA1 on tumor growth and immune cells in vivo was evaluated by xenograft model combined with immunohistochemistry. RESULTS: HNRNPA1 was upregulated in a majority of malignancies and correlated with immunosuppressive status of the tumor immune microenvironment. Downregulation of HNRNPA1 could induce the activation of immune-related pathways and biological processes. Disruption of HNRNPA1 resulted in aberrant alternative splicing events and generation of immunogenic neoantigens. Downregulation of HNRNPA1 inhibited tumor growth and increased CD8+ T cell infiltration in vivo. CONCLUSION: Our study demonstrated that targeting HNRNPA1 could produce immunogenic neoantigens that elicit anti-tumor immunity by inducing abnormal mRNA splicing. It suggests that HNRNPA1 may be a potential target for immunotherapy.

IL-2-free tumor-infiltrating lymphocyte therapy with PD-1 blockade demonstrates potent efficacy in advanced gynecologic cancer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy has been restricted by intensive lymphodepletion and high-dose intravenous interleukin-2 (IL-2) administration. To address these limitations, we conducted preclinical and clinical studies to evaluate the safety, antitumor activity, and pharmacokinetics of an innovative modified regimen in patients with advanced gynecologic cancer. METHODS: Patient-derived xenografts (PDX) were established from a local recurrent cervical cancer patient. TILs were expanded ex vivo from minced tumors without feeder cells in the modified TIL therapy regimen. Patients underwent low-dose cyclophosphamide lymphodepletion followed by TIL infusion without intravenous IL-2. The primary endpoint was safety; the secondary endpoints included objective response rate, duration of response, and T cell persistence. RESULTS: In matched patient-derived xenografts (PDX) models, homologous TILs efficiently reduced tumor size (p < 0.0001) and underwent IL-2 absence in vivo. In the clinical section, all enrolled patients received TIL infusion using a modified TIL therapy regimen successfully with a manageable safety profile. Five (36%, 95% CI 16.3-61.2) out of 14 evaluable patients experienced objective responses, and three complete responses were ongoing at 19.5, 15.4, and 5.2 months, respectively. Responders had longer overall survival (OS) than non-responders (p = 0.036). Infused TILs showed continuous proliferation and long-term persistence in all patients and showed greater proliferation in responders which was indicated by the Morisita overlap index (MOI) of TCR clonotypes between infused TILs and peripheral T cells on day 14 (p = 0.004) and day 30 (p = 0.004). Higher alteration of the CD8+/CD4+ ratio on day 14 indicated a longer OS (p = 0.010). CONCLUSIONS: Our modified TIL therapy regimen demonstrated manageable safety, and TILs could survive and proliferate without IL-2 intravenous administration, showing potent efficacy in patients with advanced gynecologic cancer. TRIAL REGISTRATION: NCT04766320, Jan 04, 2021.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

Comprehensive analysis of lncRNA-miRNA-mRNA ceRNA network and key genes in granulosa cells of patients with biochemical primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is a common condition leading to the pathological decline of ovarian function in women of reproductive age, resulting in amenorrhea, hypogonadism, and infertility. Biochemical premature ovarian insufficiency (bPOI) is an intermediate stage in the pathogenesis of POI in which the fertility of patients has been reduced. Previous studies suggest that granulosa cells (GCs) play an essential role in the pathogenesis of POI, but their pathogenetic mechanisms remain unclear. To further explore the potential pathophysiological mechanisms of GCs in POI, we constructed a molecular long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) network using GC expression data collected from biochemical premature ovarian failure (bPOI) patients in the GEO database. We discovered that the GCs of bPOI patients had differential expression of 131 mRNAs, 191 lncRNAs, and 28 miRNAs. By systematic network analysis, we identified six key genes, including SRSF1, PDIA5, NEURL1B, UNK, CELF2, and CFL2, and five hub miRNAs, namely hsa-miR-27a-3p, hsa-miR-24-3p, hsa-miR-22-3p, hsa-miR-129-5p, and hsa-miR-17-5p, and the results suggest that the expression of these key genes may be regulated by two hub miRNAs, hsa-miR-27a-3p and hsa-miR-17-5p. Additionally, a POI model in vitro was created to confirm the expression of a few important genes. In this study, we discovered a unique lncRNA-miRNA-mRNA network based on the ceRNA mechanism in bPOI for the first time, and we screened important associated molecules, providing a partial theoretical foundation to better understand the pathogenesis of POI.

