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Objective:To investigate the effect of transcription factor nuclear factor IB (NFIB) on cell proliferation and invasion in breast cancer.Methods:The lentivirus pLKO.1-shNFIB plasmid was constructed, packaged and infected with human estrogen receptor positive (ER + ) breast cancer cell line MCF-7 and triple-negative breast cancer (TNBC) cell line MDA-MB-231, respectively, NFIB was stably knocked down and verified by Western blot method; Cell count test (CCK-8) and clone formation test were used to investigate the effect of knockdown NFIB on the growth and proliferation of breast cancer cells; The transwell experiment and Western blot method were performed to detect the expression of epithelial mesenchymal transition protein markers. The effect of knockdown NFIB on the invasive ability of triple-negative breast cancer cells was explored; Kaplan-Meier survival was used to analyze web data (http: //kmplot.com/analysis/) to explore the effect of NFIB on the prognosis of ER + breast cancer and triple-negative breast cancer patients. Results:In MCF-7 and MDA-MB-231 breast cancer cells, knocking down NFIB inhibited cell growth and proliferation; In triple-negative breast cancer MDA-MB-231 cells, knocking down NFIB promoted the expression of interstitial marker fibronectin and promoted cell invasion; The lower the expression of NFIB, the worse the prognosis of triple negative breast cancer patients, while the expression of NFIB had no effect on the prognosis of ER + breast cancer patients. Conclusions:Knocking down NFIB inhibits the proliferation of MCF-7 cells, and the expression level of NFIB is not related to the prognosis of ER + breast cancer patients; Knocking down NFIB inhibits the proliferation of MDA-MB-231 cells but promotes their invasion; The low expression of NFIB is associated with the poor prognosis of triple-negative breast cancer patients.
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Objective:To establish a melanoma cell line derived from a Chinese female patient of Han nationality with metastatic melanoma, and to study its basic biological characteristics.Methods:Metastatic melanoma cells were isolated from axillary lymph nodes of a 17-year-old female patient with malignant melanoma, and subjected to culture in vitro to establish a cell line. Short tandem repeat (STR) genotyping was performed to compare the information about the cell line and its derived tissue, and to detect gene mutations; cell counting kit-8 (CCK8) assay was conducted to assess the cellular proliferative activity, and soft agar cloning assay to assess the anchorage-independent proliferation; karyotype analysis was performed to determine the number and structure of chromosomes; with the highly aggressive melanoma cell line A2058 and keratinocyte line HaCaT serving as controls, Transwell assay was conducted to detect differences in cell migratory and invasive abilities, cell immunofluorescence assay and Western blot analysis were performed to determine the protein expression of melanoma-specific markers HMB45, S100 and Melan-A; the tumorigenicity was evaluated in vivo in a tumor-bearing mouse model. Results:A melanoma cell line was successfully established and named ZJMM-45, which was cultured for more than 70 passages over a 1-year period, and showed a stable shape and proliferative activity. The cells were spindle-shaped or polygonal, and could produce melanin. STR matching analysis showed that the ZJMM-45 sample was 96% matched with the cryopreserved lymph node tissue of the patient, suggesting that they were from the same source. A tumor-related gene BRAF V600E mutation (c.1799T>A) was identified in the ZJMM-45 cell line; karyotype analysis revealed that ZJMM-45 cells had triploid chromosomes and abnormal structures. ZJMM-45 cells grew exponentially in vitro and reached a plateau-phase in growth on day 5; the cells grew clonally and formed colonies in agar, showing anchorage-independent and malignant proliferative activity. Cell immunofluorescence assay and Western blot analysis showed that both ZJMM-45 and A2058 cells expressed HMB45, S100 and Melan-A; Transwell assay revealed that the numbers of invasive and migratory ZJMM-45 cells (300 ± 14, 260 ± 14, respectively) were significantly higher than those of invasive and migratory A2058 cells (150 ± 6, 160 ± 19, t = 13.25, 11.76, respectively, both P < 0.001) . The tumor-bearing mouse experiment showed that all 5 nude mice developed tumors with an inner diameter of 1.0 cm after 4 weeks, and the tumors were histopathologically characterized by proliferating hyperchromatic melanoma cell nuclei and formation of small nests, which were similar to primary solid tumors. Conclusion:The metastatic melanoma cell line ZJMM-45 derived from a Chinese patient with melanoma was successfully constructed, which carried the BRAF V600E mutation and expressed melanoma-specific markers, and was characterized by rapid proliferation, invasion and metastasis in both in vivo and in vitro culture, as well as obvious tumorigenicity in the in vivo experiment.
