Inhibition of ATM promotes PD-L1 expression by activating JNK/c-Jun/TNF-α signaling axis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer. Anti-PD-1/PD-L1 treatment for advanced TNBC is still limited to PD-L1-positive patients. Ataxia telangiectasia mutated (ATM) is a switch molecule for homologous recombination and repair. In this study, we found a significant negative correlation between ATM and PD-L1 in 4 TNBC clinical specimens by single-cell RNA sequencing (scRNA-seq), which was confirmed by immunochemical staining in 86 TNBC specimens. We then established ATM knockdown TNBC stable cell lines to perform in vitro studies and animal experiments, proving the negative regulation of PD-L1 by ATM via suppression of tumor necrosis factor-alpha (TNF-α), which was confirmed by cytokine array analysis of TNBC cell line and analysis of clinical specimens. We further found that ATM inhibits TNF-α via inactivating JNK/c-Jun by scRNA-seq, Western blot and luciferase reporter assays. Finally, we identified a negative correlation between changes in phospho-ATMS1981 and PD-L1 levels in TNBC post- and pre-neoadjuvant therapy. This study reveals a novel mechanism by which ATM negatively regulates PD-L1 by downregulating JNK/c-Jun/TNF-α in TNBC, shedding light on the wide application of immune checkpoint blockade therapy for treating multi-line-resistant TNBC.

[Genetic analysis of a child with Complex cortical dysplasia with other brain malformations type 6 due to a p.M73V variant of TUBB gene].

OBJECTIVE: To explore the genetic basis for a child with multiple malformations. METHODS: A child who had presented at Shanxi Provincial Children's Hospital in February 2021 was selected as the study subject. Clinical data of the patient was collected, and whole exome sequencing (WES) was carried out to screen pathogenic variants associated with the phenotype. Candidate variant was validated by Sanger sequencing of her family members. RESULTS: The child had normal skin, but right ear defect, hemivertebral deformity, ventricular septal defect, arterial duct and patent foramen ovale, and separation of collecting system of the left kidney. Cranial MRI showed irregular enlargement of bilateral ventricles and widening of the distance between the cerebral cortex and temporal meninges. Genetic testing revealed that she has harbored a heterozygous variant of NM_178014.4: c.217A>G (p.Met73Val) in the TUBB gene, which was unreported previously and predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). The child was diagnosed with Complex cortical dysplasia with other brain malformations 6 (CDCBM6). CONCLUSION: CDCBM is a rare and serious disease with great genetic heterogeneity, and CDCBM6 caused by mutations of the TUBB gene is even rarer. Above finding has enriched the variant and phenotypic spectrum of the TUBB gene, and provided important reference for summarizing the genotype-phenotype correlation of the CDCBM6.

Correlation of TGF-ß signaling pathway gene polymorphisms with unexplained recurrent spontaneous abortion.

BACKGROUND: The association of key genes in the transforming growth factor-ß (TGF-ß) signaling pathway and their gene polymorphisms with unexplained recurrent spontaneous abortion (URSA) is unclear. OBJECTIVE: To investigate the association of gene polymorphisms related to the TGF-ß signaling pathway in URSA women. METHODS: The study population consisted of 80 women with URSA and 90 normal control women, of which 10 women with URSA and 10 normal control women underwent high-throughput sequencing to select loci, and the remaining 70 women with URSA and 80 normal control women underwent flight mass spectrometry experiments to verify gene loci polymorphism. A total of 7 polymorphic loci in interleukin-6 (IL-6), TGF-ß1, TNF-α, SMAD1, and TNFRSF4 genes were screened by high-throughput sequencing combined with a review of databases. An SNP flight mass spectrometer (Mass ARRAY detection system) was applied to detect the polymorphisms and their frequencies in 70 women with URSA and 80 normal control women at the 7 gene loci. RESULTS: Among the 7 loci of IL-6, TGF-ß1, TNF-α, SMAD1, and TNFRSF4 genes, 2 loci were found to have significantly different allele and genotype frequency distributions between the 70 URSA and 80 normal controls, one was the IL-6 gene -174G/C locus (rs1800795), the risk of disease was 2.636 and 3.231 times higher in individuals carrying the C allele and CC genotype than in those carrying the G allele and GG genotype, respectively; the other was the TGF-ß1 gene -509T/C locus (rs1800469), and the risk of disease was 1.959 and 3.609 times higher in individuals carrying the T allele and TT genotype than in those carrying the C allele and CC genotype, respectively. The remaining 5 genetic loci have no statistically significant. CONCLUSION: IL-6 gene -174G/C locus (rs1800795) genotype CC and allele C may be the causative factor of URSA, TGF-ß1 gene -509T/C locus (rs1800469) genotype TT and allele T may be the causative factor of URSA, and polymorphisms of the 2 loci may be associated with URSA.

