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Abstract Objectives Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. Method TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. Results Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. Conclusion Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.
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Objective:To investigate the effects of miRNA-30a-5p (miR-30a-5p) and metadherin (MTDH) on the proliferation, invasion and migration abilities of human breast cancer cells in vitro.Methods:The expression of MTDH in cancer and paracancerous tissues of 112 breast cancer patients in the database and miR-30a-5p in cancer and paracancerous tissues of 103 breast cancer patients in the database were analyzed using data from The Cancer Genome Atlas (TCGA) database. Pearson correlation analysis was used to analyze the correlation between miR-30a-5p and MTDH in 1 222 breast cancer patients in the database; the data were updated to August 2022. Breast cancer MDA-MB-231 cells were divided into negative control group (transfected with negative control sequence), miR-30a-5p overexpression group (transfected with miR-30a-5p mimics), siMTDH group [transfected with small interfering RNA against MTDH (siMTDH)], siMTDH+miR-30a-5p overexpression group (transfected with both siMTDH and miR-30a-5p mimics); cell proliferation ability was detected by methyl thiazol tetrazolium (MTT) assay, cell migration ability was detected by cell scratch assay, cell invasion ability was detected by Transwell assay. The relative expressions of miR-30a-5p, MTDH, matrix metalloproteinase (MMP)-2, MMP-9, vimentin and β-catenin mRNA in cells were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR), and the expressions of MTDH, N-cadherin (N-cad), β-catenin, Snail and MMP-9 proteins were detected by Western blotting.Results:In the TCGA database, MTDH expression level was higher and miR-30a-5p expression level was lower in breast cancer tissues compared with paracancerous tissues, and the differences were statistically significant (both P < 0.001). There was a negative correlation between MTDH and miR-30a-5p expressions in 1 222 patients with breast cancer ( r=-0.134, P < 0.001). Compared with the negative control group, the cell proliferation ability was reduced in both siMTDH group and miR-30a-5p overexpression group at 24, 48 and 72 h (all P < 0.001). The cell scratch healing rate in miR-30a-5p overexpression group and siMTDH group was lower than that in negative control group [(61.6±1.6)%, (54.7±5.9)% vs. (80.3±3.0)%] (both P < 0.05). Compared with the negative control group, The number of migrated cells in miR-30a-5p overexpression group and siMTDH group was less than that in negative control group (881±50, 725±63 vs. 1 172±66) (both P < 0.05). Compared with the negative control group, the relative expressions of MMP-2, MMP-9, vimentin and β-catenin mRNA were all down-regulated in MDA-MB-231 cells of miR-30a-5p overexpression group and siMTDH group (all P < 0.05). Compared with the negative control group, the relative expressions of N-cad, β-catenin, Snail and MMP-9 proteins were down-regulated in MDA-MB-231 cells of miR-30a-5p overexpression group and siMTDH group (all P < 0.05). There was no statistical difference in the number of migrated MDA-MB-231 cells between siMTDH+miR-30a-5p overexpression group and siMTDH group (476±5 vs. 389±46, t = 3.37, P = 0.078). There was no statistical difference in the number of migrated cells between siMTDH+miR-30a-5p overexpression group and miR-30a-5p overexpression group (476±5 vs. 477±22, t = 0.02, P = 0.983). Conclusions:The expression of miR-30a-5p is negatively correlated with the expression of MTDH in breast cancer tissues, and either overexpression of miR-30a-5p or silence of MTDH in breast cancer MDA-MB-231 cells in vitro can inhibit cell proliferation, invasion and migration, but MTDH may not be a target gene of miR-30a-5p.
