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Abstract Objectives Melanoma is one of the leading causes of cancer death. Kinesin Family member 22 (KIF22) is essential for the invasion of melanoma cells, but the role and mechanism of KIF22 in the proliferation and glycolysis in melanoma remains unknown. Methods KIF22 expression in melanoma tissues and the relationship between KIF22 high expression and overall survival rate in patients with melanoma were analyzed using the Tnmplot database. KIF22 expression in melanoma cells was examined by western blot. Then, KIF22 was silenced and CCK-8 assay, EDU staining and flow cytometry analysis were adopted for assessing cell proliferation and apoptosis. In addition, the glycolysis metabolism of melanoma cells was reflected by detecting Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR). The expression of proteins related to apoptosis, glycolysis and EGFR/STAT3 signaling was tested by western blot. Subsequently, melanoma cells were treated with EGF or Colivelin to further elucidate the regulatory effect of KIF22 on EGFR/STAT3 signaling. Results KIF22 expression was notably upregulated in melanoma tissues and cells, and KIF22 high expression was associated with a poor prognosis. Moreover, KIF22 insufficiency suppressed proliferation and accelerated apoptosis of melanoma cells. Additionally, glycolysis was reduced by KIF22 depletion, evidenced by the decreased ECAR and increased OCR, accompanied by the downregulated expression of HK2, PKM2 and LDHA. Importantly, the impacts of KIF22 depletion on the progression of melanoma were partially attenuated after EGF or Colivelin treatment. Conclusion Collectively, KIF22 knockdown suppressed the proliferation and glycolysis and facilitated the apoptosis of melanoma cells by inactivating EGFR/STAT3 signaling.
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Objective:To investigate the expression of zinc finger protein 22 (ZNF22) gene in hepatocellular carcinoma (HCC) and its effect on tumor proliferation, apoptosis, invasion and metastasis of HCC.Methods:The expression of ZNF22 in 32 HCC specimens, and 371 HCC samples from the cancer genome atlas database were analyzed. ZNF22 knockdown and negative control SNU-449 and JHH-7 HCC cell lines were constructed. The effects of ZNF22 on HCC cells were observed by cell proliferation assay, plate clone formation assay, apoptosis assay, scratch healing assay, Transwell invasion assay, subcutaneous tumor formation, tail vein injection transfer, and small animal live imaging assay in nude mice.Results:The expression of ZNF22 gene is higher in HCC tissues than in paracellular carcinoma tissues, and the difference was statistically significant ( P<0.001). The growth rate of SNU-449 and JHH-7 cells in ZNF22 knockdown group was lower than that in control group, and the difference was statistically significant ( P<0.001). Compared with negative control group, the clone number formed by SNU-449 cells in ZNF22 knockdown group decreased (26±8 vs. 59±5, P<0.01), the level of apoptosis increased (6.60%±0.22% vs. 2.38%±0.30%, P<0.001), the migration rate decreased (14.47%±6.42% vs. 68.84%±8.01%, P<0.001), and the number of invasive cells decreased (48.00±2.23 vs. 179.00±4.81, P<0.001). There was no obvious tumor growth after subcutaneous injection of JHH-7 cells into nude mice in ZNF22 knockdown group, and the systemic fluorescence expression was lower than that of the negative control group, and the difference was statistically significant ( P<0.05). No metastases were observed on autopsy in knockdown group nude mice. Conclusion:ZNF22 is highly expressed in HCC while knockdowing ZNF22 gene inhibited the growth, proliferation, invasion, metastasis of HCC cells, and induced apoptosis of HCC cells.
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Objective:To explore the effect of down-regulation of long non-coding RNA (lncRNA) CTB-191K22.5 on the proliferation and invasion of colorectal cancer SW480 cells and the molecular mechanism.Methods:The TCGA database was used to analyze the expression differences of CTB-191K22.5 in colorectal cancer tissues and normal tissues. The CTB-191K22.5 inhibitor (Anti-CTB-191K22.5) and negative inhibitor (Control) were transfected into colorectal cancer SW480 cells, denoted as Observation group and Control group, real-time quantitative polymerase chain reaction (qRT) -PCR) was used to evaluate the inhibitory effect. MTT method and Transwell chamber method were used to evaluate the proliferation and invasion of SW480 cells. Western blot was used to evaluate the protein levels of PI 3K/AKT/mTOR signaling pathway in SW480 cells. The bioinformatics software starBase v2.0 was used to predict the target genes of CTB-191K22.5. qRT-PCR was used to evaluate the expression of CTB-191K22.5 target gene in SW480 cells. Measurement data were expressed as Mean±SD, and t-test was used for comparison between two groups. Results:Compared with normal tissues, the expression of CTB-191K22.5 in colorectal cancer tissues was significantly increased ( P<0.01). The expression of CTB-191K22.5 in SW480 cells of the Control group and Observation group were 6.60±0.85 and 1.08±0.21, respectively. The expression level of CTB-191K22.5 decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the SW480 cell proliferation ability of the Observation group decreased ( P<0.01). The invasion numbers of SW480 cells in the Control group and Observation group were (135.4 ± 16.29) and (42.24±14.59), respectively. The invasion ability of SW480 cells decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the expression levels of PI 3K/AKT/mTOR signaling pathway protein in SW480 cells in the Observation group decreased. miR-326 may be the target gene of CTB-191K22.5. Compared with the Control group, transfection with Anti-CTB-191K22.5 significantly increased the expression level of miR-326 in SW480 cells ( P<0.01). Conclusion:CTB-191K22.5 is highly expressed in colorectal cancer tissues, and down-regulation of CTB-191K22.5 may inhibit the proliferation and invasion of colorectal cancer SW480 cells by targeting miR-326.