Effects of tumor-infiltrating lymphocytes on nonresponse rate of neoadjuvant chemotherapy in patients with invasive breast cancer.

High level of tumor-infiltrating lymphocytes (TILs) can predict the rate of total pathological complete remission (tpCR) of breast cancer patients who receive neoadjuvant chemotherapy (NACT). This study focused on evaluating the data of patients whose primary tumor and/or lymph node metastasis show nonresponse (NR) to NACT, trying to provide a basis for the clinical decision which patients will develop NACT resistance. The study included breast cancers from 991 patients who received NACT. ROC curve analysis confirmed that TILs showed significant predictive value for NR of hormone receptor (HR)+HER2- and triple-negative breast cancer (TNBC). Among HR+HER2- breast cancer, TILs ≥ 10% was an independent predictor for low NR rate. Furthermore, positive correlation of TILs with Ki67 index and Miller-Payne grade, and negative correlation with ER and PR H-scores were only identified in this subgroup. In TNBC, TILs ≥ 17.5% was an independent predictor for low NR rate. The predictive value of low TILs on NR may facilitate to screen patients with HR+HER2- or TNBC who may not benefit from NACT. HR+HER2- breast cancer with low levels of TILs should be carefully treated with neoadjuvant chemotherapy, and other alternatives such as neoadjuvant endocrine therapy can be considered.

STAT3 and PD-L1 are negatively correlated with ATM and have impact on the prognosis of triple-negative breast cancer patients with low ATM expression.

INTRODUCTION: Triple-negative breast cancer (TNBC) is known for its aggressive behaviors and lacking of effective treatment. Programmed cell death ligand-1 (PD-L1) inhibitor has just been approved for using in the management of advanced TNBC. To accurately screen TNBC sensitive to anti-PD-L1 treatment and to explore the feasibility of the ataxia-telangiectasia mutation protein (ATM) inhibitor combined with PD-L1 inhibitor, radiotherapy and chemotherapy, we focus on whether ATM participates in the regulation of PD-L1 and affects the prognosis of patients through c-Src, signal transducer and activator of transcription 1&3 (STAT1 and STAT3). MATERIALS AND METHODS: We used immunohistochemical staining to explore the relationship of ATM with c-Src, STAT1, STAT3, PD-1/PD-L1, Tumor-infiltrating lymphocytes (TILs), as well as other clinicopathologic features in 86 pathological stage III TNBCs. Their impact on prognosis was also explored. RESULTS: We found ATM expression was negatively correlated with STAT1, STAT3, PD-L1, TILs and CD8 + cells in TNBC. STAT1 positively correlated the expression of PD-L1. In TNBC with ATM low expression, STAT3 was an independent factor for improved prognosis, while PD-L1 was an independent negative prognostic factor. Furthermore, in low ATM group, the phosphorylation of tyrosine at position 419 of c-Src (p-c-src Y419) was correlated with the overexpression of STAT3. CONCLUSION: Locally advanced TNBC with low ATM expression may be more likely to benefit from anti-PD-L1 inhibitors. The feasibility of ATM functional inhibitor combined with immune checkpoint blockade therapies in the treatment of TNBC is also worthy of further exploration. Our study suggests that STAT3 has different impacts on tumor progression in different tumors.

miR-30 inhibits the progression of osteosarcoma by targeting MTA1.