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Objective:To determine the expression of silent information regulator 1 (Sirt1) , Sirt3 and hypoxia-inducible factor 1α (HIF-1α) in cutaneous squamous cell carcinoma (CSCC) tissues and cells, and to explore their role in the occurrence and development of CSCC.Methods:From January 2019 to December 2020, 30 lesional skin tissues were obtained from patients with histopathologically confirmed poorly-, moderately- or well-differentiated CSCC, and 30 normal skin tissues were obtained from patients with non-cancerous diseases in Department of Dermatology, General Hospital of Ningxia Medical University. A CSCC cell line A431 and a human keratinocyte cell line HaCaT were cultured. Immunohistochemical study, Western blot analysis and real-time quantitative PCR (RT-PCR) were performed to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in CSCC tissues of different grades of differentiation and normal skin tissues, cytochemical and immunofluorescence staining and RT-PCR were conducted to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in A431 and HaCaT cells, respectively. Comparisons of measurement data among multiple groups were performed by using one-way analysis of variance, and comparisons between two groups by using t test. Results:Immunohistochemical study showed that the expression level of Sirt3 (expressed as the average optical density) was 100 ± 12.12, 117.72 ± 26.23, 127.32 ± 24.45, 132.71 ± 31.61 in the normal skin tissues and well-, moderately- and poorly-differentiated CSCC tissues respectively, and there was a significant difference among these groups ( F = 20.14, P < 0.001) ; the expression of Sirt1 and HIF-1α increased in turn from the normal skin tissues to the well-, moderately- and poorly-differentiated CSCC tissues, and significantly differred in these groups ( F = 174.50, 225.00, respectively, both P < 0.001) . As Western blot analysis revealed, the expression level of Sirt3 significantly differed among the normal skin tissues, well-, moderately- and poorly-differentiated CSCC tissues (expressed as relative gray value: 1.000 ± 0.132, 1.403 ± 0.411, 1.387 ± 0.393, 1.677 ± 0.683, respectively; F = 34.97, P < 0.001) , and so did the expression levels of Sirt1 and HIF-1α ( F = 69.29, 199.90, respectively, both P < 0.00l) , with a gradually increasing trend in their expression levels from the the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues. RT-PCR showed that the mRNA expression of Sirt3, Sirt1 and HIF-1α was sequentially increased from the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues, and significant differences were observed among these groups ( F = 113.00, 174.50, 50.33, respectively, all P < 0.001) . The protein expression levels of Sirt3, Sirt1 and HIF-1α were significantly higher in the A431 cells than in the HaCaT cells ( t = 16.75, 18.34, 27.76, respectively, all P < 0.001) , and so were their mRNA expression levels ( t= 14.22, 9.62, 16.86, respectively, all P < 0.001) . Conclusion:Increased expression of Sirt3, Sirt1 and HIF-1α was observed in CSCC tissues and cells, which may promote the occurrence and development of CSCC.
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Objective:To analyze the expression and clinical significance of reticulocalbin 3 (RCN3) in colon cancer by bioinformatics database and biological experiments.Methods:Colon cancer HT29 and SW620 cells and colon normal mucosal cells FHC were cultured. The expression of RCN3 in cells was verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot. The expression data of RCN3 in normal colon tissue and colon cancer tissue were obtained by Ualcan database. The co-expressed gene information of RCN3 from LinkedOmics database was obtained, and the biological processes and related functions of these RCN3 co-expressed genes through were analyzed by gene ontology analysis (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The protein-protein interaction network of RCN3 related coding genes was constructed by using STRING database. Finally, the relationship between the expression of RCN3 and the clinical prognosis of patients with colon cancer was compared and analyzed according to GEPIA, Ualcan and Linked Omics biological database.Results:Western blot and qRT-PCR results showed that the mRNA and protein expression of RCN3 in HT29 and SW620 colon cancer cells was significantly higher than those in FHCcells ( all P<0.05). The analysis of biological database showed that the expression level of RCN3 in colon cancer tissue was higher than that in normal colon tissue ( P<0.05). GO enrichment analysis showed that RCN3 co-expression genes were mainly involved in the composition of extracellular matrix and extracellular domain structure, the binding process of extracellular matrix and multiple receptors, and the biological processes related to tumor development such as cell adhesion, immune response, and angiogenesis through extracellular domain structure. KEGG pathway analysis showed that RCN3 co-expression genes were mainly involved in ECM receptor interaction, cytokine receptor interaction, chemokine signaling pathway, phosphatidylinositol-3-kinase protein kinase B (PI3K-Akt) signaling pathway, phagosome signal, IgA related intestinal immune network signal, these signaling pathways always related to tumor invasion, migration and inflammatory immune response. The protein-protein interaction network analysis showed that the coding protein genes that directly interacted with RCN3 protein that included PRDX6, NOSIP, PCSK6, IMMP1L, PRRG2, FBXO47, FCGRT, FKBP9, PCDHGA12, and PNMAL1, which were mainly involved in the occurrence and development of colorectal cancer, liver cancer, gastric cancer, breast cancer, lung cancer, and ovarian cancer. Survival curve analysis showed that the overall survival rate of colon cancer patients with high expression of RCN3 was significantly lower than that of patients with low expression of RCN3 ( P<0.05). Conclusions:RCN3 is highly expressed in colon cancer tissues and cells, which is closely related to the occurrence, development and prognosis of colon cancer. It can be used as one of the markers for early screening and prognosis prediction of colon cancer.
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Objective:To clarify the clinical significance, diagnostic and prognostic values of circular RNA circ_0141633 and circ_0008234 in peripheral blood of pancreatic cancer, and analyze their impact on the biological function of pancreatic cancer cells.Methods:The peripheral blood samples of 97 pancreatic cancer patients and 71 healthy controls were collected, and the expression of circ_0141633 and circ_0008234 was analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). The relationships between the expression of circ_0141633 and circ_0008234 and clinicopathological characteristics and prognosis of pancreatic cancer were analyzed by chi-square test, K-M survival curves and Cox proportional hazards regression model. The area under curve (AUC), sensitivity and specificity of circ_0141633 and circ_0008234 in the diagnosis of pancreatic cancer were analyzed by receiver operating characteristic (ROC) curve. The effects of circ_0141633 and circ_0008234 on the proliferation, migration, invasion and epithelial mesenchymal transformation (EMT) of Bxpc-3 cells were analyzed by methyl thiazolyl tetrazolium (MTT) method, cell scratch test, Transwell invasion and Western blot.Results:The expression of circ_0141633 and circ_0008234 in peripheral blood of pancreatic cancer patients was higher than those of healthy controls (all P<0.05). High expression of circ_0141633 and circ_0008234 was associated with higher clinical stage, lymph node metastasis and venous invasion, and were independent risk factors for poor prognosis in pancreatic cancer (all P<0.05). The overall survival rate of patients with high expression of circ_0141633 and circ_0008234 was significantly lower than that of patients with low expression of circ_0141633 and circ_0008234 (all P<0.05). The AUC of circ_0141633, circ_0008234, the combination of circ_0141633 and circ_0008234, CA19-9 in the diagnosis of pancreatic cancer was 0.70, 0.67, 0.88 and 0.82, with sensitivity of 64.32%, 60.79%, 78.22% and 73.97%, respectively. The specificity was 68.54%, 65.46%, 81.65% and 79.41%, respectively. The diagnostic efficiency of combination was superior to CA19-9, circ_0141633 and circ_0008234 alone (all P<0.05). Interfering with circ_0141633 and circ_0008234 alone could inhibit the proliferation, migration, invasion and EMT of Bxpc-3 cells, and the above inhibitory effect was more obvious after interfering with both of circ_0141633 and circ_0008234 (all P<0.05). Conclusions:The high expression of circ_0141633 and circ_0008234 in peripheral blood could be used as potential diagnostic and prognostic biomarkers of pancreatic cancer, and could promote the progression of pancreatic cancer.
