OBJECTIVE To determine the contents of 15 components in Aurantii Fructus Immaturus from different origins （Citrus aurantium， C. junos， C. aurantium Linn.， C. sinensis Osb.， C. sinensis）， screen differential components， and provide references for the quality evaluation of Aurantii Fructus Immaturus. METHODS HPLC method was adopted to determine the contents of synephrine， N-methyltyramine， 5，7-dihydroxychromone-7-neohesperidoside， neoponcirin， narirutin， naringin， hesperidin， neohesperidin， naringenin， hesperetin， sinensetin， nobiletin， tangeretin， 5-demethylnobiletin， and auraptene in 46 batches of Aurantii Fructus Immaturus from different origins. The determination was performed on Waters Symmetry C18 column with mobile phase consisted of acetonitrile-0.1% formic acid （gradient elution） at the flow rate of 1.0 mL/min； column temperature was set at 40 ℃ ， detection wavelength was 284 nm， and sample injection volume was 5 μL. The differences between different origins of Aurantii Fructus Immaturus were analyzed by cluster analysis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA）， and differential components were screened. RESULTS The linear relationships of the aforementioned 15 components were all good within the tested mass concentration ranges （all r＞0.999 0）. The RSDs for precision， stability （24 h）， and repeatability tests were all less than 2.00%. The average recovery rate ranged from 91.1% to 103.9% （all RSDs＜3.10%）. Cluster analysis， PCA， and OPLS-DA revealed that C. sinensis Osb. and C. sinensis were clustered into one category， while C. aurantium，C. junos and C. aurantium Linn. were clustered into another category. The variable importance projection values for neohesperidin， auraptene， naringin， neoponcirin， tangeretin， hesperidin， sinensetin， and 5，7-dihydroxychromone-7-neohesperidoside were all greater than 1. CONCLUSIONS In this study， the contents of 15 components in Aurantii Fructus Immaturus from different origins are determined， and 8 differential components， including neohesperidin， auraptene， naringin， and neoponcirin， are screened out.

Trophoblast cells serve as the foundation for placental development. We analyzed published multiomics sequencing data and found that trophoblast cells highly expressed RRS1 compared to primitive endoderm and epiblast. We used HTR-8/SVneo cells for further investigation, and Western blot and immunofluorescence staining confirmed that HTR-8/SVneo cells highly expressed RRS1. RRS1 was successfully knocked down in HTR-8/SVneo cells using siRNA. Using IncuCyte S3 live-cell analysis system based on continuous live-cell imaging and real-time data, we observed that proliferation, migration, and invasion abilities were all significantly decreased in RRS1-knockdown cells. RNA-seq revealed that knockdown of RRS1 affected the gene transcription, and upregulated pathways in extracellular matrix organization, DNA damage response, and intrinsic apoptotic signaling, downregulated pathways in embryo implantation, trophoblast cell migration, and wound healing. Differentially expressed genes were enriched in diseases related to placental development. Consistent with these findings, human chorionic villus samples collected from spontaneous abortion cases exhibited significantly reduced RRS1 expression compared to normal controls. Our results highlight the functional importance of RRS1 in human trophoblasts and suggest that its deficiency contributes to early pregnancy loss.
Humans ; Trophoblasts/physiology* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Female ; Pregnancy ; Abortion, Spontaneous/metabolism* ; Cell Line ; Placentation/genetics*

Abstract Alternative splicing(AS) is one of the important ways of post-transcriptional regulation, which can generate multiple mRNA isoforms from a single gene. In recent years, many studies have shown that AS can occur in almost all types of tumors, and the abnormal expression of splicing factors is related to the disease progression of tumor patients, which may become a potential marker for judging tumor progression. Therefore, this review summarizes the role, molecular mechanism, clinical relevance and treatment response of AS in tumors. It is found that AS can participate in the progression of malignant tumors by regulating tumor invasion, metastasis, apoptosis, cell metabolism, and promoting tumor immune escape and treatment resistance, which provides a theoretical basis for the clinical application of AS. The precise identification of tumor-specific AS and the development of small molecule inhibitors targeting specific AS isoforms may provide a new direction for precise and personalized cancer treatment.