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Objective:To investigate the relationship between long non-coding RNA (lncRNA) DHRS4-AS1 and disease-free survival in osteosarcoma patients and the mechanisms of its effect on proliferation and migration of osteosarcoma cells in vitro.Methods:The data of DHRS4-AS1 transcriptome levels and survival status of osteosarcoma patients in GEPIA database were collected since the database was established, and the patients were divided into high DHRS4-AS1 expression group and low DHRS4-AS1 expression group based on the median DHRS4-AS1 transcriptome level, with 59 cases in each group, and the Kaplan-Meier method was used to analyze the disease-free survival of the two groups. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of DHRS4-AS1 in osteosarcoma cell lines MG-63, HOS, 143B, U-2OS, Saos2 and normal osteoblast cell line hFOB1.19, and the osteosarcoma cell line with the lowest DHRS4-AS1 expression level was selected for subsequent experiments. The plasmid carrying DHRS4-AS1 sequence and the plasmid carrying negative control sequence were transfected into the selected osteosarcoma cells as DHRS4-AS1 group and control group. CCK-8 method was used to detect the proliferation of each group of cells, and the absorbance value was used as the cell proliferation ability; cell scratch assay was used to detect the migration of each group of cells. The bioinformatics website starBase V2.0 was used to predict the target genes of DHRS4-AS1, and the dual luciferase reporter gene assay was used to verify the targeting relationship between DHRS4-AS1 and the target genes. The expression levels of target genes and downstream genes of osteosarcoma cells in control group and DHRS4-AS1 group were detected by qRT-PCR and Western blotting.Results:Survival analysis showed that the disease-free survival of osteosarcoma patients in the high DHRS4-AS1 expression group in GEPIA database was superior to that of the low DHRS4-AS1 expression group ( P < 0.001). Compared with normal osteoblastic hFOB1.19 cells, the expression level of DHRS4-AS1 was low in all osteosarcoma cells (all P < 0.01), with the lowest expression level of DHRS4-AS1 in U-2OS cells ( P < 0.001). Cell proliferation ability was reduced in U-2OS cells of the DHRS4-AS1 group after 1, 2, 3 and 4 d of culture compared with the control group (all P < 0.05). The migration rate of U-2OS cells in the DHRS4-AS1 group was lower than that in the control group [(31±6)% vs. (63±4)%, t = 4.38, P = 0.005]. starBase V2.0 website predicted that DHRS4-AS1 complementarily bound to miRNA-411-3p (miR-411-3p); dual luciferase reporter gene assay showed that miR-411-3p overexpression reduced the luciferase activity of the wild-type DHRS4-AS1 reporter gene ( P < 0.001), but had no effect on the luciferase activity of the mutant DHRS4-AS1 reporter gene ( P > 0.05). qRT-PCR showed that the relative expression of miR-411-3p in U-2OS cells of the DHRS4-AS1 group was low (0.22±0.06 vs. 1.06±0.23, t = 3.55, P = 0.012) and the relative expression of metastasis suppressor MTSS1 mRNA was high (5.58±1.03 vs. 1.06±0.22, t = 4.28, P = 0.005) compared with the control group; Western blotting showed that MTSS1 expression was elevated, and the expression levels of cell proliferation phenotype proteins CDK3 and cyclin C and cell migration phenotype proteins ZEB2 and KLF8 were low. Conclusions:Osteosarcoma patients with high expression of lncRNA DHRS4-AS1 have better disease-free survival, and its expression is low in osteosarcoma cell lines. DHRS4-AS1 may promote MTSS1 gene expression and inhibit cell proliferation and migration by targeting and down-regulating miR-411-3p expression in osteosarcoma cells.
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Objective:To investigate the characteristics related to proliferation, migration and invasion of radiation-induced polyploid colon cancer SW1116 cells and their progeny.Methods:Colon cancer SW1116 cells were conventionally cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum. SW1116 cells at logarithmic growth stage were irradiated with 7 Gy X-ray, and the morphological changes of the cells were observed by inverted microscope on days 3, 5, 10 and 19 after radiation induction. According to the morphological changes of the cells, the cells at day 3 after radiation induction were labeled as polyploid giant cancer cell (PGCC) group, and the cells at day 19 were recorded as PGCC progeny group. SW1116 cells without radiation induction were used as control group. Flow cytometry was used to detect cell ploidy in the control, PGCC and PGCC progeny groups, CCK-8 assay was used to detect the proliferation ability of the three groups, cell migration and invasion abilities of the three groups were detected by cell scratch assay and Transwell assay, and Western blotting was used to detect the expressions of cell cycle and proliferation-related proteins and epithelial-mesenchymal transition (EMT) marker N-cadherin (N-cad) in the three groups.Results:The volume of SW1116 cells gradually became larger on days 3, 5 and 10 after radiation induction, and returned to normal on day 19. The proportions of polyploid (DNA content >4N) cell subsets in the control group, PGCC group and PGCC progeny group were (2.3±1.1)%, (23.1±8.1)% and (3.2±0.5)%, the difference was statistically significant ( F = 18.52, P < 0.05), and the proportion of polyploid cell subpopulations in the PGCC group was higher than that in the control group ( t = 5.38, P < 0.01), but the differences between the PGCC progeny group and the control group were not statistically significant ( t = 0.22, P > 0.05). After 72 h of culture, the cell proliferation rates of the control, PGCC and PGCC progeny groups were (100.0±4.1)%, (73.5±0.7)% and (123.9±3.5)%, and the difference was statistically significant ( F = 190.27, P < 0.001). After 48 h of cell scratching, the scratch healing rates in the control, PGCC and PGCC progeny groups were (38.0±2.7)%, (41.5±4.0)% and (63.7±4.2)%, and the difference was statistically significant ( F = 43.05, P < 0.001). After 24 h of culture, the number of invasive cells in the control, PGCC and PGCC progeny groups was 12.9±1.2, 3.4±0.6 and 23.7±1.5, and the difference was statistically significant ( F = 63.64, P < 0.001). The expression levels of cell cycle-related proteins P-cdc25c, cdc25c and cdc2 in the PGCC group were lower than those in the control group (all P < 0.05), and the expression levels of transcription factor-related proteins E2F-2, E2F-3 and EMT marker N-cad were downregulated compared with the control group (all P < 0.05); the expression levels of P-cdc25c, cdc25c, cdc2, E2F-2, E2F-3 and N-cad proteins in the PGCC progeny group were higher than those in the control group (all P < 0.05). Conclusions:Radiation can induce colon cancer SW1116 cells to produce polyploid, which may then generate daughter cells through asymmetric mitosis and gain new life, and then promote the recurrence and metastasis of colon cancer.