OBJECTIVES: MicroRNAs (miRNAs) have been considered as a new class of novel diagnostic and predictive biomarker in many diseases. However, there are few studies on miRNA in osteosarcoma (OS). This study aimed to investigate the roles of miR-30 on OS occurrence and development. METHODS: PCR was used to detect mRNA levels of miR-30 and MTA1 in cancer tissues, adjacent non-cancerous tissues from OS patients. Western blot was used to detect MTA1 protein expression in all tissues and cell lines (hFOb1.19,Saos-2, MG63, and U2OS). The correlation between miR-30 and MTA1 was predicted through bioinformatics software, and identified by a luciferase reporting experiment. In vitro, functional test detected the specific effects of miR-30 and MTA1 on the development of OS. RESULTS: miR-30 expression was significantly reduced, while the expression of MTA1 was increased in OS tissues and cells. Luciferase reporting experiment showed that miR-30 sponged MTA1 which was negatively correlated with miR-30 expression. Furthermore, rescue tests revealed that MTA1 restrained the functions of miR-30 on cell proliferation and migration of OS. CONCLUSION: Our finding showed that miR-30 modulated the proliferation and migration by targeting MTA1 in OS.

Carcinoma-associated fibroblasts promote the proliferation and metastasis of osteosarcoma by transferring exosomal LncRNA SNHG17.

Cancer-associated fibroblasts (CAFs) serve as a predominant regulator in the tumor microenvironment. However, the crosstalk between CAFs and OS cells remains mostly unclear. Recent studies explored that long non-coding RNA (LncRNAs) involved in regulating osteosarcoma (OS) formation and development, but their functions in CAFs are unknown. Here, we first investigated the SNHG17 was upregulated in OS tissues and correlated with the poor prognosis through the integrating clinical data. We then evaluated the function of SNHG17 in vitro using the stable SNHG17-depleted OS cells. HOS cells with SNHG17 knocked down were performed to generate the OS xenograft model. Through immunohistochemistry assay and TUNEL apoptosis assay, the role of SNHG17 on OS development was assessed in vivo. We then examined the SNHG17 expression in exosomes derived from CAFs, normal fibroblasts (NFs), and tumor tissues from the OS clinical samples. The interaction among SNHG17, miR-2861, and MMP2 was predicted by bioinformatics analysis and identified by RIP and luciferase assays. The cell proliferation, migration, and apoptosis of SJSA-1 and HOS cells co-cultured with CAFs-derived exosomes were assessed by CCK-8 and colony formation assays. We found that SNHG17 was upregulated in the tumor tissues and presented a pro-tumorigenic effect on OS both in vitro and in vivo. It also was an essential exosomal cargo of CAFs and could affect OS cell proliferation and migration in vitro. CAFs-released exosomal SNHG17 acted as an essential molecular sponge for miR-2861 in OS cells. Moreover, MMP2 was a direct target of miR-2861 and was regulated by SNHG17. Overall, our findings identified that SNHG17 was an essential exosomal cargo of OS-related CAFs that contributes to proliferation and metastasis of OS, supporting the therapeutic potency of targeting the crosstalk between cancer cells and CAFs.

[Wiedemann-Steiner syndrome due to novel nonsense variant of KMT2A gene in a case].

OBJECTIVE: To explore the genetic basis for a child with unexplained global developmental delay (GDD), seizure, and facial deformity. METHODS: Whole exome sequencing (WES) was carried out for the patient. Candidate variants were verified by Sanger sequencing of the patient and his parents. RESULTS: WES revealed that the patient has carried a previously unreported de novo heterozygous nonsense c.4906C>T (p.Arg1636Ter) variant of the KMT2A gene, Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.4906C>T variant of KMT2A gene was predicted to be pathogenic (PVS1+ PS2+ PM2+PP3). CONCLUSION: The heterozygous nonsense c.4906C>T (p.Arg1636Ter) variant of the KMT2A gene probably underlay the disease in the child. Above finding has enriched the spectrum of pathogenic variants of the KMT2A gene.

lncRNA TUSC7 inhibits osteosarcoma progression through the miR­181a/RASSF6 axis.