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Objective To investigate the regulation of miR-939-5p on USP22 gene expression and its effect on liver cancer migration and proliferation.Methods The expression of miR-939-5p in hepatoma cell lines (HepG2,MHCC-97H,SMMC-7721,BEL-7404 and Huh7) and normal liver cell line LO2 was detected by realtime PCR (qPCR).The liver cancer cells with the lowest expression were used as experimental subjects,and transfected with miR-939-5p (Experimental group) or miR-NC (Control group).qPCR was used to detect the transfection efficiency of miR-939-5p.Transwell migration assay and MTT proliferation assay were used to detect the migration and proliferation of hepatoma cells after transfected miR-939-5p.Bioinformatics software predicted target genes for miR-939-5p.The dual luciferase reporter gene verified the interaction of miR-939-5p with the target gene.qPCR and Western blotting were used to detect the expression of target genes at mRNA and protein levels after over-expression of miR-939-5p.Measurement data were expressed as (Mean ± SD),and t test was used for comparison between groups.Results The expression of miR-939-5p was significantly lower in hepatoma cell lines than in normal hepatocytes (P ＜0.01),and the expression of miR-939-5p was the lowest in SMMC-7721 cells (P＜0.01).miR-939-5p was efficiently transfected into SMMC-7721 cells [(1.01 ±0.07) vs (20.12 ± 1.27),P ＜0.01].High expression of miR-939-5p inhibited the migration ability (P ＜ 0.01) and proliferative capacity of liver cancer SMMC-7721 cells (P ＜0.05).The USP22 gene may be a target gene of miR-939-5p.The luciferase reporter gene confirmed that miR-939-5p specifically binds to the 3'-UTR of USP22 mRNA (P ＜ 0.01).USP22 expression was decreased at mRNA and protein levels after high expression of miR-939-5p (P ＜ 0.01).Conclusions The expression of miR-939-5p was down-regulated in hepatocellular carcinoma cell lines,and miR-939-5p inhibited the migration and proliferation of liver cancer SMMC-7721 cells.The molecular mechanism was to interfere with the expression of USP22 gene.
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Objective To explore the effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells.Methods MLAA-22 gene was down-regulated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)system in U937 cells.The activity of single guide RNA (sgRNA)was detected by CruiserTMenzyme digestion assay.The mutation rate of MLAA-22 gene was analyzed by TA cloning and sequencing assay of PCR products of the gene mutation region.Cell proliferation was evaluated by CCK-8 assay.Expression of CD11b was tested by flow cytometry to evaluate cell differentiation. Results CruiserTMenzyme digestion assay showed the sgRNA of the CRISPR/Cas9 system identified the target spot efficiently.TA cloning and sequencing assay displayed the mutation rate of MLAA-22 gene was 61.3%.CCK-8 assay demonstrated that the proliferation was obviously inhibited in MLAA-22-knockdown U937 cells.In addition, flow cytometry assay indicated CD11b-positive cells significantly increased in MLAA-22-knockdown U937 cells. Conclusion MLAA-22 gene regulates the proliferation of U937 cells probably by regulating their differentiation, thus promoting the occurrence and development of acute monocytic leukemia.
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Objective To investigate the expression of USP22 in cervical cancer cells,and the effects of USP22 on cell proliferation and chemosensitivity to cisplatin in cervical cancer cells. Methods Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to detect the expression of USP22 mRNA in cervical cancer cells.Cell count kit-8,flow cytometry,and tumorigenesis were performed to detect the effects of USP22 on the proliferation,apoptosis,cycle cycle,and chemosensitivity of cervical cancer cells to cisplatin in vitro and in vivo. Results USP22 was upregulated in cervical cancer cells compared to human immortalized epidermal cells. Cell proliferation was inhibited,cell apoptosis was promoted,cell cycle was arrested in G1 stage and chemosensi-tivity to cisplatin was enhanced in cervical cancer cells transfected with USP22-siRNAs.Furthermore,tumorigene-sis assays proved tumor growth was inhibited and chemosensitivity to cisplatin was enhanced when USP22 was silenced in HeLa cells. Conclusion USP22 expression is upregulated in cervical cancer cells,and USP22 could promote cell proliferation and inhibit chemosensitivity to cisplatin in cervical cancer cells.