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Objective:To investigate effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts.Methods:Immunohistochemical technique was used to detect ROCK1 protein expression in human keloids and normal skin tissues, and Western blot analysis was performed to detect the expression of ROCK1, transforming growth factor β1 (TGF-β1) and E-cadherin in keloid tissues. In vitro cultured human keloid fibroblasts (HKFs) were divided into 4 groups: ROCK1 gene overexpression control group (ROCK1 NC group) transfected with ROCK1 gene overexpression control vectors, ROCK1 gene overexpression group (ROCK1 OE group) transfected with ROCK1 gene overexpression vectors, ROCK1 gene knockdown control group (sh NC group) transfected with ROCK1 gene knockdown control vectors, and ROCK1 gene knockdown group (shROCK1 group) transfected with ROCK1 gene knockdown vectors. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of ROCK1 gene on the survival rate of HKFs, Transwell assay to evaluate the effect on the migration of HKFs, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of ROCK1, TGF-β1 and E-cadherin, respectively. Results:Immunohistochemical study showed that ROCK1 protein expression decreased significantly in the human keloid tissues compared with the normal tissues ( t = 6.47, P = 0.003) ; Western blot analysis showed that the expression levels of ROCK1 and E-cadherin significantly decreased ( t = 14.02, 162.20, respectively, both P < 0.001), while TGF-β1 expression significantly increased ( t = 76.01, P < 0.001) in the keloid tissues compared with the expression levels of corresponding proteins in the normal tissues. CCK8 assay showed that the cell activity was significantly lower in the ROCK1 OE group than in the ROCK1 NC group after 24-hour transfection ( t = 3.25, 3.78, P = 0.031, 0.019, respectively), and significantly higher in the shROCK1 group than in the sh NC group ( t = 3.12, 2.79, P = 0.036, 0.049, respectively). Transwell assay showed that the number of migratory cells was significantly lower in the ROCK1 OE group than in the ROCK1 NC group ( t = 5.17, P = 0.004), and significantly higher in the shROCK1 group than in the sh NC group ( t = 9.28, P < 0.001). Compared with the ROCK1 NC group, the ROCK1 OE group showed significantly increased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but decreased mRNA and protein expression levels of TGF-β1 (both P < 0.001) ; compared with the sh NC group, the shROCK1 group showed significantly decreased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but significantly increased mRNA and protein expression levels of TGF-β1 ( P = 0.005 or < 0.001) . Conclusions:The ROCK1 gene can inhibit the proliferation and migration of HKFs. Overexpression of the ROCK1 gene can down-regulate the TGF-β1 gene expression and up-regulate the E-cadherin gene expression in HKFs.