Objective : To explore the role of semaphoring 6d(SEMA6D) in the malignant progression of triple-negative breast cancer(TNBC). Methods : Bioinformatics and Immunohistochemistry(IHC) were used to analyze the expression level of SEMA6D in TNBC and paracancer non-tumor tissues and its relationship with patients′ clinicopathological features. MDA-MB-231 cell line stably knocking down the expression of SEMA6D was constructed, and the effects of SEMA6D on migration and invasion of TNBC cells were investigated by Wound-healing assays and Transwell assays. cBioPortal and GEPIA2 databases were used to screen out the gene negatively associated with it, namely aurora kinase A(AURKA). Bioinformatics and IHC were used to analyze the expression level of AURKA in TNBC and paracancer non-tumor tissues and its relationship with patients' clinicopathological features. Western blot assay was used to analyze the expression of AURKA and the effect of epithelial-mesenchymal transition(EMT) makers Claudin-1, N-cadherin and Vimentin after knocking downSEMA6D. Results: Bioinformatics analysis and IHC results showed that the expression of SEMA6D in TNBC tissues was significantly lower than that in paracancer non-tumor tissues(bothP<0.05). The expression of AURKA in TNBC tissues was significantly higher than that in paracancer non-tumor tissues(bothP<0.05), SEMA6D and AURKA were significantly negatively correlated in TNBC(P<0.01). Both low expression of SEMA6D and high expression of AURKA were positively correlated with tumor size, tumor histological grade, clinical stage and lymph node metastasis in TNBC patients(allP<0.05). The knockdown ofSEMA6Dsignificantly promoted the migration and invasion ability of TNBC cells(bothP<0.01). Western blot results showed that the knockdown ofSEMA6Dupregulated AURKA expression, promoted the expression of N-cadherin and Vimentin, and inhibited the expression of Claudin-1 in tumor cells. Conclusion Down-regulation of SEMA6D expression in TNBC may be involved in the malignant progression of TNBC through up-regulation of AURKA expression and promotion of EMT.

Objective : To explore the expression of prohibitin2(PHB2) in breast cancer and its effect on the biological behaviors of tumor cells. Methods : Immunohistochemistry was used to detect the expression of PHB2 protein in breast cancer tissues and its relationship with clinicopathologic features. Breast cancer stable transient cell lines were constructed with knockdown and overexpression ofPHB2, respectively. The effects of PHB2 on cell proliferation, migration and invasion ability were detected by clone formation assay, scratch assay and Transwell assay. Western blot(WB) was used to detect the effects of PHB2 on the expression of epithelial-mesenchymal transition(EMT)-related markers, including E-cadherin, N-cadherin, Snail family transcriptional repressor 1(Snail) protein, Vimentin, and Claudin-1. The effect of PHB2 on tumorigenicityin vivowas detected by subcutaneous tumor formation assay in nude mice. Results: The result of immunohistochemical showed that PHB2 was highly expressed in breast cancer and the expression of PHB2 was significantly positive correlated with tumor size, human epidermal growth factor receptor-2(HER-2) status and proliferation index Ki-67 levels(P<0.05). Clone formation assay, scratch assay and Transwell assay revealed that knockdown ofPBH2significantly inhibited the proliferation, migration and invasion ability of breast cancer cells(P<0.01), while the overexpression ofPHB2significantly promoted cell proliferation, migration and invasion(P<0.01). The result of subcutaneous tumor formation experiment in nude mice revealed a significant decrease in tumor volume and weight in knockdownPHB2mice(P<0.000 1), whilePHB2overexpression tumors significantly increased in volume and weight(P<0.001).WB assay showed that the protein expression of epithelial marker E-cadherin increased, while the expressions of mesenchymal markers N-cadherin, Snail and Vimentin decreased significantly afterPHB2knockdown with them in control cells(P<0.01). The expression of Claudin-1 decreased, while the expressions of N-cadherin, Snail and Vimentin increased significantly inPHB2overexpression cells(P<0.05). Conclusion PHB2 is highly expressed in breast cancer and promotes multiple malignant biological behaviors in tumor cells, suggesting PHB2 may be a potential target for breast cancer diagnosis and treatment.