Sohlh1 and Lhx8 are prominent biomarkers to estimate the primordial follicle pool in mice.

Efficient evaluation of the primordial follicle pool (PFP) of mammalian models is an essential subject in biomedical research relating to ovarian physiology and pathogenesis. Our recent study has identified a gene signature including Sohlh1, Nobox, Lhx8, Tbpl2, Stk31, Padi6, and Vrtn strongly correlated with ovarian reserve by using bioinformatics analysis. Aimed to investigate the validity of these candidate biomarkers for evaluating the PFP, we utilized an OR comparison model to decode the relationship between the numbers of PFP and candidate biomarkers in the present study. Our results suggest that these biomarkers Sohlh1, Nobox, Lhx8, Tbpl2, Stk31, Padi6, and Vrtn possess independent potential to evaluate the number of the PFP. And the combination of Sohlh1 and Lhx8 can be used as the optimal biomarkers for rapid assessment of the PFP in the murine ovary. Our findings provide a new perspective for evaluating the PFP of the ovary in animal studies and the clinic.

Expression of high-mobility group box-1 in eutopic/ectopic endometrium and correlations with inflammation-related factors in adenomyosis.

OBJECTIVE: To investigate the expression of HMGB1 and toll-like receptor 4 (TLR4) in adenomyosis eutopic/ectopic endometrium. METHODS: Twenty patients with adenomyosis and 20 controls, all undergoing laparoscopy, were recruited from September 2015 to July 2016. Samples were collected from the endometrium without adenomyosis (CE), the eutopic endometrium with adenomyosis (EuE), and the ectopic endometrium with adenomyosis (EE). The mRNA and protein expression of HMGB1 and TLR4, and interleukin-6 (IL-6) and interleukin-8 (IL-8) RNA expression levels were measured. RESULTS: The average age of the adenomyosis women was 43.4 ± 5.3 years; their BMI was 23.3 ± 2.3 kg/m2. The control group included women aged 38.8 ± 9.8 years, with BMI 22.2 ± 3.4 kg/m2. The mRNA expression levels of HMGB1, TLR4, IL-6, and IL-8 in the EE and EuE groups were higher than those in the CE group (p < .01), and those in the EE group were higher than those in the EuE group (p < .01). The protein expression levels of HMGB1 and TLR4 in the EE and EuE groups were higher than those in the CE group (p < .01); they were higher in the EE group than the ones in the EuE group (p < .01). HMGB1 mRNA was significantly positively correlated with TLR4 in EuE and EC patients (r = 0.538 and r = 0.916, p < .01), as well as with IL-6 (r = 0.470 and r = 0.976, p < .01) and IL-8 (r = 0.574 and r = 0.650, p < .01). CONCLUSIONS: The overexpression of HMGB1 and TLR4 in EuE and EE is positively correlated with IL-6 and IL-8 expression. The HMGB1 signaling-mediated immune-inflammatory system might be involved in the development of adenomyosis.

A Rare Secondary Dysmenorrhea Resulted from Separation of Corpus Uteri from Cervix: A Case Report and Literature Review.

Secondary dysmenorrhea is a pain associated with disease such as endometriosis, pelvic inflammatory disease, leiomyomas, and interstitial cystitis. Treatment of secondary dysmenorrhea always focuses on the causative pelvic pathology or medical condition. Here, we found a rare case with secondary dysmenorrhea that resulted from traumatic separation of the uterine corpus from the cervix. In this case, the patient experienced a childhood blunt trauma of the pelvic crush and was successfully diagnosed by magnetic resonance imaging and 3-dimensional ultrasonography. Moreover, laparoscopic anastomosis could be a minimally invasive way to resolve this problem.

Progress in the Application of Magnetic Nanoparticles in Forensic Trace Analysis.