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Objective:To explore the effect of the mucin modulator Talniflumate (Tal) on breast cancer cells and its synergistic effect after combined with the chemotherapy drug paclitaxel (PTX).Methods:The breast cancer cells were cultured in vitro. Lymphocyte proliferation activity assay (MTS) was used to detect the effects of different concentrations of Talniflumate alone and paclitaxel on the survival rate of breast cancer cells. The effects of the above drugs on the apoptosis of breast cancer cells were detected by flow cytometry. Western blot was used to detect the expression of glucosamine transferase (GCNT3) (the target of Talniflumate) in breast cancer cells before and after the treatment with Talniflumate. Transcriptome sequencing clarified the changes in related signaling pathways after treatment with Talniflumate. Results:Talniflumate promoted the apoptosis of breast cancer cells MCF7 and MDA-MB-231 cells in a concentration-dependent manner. The combination of Talniflumate and paclitaxel had a significant synergistic killing effect in MCF7 cells but not MDA-MB-231 cells. Western blot indicated that GCNT3 was highly expressed in MCF7 cells, while almost no expression in MDA-MB-231 cells; Talniflumate could reduce the expression of GCNT3 in MCF7 cells and after combined with paclitaxel, the expression of GCNT3 was downregulated more significantly. Transcriptome sequencing suggested that Talniflumate can regulate the expression of multiple signaling pathways such as TNF, p53, and SNARE.Conclusions:Talniflumate could induce apoptosis of breast cancer cell. Talniflumate combined with paclitaxel has a significant synergistic effect in killing tumor cells in breast cancer cells with high GCNT3 expression such as MCF7. The mechanism of Talniflumate induce apoptosis of breast cancer cells may be related to multiple signaling pathways such as TNF, p53, and SNARE.
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Objective:To investigate the effects of poly adenosine diphosphate ribose polymerase-1(PARP-1) inhibitor fluzoparib on proliferation, apoptosis and migration of pancreatic cancer PANC1 cells.Methods:PANC1 cells cultured in conventional culture medium were used as control group, and PANC1 cells cultured in the medium containing fluzoparib were used as fluzoparib group. The effects of fluzoparib with different concentrations on the proliferation of PANC1 cells were detected by CCK8 method, and the half inhibitory concentration (IC 50) of fluzoparib on PANC1 cells was calculated. The effect of fluzoparib on apoptosis and cell cycle of PANC1 cells was detected by flow cytometry, and the migration ability of PANC1 cells was detected by cell scratch test and Transwell chamber. Results:Compared with control group, with the increase of fluzoparib concentration and the prolongation of the action time, the cell proliferation activity of PANC1 in fluzoparib group was significantly decreased, and the differences were statistically significant (all P values <0.05). IC 50 of fluzoparib on PANC1 cells cultured for 24 h was 0.03 mmol/L. After 24 h culture, the IC 50 apoptosis rate of fluzoparib group was (32.19±2.48)%, and the apoptosis rate of control group was (21.99±6.30)%. The former was greatly higher than the latter, and the difference was statistically significant ( P<0.05). The proportion of cells in G 2/M phase was (16.28±0.62)% in the fluzoparib group and (11.64±0.88)% in the control group, and the difference between the two groups was statistically significant ( P<0.05). The migration rates of PANC1 cells in IC 50 fluzoparib group in 12 h and 24 h culture were (2.59±1.46)% and (19.76±7.84)%; and those in control group were (27.08±2.17)% and (45.92±3.61)%, respectively. The number of transmembrane cells was (348±19) cells/10 visual field in the fluzoparib group and (587±14) cells/10 visual field in the control group. The migration ability of PANC1 cells in fluzoparib group was significantly lower than that in control group ( P<0.05). Conclusions:Fluzoparib can inhibit the proliferation and migration of PANC1 cells and promote the apoptosis of PANC1 in vitro, which may be an effective drug for the treatment of pancreatic cancer.