Objective : To investigate the expression of Keratin 14(KRT14) in Basal-like Breast Cancer(BLBC) and its biological functions and mechanisms. Methods : The expression levels of KRT14 mRNA in BLBC and para-cancer breast tissues were analyzed using The Cancer Genome Atlas(TCGA) database. qPCR, Western blot(WB), and immunohistochemistry were employed to detect KRT14 expression in BLBC and adjacent normal tissues, and its correlation with clinicopathological features was analyzed. KRT14 overexpression and knockdown were performed in breast cancer cells, and cell scratch and transwell assays were performed to evaluate changes in migration and invasion abilities. To investigate the expression of proteins related to the Wnt/β-catenin signaling pathway, including catenin Beta 1(β-catenin), wingless-type MMTV integration site family, member 1(Wnt1), matrix metallopeptidase 7(MMP7), and cellular myelocytomatosis viral oncogene homolog(c-Myc), as well as the cellular localization of β-catenin, WB and immunofluorescence(IF) techniques were employed. Additionally, a Wnt/β-catenin signaling pathway inhibitor was used to verify the mechanism of action of KRT14. Results : The expression of KRT14 was significantly higher in BLBC tissues compared to normal tissues(P<0.05), and was associated with higher T stage and histological grade(P<0.05). The overexpression of KRT14 significantly enhanced the migration and invasion abilities of breast cancer cells, while the knockdown of KRT14 significantly reduced those abilities(P<0.01). The overexpression of KRT14 can increase the expression levels of Wnt/β-catenin pathway-related proteins β-catenin, Wnt1, MMP7, and c-Myc, thereby activating the Wnt/β-catenin pathway. Moreover, the inhibition of this pathway can eliminate the effects of KRT14 on cell migration and invasion. Conclusion The high expression of KRT14 in BLBC may promote the migration and invasion of breast cancer cells through the Wnt/β-catenin signaling pathway.

Objective : To investigate the expression of(heat shock protein a member 8,HSPA8) in breast cancer and its effect on tumor biological behaviors. Methods: Bioinformatics analysis and immunohistochemistry assays were used to detect the expression of HSPA8 in breast cancer and adjacent non-tumor breast tissues,and the relationship between its expression and clinicopathological characteristics of breast cancer patients was analyzed.Correlation between HSPA8 expression and prognosis of breast cancer patients was analyzed by Kaplan-Meier Plotter database.HSPA8 knockdown and over expression breast cancer stabilized cells were constructed,respectively.CCK-8,clone formation,Transwell,cell scratch,Western blot and immunofluorescence assay were used to detect the effects of HSPA8 on the proliferation,invasion and migration of breast cancer cells,and its effect on epithelial-mesenchymal transition(EMT). Results : Bioinformatics analysis and immunohistochemistry assay revealed that the expression of HSPA8 in breast cancer tissues was higher than that in adjacent non-tumour tissues(P<0.05),and its expression level of the protein was significantly and positively correlated with the tumor size,histological grade,lymph node metastasis and Ki-67 proliferation index(P<0.05).The Kaplan-Meier survival curve showed that high expression of HSPA8 was significantly associated with poor prognosis in breast cancer patients(P<0.05).CCK-8,clone formation,transwell,cell scratch,Western blot and immunofluorescence assay showed that knockdown of HSPA8 expression could significantly inhibit the proliferation,invasion,migration function and EMT of breast cancer cells(P<0.05),while overexpression of HSPA8 could significantly promote the proliferation,invasion,migration function and EMT of breast cancer cells(P<0.05). Conclusion HSPA8 is highly expressed in breast cancer tissues,which is closely related to disease progression and the malignant phenotype of breast cancer,suggesting that HSPA8 may be a potential biological target for breast cancer treatment.