Given the complexity of biological samples and the trace nature of target materials in forensic trace analysis, a simple and effective method is needed to obtain sufficient target materials from complex substrates. Magnetic nanoparticles (MNPs) have shown a wide range of application value in many research fields, such as biomedicine, drug delivery and separation, due to their unique superparamagnetic properties, stable physical and chemical properties, biocompatibility, small size, high specific surface area and other characteristics. To apply MNPs in the pretreatment of forensic materials, maximize the extraction rate of the target materials, and minimize interference factors to meet the requirements of trace analysis of the target materials, this paper reviews the application of MNPs in the fields of forensic toxicological analysis, environmental forensic science, trace evidence analysis and criminal investigation in recent years, and provides research ideas for the application of MNPs in forensic trace analysis.

MCM2 in human cancer: functions, mechanisms, and clinical significance.

BACKGROUND: Aberrant DNA replication is the main source of genomic instability that leads to tumorigenesis and progression. MCM2, a core subunit of eukaryotic helicase, plays a vital role in DNA replication. The dysfunction of MCM2 results in the occurrence and progression of multiple cancers through impairing DNA replication and cell proliferation. CONCLUSIONS: MCM2 is a vital regulator in DNA replication. The overexpression of MCM2 was detected in multiple types of cancers, and the dysfunction of MCM2 was correlated with the progression and poor prognoses of malignant tumors. According to the altered expression of MCM2 and its correlation with clinicopathological features of cancer patients, MCM2 was thought to be a sensitive biomarker for cancer diagnosis, prognosis, and chemotherapy response. The anti-tumor effect induced by MCM2 inhibition implies the potential of MCM2 to be a novel therapeutic target for cancer treatment. Since DNA replication stress, which may stimulate anti-tumor immunity, frequently occurs in MCM2 deficient cells, it also proposes the possibility that MCM2 targeting improves the effect of tumor immunotherapy.

Hsa_circ_0000497 and hsa_circ_0000918 contributed to peritoneal metastasis of ovarian cancer via ascites.

PURPOSE: As a common complication of epithelial ovarian cancer (EOC), malignant ascites contributes to the peritoneal metastasis of EOC. CircRNAs play essential roles in tumor metastasis. However, no circRNAs have been reported to be involved in EOC peritoneal metastasis via ascites. METHODS: Total of 22 samples from 9 EOC patients containing primary lesions (T), tumor cells from ascites (ASC), and metastatic lesions (M) were included for RNA sequencing to identify differentially expressed circRNAs and mRNAs among different tumors. Bioinformatic analyses, including single-sample Gene Set Enrichment Analysis and soft cluster analysis, were performed to find circRNAs potentially correlated with ascitic metastasis. Wound healing and transwell analysis were performed to evaluate tumor cells metastasis in vitro. Quantitative real-time PCR and western-blot were used for gene expression evaluation. RESULTS: According to transcriptomic analysis, ASC showed mesenchymal phenotype while T and M showed epithelial phenotype. 10 circRNAs were differentially expressed among ASC, T, and M. Among them, hsa_circ_0000497 and hsa_circ_0000918 were significantly up-regulated in ASC. Functional analysis showed that both hsa_circ_0000497 and hsa_circ_0000918 promoted metastasis of EOC via epithelial-mesenchymal transition (EMT) in vitro. The regulatory network construction identified 8 miRNAs and 19 mRNAs, and 7 miRNAs and 17 mRNAs as potential downstream target genes of hsa_circ_0000497 and hsa_circ_0000918, respectively, which may play pivotal roles in EOC ascitic metastasis. CONCLUSIONS: circRNAs (hsa_circ_0000497 and hsa_circ_0000918) contribute to metastasis of EOC via ascites by regulating EMT. These circRNAs may serve as novel potential therapeutic targets or prognostic biomarkers for EOC peritoneal metastasis.

miR-133a targets YES1 to reduce cisplatin resistance in ovarian cancer by regulating cell autophagy.