Osteosarcoma (OS) is one of the most aggressive malignancies, accompanied by an elevated incidence and a decreased rate of healing. Recently, several long non­coding RNAs (lncRNAs) have been reported to be involved in OS progression. Although tumor suppressor candidate 7 (TUSC7) was reported as a novel lncRNA, little is known about its biological functions in OS. The present study was designed to explore whether TUSC7 was involved in the pathological development of OS using various methods, including hematoxylin and eosin staining, Cell Counting Kit­8 assay, colony formation assay and Transwell assay. The present study revealed that TUSC7 expression was downregulated in OS tissues and cell lines compared with in normal tissues and cell lines. Functionally, the current results revealed that overexpression of TUSC7 inhibited OS cell proliferation, migration and invasion, while promoting apoptosis in vitro and in vivo. Next, the subcellular distribution of TUSC7 was examined by nuclear/cytoplasmic RNA fractionation and reverse transcription­quantitative PCR. Mechanistic studies revealed that TUSC7 exerted its role by sponging microRNA (miR)­181a in OS cell lines. Ras association domain family member 6 (RASSF6) was confirmed as a target gene of miR­181a, and the expression levels of RASSF6 were negatively regulated by miR­181a. Additionally, the results of rescue experiments suggested that overexpression of miR­181a neutralized the inhibitory effects of TUSC7 overexpression on OS cells. Overall, the present study demonstrated that the tumor suppressor role of TUSC7 in OS progression was mediated through the miR­181a/RASSF6 axis, which may represent a new therapeutic target for OS.

The Comparison of Intrathecal Ropivacaine with Bupivacaine for Knee Arthroscopy: A Meta-analysis of Randomized Controlled Trials.

The comparison of intrathecal ropivacaine with bupivacaine for knee arthroscopy remains controversial. We conduct a systematic review and meta-analysis to explore the efficacy of intrathecal ropivacaine versus bupivacaine for knee arthroscopy. We search PubMed, EMbase, Web of science, EBSCO, and Cochrane library databases through August 2019 for randomized controlled trials (RCTs) assessing the effect of intrathecal ropivacaine versus bupivacaine for knee arthroscopy. This meta-analysis is performed using the random effects model. Five RCTs are included in the meta-analysis. Overall, compared with intrathecal bupivacaine for knee arthroscopy, intrathecal ropivacaine is associated with increased onset time of motor block (mean difference [MD] = 2.05, 95% CI: 1.43-2.67, p < 0.00001) and decreased duration of sensory block (MD = -26.82, 95% CI: -31.96 to -21.67, p < 0.00001) but shows no remarkable influence on onset time of sensory block (MD = -0.09; 95% CI: -1.89 to 1.70, p = 0.92), duration of motor block (MD = -59.76; 95% CI: -124.44 to 4.91, p = 0.07), time to maximum block (MD = 2.35; 95% CI: -0.16 to 4.86, p = 0.07), first urination time (MD = -26.42, 95% CI: -57.34 to 4.51, p = 0.09), or first ambulation time (MD = 3.63, 95% CI: -25.20 to 32.47, p = 0.80).Intrathecal ropivacaine can substantially increase onset time of motor block and decrease the duration of sensory block than intrathecal bupivacaine for knee arthroscopy.

Mutation analysis of TCOF1 gene in Chinese Treacher Collins syndrome patients.

BACKGROUND: Treacher Collins syndrome (TCS) is a rare autosomal dominant or recessive disorder, that involves unique bilateral craniofacial malformations. The phenotypes of TCS are extremely diverse. Interventional surgery can improve hearing loss and facial deformity in TCS patients. METHOD: We recruited seven TCS families. Variant screening in probands was performed by targeted next-generation sequencing (NGS). The variants identified were confirmed by Sanger sequencing. The pathogenicity of all the mutations was evaluated using the guidelines of the American College of Medical Genetics and Genomics (ACMG) and InterVar software. RESULTS: Three frameshift variants, two nonsense variants, one missense variant, and one splicing variant of TCOF1 were identified in the seven TCS probands. Five variants including c.1393C > T, c.4111 + 5G>C, c.1142delC, c.2285_2286delCT, and c.1719delG had not been previously reported. Furthermore, we report the c.149A > G variant for the first time in a Chinese TCS patient. We provided prenatal diagnosis for family 4. Proband 7 chose interventional surgery. CONCLUSION: We identified five novel variants in TCOF1 in Chinese patients with TCS, which expands the mutation spectrum of TCOF1 in TCS. Bone conduction hearing rehabilitation can improve hearing for TCS patients and prenatal diagnosis can provide fertility guidance for TCS families.