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Objective:To investigate the inhibitory effect of combined strategy of poly adenosine diphosphate ribose polymerase (PARP) inhibitor and interleukin-1β (IL-1β) inhibitor on homologous recombination deficiency (HRD)-proficient ovarian cancer cells.Methods:(1) HRD-proficient ovarian cancer cell lines OVCAR3 and CAOV3 were treated with PARP inhibitor olaparib. Screening by RNA sequencing analysis, the expression level of IL-1β was validated by enzyme-linked immunosorbent assay (ELISA) and western blot. (2) The dose-response curves of IL-1β inhibitor diacerein were evaluated by cell counting kit-8 (CCK-8) assays in OVCAR3 and CAOV3 cells. CCK-8 assays were further applied to determine the viabilities of OVCAR3 and CAOV3 cells. (3) To evaluate the synergistic effects of olaparib and IL-1β inhibitor in vivo, the transplanted ovarian cancer model was constructed. BALB/c-nude mice ( n=16) were injected intraperitoneally with 1×10 7 OVACR3 cells labelled with luciferase (OVCAR3-Luc). Immunohistochemistry (IHC) assay was performed to determine nuclear antigen associated with cell proliferation (Ki-67) expression. (4) Blood routine tests, kidney and liver function tests were performed to analyze the toxic reaction of different drug treatments. The potential drug-induced injuries of vital organs including heart, liver, spleen, lungs and kidneys of nude mice were determined by hematoxylin-eosin (HE) staining. Results:(1) The RNA sequencing results showed that the mRNA level of IL-1β was the most significantly increased among the 25 differentially expressed genes in OVCAR3 cells treated with olaparib, compared to the negative control group. Olaparib treatment significantly promoted the secretion and expression of IL-1β protein in both OVACR3 and CAOV3 cells by ELISA [(36.2±3.5) and (49.5±3.5) pg/ml, respectively; all P<0.001] and western bolt (2.87±0.37 and 2.05±0.08, respectively; all P<0.01). (2) The half maximal inhibitory concentration (IC 50) value of IL-1β inhibitor was determined as follows: 75 μmol/L for OVACR3 cells and 100 μmol/L for CAOV3 cells. The treatments were divided into four groups including control group, olaparib monotherapy group, IL-1β inhibitor monotherapy group and the combination therapy group. The cell viabilities of each group in OVCAR3 and CAOV3 were determined by CCK-8 assay. The data in each group were showed as follows for OVCAR3 and CAOV3 cells: (100.0±0.4)% and (100.0±3.5)% in control group; (63.1±6.2)% and (63.3±3.8)% in olaparib monotherapy group; (61.6±4.7)% and (63.8±3.5)% in IL-1β inhibitor monotherapy group; and (32.9±5.2)% and (30.0±1.3)% in the combination therapy group. The viability assay showed that the combined strategy exhibited a significant inhibition effect on OVACR3 and CAOV3 cells, compared to the monotherapy group and the control group (all P<0.01). (3) All mice with transplanted tumors of HRD-proficient ovarian cancer cells were randomly divided into four groups, and treated with four different treatments as mentioned above, respectively. After 4 weeks (on day 29), the vivo fluorescence imaging were determined. The results showed that the amount of fluorescence of transplanted tumors was mostly decreased in the combination therapy group [(0.5±0.4)×10 10 p/s], compared to the control group [(4.2±1.0)×10 10 p/s] or the groups treated with any single drug [(3.1±0.9)×10 10, (2.2±0.9)×10 10 p/s; all P<0.05]. Mice were then sacrificed under anesthesia, and all transplanted tumors detached and weighed for further investigation. The weight of transplanted tumors was significantly decreased in the combination therapy group [(0.09±0.03) g], compared to that in control group [(0.25±0.05) g] or groups treated with any single drug [(0.17±0.03), (0.19±0.04) g; all P<0.05]. The measurement of the expression of Ki-67 showed that it was significantly decreased in the combination therapy group (0.33±0.10), compared to that in the control group (1.00±0.20) or monotherapy groups (0.76±0.07, 0.77±0.12; all P<0.05). (4) There were no significant differences of body weights, blood routine test, renal and liver function tests among mice with different treatments (all P>0.05). Moreover, no significant injuries were observed in the vital organs among the four groups. Conclusions:The combination of olaparib and IL-1β inhibitor synergistically exhibits significant cytotoxicity in HRD-proficient ovarian cancer cells. Moreover, the blood routine and blood biochemistry results confirmed the biosafety of the combination of olaparib and IL-1β inhibitor.
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Objective:To investigate the regulation effect of miR-125b in the gastric cancer cell growth mediated by apoptosis related protein (Fas)/apoptosis related protein ligand (FasL) signal.Methods:Gastric cancer SGC-7901 cells were cultured in vitro. MiR-125b inhibitor sequence, NC sequence and transfection reagent were transfected into SGC-7901 cells and divided into three groups: miR-125b inhibited group, NC group and control group. The expression of miR-125b in transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The colony formation was detected by plate cell clone formation assay. Cell apoptosis and cycle were detected by flow cytometry. The protein expression of Fas and FasL was detected by Western blot. The targeted regulation of Fas by miR-125b was detected by luciferase activity assay. Results:The expression level of miR-125 and the number of cell colony in miR-125b inhibited group was significantly lower than those in control group and NC group, and the inhibition rate of cell proliferation and apoptosis rate were significantly higher than that in control group and NC group (all P<0.05). The DNA content in G 1 phase in miR-125b inhibited group was significantly higher than that in control group and NC group, and the DNA content in S phase in miR-125b inhibited group was significantly lower than that in control group and NC group (all P<0.05). The expression of Fas and FasL protein in miR-125b inhibited group was significantly higher than that in control group and NC group (all P<0.05). The target site of miR-125b was found in 3′-UTR of Fas mRNA, and compared with the NC+ Fas 3′UTR-Wt group, the activity of luciferase in the miR-125b inhibited group+ Fas 3′-UTR-Wt group decreased significantly ( P<0.05). Conclusions:Inhibition of miR-125b expression can activate Fas/FasL signal and inhibit SGC-7901 cell proliferation, induce G 1 phase arrest of cell cycle and promote apoptosis.