BACKGROUND: Accumulating evidence has revealed that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. Emerging evidence has demonstrated that miR-133a participates in the tumorigenesis of various cancers. However, whether miR-133a is associated with cisplatin resistance in ovarian cancer remains unclear. OBJECTIVE: To investigate the role of miR-133a in the development of cisplatin resistance in ovarian cancer. METHODS: MiR-133a expression in cisplatin-resistant ovarian cancer cell lines was assessed by reverse-transcription quantitative PCR (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to evaluate the viability of tumour cells treated with cisplatin in the presence or absence of miR-133a. A luciferase reporter assay was used to analyse the binding of miR-133a with the 3' untranslated region (3'UTR) of YES proto-oncogene 1 (YES1). The YES1 expression level was analysed using a dataset from the International Cancer Genome Consortium (ICGC) and assessed by RT-qPCR and western blotting in vitro. The roles and mechanisms of YES1 in cell functions were further probed via gain- and loss-of-function analysis. RESULTS: The expression of miR-133a was significantly decreased in cisplatin-resistant ovarian cancer cell lines (A2780-DDP and SKOV3-DDP), and the overexpression of the miR-133a mimic reduced cisplatin resistance in A2780-DDP and SKOV3-DDP cells. Treatment with the miR-133a inhibitor increased cisplatin sensitivity in normal A2780 and SKOV3 cells. MiR-133a binds the 3'UTR of YES1 and downregulates its expression. Bioinformatics analysis revealed that YES1 expression was upregulated in recurrent cisplatin-resistant ovarian cancer tissue, and in vitro experiments also verified its upregulation in cisplatin-resistant cell lines. Furthermore, we discovered that miR-133a downregulated the expression of YES1 and thus inhibited cell autophagy to reduce cisplatin resistance. Yes1 knockdown significantly suppressed the cisplatin resistance of ovarian cancer cells by inhibiting autophagy in vitro. Xenograft tumour implantation further demonstrated that Yes1 overexpression promoted ovarian tumour development and cisplatin resistance. CONCLUSIONS: Our results suggest that the miR-133a/YES1 axis plays a critical role in cisplatin resistance in human ovarian cancer by regulating cell autophagy, which might serve as a promising therapeutic target for ovarian cancer chemotherapy treatment in the future.

Kaempferol Mediated AMPK/mTOR Signal Pathway Has a Protective Effect on Cerebral Ischemic-Reperfusion Injury in Rats by Inducing Autophagy.

Ischemia/reperfusion (I/R) caused by ischemic stroke treatments leads to brain injury and its pathological mechanism is related to autophagy. The underlying mechanism of kaempferol on cerebral I/R injury needs to be explored. To establish I/R injury, we used a middle cerebral artery occlusion-reperfusion (MCAO) model in rats. MCAO rats were treated with the same amount of saline (I/R group); Treatment group rats were treated orally with kaempferol (50, 100, 200 mg/kg) for 7 days before surgery. After reperfusion for 24 h, the scores of neurological deficits and infarct volume in each group were evaluated. LC3, Beclin-1 p62, AMPK and mTOR protein expression levels were examined by TTC staining, immunofluorescence staining, qRT-PCR and western blotting assay. H&E and TTC staining showed that compared with model group, the infarction size of rats in kaempferol group was markedly reduced. Meanwhile, the results showed that kaempferol had a dose-dependent nerve function repairability. Nissl and TUNEL staining showed that kaempferol could reduce neuronal apoptosis and ameliorate neuronal impairment after I/R. Western blotting and qRT-PCR results showed that kaempferol could protect the brain from ischemia reperfusion by activating autophagy. In addition, add AMPK inhibitor, western blotting and immumohistochemical staining showed that kaempferol mediated AMPK/mTOR signal pathway in MCAO rats. Kaempferol could mediate the AMPK signal pathway to regulate autophagy and inhibit apoptosis to protect brain against I/R injury.

Optimization of endometrial cancer organoids establishment by cancer-associated fibroblasts.

Most endometrial cancers (EC) are diagnosed at an early stage with a favorable prognosis. However, for patients with advanced or recurrent disease, the chemotherapy response rate and overall survival remain poor. A novel in vitro model, tumor organoids, has important value in providing a more individualized treatment plan for tumor patients. However, the slow growth of the established EC organoid seriously hinders the application of EC organoids. Cancer-associated fibroblasts (CAFs), the main component of tumor stroma, have been reported to promote the proliferation of endometrial cancer cell lines and primary endometrial cancer cells in vivo and in vitro. Therefore, we optimized the current endometrial cancer organoid by introducing CAFs isolated from EC lesions. Here we developed long-term expandable organoids from endometrial cancer lesions, which show disease-associated traits and cancer-linked mutations. Based on the co-culture of CAFs and endometrial cancer organoids, we found that CAFs could promote the growth of endometrial cancer organoids, might by secreting factors according to the result that CAFs could also promote the growth. Our research provided a more promising model for the basic and preclinical study of endometrial cancer.