MicroRNA­22 mediates the cisplatin resistance of osteosarcoma cells by inhibiting autophagy via the PI3K/Akt/mTOR pathway.

Osteosarcoma (OS) is the most common primary malignant tumor of the bone affecting children and adolescents. Chemotherapy is now considered as a standard component of OS treatment, not only for children, but also for adults. However, chemoresistance continues to pose a challenge to therapy. Inhibition of autophagy has been demonstrated to decrease chemoresistance in OS. Moreover, microRNA­22 (miR­22) inhibits autophagy, leading to an improvement in the sensitivity of cisplatin (CDDP) in OS. The aim of the present study was therefore to investigate whether miR­22 could mediate the CDDP resistance of OS cells by inhibiting autophagy via the phosphoinositide 3­kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Cell proliferation assay, LC3 flow cytometry assay and monodansylcadaverine staining in MG63 cells and CDDP resistance cells (MG63/CDDP) were performed to explore to role of miR­22 and CDDP in OS chemoresistance. Inoculation of tumor cells in an in vivo model, reverse transcription­quantitative PCR (RT­qPCR) assay, western blot analysis, and immunohistochemistry analysis were performed to investigate the role of miR­22 and CDDP in the PI3K/Akt/mTOR pathway as it is affected by autophagy. The results revealed that miR­22 inhibited the proliferation of MG63 and MG63/CDDP cells, and enhanced the anti­proliferative ability of CDDP in vivo and in vitro. miR­22 mediated the CDDP resistance of OS cells by inhibiting autophagy and decreasing CDDP­induced autophagy via downregulation of the expression of PI3K, Akt, and mTOR at the mRNA level, and the expression of PI3K, phosphorylated (p)­Akt, and p­mTOR at the protein level. It was also convincingly demonstrated that miR­22 mediates the CDDP resistance of OS by inhibiting autophagy via the PI3K/Akt/mTOR pathway. Furthermore, in the MG63 cells that were affected by CDDP, the role of miR­22 was shown to be similar to that of the investigated inhibitor of PI3K (wortmannin) in terms of regulating the PI3K/Akt/mTOR pathway, and wortmannin could also promote the effect of miR­22. Interestingly, CDDP was demonstrated to induce autophagy by inhibiting the PI3K/Akt/mTOR pathway, whereas the pathway was upregulated in the state of chemoresistance. In conclusion, downregulation of the PI3K/Akt/mTOR pathway was shown to assist in the process of preventing chemoresistance.

The expression and prognostic role of EBP1 and relationship with AR in HER2+ breast cancer.

Human epidermal growth factor receptor (HER)-2 positive (HER2+) breast cancer (BC) has a poor survival rate and is more aggressive in nature. HER2-targeting agents could be beneficial for patients with HER2+ BC. In addition, targeted therapy and chemotherapy have been successfully used. However, a few patients are resistant to treatment. ErbB3 binding protein 1 (EBP1) binds to HER3 and inhibits the proliferation and invasive potential of tumor cells. However, its role in HER2+ BC has not been demonstrated. In this study, we aimed to analyze the relationship between androgen receptor (AR) and EBP1 expression in HER2+ BC. A total of 282 cases (140 cases of HER2+ invasive BC and 142 HER2-negative invasive BC) were included in this study. We performed immunohistochemistry (IHC) to analyze the expression of AR and EBP1; thereafter, we evaluated the relationship between these two biomarkers and estrogen receptor (ER), progesterone receptor (PR), HER2, p53, Ki67 expression, and other clinicopathological parameters. Of the HER2+ cases, 67 (47.9%) showed high expression of EBP1 (EBP1high) and 73 (52.1%) showed low/no expression of EBP1 (EBP1low/no). EBP1 expression was correlated with AR expression, histological grade, and lymphatic metastasis (p < 0.001, < 0.001, and 0.013, respectively). Kaplan-Meier analysis revealed that AR+ and EBP1low/no group had poorer overall survival (OS) and disease-free survival (DFS) compared with other groups (AR- and EBP1low/no, AR+ and EBP1high, and AR- and EBP1high). AR+ and EBP1low/no expression were independent prognostic factors for OS and DFS in HER2+ BC. This study showed the clinicopathological role of EBP1 and AR in HER2+ BC. Targeting EBP1 may be an effective treatment strategy for patients with AR+ HER2+ BC.

