Objective: To observe the effects of exosomes derived from human umbilical cord mesenchymal stem cells on the proliferation and invasion of pancreatic cancer cells, and to analyze the contents of exosomes and explore the mechanisms affecting pancreatic cancer cells. Methods: Exosomes extracted from human umbilical cord mesenchymal stem cells were added to pancreatic cancer cells BxPC3, Panc-1 and mouse models of pancreatic cancer, respectively. The proliferative activity and invasion abilities of BxPC3 and Panc-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The expressions of miRNAs in exosomes were detected by high-throughput sequencing. GO and KEGG were used to analyze the related functions and the main metabolic pathways of target genes with high expressions of miRNAs. Results: The results of CCK-8 cell proliferation assay showed that the absorbance of BxPC3 and Panc-1 cells in the hucMSCs-exo group was significantly higher than that in the control group [(4.68±0.09) vs. (3.68±0.01), P<0.05; (5.20±0.20) vs. (3.45±0.17), P<0.05]. Transwell test results showed that the number of invasion cells of BxPC3 and Panc-1 in hucMSCs-exo group was significantly higher than that in the control group (129.40±6.02) vs. (89.40±4.39), P<0.05; (134.40±7.02) vs. (97.00±6.08), P<0.05. In vivo experimental results showed that the tumor volume and weight in the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) group were significantly greater than that in the control group [(884.57±59.70) mm(3) vs. (695.09±57.81) mm(3), P<0.05; (0.94±0.21) g vs. (0.60±0.13) g, P<0.05]. High-throughput sequencing results showed that miR-148a-3p, miR-100-5p, miR-143-3p, miR-21-5p and miR-92a-3p were highly expressed. GO and KEGG analysis showed that the target genes of these miRNAs were mainly involved in the regulation of glucosaldehylation, and the main metabolic pathways were ascorbic acid and aldehyde acid metabolism, which were closely related to the development of pancreatic cancer. Conclusion: Exosomes derived from human umbilical cord mesenchymal stem cells can promote the growth of pancreatic cancer cells and the mechanism is related to miRNAs that are highly expressed in exosomes.
Mice ; Animals ; Humans ; MicroRNAs/metabolism* ; Exosomes/genetics* ; Sincalide/metabolism* ; Pancreatic Neoplasms/metabolism* ; Carcinoma, Pancreatic Ductal/genetics* ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.
Humans ; Atorvastatin/pharmacology* ; PTEN Phosphohydrolase/pharmacology* ; Sincalide/metabolism* ; Drug Resistance, Neoplasm/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Leukemia, Promyelocytic, Acute/drug therapy* ; Doxorubicin/pharmacology* ; Apoptosis ; RNA, Small Interfering/pharmacology* ; Glycolysis ; Glucose/therapeutic use* ; Cell Proliferation

OBJECTIVE: To screen the differentially expressed long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells. METHODS: The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR. RESULTS: The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P < 0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P < 0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D-AS1 showed the highest increase of expression after acquired osimertinib resistance (P < 0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P < 0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P < 0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P < 0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway. CONCLUSION The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinibresistant NSCLC.
Humans ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Lung Neoplasms/genetics* ; RNA, Long Noncoding/metabolism* ; Sincalide/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Movement ; MicroRNAs/genetics* ; Gene Expression Regulation, Neoplastic ; Membrane Proteins/metabolism*

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC. METHODS: PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8. RESULTS: CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05). CONCLUSIONS The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.
Humans ; Gefitinib/therapeutic use* ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Sirtuin 3/therapeutic use* ; Lung Neoplasms/metabolism* ; Reactive Oxygen Species/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Cigarette Smoking ; Sincalide/therapeutic use* ; ErbB Receptors/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Cell Line, Tumor

OBJECTIVES: Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. METHODS: The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. RESULTS: Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). CONCLUSIONS HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.
Humans ; Carcinoma, Squamous Cell/genetics* ; Sincalide/metabolism* ; Tongue Neoplasms/genetics* ; Mouth Neoplasms ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism* ; RNA, Messenger ; Tongue/metabolism* ; Cell Line, Tumor

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.
Humans ; Carcinoma, Hepatocellular/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Liver Neoplasms/genetics* ; Molecular Docking Simulation ; Sincalide/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Hep G2 Cells ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

OBJECTIVE: To investigate the effect and molecular mechanism of lncRNA X-inactive specific transcript (XIST) on the proliferation and apoptosis of acute myeloid leukemia cells KG1a. METHODS: Forty-one patients with acute myeloid leukemia from January 2017 to May 2019 treated in Beijing Aerospace Center Hospital were collected, as well as 20 patients who conformed to the international standard of iron deficiency anemia as control group. KG1a cells were divided into pcDNA group, pcDNA-XIST group, pcDNA-XIST+miR-NC group, and pcDNA-XIST+miR-196b group. Real-time fluorescence quantitative PCR was used to detect the expressions of XIST and miR-196b, CCK-8 was used to detect cell activity, flow cytometry was used to detect cell cycle and apoptosis, Western blot method was used to detect the protein expressions of cleaved-caspase3, pro-caspase3, Bax, and Bcl-2, and dual luciferase report experiment was used to detect the targeting relationship between XIST and miR-196b. RESULTS: The expression level of lncRNA XIST in bone marrow cells in the AML group was significantly lower than that in the iron deficiency anemia group (P<0.001). Compared with pcDNA group, the expression level of lncRNA XIST, proportion of cells in G₀/G₁ phase, apoptosis rate, and the expression levels of cleaved-caspase3 and Bax in the pcDNA-XIST group of KG1a cells were significantly increased (all P<0.001), while the expression level of miR-196b, cell viability, the proportion of S-phase cells, and the expression levels of pro-caspase3 and Bcl-2 were significantly decreased (all P<0.001). Compared with pcDNA-XIST group, the cell activity, proportion of S-phase cells, and the expression levels of pro-caspase3 and Bcl-2 in the pcDNA-XIST+miR-196b group were significantly increased (all P<0.001), while the proportion of cells in the G₀/G₁ phase, apoptosis rate, and the expression levels of cleaved-caspase3 and Bax decreased (all P<0.001). CONCLUSION Overexpression of lncRNA XIST can inhibit the proliferation of acute myeloid leukemia cells KG1a and promote apoptosis by down-regulating the expression of miR-196b.
Anemia ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Leukemia, Myeloid, Acute/metabolism* ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Long Noncoding/genetics* ; Sincalide ; bcl-2-Associated X Protein