Characterization and Identification of a Ripening-Related Gene AaPG18 in Actinidia arguta.

Actinidia arguta (A. arguta) is a kind of climacteric fruit that quickly softens and limits fruit shelf-life and commercial value. Therefore, it is of great significance to develop kiwifruit genotypes with an extended shelf-life of fruit. However, the ripening and softening mechanisms remain unclear in A. arguta. Here, we demonstrated that a key polygalacturonase (PG)-encoding gene AaPG18 was involved in A. arguta ripening through the degradation of the cell wall. Fruits were harvested at three developmental stages (S1, S2, and S3) for high-throughput transcriptome sequencing, based on which two candidate transcripts c109562_g1 and c111961_g1 were screened. The genome-wide identification of the PG gene family assigned c109562_g1 and c111961_g1 to correspond to AaPG4 and AaPG18, respectively. The expression profiles of candidate genes at six preharvest stages of fruit showed significantly higher expression levels of AaPG18 than AaPG4, indicating AaPG18 might be a key gene during fruit ripening processes. The subcellular localization displayed AaPG18 was located at the cytoplasmic membrane. The transient overexpression of AaPG18 in strawberry and the following morphological observation suggested AaPG18 played a key role in maintaining the stability of cell morphology. The homologous transient transformation in A. arguta "RB-4" proved the crucial function of AaPG18 in fruit ripening processes by causing the rapid redness of the fruit, which was an indicator of fruit maturity. All in all, our results identified AaPG18 as a key candidate gene involved in cell wall degeneration, which provides a basis for the subsequent exploration of the molecular mechanisms underlying the ripening and softening of A. arguta fruit.

Laparoscopic Uterine Artery Occlusion Combined with Uterine-sparing Pelvic Plexus Block and Partial Adenomyomectomy for Adenomyosis: A Video Case Report.