Rosmarinic Acid, the Main Effective Constituent of Orthosiphon stamineus, Inhibits Intestinal Epithelial Apoptosis Via Regulation of the Nrf2 Pathway in Mice.

Many studies have shown that Orthosiphon stamineus extract (OE) has antioxidant activity, and we previously reported that OE protects the intestine against injury from a high-fat diet. However, the molecular mechanism underlying this protective effect of OE was unclear. Here, OE was separated according to polarity and molecular weight, and the antioxidant activity of each component was compared. The components with the highest antioxidant activity were analyzed by HPLC, which confirmed that rosmarinic acid (RA) was the main effective constituent in OE. OE and RA were then tested in a mouse high-fat diet-induced intestinal injury model. The antioxidant indices and morphological characteristics of the mouse jejunum were measured, and activation of the nuclear factor E2-related factor 2 (Nrf2) pathway and apoptosis of jejunal epithelial cells were analyzed. Of all the constituents in OE, RA contributed the most. Both RA and OE activated the Nrf2 pathway and increased downstream antioxidant enzyme activity. RA and OE protected the mouse intestine against high-fat diet-induced oxidative stress by preventing intestinal epithelial cell apoptosis via both extracellular and intracellular pathways. Thus, RA, the main effective constituent in OE, inhibits intestinal epithelial apoptosis by regulating the Nrf2 pathway in mice.

A comparative study of the antioxidant and intestinal protective effects of extracts from different parts of Java tea (Orthosiphon stamineus).

The aim of this study was to compare the free radical scavenging ability and intestinal epithelial cell protective effects of Java tea (Orthosiphon stamineus) root extracts (ORE), stem extracts (OSE), and leaf extracts (OLE) to determine the potential of Java tea by-products. The Java tea extracts were prepared using a standard water-ethanol method. The antioxidant activity and intestinal protective effects were tested by H2O2-induced cell model and high-fat diet-induced mice model, respectively. The results showed that the total phenolic acid and flavonoid content and relative content were different in the ORE, OSE, and OLE. ORE had the highest total polyphenol and flavonoid content, the highest free radical scavenging rate, and the highest intracellular free radical scavenging rate. However, the yeast content in the ORE was lower than that in the OSE and OLE. All the Java tea extracts protected mouse intestine from high-fat diet-induced oxidative injury. This study indicates the potential of Java tea extracts as food or feed additives to protect the intestine from oxidative stress.

Robot-assisted versus conventional laparoscopic surgery for endometrial cancer staging: A meta-analysis.

This meta-analysis broadly compared the safety and efficacy of robot-assisted laparoscopy (RAL) with that of conventional laparoscopy (CL) for endometrial cancer staging. The advantages of RAL were evaluated through the outcomes in terms of conversion rates, complications, length of operation, blood loss, number of lymph nodes harvested, and length of hospitalization. Three electronic databases (PubMed, MEDLINE, and EmBASE) were searched to identify eligible studies. We selected all retrospective studies documenting a comparison between RAL and CL for endometrial cancer staging between 2005 and 2015, and tallied with meta-analyses criteria. Only studies published in English were included in this analysis. The outcomes of the extracted data were pooled and estimated by the Review Manager version 5.1 software. Seventeen studies met the eligibility criteria. Among the 2105 patients reported, 912 underwent RAL and the other 1193 underwent CL for endometrial cancer staging. Compared with CL, RAL had lower conversion rates [risk ratio, 0.4; 95% confidence interval (CI), 0.25-0.64; p = 0.0002]. Its complications were also less than that of CL (risk ratio, 0.72; 95% CI, 0.56-0.94; p = 0.02). RAL was associated with significantly less intraoperative blood loss (weighted mean difference, -79.2 mL; 95% CI, from -103.43 to -54.97; p < 0.00001) and a shorter length of hospitalization (weighted mean difference, -0.37 days; 95% CI, from -0.57 to -0.17; p = 0.0003). We found no significant differences in the length of operation and number of lymph nodes harvested between the two groups. From our meta-analysis results, RAL is a safe and effective alternative to CL for endometrial cancer staging. Further studies are required to determine potential advantages or disadvantages of RAL.