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Objective:To investigate the effect of ring finger protein 187 (RNF187) on cell pro-liferation, migration and invasion of hepatocellular carcinoma (HCC).Methods:Messenger RNA (mRNA) level of RNF187 in HCC was analyzed by bioinformatics. Huh7 cells transfected with small interfering RNA (siRNA) of negative control or target gene respectively were classified as non-transfection (NC) group and RNF187 knockdown group. After 24 hours of transfection, the above two groups dimethyl sulfoxide (DMSO) were used as NC+ DMSO group and RNF187 knockdown + DMSO group. 24 hours after transfection with siRNA of target gene, the cells dealt with bafliomycin A1 (BFA) were set as RNF187 knockdown + BFA group. The regulation of RNF187 on malignant biological behavior and autophagy level of HCC cells were explored by cell counting kit-8 (CCK8) proliferation assay, cell scratch assay, transwell assay and western blot.Results:Compared with normal liver tissue, the mRNA level of RNF187 was higher in HCC tissue ( P<0.05). Compared with NC group, the absorbance at 48 h and 72 h and the scratch healing rate at 12 h and 24 h of RNF187 knockdown group were all lower, the differences were statistically significant (all P<0.001). The number of transmembrane cells in RNF187 knockdown group (39.50±5.57) at 24 h was lower than that in NC group (128.25±17.35), the differences were statistically significant ( t=9.74, P<0.001). Compared with NC group, the relative expression of total LC3 and Beclin-1 in RNF187 knockdown group all increased, while the relative expression of phosphorylated mammalian target of rapamycin decreased, the difference were statistically significant (all P<0.05). Compared with RNF187 knockdown+ DMSO group, the autophagy flow level, the 48 h and 72 h absorbance, the scratch healing rate at 24 h in RNF187 knockdown + BFA group were higher, the differences were statistically significant (all P<0.001). The number of transmembrane cells in RNF187 knockdown + BFA group (119.00±2.65) was more than that in RNF187 knockdown + DMSO group (57.67±2.52), the differences were statistically significant ( t=29.09, P<0.001). Conclusion:RNF187 is highly expressed in HCC tissue and knockdown of RNF187 inhibits the malignant biological behavior of HCC by enhancing the level of autophagy.
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Objective:To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results:Compared with the control group, cell viability ( t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate ( t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant ( t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant ( t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion:BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
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Objective:To detect the expression of MYBL2 gene in gastric adenocarcinoma tissue and its effects on cell proliferation and invasion. Methods:A total of 100 cases of gastric adenocarcinoma tissue and 100 cases of paracancerous tissue were selected from patients who received surgery in The People's Hospital of Yuhuan between January 2017 and December 2020. Gastric adenocarcinoma cell lines MGC-803 were transfected with MYBL2 siRNA and siRNA control. The cells not transfected were used as controls. MYBL2 gene expression in gastric adenocarcinoma tissue and paracancerous tissue as well as MGC-803 were determined by quantitative real time-polymerase chain reaction. MGC-803 cell proliferation was determined by MTT. The invasive ability of MGC-803 cells was determined by Transwell assay. The migration ability of MGC-803 cells was determined by Scratch testing. MYBL2 protein expression in gastric adenocarcinoma tissue and paracancerous tissue as well as MGC-803 cells was determined by western blotting. Results:The relative mRNA expression of MYBL2 in gastric adenocarcinoma tissue was significantly higher than that in paracancerous tissue [(0.65 ± 0.17) vs. (0.18 ± 0.05), t = 26.52, P < 0.05). The relative mRNA expression of MYBL2 in the MYBL2 siRNA group (0.29 ± 0.07) was significantly lower than that in the control group (0.73 ± 0.12) and siRNA group (0.71 ± 0.16, t = 5.48, 4.16, both P < 0.05). MTT assay showed that after 24 and 48 hours of culture, MGC-803 cell proliferation rate in the MYBL2 siRNA group [(40.95 ± 5.46)%, (52.12 ± 12.27)%] was significantly lower than that in the control group [(67.84 ± 6.45)%, (87.83 ± 9.96)%] and siRNA group [(66.98 ± 7.85)%, (85.98 ± 10.24)%, t = 5.51, 3.91, 4.71, 3.67, all P < 0.05]. MGC-803 cell invasion rate in the MYBL2 siRNA group [ (62.12 ± 6.43)%] was significantly lower than that in the control group [(89.74 ± 6.56)%] and siRNA group [(88.83 ± 7.85)%, t = 5.20, 4.55, both P < 0.05]. The number of MGC-803 cells migrated in the MYBL2 siRNA group [(4.32 ± 0.84) × 10 3] was significantly lower than that in the control group [(8.95 ± 1.64) × 10 3] and siRNA group [(8.83 ± 1.78) × 10 3, t = 4.35, 3.96, both P < 0.05]. The gray value of MYBL2 protein in the gastric adenocarcinoma tissue was (0.56 ± 0.15), which was significantly higher than that in the paracancerous tissue [(0.23 ± 0.07), t = 19.93, P < 0.001]. The gray value of MYBL2 protein in the MYBL2 siRNA group was (0.21 ± 0.03), which was significantly lower than that in the control group (0.67 ± 0.15) and siRNA group (0.65 ± 0.19) ( t = 5.20, 3.96, both P < 0.05). Conclusion:MYBL2 gene is highly expressed in gastric adenocarcinoma tissue. siRNA silencing MYBL2 can decrease the ability of MGC-803 cells to proliferate, invade and migrate and downregulate MYBL2 expression. This study is highly innovative and scientific.