OBJECTIVE: Adenomyosis usually causes dysmenorrhea and anemia. Clinically, it is difficult to be treated with medicine or by traditional surgery, however, hysterectomy is always performed for radical treatment. In this article, we introduce a new method that could control the dysmenorrhea and the anemia through laparoscopic uterine artery occlusion (LUAO) combined with uterine-sparing pelvic plexus block and partial adenomyomectomy for uterus preservation. DESIGN: Surgical video article. Local institutional review board approval for the video reproduction was obtained. SETTING: A 42-year-old patient, who had a history of a previous cesarean delivery, was admitted to our department with complaints of progressive dysmenorrhea for more than 5 years and aggravated with anemia for 1 year. The patient had failed treatment with traditional Chinese medicine and gonadotropin-releasing hormone and had to take painkillers for nearly half a year. The patient had no desire for another pregnancy. After careful consideration, the patient strongly rejected hysterectomy and demanded the preservation of the uterus, insisting on the integrity of the organs. A gynecologic examination showed that the uterus was hard and enlarged similar to one that is more than 8 gestational weeks, without tender nodules in the rectouterine pouch. The visual analog scale pain score was 7, and her hemoglobin was 93 g/L (after correction). The preoperative magnetic resonance imaging implied that there was 1 lesion in the posterior wall and the maximum diameter of the lesion was 7.8 cm. INTERVENTIONS: We performed laparoscopic partial adenomyomectomy combined with occlusion of uterine artery to limit the amount of intraoperative bleeding, dissected the uterine branch of pelvic plexus nerve, and performed electrocoagulation blocking to relieve the dysmenorrhea. The specific operation procedures are as follows (Video): Firstly, we opened the peritoneum through Cheng's triangle, which contained the external iliac blood vessels, the round ligament, and the infundibulopelvic ligament (Fig. 1). Secondly, we separated the lateral rectal space and exposed the ureter, the internal iliac artery, the uterine artery, and the deep uterine vein. Thirdly, we found that the pelvic plexus was located on the outside of the sacral ligament and was approximately 2 to 3 cm below the ureter, going against the sacral ligament and passing through below the deep uterine vein (Supplemental Video 1). Fourthly, we separated the 4 layers of the paracervix [1]. The first layer included the internal iliac artery and the uterine artery. The second layer was the ureter. The third layer was the deep uterine vein. The last layer was the pelvic plexus, which involved the forward-going bladder branch, the inward-going uterine branch, and the downward-going rectal branch (Supplemental Video 2). These anatomic structures are similar to the complex architecture of an overpass called the Cheng's Cross [2] (Fig. 2). In this operation, only the uterine artery and the uterine branch would be blocked. Finally, we performed the partial adenomyomectomy. The endometrium, the myometrial tissues, and the serosa were repaired in some layers with continuous suture, depending on the depth of incision. The operation time was 92 minutes, and the intraoperative hemorrhage was approximately 50 mL. The patient was able to get out of bed on the first day after the operation and urinate after removing the catheter. On the second day after the surgery, the patient had exhaustion and defecation. From the third day after the surgery, gonadotropin-releasing hormone (Goserelin Acetate Sustained-Release Depot,3.6mg each, subcutaneous injection, name of the enterprise: AstraZeneca UK Limited) was used every 4 weeks, with a total of 3 times. Menstruation began on the 67th day after withdrawal of the drug. The results of postoperative condition of the patient followed up at 6 months after surgery were collected as follows: dysmenorrhea was significantly relieved (visual analog scale score was 2), hemoglobin was 123 g/L, and uterine volume was reduced to 43% of preoperative volume. The comparison of the patient's preoperative and postoperative magnetic resonance imaging showed that the uterus was approximately the same size as that of a woman of the same age, and the incision healed well (Fig. 3). CONCLUSION: Adenomyosis is a common gynecologic disease, mainly occurring in women of childbearing age. Adenomyosis is defined as endometrial glands and stroma that invade the myometrium and is surrounded by chronical inflammation in the endometrium [3]. Secondary dysmenorrhea and menorrhagia are the most common chief complaints in patients with adenomyosis, among which dysmenorrhea is the most unbearable symptom [2]. In the past, we had always treated adenomyosis by hysterectomy [4]. With the continuous pursuit of quality of life, it is difficult to meet clinical needs through drugs and traditional surgical methods. Uterine sparing surgery is a current trend in the treatment of adenomyosis, which enables women to maintain fertility and avoid the effects of hysterectomy on sexual function and mental discomfort. Dysmenorrhea can be divided into peripheral dysmenorrhea and central dysmenorrhea. According to our previous studies on dysmenorrhea, the uterine branch nerve has a controlling effect on dysmenorrhea [2]. The purpose of pelvic plexus uterine branch ablation is to further relieve dysmenorrhea by blocking nerve conduction pathways. Therefore, we selectively blocked the uterine branch nerve to alleviate the dysmenorrhea of adenomyosis. The uterine artery controls 90% of uterine blood flow. According to our team research, LUAO is an effective method to treat symptomatic uterine myomas and adenomyosis. We investigated the morphologic change and apoptosis occurring in myomal and adjacent myometrial tissues after LUAO. We concluded that apoptosis through mitochondrial pathways may lead to reduction of the volume of myoma and myometrium and eventually relief of symptoms [5,6]. We speculated "single organ shock uterine" to explain uterine artery occlusion (UAO) mechanism, which was different from uterine artery embolization. The single organ shock theory of UAO can still inhibit the growth of myomas effectively. It is difficult to completely remove adenomyosis lesions during surgery, especially for diffuse adenomyosis. Therefore, in our team, we performed UAO combined with resection of focal lesions in key areas for patients with diffuse adenomyosis, instead of pursuing radical resection [7,8]. The purpose of UAO is to reduce the amount of bleeding during surgery and further atrophy of residual and scattered adenomyosis lesions in utero [5,6]. The intraoperative blocking of the uterine artery can reduce intraoperative bleeding and operation time, improve operation quality, and decrease recurrence rate. In our team, this technique has been used in clinic for more than 10 years. Our previous studies have shown that LUAO combined with pelvic plexus uterine branch nerve block and resection of most of the adenomyosis has achieved satisfactory clinical efficacy as a treatment for adenomyosis [2,3]. With this procedure, we can help patients with adenomyosis retain their uterus and relieve the anxiety caused by hysterectomy. In conclusion, UAO and uterine branch ablation in uterine sparing laparoscopic treatment is a safe and effective method, which may be considered as a good choice for symptomatic adenomyosis.