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Objective:To investigate the expression of zinc finger protein 580 (ZNF580) in oxygen-glucose deprivation (OGD) model of SH-SY5Y cell line and its overexpression on the apoptosis of hypoxic-ischemic neurons and the possible mechanism.Methods:The study was divided into two parts: (1) Human neuroblastoma SH-SY5Y cell line was cultured and divided into the model group and control group. The model group was incubated at 37 ℃ for 6 h in a three-gas incubator of 95% N 2, 5% CO 2, and 0.1% O 2 to establish OGD model, and proteins were extracted at 6, 12, and 24 h after OGD. The expression of ZNF580 was quantified by Western blot. (2) Effects of ZNF580 overexpressed with lentivirus transfection on the apoptosis and cleaved caspase-3 expression: Cells were collected from the control group and model group 24 h after OGD. Overexpressed ZNF580 cells were constructed by lentivirus transfection as the overexpression group and then treated with OGD. Flow cytometry was used to detect the apoptosis rate in the three groups and Western blot was used to detect the expression of cleaved caspase-3. Two independent sample t-test, one-way variance analysis, and LSD- t for pairwise comparison were used for statistical analysis. Results:(1) ZNF580 expression was significantly increased at 6, 12, and 24 h after OGD compared with the control group (1.36±0.05, 2.12±0.07, 1.69±0.05 vs 1.00, LSD- t=9.20, 28.26, and 19.21, all P<0.001). (2) Apoptosis rates of the control, model, and overexpression groups were (1.07±0.56)%, (21.51±1.65)%, and (3.42±0.93)%, respectively, and relative expression levels of cleaved caspase-3 were 1.00, 2.47±0.59, and 1.70±0.25, respectively. Compared with the control group, apoptosis rate and cleaved caspase-3 relative expression level were significantly increased in the model group (LSD- t=21.98 and 8.17, both P=0.001), while the two figures were significantly decreased in the overexpression group when compared with the model group (LSD- t=19.45, P=0.001; LSD- t=4.28, P=0.005). Conclusion:Hypoxia and ischemia could lead to the overexpression of ZNF580, which may reduce the apoptosis of hypoxic-ischemic neurons by inhibiting the expression of cleaved caspase-3 and affecting its enzymatic activation.
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Objective:To observe the expression of microRNA (miRNA, miR)-5787 in breast cancer tissues and various cell lines, analyze the effect of overexpression of miR-5787 on breast cancer cell invasion and proliferation, and explore its possible molecular mechanism.Methods:Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression of miR-5787 in 47 breast cancer tissues and adjacent tissues, 4 breast cancer cell lines and normal breast epithelial cells. Breast cancer cell lines with the lowest miR-5787 expression were selected and transfected with miR-5787 mimics (experimental group) and control NC mimics (control group), respectively. qRT-PCR was used to detect the expression of miR-5787 in cells of the two groups. Transwell invasion experiment and cell counting kit-8 (CCK-8) were used to detect the effect of miR-5787 overexpression on breast cancer cell invasion and proliferation. Bioinformatics software and dual luciferase reporter gene experiments were used to predict and verify the target genes that miR-5787 could complementally bind. qRT-PCR and Western blot were used to detect the expression of target gene mRNA and protein.Results:Compared with adjacent tissues (5.05±0.82), the expression of miR-5787 in breast cancer tissues (1.32±0.33) was significantly reduced ( P<0.01). Compared with normal breast epithelial cells, the expression of miR-5787 in the four breast cancer cell lines was reduced ( P<0.05), and the expression in HCC1937 cells was the lowest ( P<0.01). After transfection of miR-5787 mimics, the expression of miR-5787 in HCC1937 cells in the experimental group was significantly higher than that in the control group ( P<0.01). Overexpression of miR-5787 could inhibit the invasion ( P<0.05) and proliferation ( P<0.05) of breast cancer HCC1937 cells. Bioinformatics software showed that the target gene of miR-5787 might be heparan sulfate proteoglycan 2 (HSPG2), and miR-5787 could complement HSPG2 mRNA ( P<0.01). qRT-PCR and Western blot indicated that overexpression of miR-5787 could significantly inhibit the expression of HSPG2 gene ( P<0.01), with the decreased expression of Vimentin, N-cadherin, Ki67 and PCNA. Conclusions:miR-5787 expression was low in breast cancer tissues and cell lines. Overexpression of miR-5787 could inhibit the invasion and proliferation of breast cancer HCC1937 cells by interfering with the expression of HSPG2 gene.
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Objective:To detect the expression of miR-378 in cervical cancer and investigate its effects on the proliferation and invasion of cancer cells as well as the underlying mechanism.Methods:A total of 185 cervical tissue samples of women who received gynecological examination in Qilu Hospital of Shandong University from January 2012 to January 2016 were included in this study. Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-378 in cervical tissue and C-33A cells. Western blot assay was performed to detect the expression of different cancer genes ATG12, CCND1 and pRb in C-33A cells. BrdU cell proliferation and Transwell invasion assay were performed to determine cell proliferation and invasion. Target Scan was used to predict and screen miR-378 gene targets and verified by a dual-luciferase reporter assay system.Results:The expression of miR-378 in cervical intraepithelial neoplasia (CIN) III lesioned tissue and cervical cancer tissue was significantly higher than that in normal cervical tissues ( F = 103.091, t = 9.381, 8.936, both P < 0.05). The expression of miR-378 in cervical cancer tissues with positive lymph node metastasis was significantly higher than that in cervical cancer tissues with negative lymph node metastasis ( t = 1.007, P < 0.01). The overexpression of miR-378 in cervical cancer tissues significantly promoted the migration and invasion of C-33A cells ( t = 5.285, P < 0.05), while low expression of miR-378 in cervical cancer tissues significantly inhibited the migration and invasion of HeLa cells ( t = 2.941, P < 0.05). The overexpression of miR-378 in C-33A cells significantly decreased the expression of ATG12, CCND1and pRb ( t = 1.382, 1.431 and 2.086, all P < 0.05). The low expression of miR-378 in C-33A cells significantly increased the expression of ATG12, CCND1 and pRb ( t = 3.961, 3.062 and 2.894, all P < 0.05). Conclusion:miR-378 can greatly promote the metastasis of cervical cancer cells. ATG12, as a direct target of miR-378, provides new insights into the molecular mechanism underlying cervical cancer pathology and therapeutic target.