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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 explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs).Methods:A experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by realtime quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups.Results:The ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant ( F=53.40, 50.30; P<0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant ( F=54.35, 52.84, 84.35; P<0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant ( F=53.66, 83.54; P<0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant ( F=24.87, 53.84; P<0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P<0.001; BMP4 group: F=56.87, 59.35, 58.35; P<0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group ( F=48.32, 56.33, 55.01; P>0.05). Conclusion:BMP4 induces the proliferation and migration of hRMECs through glycolysis.
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Objective:To evaluate the effect of propofol on proliferation, invasion and migration of human melanoma cells and role of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2)/matrix metalloproteinase (MMP) signaling pathway.Methods:SKMEL-5 cells were cultured in vitro and divided into 4 groups ( n=36 each) using the random number table method: control group (group C), propofol group (group P), COX-2 overexpression group (group COX-2), and COX-2 overexpression plus propofol group (group COX-2+ P). Propofol at the final concentration of 60 μmol/L was added in group P. The COX-2 overexpression plasmid pcDNA3.1-COX-2 was transfected into SKMEL-5 cells in group COX-2 and group COX-2+ P, and propofol at the final concentration of 60 μmol/L was added in group COX-2+ P.After incubation for 48 h, the cell proliferation rate was determined by CCK-8 method, the cell invasion and migration ability was determined by Transwell assay, the expression of COX-2 in cells was detected by Western blot, the expression of COX-2 mRNA in cells was detected by quantitative real-time polymerase chain reaction, and the concentrations of serum PGE2, MMP-2 and MMP-9 were determined by enzyme-linked immunosorbent assay. Results:Compared with group C, the cell proliferation rate was significantly decreased, the number of cell invasion and migration was decreased, the expression of COX-2 protein and mRNA was down-regulated, and the concentrations of PGE2, MMP-2 and MMP-9 in the supernatant were decreased in group P, and the cell proliferation rate was significantly increased, and the number of cell invasion and migration was increased, the expression of COX-2 protein and mRNA was up-regulated, and the concentrations of PGE2, MMP-2 and MMP-9 in the supernatant were increased in group COX-2 ( P<0.05). Compared with group P, the cell proliferation rate was significantly increased, and the number of cell invasion and migration was increased, the expression of COX-2 protein and mRNA was up-regulated, and the concentrations of PGE2, MMP-2 and MMP-9 in the supernatant were increased in group COX-2+ P ( P<0.05). Conclusions:Propofol can inhibit the proliferation, invasion and migration of human melanoma cells, and the mechanism may be related to inhibition of the COX-2/PGE2/MMP signaling pathway.
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Objective:To investigate the expression of PC4 and SFRS1 interacting protein 1 (PSIP1) in oral squamous cell carcinoma cells and the effects of PSIP1 silencing on the migration and invasion of oral squamous cell carcinoma cells, and to preliminarily explore its mechanism.Methods:The PSIP1 gene of oral squamous cell carcinoma cell line HN30 was silenced by RNA interference technique. HN30 cells were divided into si-NC group (transfected with siRNA-NC) and si-PSIP1 group (transfected with siRNA-PSIP1). Quantitative real-time PCR was used to detect the expression of PSIP1 mRNA. Scratch test and Transwell invasion test were used to detect the migration and invasion abilities of HN30 cells, and Western blotting was used to detect the expression levels of epithelial-mesenchymal transformation (EMT) related proteins in HN30 cells of the two groups.Results:The relative expression levels of PSIP1 of HN30 cells in the si-NC group and si-PSIP1 group were 1.00±0.00 and 0.21±0.06 respectively, with a statistically significant difference ( t=22.30, P=0.002). The scratch healing rates of the si-NC group and si-PSIP1 were (48.21±4.66)% and (42.05±11.74)% at 12 h respectively, with no statistically significant difference ( t=1.46, P=0.173), and the scratch healing rates of the two groups were (86.61±6.06)% and (67.76±3.62)% at 24 h respectively, with a statistically significant difference ( t=8.01, P<0.001). The invasion numbers of HN30 cells in the si-NC group and si-PSIP1 group were 91.00±7.05 and 23.34±4.98, and there was a statistically significantly difference ( t=19.20, P<0.001). Compared with the si-NC group, the migration and invasion abilities of HN30 cells in the si-PSIP1 group decreased significantly (all P<0.001). The expression levels of E-cadherin of the si-NC group and si-PSIP1 group were 1.06±0.02 and 1.43±0.13 respectively, with a statistically significant difference ( t=-4.94, P=0.036), and the expression levels of N-cadherin were 1.00±0.04 and 0.57±0.14 respectively, with a statistically significant difference ( t=5.03, P=0.007). Compared with the si-NC group, the expression level of E-cadherin in the si-PSIP1 group increased, while the expression level of N-cadherin decreased. Conclusion:Silencing the expression of PSIP1 can significantly inhibit the migration and invasion of HN30 cells, and the mechanism may be related to the effect of PSIP1 on the EMT pathway of oral squamous cell carcinoma.