CXCL12 promotes human ovarian cancer cell invasion through suppressing ARHGAP10 expression.

The CXCL12/CXCR4 axis is strongly implicated as key determinant of tumor invasion and metastasis in ovarian cancer. However, little is known about the potential downstream signals of the CXCL12/CXCR4 axis that contribute to ovarian cancer cell invasion and metastasis. ARHGAP10, a member of Rho GTPase activating proteins is a potential tumor suppressor gene in ovarian cancer. In this study, a negative correlation between the protein levels of CXCL12, CXCR4, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR2) and ARHGAP10 was uncovered in ovarian cancer tissues and paired adjacent noncancerous tissues. CXCL12 stimulation reduced the expression of ARHGAP10. Furthermore, the pretreatment of CXCR4 inhibitor (AMD3100) or the vascular endothelial growth factor receptor-2 (VEGFR2) inhibitor (SU1498) abrogated the CXCL12-deduced expression of ARHGAP10. Finally, an in vitro functional assay revealed that CXCL12 did not stimulate ovarian cancer cell invasion when ARHGAP10 was overexpressed or when ovarian cancer cells were pre-treated with AMD3100 or SU1498. Knockdown of ARHGAP10 significantly suppressed the inhibitory effects of SU1498 on ovarian cancer cell invasion and lung metastasis. In summary, these findings suggest that CXCL12/CXCR4 promotes ovarian cancer cell invasion by suppressing ARHGAP10 expression, which is mediated by VEGF/VEGFR2 signaling.

Wogonin Increases Cisplatin Sensitivity in Ovarian Cancer Cells Through Inhibition of the Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway.

BACKGROUND Wogonin (5,7-dihydroxy-8-methoxyflavone), one of flavonoids isolated from the Scutellaria baicalensis, has been regarded as an anticancer candidate because of its maximal efficacy in cancer cells. This study aimed to explore the possible mechanism that wogonin uses to enhance the sensitivity of ovarian cancer cells to cisplatin chemotherapy. MATERIAL AND METHODS The growth inhibition rates of ovarian cancer cells SKOV3/DDP and C13* were assessed by Cell Counting Kit-8 (CCK-8) assay. The apoptosis was assessed under a fluorescence microscope following staining with Hoechst. We further analyzed the expression of Bcl-2, cleaved caspases-3, cleaved-PARP, and phospho-Akt by western blotting. RESULTS In the present study, we found that wogonin reduced proliferation of ovarian cancer cells SKOV3, SKOV3/DDP, OV2008, and C13* in dose- and time-dependent manners and it sensitized cisplatin-induced cytotoxicity. Moreover, treatment with wogonin also increased cisplatin-resistant SKOV3/DDP and C13* cells to low dose cisplatin-induced cell apoptosis. Additionally, such treatment resulted in a significant decrease in phosphorylated Akt. CONCLUSIONS Wogonin could significantly increase the sensitivity of cisplatin-resistant ovarian cancer cells to cisplatin by downregulating the PI3K/Akt pathway.

Berberine in combination with cisplatin induces necroptosis and apoptosis in ovarian cancer cells.

