Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the Transwell^TM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
Humans ; Cyclin D1/genetics* ; HEK293 Cells ; Interferon-gamma ; RNA, Small Interfering ; Apoptosis/genetics* ; Cadherins ; Cell Proliferation/genetics* ; Glioma/genetics* ; Proto-Oncogene Proteins c-bcl-2 ; Oxidoreductases Acting on Sulfur Group Donors ; STAT1 Transcription Factor/genetics*

BACKGROUND: Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1). METHODS: The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group. RESULTS: Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05). CONCLUSIONS FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.
Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/genetics* ; Dual Specificity Phosphatase 1/pharmacology* ; Cell Proliferation ; p38 Mitogen-Activated Protein Kinases/pharmacology* ; Methylation ; Apoptosis ; Cell Line, Tumor

Humans ; Myocardial Reperfusion Injury/genetics* ; RNA, Long Noncoding/genetics* ; Myocytes, Cardiac ; Apoptosis ; Myocardial Ischemia

Humans ; Myocardial Reperfusion Injury/genetics* ; RNA, Long Noncoding/genetics* ; Myocytes, Cardiac ; Apoptosis ; Myocardial Ischemia

Objective: To investigate the effect of long non-coding RNA urothelial carcinoma-associated 1 (UCA1) gene on the proliferation, migration, apoptosis and immune escape of endometrial cancer cells and its molecular mechanism. Methods: Endometrial cancer tissues and adjacent normal tissues of patients with endometrioid adenocarcinoma who underwent total or partial hysterectomy in Henan Provincial People's Hospital from 2017 to 2019 were collected. The expressions of UCA1 and miR-204-5p were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the cell proliferation, migration and apoptosis were detected by cell counting kit 8 (CCK8) method, Transwell method, flow cytometry, and dual-luciferase reporter assay was used to explore the target relationship between UCA1 and miR-204-5p. HEC-1A-sh-NC or HEC-1A-sh-UCA1 cells were co-cultured with peripheral blood mononuclear cells (PBMC) or cytokine-induced killer cells in vitro to explore the role of UCA1 in immune escape. Results: The expression level of UCA1 in endometrial cancer tissue (17.08±0.84) was higher than that in adjacent normal endometrial tissue (3.00±0.37), and the expression level of miR-204-5p (0.98±0.16) was lower than that in adjacent normal endometrial tissue (2.00±0.20, P<0.05). Pearson correlation analysis showed that the expression of miR-204-5p was negatively correlated with the expression of UCA1 (r=-0.330, P=0.030). The expressions of UCA1 and miR-204-5p were associated with the International Federation of Gynecology and Obstetrics stage of endometrial cancer, lymph node metastasis and vascular invasion (P<0.05). The relative ratio of absorbance (0.58±0.11) and the number of cell migration [(199.68±18.44)] in the sh-UCA1 group were lower than those in the sh-NC group (1.24±0.17 and 374.76±24.83), respectively. The apoptosis rate of sh-UCA1 group [(28.64±7.80)%] was higher than that of sh-NC group [(14.27±4.38)%, P<0.05]. After different ratios of effector cells and target cells were cultured, the cell survival rate of HEC-1A-sh-UCA1 group was lower than that of HEC-1A-sh-NC group, and the difference was statistically significant (P<0.05). UCA1 had a binding site for miR-204-5p. The relative ratio of absorbance (1.74±0.08) and the number of cell migration (426.00±18.00) cells in the UCA1+ anti-miR-204-5p group were higher than those in the control group [1.00±0.03 and (284.00±8.00) cells, respectively]. The apoptosis rate of UCA1+ anti-miR-204-5p group [(5.42±0.93)%] was lower than that of control group [(14.82±1.48)%, P<0.05]. HEC-1A-sh-UCA1 cells could induce higher interferon gamma (IFN-γ) expression when co-cultured with PBMC, and the levels of IFN-γ expression in PHA group and PHA+ pre-miR-204-5p group cells were 2.42±0.49 and 1.88±0.26, which were higher than that in the PHA+ pre-NC group (0.85±0.10, P<0.05). When co-cultured with cytokine-induced killer cells (different ratios) in vitro, the HEC-1A-sh-UCA1 group and the HEC-1A-pre-miR-204-5p group had lower survival rates than that in the HEC-1A-pre-miR-204-5p group. In the HEC-1A-pre-NC group, the differences were statistically significant (P<0.05). Conclusion: UCA1/miR-204-5p may play an important role in human endometrial cancer.
Female ; Humans ; MicroRNAs/metabolism* ; RNA, Long Noncoding/genetics* ; Leukocytes, Mononuclear ; Carcinoma, Transitional Cell ; Antagomirs ; Cell Line, Tumor ; Urinary Bladder Neoplasms ; Cell Proliferation ; Endometrial Neoplasms/genetics* ; Apoptosis/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic