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Objective:To detect the expressions of anterior gradient protein 2 (AGR2) in the cultures of three colon cancer cell lines SW480, SW620 and COLO205, and to investigate the effects of different concentrations of exogenous AGR2 on the proliferation, migration and invasion abilities of SW620 cells.Methods:Western blotting was used to detect the expression levels of AGR2 protein in SW480, SW620 and COLO205 colon cancer cell cultures. SW620 cells were divided into blank control group, anterior gradient protein 2 homologous human recombinant protein (rAGR2) low concentration group (100 μg/ml) and rAGR2 high concentration group (200 μg/ml), and CCK-8 assay, cell scratch assay and Transwell migration and invasion assay were used to detect the effects of different concentrations of rAGR2 on the biological behaviors of SW620 cells.Results:Western blotting results showed that the expression levels of AGR2 protein in SW480, SW620 and COLO205 cells were 0.545±0.097, 0.662±0.040 and 0.882±0.156 respectively, with a statistically significant difference ( F=7.46, P=0.024). The level of AGR2 protein in COLO205 cell line was significantly higher than that in SW480 and SW620 cell lines ( P=0.009; P=0.047). The results of CCK-8 experiment showed that the proliferative activities of SW620 cells in the blank control group, rAGR2 low concentration group and rAGR2 high concentration group were 0.422±0.031, 0.542±0.040 and 0.574±0.033 respectively, with a statistically significant difference ( F=26.35, P<0.001), and the rAGR2 low concentration group and rAGR2 high concentration group were significantly higher than the blank control group (both P<0.001). The results of cell scratching assay showed that the percentage of 36 h cell scratching area was (28.029±2.107)%, (20.642±0.983)% and (16.951±1.608)% for the three groups of cells respectively, with a statistically significant difference ( F=35.85, P<0.001), the rAGR2 low concentration group was higher than the blank control group ( P=0.001), and the rAGR2 high concentration group was higher than the rAGR2 low concentration group ( P=0.032). The results of cell migration assay showed that the number of cells migrated in the three groups was 447.1±32.3, 513.1±55.8 and 632.4±50.3 respectively, with a statistically significant difference ( F=35.62, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P=0.007), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P<0.001). The results of the invasion assay showed that the number of cells invaded in the three groups was 369.1±56.1, 505.1±34.4 and 579.0±71.5 respectively, with a statistically significant difference ( F=32.40, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P<0.001), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P=0.010). Conclusion:The expression of AGR2 protein varies in the extracellular fluid of different invasive colon cancer cells and increases with the invasive ability. AGR2 protein can increase the proliferation, migration and invasive abilities of colon cancer cells SW620.
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Objective:To study the effect and mechanism of Rab4A knockout expression on proliferation, migration and invasion of gastric cancer cells. Methods:The expression of Rab4A in four human gastric cancer cell lines MGC-803, SGC-790, MKN45 and AGS was detected by Western blot. Rab4A was knocked out in AGS cells with the highest expression level, and untransfected gastric cancer cells were used as control group. Cell proliferation, migration and invasion were detected by CCK8 and Transwell assay, respectively. Western blot analysis was used to investigate the expression changes of epidermal growth factor receptor (EGFR), downstream pathway proteins AKT and β-catenin induced by Rab4A knockout. The interaction between Rab4A and MiR- 496 was detected by dual luciferase reporter gene, and the effect of MiR- 496 transfection on Rab4A expression was detected by qPCR and Western blot. GraphPad Prism 9 software was used for data analysis, t-test was used for comparison between the two groups, and normal distribution measurement data were expressed as mean ± standard deviation ( ± s). Results:The expression of Rab4A was the highest in AGS cells, and the knockdown of Rab4A inhibited the proliferation, migration and invasion of AGS cells ( P<0.05). In Rab4A knockout gastric cancer cells, the surface expression of epidermal growth factor receptor (EGFR) was significantly decreased, and the expression of downstream pathway proteins p-AKT and β-catenin was also inhibited ( P<0.05). The luciferase reporter showed that MiR- 496 could bind the 3′UTR of Rab4A. In addition, MiR- 496 down-regulated the expression of Rab4A in AGS cells( P<0.05). Conclusion:The expression of Rab4A is inhibited by MiR- 496, and the proliferation, migration and invasion of gastric cancer cells can be inhibited by down-regulating the surface expression of EGFR after inhibiting Rab4A expression.