【Objective】 To investigate the frequency of platelet antibodies in voluntary blood donors and patients in Zhongshan, Guangdong Province, and to study the specificity and cross-matching of platelet antibodies. 【Methods】 Platelet antibodies of blood donors and patients were screened by solid-phase immunoadsorption (SPIA), rechecked by flow cytometry (FCM), and antibody specificity was identified by PakPlus enzyme immunoassay, and platelet cross-matching was simulated by SPIA. 【Results】 A total of 1 049 blood donor samples and 598 patient samples were tested, with 6 (0.57%) and 49 (8.19%) samples positive for SPIA,respectively(P<0.05); In SPIA positive samples, the positive concordance rate of FCM in blood donors and patients was 100% vs 95%, and that of enzyme immunoassay was 100% vs 88%. Among the initial screening positive samples of blood donors, 5 were anti-HLA Ⅰ antibodies, accounting for 83%, and 1 was anti CD36 antibody, accounting for 17%, with an incidence rate of 0.10%. Among the 14 samples of enzyme immunoassay positive patients, 2 were anti-GP Ⅱb/Ⅲa, 1 was anti-GP Ⅱa/Ⅱa, 8 were anti HLA Ⅰ, and 3 were mixed antibodies (HLA Ⅰ, GP Ⅱb/Ⅲa, GP Ⅰa/Ⅱa). According to the types of antibodies, HLA Ⅰ antibodies were the most common, accounting for 65% (11/17), followed by HPA related anti GP, accounting for 35% (6/17). The majority of patients had a platelet antibody positive typing rate below 30%, accounting for 71.4% (10/14). 【Conclusions】 The positive rate of platelet antibody of patients in Zhongshan area is significantly higher than that of voluntary blood donors, and most of them are anti-HLA Ⅰ and anti-GP, and the incidence of anti-CD36 is extremely low. Therefore, it is necessary to establish a known platelet antigen donor bank, and at the same time, carry out platelet antibody testing and matching of patients, which is helpful to solve the issue of platelet transfusion refractoriness.

Purpose To explore the expression of autoph-agy-related 7(ATG7)in breast cancer and its effect on the breast cancer development.Methods Immunohistochemistry(IHC)was used to detect ATG7 protein expression in breast cancer tissues and the relationship between ATG7 and clinico-pathological features was analyzed.ShRNA was used to interfere with the expression of ATG7 in breast cancer cell line MCF-7.Puromycin was used to screen for stably transfected cells and Western blot was used to detect transfection efficiency.The effect of ATG7 knockdown cells on proliferation ability was de-tected by CCK8 and clone formation experiments.The effect of ATG7 knockdown cells on tumorigenicity in vivo was detected by subcutaneous tumor formation experiment in nude mice.Results IHC showed that ATG7 expression in breast cancer tissues was mainly localized in cytoplasm,and its expression was significant-ly correlated with tumor size and Ki67 expression(P＜0.05).ATG7-shRNA significantly interfered with ATG7 expression in breast cancer cells MCF-7.CCK8 and clone formation experi-ments showed that ATG7 knockdown promoted the cell prolifera-tion compared with the control group.The experiment of subcu-taneous tumor formation in nude mice showed that the tumor for-mation ability of mice was significantly increased after ATG7 knockdown compared with the control group.Conclusion ATG7 may inhibit the proliferation capacity of breast cancer and could be a potential target for breast cancer therapy.