Objective: To investigate the effect of rigosertib (RGS) combined with classic chemotherapy drugs including 5-fluorouracil, oxaliplatin, and irinotecan in colorectal cancer. Methods: Explore the synergy effects of RGS and 5-fluorouracil (5-FU), oxaliplatin (OXA), and irinotecan (IRI) on colorectal cancer by subcutaneously transplanted tumor models of mice. The mice were randomly divided into control group, RGS group, 5-FU group, OXA group, IRI group, 5-FU+ RGS group, OXA+ RGS group and IRI+ RGS group. The synergy effects of RGS and OXA on KRAS mutant colorectal cancer cell lines in vitro was detected by CCK-8. Ki-67 immunohistochemistry and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed on the mouse tumor tissue sections, and the extracted tumor tissue was analyzed by western blot. The blood samples of mice after chemotherapy and RGS treatment were collected, blood routine and liver and kidney function analysis were conducted, and H&E staining on liver sections was performed to observe the side effects of chemotherapy and RGS. Results: The subcutaneously transplanted tumor models were established successfully in all groups. 55 days after administration, the fold change of tumor size of OXA+ RGS group was 37.019±8.634, which is significantly smaller than 77.571±15.387 of RGS group (P=0.029) and 92.500±13.279 of OXA group (P=0.008). Immunohistochemical staining showed that the Ki-67 index of tumor tissue in control group, OXA group, RGS group and OXA+ RGS group were (100.0±16.8)%, (35.6±11.3)%, (54.5±18.1)% and (15.4±3.9)%, respectively. The Ki-67 index of OXA+ RGS group was significantly lower than that in control group (P=0.014), but there was no significant difference compared to OXA group and RGS group (OXA: P=0.549; RGS: P=0.218). TUNEL fluorescence staining showed that the apoptotic level of OXA+ RGS group was 3.878±0.547, which was significantly higher than 1.515±0.442 of OXA group (P=0.005) and 1.966±0.261 of RGS group (P=0.008). Western blot showed that the expressions of apoptosis related proteins such as cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 8 in the tumor tissues of mice in the OXA+ RGS group were higher than those in control group, OXA group and RGS group. After the mice received RGS combined with chemotherapy drugs, there was no significant effect on liver and kidney function indexes, but the combined use of oxaliplatin and RGS significantly reduced the white blood cells [(0.385±0.215)×10(9)/L vs (5.598±0.605)×10(9)/L, P<0.001] and hemoglobin[(56.000±24.000)g/L vs (153.333±2.231)g/L, P=0.001] of the mice. RGS, chemotherapy combined with RGS and chemotherapy alone did not significantly increase the damage to liver cells. Conclusions: The combination of RGS and oxaliplatin has a stronger anti-tumor effect on KRAS mutant colorectal cancer. RGS single agent will not cause significant bone marrow suppression and hepatorenal injury in mice, but its side effects may increase correspondingly after combined with chemotherapy.
Animals ; Mice ; Antineoplastic Combined Chemotherapy Protocols ; Apoptosis Regulatory Proteins ; Colorectal Neoplasms/genetics* ; Fluorouracil/pharmacology* ; Irinotecan/therapeutic use* ; Ki-67 Antigen ; Oxaliplatin ; Proto-Oncogene Proteins p21(ras)/therapeutic use*

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.
Humans ; MicroRNAs/metabolism* ; Retinoblastoma/pathology* ; Caspase 3/metabolism* ; RNA, Long Noncoding/metabolism* ; Antagomirs/pharmacology* ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis/genetics* ; Retinal Neoplasms/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics*

BACKGROUND: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. METHODS: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. RESULTS: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. CONCLUSIONS Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.
Animals ; Humans ; Mice ; Apoptosis ; Bone Marrow Cells ; Cell Communication ; Connexin 43/genetics* ; Gap Junctions/metabolism* ; Imatinib Mesylate/therapeutic use* ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology* ; Mesenchymal Stem Cells/metabolism* ; Tumor Microenvironment ; Calcium/metabolism*

OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*