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Objective:To investigate the expression of long non-coding RNA (lncRNA) ALMS1-IT1 in colorectal cancer tissues and the molecular mechanism of its effect on the proliferation and migration of colorectal cancer HT-29 cells in vitro.Methods:The cancer tissue specimens and paracancerous tissue (>5 cm from the edge of the tumor) specimens were collected from 40 colorectal cancer patients who were diagnosed by pathological examination after surgical resection in Hubei 672 Orthopedic Hospital of Integrated Traditional Chinese and Western Medicine from July 2018 to November 2020. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of ALMS1-IT1 in colorectal cancer tissues and paracancerous tissues, when the relative expression of ALMS1-IT1 was higher than or equal to its median relative expression, ALMS1-IT1 was highly expressed, and the correlation of ALMS1-IT1 expression with the clinicopathological characteristics of patients was analyzed. HT-29 cells were infected with the empty lentivirus and the lentivirus carrying the ALMS1-IT1 silence sequence, and named control group and si-ALMS1-IT1 group. qRT-PCR was used to detect the expression of ALMS1-IT1 in the two groups of HT-29 cells. CCK-8 method and Transwell experiment were used to detect the proliferation and migration ability of the two groups of HT-29 cells. The starBase v2.0 online database was used to predict ALMS1-IT1 interacting molecules, and qRT-PCR and Western blot were used to detect the expression of these molecules.Results:The relative expression of ALMS1-IT1 in colorectal cancer tissues was higher than that in paracancerous tissues (4.54±0.61 vs. 1.19±0.31, t = 34.89, P < 0.01). The median relative expression of ALMS1-IT1 in cancer tissues of 40 patients was 2.93, and the high expression rate of ALMS1-IT1 was 50.0% (20/40). The high expression rate of ALMS1-IT1 in cancer tissues of TNM stage Ⅲ patients was higher than that in TNM stage Ⅰ-Ⅱ patients, the high expression rate of ALMS1-IT1 in poorly-differentiated patients was higher than that in well- and moderately-differentiated patients, and the high expression rate of ALMS1-IT1 in patients with lymph node metastasis was higher than that in patients without lymph node metastasis (all P < 0.01). The cell proliferation capacity (absorbance value) of HT-29 cells in the si-ALMS1-IT1 group after cultured for 2, 3, 4, and 5 days was lower than that in the control group (all P < 0.05). The number of cell migration at 24 h in HT-29 cells of the si-ALMS1-IT1 group was less than that of the control group (45±7 vs. 112±18, t = 3.45, P < 0.05). Using starBase v2.0 online database to predict that the target gene of ALMS1-IT1 may be miRNA-889-3p (miR-889-3p), and the target gene of miR-889-3p may be ATAD2. Compared with the control group, the relative expression of miR-889-3p in HT-29 cells of the si-ALMS1-IT1 group increased (4.24±0.46 vs. 1.01±0.11, t = 6.81, P < 0.01). Compared with the control group, ATAD2 mRNA ( P < 0.01) and protein expression levels in the si-ALMS1-IT1 group were reduced. Conclusions:ALMS1-IT1 is highly expressed in colorectal cancer tissues, and the ALMS1-IT1 expression is related to the TNM stage, degree of tumor differentiation and lymph node metastasis of patients. Down-regulation of ALMS1-IT1 in vitro may inhibit the proliferation and migration of colorectal cancer HT-29 cells by regulating the miR-889-3p-ATAD2 axis. ALMS1-IT1 may be a therapeutic target for colorectal cancer.