OBJECTIVE: To investigate the expression of ROBO3 in pediatric AML patients and explore its function on cell proliferation and apoptosis. METHODS: The expression of ROBO3 in pediatric AML patients at different treatment stage was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The relationship between the expression of ROBO3 and clinic pathological characteristics in newly diagnosed pediatric AML patients was analyzed. Moreover, the effects of ROBO3 on the proliferation and apoptosis of AML cell lines HL-60 and THP-1 were estimated by using CCK-8 and flow cytometry after transfection with ROBO3 siRNA. RESULTS: It was found that ROBO3 expression was significantly increased in most of newly diagnosed pediatric AML patients, especially in non-M3 subtype, younger patients (<10 years old), and high risk group, compared to corresponding controls. Furthermore, the expression level of ROBO3 was sharply decreased in patients who achieved complete remission. Targeting ROBO3 significantly inhibited AML cell proliferation, as well as increased apoptosis by ROBO3 siRNA transfection in vitro. CONCLUSION ROBO3 is differentially expressed within distinct subtypes of the pediatric AML patients, which suggested that ROBO3 may be a potential biomarker and a new therapeutic target for pediatric AML.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Child ; Humans ; Leukemia, Myeloid, Acute/genetics* ; MicroRNAs/genetics* ; RNA, Small Interfering ; Receptors, Cell Surface ; Sincalide

OBJECTIVE: To observe the mutation and expression of TCF3 gene in Burkitt's lymphoma (BL), and explore its effect on the proliferation of BL cells and clinical efficacy and prognosis. METHODS: The mutation and expression of TCF3 in tumor tissues from BL patients were observed by the second-generation sequencing and real-time quantitative PCR. The proliferation and apoptosis of lymphoma cells after TCF3 knocked down were observed by siRNA interference technique and CCK-8 method. Survival analysis was used to observe the relationship between TCF3 mutation and the treatment efficacy and prognosis of BL patients. RESULTS: There were high frequency mutation rate (mutation rate was 23.7%) and high expression of TCF3 in BL patients. After TCF3 knocked down, cell proliferation was inhibited and apoptosis was promoted. In TCF3-siRNA group and control group, the cell proliferation rate at 48 h was (50.2±5.9)% and (96.6±11.4)%, and apoptosis rate was 30.1% and 1.5%, respectively, which showed significantly different between the two groups (P<0.001, P=0.005). The complete remission rate of patients with TCF3 mutation was low. The complete remission rate of mutant group and wild-type group was 44.4% and 82.8%, respectively (P=0.023). The 2-year progression-free survival rate and overall survival rate of the patients with TCF3 mutation was 55.6% and 61.0%, respectively, which was lower than 83.2% and 85.2% of the patients without mutation, but the differences were not statistically significant. CONCLUSION There are mutation and abnormal expression of TCF3 in patients with BL. Patients with TCF3 mutations have low remission rate and poor prognosis.
Apoptosis ; Basic Helix-Loop-Helix Transcription Factors/therapeutic use* ; Burkitt Lymphoma/genetics* ; Humans ; Prognosis ; RNA, Small Interfering/therapeutic use* ; Sincalide/therapeutic use*

OBJECTIVE: To investigate the effect of BECN1 expression on proliferation and invasion of human multiple myeloma (MM) cell line RPMI-8226. METHODS: RPMI-8226 cells were cultured in vitro, the recombinant plasmid pcDNA3.1-BECN1 was constructed and transfected into RPMI-8226 cells, then the cells were divided into three groups: control group, negative transfection group and BECN1 transfection group. The expression of BECN1 mRNA in cells of each group was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the effect of BECN1 overexpression on cell proliferation inhibition rate was detected by cell counting kit 8 (CCK-8); the effect of BECN1 overexpression on the colony formation rate was detected by plate cloning assay; the effect of BECN1 overexpression on cell invasion was detected by Transwell assay; the effects of BECN1 overexpression on the expression of cell proliferation, invasion and autophagy-related proteins were detected by Western blot. RESULTS: The expression of BECN1 mRNA in BECN1 transfection group was significantly higher than that in control group and negative transfection group (P<0.05); the inhibition rate of cell proliferation and the expression of autophagy-related proteins Beclin1, Atg5 and invasion-related protein E-cadherin in BECN1 transfection group were significantly higher than those in control group and negative transfection group; the colony formation rate, invasion number and the expression of proliferation-related proteins CyclinD1, β-catenin and invasion-related protein N-cadherin were significantly lower than those in control group and negative transfection group (P<0.05). CONCLUSION Overexpression of BECN1 can inhibit the proliferation and invasion of human MM cells RPMI-8226, which may be a potential research target of MM.
Beclin-1/metabolism* ; Cadherins/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma/genetics* ; RNA, Messenger/metabolism* ; Sincalide/metabolism* ; beta Catenin/genetics*