BACKGROUND: Berberine (BBR), a compound extracted from a variety of medicinal herbs, has been shown multiple pharmacological effects against cancer cells of different origins. Cisplatin (DDP) is known as an effective chemotherapeutic agent against cancer by inducing DNA damage and cell apoptosis. However, the effect of the combined used of BBR and DDP on cell necroptosis in ovarian cancer has not been reported. METHODS: OVCAR3 and three patient-derived primary ovarian cancer cell lines (POCCLs) were chosen as the experimental objects. To determine the potential anti-cancer activity of BBR and DDP in combination, we firstly treated OVCAR3 and POCCLs cells with BBR and/or DDP. The cell viability of OVCAR3 and POCCLs with treatment of BBR or DDP for different hours was measured by CCK-8 assay. Flow cytometry was used to analyze cell cycle distribution and changes in apoptotic cells after treatment with BBR and/or DDP. The morphological changes of OVCAR3 cells were observed by using Transmission electron microscopy (TEM) analysis. Proliferation, apoptosis and necroptosis related markers of OVCAR3 and POCCLs with treatment of BBR or DDP were measured by RT-qPCR, western blotting and immunofluorescence assay. RESULTS: Our results demonstrated that BBR significantly inhibited the proliferation of OVCAR3 and primary ovarian cancer cells in a dose- and time-dependent manner. The combination treatment of BBR and DDP had a prominent inhibitory effect on cancer cell growth and induced G0/G1 cell cycle arrest. TEM revealed that the majority of cells after BBR or DDP treatment had an increasing tendency of typical apoptotic and necrotic cell death morphology. Besides, BBR and DDP inhibited the expression of PCNA and Ki67 and enhanced the expression and activation of Caspase-3, Caspase-8, RIPK3 and MLKL. CONCLUSION: This study proposed that the combination therapy of BBR and DDP markedly enhanced more ovarian cancer cell death by inducing apoptosis and necroptosis, which may improve the anticancer effect of chemotherapy drugs. The apoptosis involved the caspase-dependent pathway, while the necroptosis involved the activation of the RIPK3-MLKL pathway. We hope our findings might provide a new insight for the potential of BBR as a therapeutic agent in the treatment of ovarian cancer.

Adipose-derived stem cells transplantation improves endometrial injury repair.

Endometrial injury is an important cause of intrauterine adhesion (IUA), amenorrhea and infertility in women, with limited effective therapies. Recently, stem cells have been used in animal experiments to repair and improve injured endometrium. To date, our understanding of adipose-derived stem cells (ADSCs) in endometrial injury repair and their further therapeutic mechanisms is incomplete. Here, we examined the benefit of ADSCs in restoration of injured endometrium by applying a rat endometrial injury model. The results revealed by immunofluorescence showed that green fluorescent protein (GFP)-labelled ADSCs can differentiate into endometrial epithelial cells in vivo. At 30 days after ADSCs transplantation, injured endometrium was significantly improved, with increased microvessel density, endometrial thickness and glands when compared with the model group. Furthermore, the fertility of rats with injured endometrium in ADSCs group was improved and had a higher conception rate (60% vs 20%, P = 0.014) compared with the control phosphate-buffered saline (PBS) group. However, there was no difference in the control group compared with the sham group. In addition, expression levels of the oestrogen receptor Eα/ß (ERα, ERß) and progesterone receptor (PR) detected by western blot and enzyme-linked immunosorbent assay (ELISA) were higher in the ADSCs group than in the PBS group. Taken together, these results suggested that ADSC transplantation could improve endometrial injury as a novel therapy for IUA.

Berberine inhibits growth and inflammatory invasive phenotypes of ectopic stromal cells: Imply the possible treatment of adenomyosis.

Adenomyosis is a common chronic gynecological disorder with some tumor-like properties, including aberrant proliferation, invasion and migration. Berberine (BBR) is an isoquinoline derivative alkaloid with diverse pharmacological activities for the treatment of a wide variety of diseases. However, the effect of BBR on adenomyosis has not been understood. This study was to evaluate the potential therapeutic effect of BBR on ectopic endometrial stromal cells (EESCs) isolated from patients with adenomyosis. Our data showed that BBR significantly inhibited the proliferation and viability of eutopic endometrial stromal cells (EuESCs) and EESCs, while slightly affected the growth of normal endometrial stromal cells (NESCs). BBR markedly exhibited a growth inhibitory effect on EESCs by triggering apoptosis and cell cycle arrest, and alleviating the expression of inflammatory invasive phenotypes (IL-6, IL-8, TGF-ß, EGF, VEGF, and MMP2). The alleviation of inflammatory invasive phenotypes partly involved nuclear translocation of NFκB/p65 and stat3 activation. Taken together, BBR markedly inhibits the growth of EESCs and might be a promising new strategy for the treatment of adenomyosis.