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Objective To investigate the expression and effect of miR-22-3p in non-small cell lung cancer (NSCLC). Methods The miR-22-3p expression level in seventy-six NSCLC tissues and para-cancer tissues was detected by qRT-PCR. The relationship between the expression of miR-22-3p and gender,age,tumor size,histolo-gy grade,pathological type and lymph node metastasis was analyzed. The function of miR-22-3p on the prolifera-tion of NSCLC cells was tested by growth curve assay. Target genes of miR-22-3p were predicted by online software Targetscan. Luciferase reporter assay and qRT-PCR was used to certificate the prediction. Results The expression of miR-22-3p was increased in NSCLC tissues than the para-cancer tissues and was correlated to lymph node metas-tasis. Overexpression of miR-22-3p could suppress the proliferation of A549 cells. Astrocyte-Elevated Gene-1(AEG-1) was predicted to be a target of miR-22-3p. MiR-22-3p was revealed to bind to AEG-13′UTR by luciferase report-er assay. Overexpression of miR-22-3p could inhibit the expression of AEG-1 in A549 cells. Suppression of miR-22-3p could increase AEG-1 expression. Conclusion MiR-22-3p could inhibit the proliferation of NSCLC by tar-geting AEG-1.
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AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells.METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR .miRNA-22 was over-expressed by trans-fection of miRNA-22 mimic.The cell viability was examined by CCK-8 assay.The cell migration was measured by wound healing test .The cell invasion was analyzed by Transwell assay .The protein expression levels of VEGF and P 53 were deter-mined by Western blot .RESULTS: Compared with the normal ovarian tissue , the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues .After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased , while the cell viability , migration and invasion were obviously decreased .Moreover , the protein expression of VEGF and P 53 was dramatically inhibited after over-expression of miRNA-22.CONCLUSION:The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer.Over-expression of miRNA-22 decreases the cell viability , migration and invasion by reducing the protein expression of VEGF and P53.
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Objective: To investigate the effect of microRNA-34b (miR-34b) on the proliferation of bladder cancer BIU-87 cells, and to exploreObjective: To investigate the effect of microRNA-34b (miR-34b) on the proliferation of bladder cancer BIU-87 cells, and to explore Methods: The expression levels of miR-34b in bladder cancer BIU-87 cells, SV-HUC-1 cells and renal tubular epithelial HK-2 cells were determined by real-time fluorescent quantitative PCR. After transfection with miR-34b mimic, the proliferation rate and clone formation rate of BIU-87 cells were detected by MTT assay and clone formation assay, respectively. After co-transfection with miR-34b mimic and ubiquitin-specific peptidase 22 (USP22)-wide type (WT) or USP22-mutation type (Mut), the specific binding ability of miR-34b to 3'-untranslated region (3'-UTR) in USP22 gene was examined by luciferase reporter system. The expression levels of USP22 mRNA and protein in BIU-87 cells after transfection with miR-34b mimic were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The expression level of miR-34b in human bladder cancer BIU-87 cells was lower than those in SV-HUC-1 cells and renal tubular epithelial HK-2 cells (both P < 0.05). The proliferation rate and clone formation rate of BIU-87 cells in miR-34b mimic transfection group were lower than those of blank control cells (BIU-87 cells without any transfection) and negative control cells [BIU-87 cells transfected with miRNA mimic negative control (miRNA mimic NC)] (all P < 0.05). The result of luciferase reporter system indicated that miR-34b targeted 3'-UTR in USP22 gene. The luciferase activity of BIU-87 cells in co-transfection with miR-34b mimic and recombinant vector USP22-WT group was decreased (P < 0.05). The expression level of USP22 protein in BIU-87 cells in miR-34b mimic transfection group were lower than those in the blank control and the negative control groups (all P < 0.05). Conclusion: miR-34b can suppress the proliferation of bladder cancer BIU-87 cells. This effect may be related to the expression of USP22.