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Objective:To investigate the effects of lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA MVIH) on proliferation, invasion and tumorigenic ability of colorectal cancer SW620 cells.Methods:SW620 cells cultured in vitro were divided into empty vector group (transfected with pcDNA3.1 empty vector plasmid), MVIH overexpression group (transfected with pcDNA3.1-MVIH overexpression vector plasmid), NC siRNA group (transfected with interference sequence negative control NC siRNA) and MVIH siRNA group (transfected with MVIH siRNA interference sequence). The mRNA expression level of MVIH in SW620 cells in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8), clone formation test and transwell chamber were used to detect the proliferation activity, clone formation ability and invasion ability of SW620 cells; The subcutaneous transplanted tumor of nude mice was constructed by subcutaneous injection of SW620 cells in each group 48 hours after transfection. The transplanted tumor volume was measured every 5 days to evaluate the tumor growth. On the 25th day, the nude mice in each group were killed and the tumor weight was measured. Results:The mRNA MVIH expression level, cell proliferation, clone formation rate and number of invasive cells of SW620 cells in the MVIH overexpression group were significantly higher than those in the empty vector group ( P<0.05); the MVIH mRNA expression level, cell proliferation , clone formation rate and number of invasive cells of SW620 cells in the MVIH-siRNA group were significantly lower than those in NC-siRNA group ( P<0.05). The results of subcutaneous transplantation tumor in nude mice showed that the growth rate of tumor in MVIH overexpression group was faster and the tumor weight was higher than those in the empty vector group ( P<0.05); the growth rate of tumor in MVIH overexpression group was slower and the tumor weight was lower than those in NC-siRNA group ( P<0.05). Conclusions:LncRNA MVIH can promote the proliferation, invasion and tumorigenesis of SW620 cells in colorectal cancer.
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Objective To investigate the effect of antisense oligonucleotides of miRNA-34a on non-small cell lung cancer (NSCLC) and its molecular mechanism.Methods The expression of miRNA34a in human non-small cell lung cancer cell line HCC827 and human normal lung cell MRC-5 was detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR).HCC827 cells were divided into three groups:blank control group,negative control group,anti-sense oligonucleotide group (liposome 2000 transfected anti-sense oligonucleotide miRNA-34a);cell counting kit-8 (CCK-8) method was used to detect cell proliferation,Jimsa staining was used to detect cell cloning ability,Transwell test was used to detect cell migration and invasion ability;RT-PCR and Western blot were used to detect phosphatase and tensin homolog (PTEN),phosphorylation-protein kinase B (p-Akt),phosphatidylinositol-3-kinase (PI3K)mRNA and protein expression.Results The relative expression of miRNA34a in HCC827 cells was significantly higher than that in human normal lung cells (P ＜ 0.01).The relative expression of miRNA34a in antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P ＜ 0.05),and there was no significant difference between negative control group and blank control group (P ＞ 0.05).At 48 h,72 h and 96 h,the proliferation level of HCC827 cells in antisense oligonucleotide miRNA-34a group was significantly lower than that in negative control group and blank control group (P ＜ 0.05).The cell cloning rate of antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P ＜ 0.01).The number of migration and invasion of HCC827 cells in antisense oligonucleotide RNA-34a group was significantly lower than that in negative control group and blank control group (P ＜0.01).The relative expression of PTEN mRNA and protein in antisense oligonucleotide miRNA-34a group was significantly higher than that in negative control group and blank control group (P ＜ 0.05);the relative expression of p-Akt,PI3K mRNA and protein in antisense oligonucleotide miRNA-34a group were significantly lower than that in negative control group and blank control group (P ＜ 0.05).Conclusions The expression level of miRNA-34a in human nonsmall cell lung cancer cells is significantly higher than that in human normal lung cells.Antisense oligonucleotides of miRNA-34a can inhibit the proliferation,cloning,migration and invasion of human non-small cell lung cancer cells.The mechanism may be related to the negative regulation of PTEN/p-Akt/PI3K signaling pathway.
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Objective To investigate the inhibitory effect of harmine on osteosarcoma cell proliferation and apoptosis by down regulating cyclooxygenase-2 (COX-2) expression.Methods Human osteosarcoma cell line U2OS was cultured in vitro and randomly divided into control group,study group 1,study group 2 and study group 3.The cells were cultured in 0,5 μmol/L,10 μmol/L and 20 μmol/L concentration of harmine for 48 hours.Cell counting kit-8 (CCK-8) method and flow cytometry were used to detect cell viability and apoptosis.The expression level of COX-2,proliferation and apoptosis related proteins and mRNA were detected by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR),respectively.Results After cultured with different concentrations of harmine for 12,24 hours and 48 hours,the cell viability of the three study groups were significantly lower than that of the control group (P ＜ 0.05),while that of the study groups 2 and 3 were significantly lower than that of the study group 1 (P ＜ 0.05).The apoptosis rate of the three study groups were significantly higher than that of the control group (P ＜0.05),while that of the two groups were significantly higher than that of the study group 1 (P ＜ 0.05).After 48 hours of culture,the levels of COX-2,cyclin D1,proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) protein and mRNA expression in study group 2 were significantly lower than those in control group,while the expression levels of cleaved caspase-3 and BCL2-Associated X (Bax) in study group 2 were significantly higher than those in control group (P ＜ 0.05).Conclusions Harmine can inhibit the proliferation and promote the apoptosis of osteosarcoma cells by inhibiting the expression of COX-2 and regulating the expression of cell cycle and apoptosis related protein.
