Aim To observe the effect of the LSD1 gene on the proliferation and apoptosis of Molt-4 cells, a kind of human acute T-lymphoblastic leukemia cells. Methods siRNA fragment based on LSD1 gene was designed, filtered out and then transfected into Molt-4 cells. The effects of LSD 1 siRNA on Molt-4 cell prolif-eration were observed by the method of MTS. The cell apoptosis was analyzed by flow cytometry. The states of histone H3K4, H3K9 methylation, histone H3 acetyla-tion, p15, DNA methyltransferase 1 (DNMT1), and apoptosis-related proteins like Bcl-2 , procaspase-3 were evaluated by Western blot. Results Silencing LSD1 gene inhibited cell proliferation. Molt-4 cell pro-liferation rate was ( 99. 65 ± 1. 21 )%, ( 83. 02 ± 1. 69)%, (65. 72 ± 2. 16)%,and (41. 15 ± 2. 23)%respectively after the treatment of Molt-4 cells with 0 , 30, 60, 120 nmol·L-1 of LSD1 siRNA after 48 hours ( P < 0. 05 ) . Cell proliferation rate was ( 99. 86 ± 1. 35)%,(65. 72 ± 2. 16)%,(48. 26 ± 1. 92)%,and ( 37. 86 ± 1. 66 )% respectively after the transfection of Molt-4 cells with 60 nmol · L-1 of LSD1 siRNA after 0 , 24 , 48 , 72 hours ( P<0. 05 ) . Cell apoptosis rate was ( 3. 35 ± 1. 26 )%, ( 12. 16 ± 1. 74 )%, ( 32. 74 ± 2. 47 )%, ( 54. 64 ± 2. 58 )% respectively after transfection of LSD1 siRNA in indicated concentrations for 48 hours ( P <0. 05 ) . At the same time, the ex-pression levels of apoptosis-related proteins like Bcl-2 , procaspase-3 decreased. LSD1 siRNA inhibited LSD1 and LSD1 mRNA, and accumulated histone mono-, and di-methylation H3K4 and histone H3 acetylation. However, alteration of H3K4 trimethylation, H3K9 methylation was not detected. LSD1 siRNA downregu-lated DNA demethylase DNMT1 and upregulated p15 . Conclusions LSD1 siRNA can inhibit Molt-4 cell proliferation and induce apoptosis. Its mechanism may be associated with epigenetic regulation. In addition, it is expected to become a new target for leukemia treat-ment.

Objective:To explore the cooperation and clinical significance of HIF-1α,ALDH1 and Hedgehog signaling pathway in the activation of cancer stem cell(CSC) in triple negative breast cancer(TNBC).Methods: ALDH1+(Aldehyde dehydrogenase1)breast cancer stem cells and ALDH1-breast cancer cells were selected from MDA-MB-231 cells by magnetic activated cell sorting system(MACS),qRT-PCR method was employed to analyze the expression differences of HIF-1α and Hedgehog signaling molecules Sonic hedgehog(SHH),patched1(PTCH1),Smoothened(SMO) and Glioma-associated oncogene homoglog1(GLI1) in ALDH1+ breast cancer stem cells and ALDH1-breast cancer cells.Immunohistochemical method was applied to study the expressions of HIF-1α and ALDH1 and the relationships among HIF-1α,ALDH1 and Hedgehog signaling molecules in TNBC.Results: The expressions of HIF-1α mRNA,SMO mRNA and GLI1 mRNA in ALDH1+ breast cancer stem cell were higher than those in ALDH1-breast cancer cell(P all<0.05).The positive expression rates of HIF-1α were 90.0% and 70.0%,and the positive rates of ALDH1 were 93.3 % and 66.7 % in TNBC and non-TNBC,respectively(P all<0.05).Spearman rank correlation analysis showed that the expression of HIF-1α was positively related with that of ALDH1 in TNBC(r=0.53,P<0.01).HIF-1α expression was correlated with lymph node metastasis and TNM stage(P all<0.05),ALDH1 expression was correlated with histological grade and TNM stage(P all<0.05).In addition,the expression of HIF-1α was positively related with that of Hedgehog signaling molecules SHH(r=0.584,P<0.01),SMO(r=0.467,P<0.01) and GLI1(r=0.439,P<0.05),the expression of ALDH1 was positively related with that of SHH(r=0.426,P<0.05) and GLI1(r=0.394,P<0.05).Conclusion: HIF-1α and Hedgehog signaling pathway were activated in ALDH1+ breast cancer stem cell.HIF-1α,ALDH1 and Hedgehog molecules may cooperate with each other to activate breast CSC to promote the malignant progression of TNBC.

MicroRNAs (miRNAs) play critical roles in the development and progression in various cancers.Dysfunctional miR-9 expression remains ambiguous,and no consensus on the metastatic progression of ovarian cancer has been reached.In this study,results from the bioinformatics analysis show that the 3'-UTR of the E-cadherin mRNA was directly regulated by miR-9.Luciferase reporter assay results confirmed that miR-9 could directly target this 3'-UTR.miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRTPCR.Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780.qRT-PCR and Western blot were performed to detect the epithelial-mesenchymal transition-associated mRNA and proteins.Immunofluorescence technique was used to analyze the expression and subcellular localization of Ecadherin,N-cadherin,and vimentin.The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones.Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers (N-cadherin and vimentin).Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer,and these processes could be effectively inhibited via miR-9 inhibitor.Thus,our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.

Tumor-associated tertiary lymphoid structures(TLSs)are ectopic lymphoid formations within tumor tissue,with mainly B and T cell populations forming the organic aggregates.The presence of TLSs in tumors has been strongly associated with patient responsiveness to immunotherapy regimens and improving tumor prognosis.Researchers have been motivated to actively explore TLSs due to their bright clinical application prospects.Various studies have attempted to decipher TLSs regarding their formation mechanism,structural composition,induction generation,predictive markers,and clinical utilization.Meanwhile,the scientific approaches to qualitative and quantitative descriptions are crucial for TLS studies.In terms of detection,hematoxylin and eosin(H&E),multiplex immunohistochemistry(mIHC),multiplex immunofluorescence(mIF),and 12-chemokine gene signature have been the top approved methods.However,no standard methods exist for the quantitative analysis of TLSs,such as absolute TLS count,analysis of TLS constituent cells,structural features,TLS spatial location,density,and maturity.This study reviews the latest research progress on TLS detection and quantification,proposes new directions for TLS assessment,and addresses issues for the quantitative application of TLSs in the clinic.