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Objective To analyze the influence of SH3 domain mutant (ABL SH3-T79Y) in BCR-ABL protein of chronic myeloid leukemia (CML) in combination with imatinib (IM) on the proliferation of CML cells in vivo and vitro, and to discuss the mechanism thereof. Methods Recombinant ABL SH3-T79Y mutant adenovirus vectors which were successfully constructed in previous work was used with IM to treat K562/G01 cells, then the cell-colony forming ability of K562/G01 cells was determined by clone formation assay, and cell cycle was assessed by flow cytometry. KCL22 cells were treated by recombinant SH3-T79Y and IM to construct subcutaneous solid tumor model in Balb/c nude mice, then the formation rate of subcutaneous tumor was estimated, the pathological examination was conducted, and the proliferation ability of KCL22 cells was assayed. K562/G01 cells were treated by SH3-T79Y and IM in combination, and the expression levels of p-BCR-ABL, BCR-ABL, p-CrkL, CrkL and Cyclin-D1 protein were determined by Western blotting. Cells treated with PBS, null recombinant adenovirus vectors or IM alone served as control groups. Results Compared to the 3 control groups, clone forming rate of K562/G01 cells decreased significantly (P<0.05) and cell cycles were arrested at S phase after being combined SH3-T79Y and IM treatment. The subcutaneous solid tumor formation rate in KCL22- Balb/c nude mice was 16.7% after combined SH3-T79Y and IM treatment, and large number of tumor cells were observed in tumor pathology examination. Western blotting revealed that the expression levels of p-BCR-ABL, p-CrkL, BCR-ABL, CrkL and Cyclin-D1 were decreased in K562/G01 cells. Conclusion Combined treatment of SH3-T79Y and imatinib may inhibit the proliferation of CML cells in vivo and in vitro by decreasing BCR-ABL and CrkL phosphorylation as well as Cyclin-D1 protein.
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AIMS@#To investigate the antitumor effects of extracts from Oxytropis falcata on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo.@*METHODS@#Cell proliferation, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and Annexin V-FITC/ PI double staining.@*RESULTS@#MTT assay revealed that essential oil and flavonoids of O. falcata (named EOOF and FOF) inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. The IC50 value of EOOF and FOF were 0.115 and 0.097 mg·mL(-1), respectively. Cell cycle was arrested at G(1) phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to FOF. Growth inhibition in H22 solid tumors transplanted mice was significantly pronounced after being treated with FOF, and the inhibition ratio were 56.1% and 70.8% at the concentration of 30 and 60 mg·kg(-1).@*CONCLUSION@#The results suggest that FOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer.
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Animals , Humans , Male , Mice , Antineoplastic Agents, Phytogenic , Apoptosis , Carcinoma, Hepatocellular , Drug Therapy , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Drugs, Chinese Herbal , Growth Inhibitors , Liver Neoplasms , Drug Therapy , Mice, Inbred ICR , Oxytropis , ChemistryABSTRACT
Objective To investigate the association between IL-22 and the pathogenesis of coronary artery atherosclerosis(AS).Methods The relative expression of IL-22 mRNA in PBMC from 30 AS patients and 8 patients without any signs of coronary artery stenosis was detected by RT-PCR.Serum IL-22 levels of 22 patients without any signs of coronary artery stenosis and 79 AS patients were detected by ELISA.CRL-1730 cells(human umbilical vein endothelial cells) were stimulated with oxidized low density lipoprotein (ox-LDL) at different dosage for 24 h,and the expression of IL-22R1 was detected by flowcytometry.The proliferation ability of CRL-1730 cells treated with IL-22(20 ng/ml) and/or ox-LDL(100 μg/ml)was measured by MTS assay,and the expression of basic fibroblast growth factor(bFGF) was detected by RTPCR and ELISA.Results Decreased IL-22 expression in PBMC and serum was observed as worsen of AS.The expression of IL-22R1 in ox-LDL treated CRL-1730 cells was increased in dose dependent manner.OxLDL decreased proliferation ability,as well as bFGF expression and releasing,of CRL-1730 cells.This effect of ox-LDL was partially rescued by IL-22.Conclusion IL-22 may have anti-atherosclerosis effect.This effect may be mediated by regulating bFGF expression and endothelial cells proliferation ability in the presence of IL-22.
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OBJECTIVE: Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells. METHODS: MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by 3H-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP. RESULTS: At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125. CONCLUSION: Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.
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Anthracenes , Blotting, Western , Butadienes , Cell Proliferation , Diterpenes , Down-Regulation , Epoxy Compounds , Imidazoles , Neurons , Nitriles , p38 Mitogen-Activated Protein Kinases , Phenanthrenes , Phosphorylation , Protein Kinases , Pyridines , RNA, Small Interfering , VanadatesABSTRACT
Interleukin-22 (IL-22) is a new kind of eytokine discovered in 2000. The major sources of IL-22 are activated T1 -cells and NK-cells. Tissue cells at outer body barriers, i.e. of the skin, kidney, the di-gestive and respiratory systems all highly express IL-22R or respond to IL-22. IL-22 functions by promoting the anti-microbial defense, inducing phase reactants, protecting against damage and enhancing natural immu-nity. Furthermore, IL-22 mediates the proliferation, differentiation and apoptesis in cancer cells, that gives us a new idea about tumor therapy.