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Objective To detect the expression of miR-373 in osteosarcoma cells and explore its effects on cell proliferation,invasion,and migration.Methods Human osteosarcoma cell line SJSA-1 and human osteoblast hFOB 1.19 cultured in vitro.Human osteosarcoma cell line SJSA-1 were randomly divided into four groups:blank control group (CK group),negative control group (NC group),miR-373 over-expressed plasmid group (miR-373 mimic group) and miR-373 inhibited plasmid group (miR-373 inhibitor group).Cells were transfected with FuGENE(R) HD transfection reagent.After 48 hours of transfection,the relative expression of miR-373 was detected by quantitative real-time polymerase chain reaction (qRT-PCR),cell proliferation by cell counting kit-8 (CCK-8) and cell invasion and migration by Transwell.Results The relative expression of miR-373 in SJSA-1 cells was significantly higher than that in hFOB-1.19 cells (P ＜ 0.05);compared with CK group and NC group,the relative expression of miR-373 in the miR-373 mimic group increased significantly (P ＜ 0.05),and the relative expression of miR-373 in the miR-373 inhibitor group was significantly lower than that in the control group (P ＜ 0.05);optical density (OD) value of miR-373 mimic group at 24 h,36 h,and 48 h were significantly higher than that of CK group and NC group (P ＜ 0.05),and OD value of miR-373 inhibitor group at 24 h,36 h,and 48 h were significantly lower than that of CK group and NC group (P ＜ 0.05);the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P ＜ 0.05),and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P ＜ 0.05);the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P ＜ 0.05),and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P ＜ 0.05).Conclusions The expression of miR-373 is up-regulated in osteosarcoma.Overexpression of miR-373 can promote the proliferation,invasion,and migration of osteosarcoma cells,while the low expression of miR-373 can inhibit the proliferation,invasion,and migration of osteosarcoma cells,which may be an important target for the diagnosis and treatment of osteosarcoma.
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Objective@#To detect the expression of miR-373 in osteosarcoma cells and explore its effects on cell proliferation, invasion, and migration.@*Methods@#Human osteosarcoma cell line SJSA-1 and human osteoblast hFOB 1.19 cultured in vitro. Human osteosarcoma cell line SJSA-1 were randomly divided into four groups: blank control group (CK group), negative control group (NC group), miR-373 over-expressed plasmid group (miR-373 mimic group) and miR-373 inhibited plasmid group (miR-373 inhibitor group). Cells were transfected with FuGENE® HD transfection reagent. After 48 hours of transfection, the relative expression of miR-373 was detected by quantitative real-time polymerase chain reaction (qRT-PCR), cell proliferation by cell counting kit-8 (CCK-8) and cell invasion and migration by Transwell.@*Results@#The relative expression of miR-373 in SJSA-1 cells was significantly higher than that in hFOB-1.19 cells (P<0.05); compared with CK group and NC group, the relative expression of miR-373 in the miR-373 mimic group increased significantly (P<0.05), and the relative expression of miR-373 in the miR-373 inhibitor group was significantly lower than that in the control group (P<0.05); optical density (OD) value of miR-373 mimic group at 24 h, 36 h, and 48 h were significantly higher than that of CK group and NC group (P<0.05), and OD value of miR-373 inhibitor group at 24 h, 36 h, and 48 h were significantly lower than that of CK group and NC group (P<0.05); the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P<0.05), and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P<0.05); the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P<0.05), and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P<0.05).@*Conclusions@#The expression of miR-373 is up-regulated in osteosarcoma. Overexpression of miR-373 can promote the proliferation, invasion, and migration of osteosarcoma cells, while the low expression of miR-373 can inhibit the proliferation, invasion, and migration of osteosarcoma cells, which may be an important target for the diagnosis and treatment of osteosarcoma.
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ObjectiveTo investigate the influence of hepatitis C virus (HCV) on the expression of lipid metabolism indices in vitro and in vivo. MethodsA total of 114 samples of patients with HCV infection who were treated in Renmin Hospital of Wuhan University from September 2017 to September 2018 were collected as experimental group, and 96 samples of healthy individuals who underwent physical examination were collected as control group. An automatic biochemical analyzer was used to measure blood lipid parameters, including triglyceride (TG), total cholesterol (TCh), high-density lipoprotein (HDL), low-density lipoprotein (LDL), small and low-density lipoprotein (sdLDL), lipoprotein (a), apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB). RT-qPCR was used to measure the mRNA expression of fatty acid synthase (FASN), acetyl-CoA carboxylase (ACACA), hydroxymethylglutarate mono-acyl CoA reductase (HMGR), ApoA1, ApoB, and low-density lipoprotein receptor (LDLR) in Huh7.5.1 cells with or without HCV infection. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. ResultsCompared with the control group, the experimental group had significantly lower serum levels of TCh (2.98±0.51 mmol/L vs 4.24±0.43 mmol/L, t=4.96, P＜0.05), HDL (0.87±0.16 mmol/L vs 1.24±0.21 mmol/L, t=5.65, P＜0.05), LDL (1.75±0.24 mmol/L vs 2.64±0.37 mmol/L, t=3.88, P＜0.05), ApoA1 (0.94±0.18 mmol/L vs 1.47±0.26 mmol/L, t=3.71, P＜0.05), and ApoB (0.67±0.31 mmol/L vs 0.98±0.14 mmol/L, t=4.41, P＜0.05). Huh7.5.1 cells with HCV infection had significantly lower mRNA expression of ApoA1 than those without HCV infection (t=-3.43, P＜0.05), as well as significantly higher mRNA expression of FASN, ACACA, HMGR, ApoB, and LDLR (t=5.40, 4.93, 3.34, 6.88, and 3.84, all P＜0.05). ConclusionHCV infection can upregulate the mRNA levels of enzymes involved in the synthesis of fatty acids and cholesterol and thus affect lipid metabolism in vivo and in vitro.