ObjectiveTo investigate the anti-inflammatory effect of the component compatibility of Gentianae Macrophyllae Radix and Clematidis Radix et Rhizoma on the rat model of rheumatoid arthritis （RA） and the mechanism. MethodSeventy-two SPF-grade SD rats （male and female） aged 5 to 6 weeks were selected. Except the blank group， the rat model of collagen-induced arthritis （CIA） was replicated by the type Ⅱ collagen induction method. The 64 rats after successfully modeling were randomly divided into model group， methotrexate group （0.375 mg·kg^-1）， gentianoside with magnoflorine group （150.454 1 mg·kg^-1+5.061 8 mg·kg^-1）， gentianoside with clematichinenoside AR group （150.454 1 mg·kg^-1+16.433 1 mg·kg^-1）， sweroside with magnoflorine group （3.455 8 mg·kg^-1+5.061 8 mg·kg^-1）， sweroside with clematichinenoside AR group （3.455 8 mg·kg^-1+16.433 1 mg·kg^-1）， swertiamarin with magnoflorine group （9.303 2 mg·kg^-1+5.061 8 mg·kg^-1）， and swertiamarin with clematichinenoside AR group （9.303 2 mg·kg^-1+16.433 1 mg·kg^-1）， with 8 rats in each group. Each group was given the corresponding medicinal solution or normal saline by gavage for 15 d. During the experiment， the general status， of rats in each group were observed and recorded. Tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， rheumatoid factor （RF）， C reactive protein （CRP）， prostaglandin E₂ （PGE₂）， and anti-cyclic peptide containing citrulline antibody （anti-CCP Ab） in the serum of rats were measured by enzyme-linked immunosorbent assay （ELISA）. The histopathological changes in rat ankle joints were observed by hematoxylin-eosin （HE） staining. Immunohistochemistry （IHC） and Western blot were used to detect the protein expression of nuclear factor-κB （NF-κB） and vascular endothelial growth factor （VEGF） in rat ankle joints. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of NF-κB and VEGF in rat ankle joints. ResultCompared with those in the blank group， rats in the model group were in poor general conditions with significant foot-plantar swelling， and the content of CRP， anti-CCP Ab， and IL-1β in the rat serum was significantly increased （P<0.01）. In the model group， the tissue structure of the ankle joint was severely damaged， and the protein and mRNA expression of NF-κB and VEGF in the rat ankle joints were significantly up-regulated （P<0.01）. As compared with the model group， the general status of rats in each administration group was significantly improved. The levels of serum TNF-α， IL-1β， RF， CRP， PGE₂， and anti-CCP Ab were reduced to different degrees in these administration groups， among which the effects of the gentianoside with clematichinenoside AR group on down-regulating serum TNF-α and IL-1β， the gentianoside with magnoflorine group on down-regulating serum RF and CRP， the sweroside with magnoflorine group on down-regulating serum PGE₂， and the swertiamarin with clematichinenoside AR group on lowering serum anti-CCP Ab were better than those of administration groups. The histopathological changes in the ankle joint were improved to different degrees. The protein and mRNA expression of NF-κB and VEGF in rat ankle joints in the administration groups was significantly down-regulated （P<0.05， P<0.01）， and the swertiamarin paired with clematichinenoside AR group had the most significant effect. ConclusionThe component compatibility of Gentianae Macrophyllae Radix and Clematidis Radix et Rhizoma exerts a good therapeutic effect on the rat model of RA， and the compatibility of components from the two medicines has a multi-channel， multi-target， and synergistic effect. The five component compatibility patterns， namely gentiobioside with magnoflorine， gentiobioside with clematichinenoside AR， sweroside with clematichinenoside AR， swertiamarin with magnoflorine， and swertiamarin with clematichinenoside AR， all have potential advantages. The mechanism may be related to the reduction of inflammatory factor secretion and the inhibition of abnormal protein and mRNA expression of NF-κB and VEGF.