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Objective@#To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.@*Methods@#Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups: blank control group, negative control group and experimental group. The blank control group was the untreated cell group, while the negative control group was the lentivirus transfected cell group by the blank vector. The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist. The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot to detect the expression of Twist. Transwell kit was used to detect cell invasion. Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation.@*Results@#⑴ The titer of lentivirus was detected. The transfection titer of lentivirus vector: shRNA-Twist vector was 3×108 TU/ml. ⑵ The results of qRT-PCR test showed that compared with the negative control group, the mRNA expression of Twist in the experimental group was decreased (q=3.177, P=0.0234). ⑶ The results of Western blot showed that compared with the negative control group, the protein expression of Twist in the experimental group was decreased (q=4.071, P=0.0304). ⑷ The results of Transwell test showed that there was significant statistical difference among the three groups (F=19.472, P=0.000). Compared with the negative control group, the number of cell imigration in the experimental group was decreased (q=3.567, P=0.0318). ⑸ The results of CCK-8 showed that there was significant statistical difference among the three groups (F=20.983, P=0.000). Compared with the negative control group, the proliferation rate in the experimental group was decreased (q=5.272, P=0.0157).@*Conclusions@#ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.
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Objective@#To evaluate the effect of spermine oxidase (SMO) inhibitor SI-4650 on the proliferation of a human malignant melanoma cell line A375, and to explore its molecular mechanism.@*Methods@#Some cultured A375 cells were divided into 6 groups to be treated with SI-4650 at concentrations of 0, 10, 20, 40, 80 and 160 μmol/L respectively for 24, 48 and 72 hours, and methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate changes in cellular proliferative activity. According to the cellular proliferative activity, 3 concentrations (0, 40, 80 μmol/L) were screened out. Some A375 cells were divided into 3 groups to be treated with 0 (control group) , 40 and 80 μmol/L SI-4650 for 48 hours. Chemiluminescence assay was conducted to detect the SMO activity in A375 cells, high-performance liquid chromatography (HPLC) analysis to determine the polyamine content in A375 cells, flow cytometry to analyze the cell cycle and detect the apoptosis, and Western blot analysis to determine the protein expression of apoptotic marker proteins Bax and c-PARP, inhibitor of apoptosis protein Bcl-2, and autophagy marker proteins Beclin-1 and LC3-Ⅱ. Statistical analysis was carried out by using one-way analysis of variance for comparison of means among several groups, and by using Student-Newman-Keuls (SNK) -q test for multiple comparisons.@*Results@#MTT assay showed that there was a significant difference in the proliferative activity of A375 cells after the treatment with different concentrations of SI-4650 for different durations (F = 977.23, 5.16 respectively, both P < 0.001) . Significant differences were observed in the SMO activity in A375 cells (F = 242.58, P < 0.001) , spermine and the total polyamine content (F = 338.02, 2 931.07 respectively, both P < 0.001) , proportion of S-phase cells (F = 31.66, P < 0.001) , proportion of apoptotic cells (F = 100.68, P < 0.001) , expression of apoptosis-related proteins Bax, c-PARP and Bcl-2 (F = 35.51, 730.11, 27.54 respectively, all P < 0.001) , and expression of autophagy marker proteins Beclin-1 and LC3-Ⅱ (F = 35.87, 425.04 respectively, P < 0.001) among the control group, 40- and 80-μmol/L SI-4650 groups. Compared with the control group, the 40- and 80-μmol/L SI-4650 groups showed significantly lower SMO activity (luminous intensity: 61 432.85 ± 2 620.92, 43 337.35 ± 1 221.25 respectively, both P < 0.05) , lower spermine (1.97 ± 0.007, 1.88 ± 0.006 respectively, both P < 0.05) and total polyamine content (3.18 ± 0.03, 2.81 ± 0.01 respectively, both P < 0.05) , higher proportions of S-phase cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) and apoptotic cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) , higher expression of apoptotic marker proteins Bax (0.83 ± 0.12, 1.18 ± 0.16 respectively, both P < 0.05) and c-PARP (0.32 ± 0.002, 0.79 ± 0.035 respectively, both P < 0.05) and autophagy marker proteins Beclin-1 (1.00 ± 0.007, 1.14 ± 0.003 respectively, both P < 0.05) and LC3-Ⅱ (0.31 ± 0.001, 0.98 ± 0.003 respectively, both P < 0.05) , and lower expression of inhibitor of apoptosis protein Bcl-2 (0.65 ± 0.09, 0.12 ± 0.002 respectively, both P < 0.05) .@*Conclusion@#SI-4650 can inhibit the proliferation of A375 cells, likely by interfering with polyamine metabolism and inducing cell cycle arrest, apoptosis and autophagy.