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Objective:To investigate the expression of long non-coding RNA (lncRNA) BDNF-AS in kidney cancer tissues, and its effect on the proliferation and migration ability of kidney cancer cells and the molecular mechanism.Methods:Real-time reverse quantitative polymerase chain reaction (rRT-PCR) was used to detect the expression levels of BDNF-AS gene in renal cancer tissues, tumor-adjacent tissues of 67 renal cancer patients and normal renal tubular epithelial cells HK-2 and renal cancer cell lines A498, ACHN, OS-RC-2, Caki-1, 786-O in Huangshi Central Hospital of Edong Medical Group from May 2017 to July 2018. The kidney cancer cell line with the lowest expression of BDNF-AS was taken as the research object. Transient transfection with BDNF-AS overexpression plasmid was treated as the experiment group or a plasmid carrying meaningless sequences was treated as the control group. rRT-PCR was used to detect transfection efficiency. After the transfection with Caki-1 for 24 h, methythiazolyl tetrazolium (MTT) method was used to detect the proliferation of cells in both groups, Transwell migration assay was applied to detect the cell migration ability, rRT-PCR was used to detect the expression level of protein tyrosine phosphatase receptor type G (PTPRG) mRNA and Western blot was used to detect the expression level of PI3K-AKT pathway related-proteins.Results:The relative expression level of BDNF-AS in kidney cancer tissues was lower than that in tumor-adjacent tissues (0.96±0.24 vs. 4.62±0.84, t = 41.76, P < 0.01). The relative expression of BDNF-AS in kidney cancer cell lines was lower than that in normal renal tubular epithelial cells HK-2 (all P < 0.05), and the relative expression in Caki-1 cells was the lowest (0.10±0.01). The relative expression of BDNF-AS in the experimental group was higher than that in the control group ( P < 0.01). From the second day of transfection, the proliferation ability of Caki-1 cells in the experimental group was lower than that in the control group (all P < 0.05). The number of Caki-1 migrated cells in the experimental group was lower than that in the control group after migration for 15 h of Caki-1 cells transfected for 24 h [(51±8) vs. (192±25), t = 5.31, P < 0.01]. After 48 h transfection, the relative expression of PTPRG mRNA in Caki-1 cells ( P < 0.01) and protein expression of the experimental group were higher than those of the control group, the expression levels of PI3K-AKT signaling pathway related-proteins p-PI3K, p-AKT, p-Tpl2 in Caki-1 cells of the experimental group were lower than those of the control group. Conclusions:The expression of BDNF-AS is down-regulated in kidney cancer tissues and cell lines. Overexpression of BDNF-AS can inhibit the proliferation and migration ability of kidney cancer Caki-1 cells. The molecular mechanism may be related to the transduction that BDNF-AS promotes PTPRG gene expression and interferes with PI3K-AKT signaling pathway.
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Objective:To study the effects of MYC-induced long non-coding RNA (MINCR) targeting miR-584-3p on the proliferation, invasion and migration of rheumatoid arthritis synovial fibroblasts (RASFs).Methods:Synovial tissue samples were collected from 25 rheumatoid arthritis (RA) patients and 25 patients with joint trauma undergoing joint replacement surgery. Expression of MINCR and miR-584-3p in synovial tissue was detected using Real-time quantitative polymerase chain reaction (PCR). RASFs were separated for in- vitro culture, and RASFs were transfected with MINCR small interfering RNA (si-MINCR), miR-584-3p mimics, si-MINCR and miR-584-3p inhibitors (anti-miR-584-3p). Changes in cellular viability, clone formation, migration and invasion were detected by cell counting kit (CCK-8), plate cloning experiment, scratch healing test, Transwell test, respectively. Dual luciferase reporter gene assay was applied to evaluate the effect of miR-584-3p on the luciferase activity of MINCR. The independent t-test was used to analyze the differences between the two groups, and the one-way analysis of variance (ANOVA) and SNK- q test were used to analyze the differences between multiple groups. Results:MINCR was significantly up-regulated in RA synovial tissue compared to normal synovial tissue [(3.27±0.36) vs (1.00±0.08), t=30.777, P<0.01], whereas miR-584-3p was significantly down-regulated in RA synovial tissue [(0.43±0.05) vs (1.00±0.06), t=36.491, P<0.01]. After interference with MINCR expression, the activity [(0.52±0.04) vs (1.05±0.09), t=16.144, P<0.01] and clone formation number [(45±5) vs (99±9), t=15.960, P<0.01], scratch healing rate [(28±3)% vs (69±6)%, t=18.013, P<0.01] and invasion number [(53±5) vs (113±12), t=14.019, P<0.01] of RASFs were significantly decreased. After overexpression of miR-584-3p, the activity [(0.65±0.05) vs (1.02±0.09), t=26.063, P<0.01], clone formation number [(52±5) vs (98±8), t=14.619, P<0.01], scratch healing rate [(35±3)% vs (68±6)%, t=13.711, P<0.01] and invasion number [(62±5) vs (117±11), t=13.346, P<0.01] of RASFs were significantly decreased. miR-584-3p could specifically bind to MINCR and significantly inhibit the luciferase activity [(0.45±0.04) vs (0.97±0.06), t=21.633, P<0.01]. Compared with interference with MINCR, the activity [(0.92±0.07) vs (0.49±0.04), t=16.000, P<0.01], the clone formation number [(86±8) vs (45±5), t=14.008, P<0.01], scratch healing rate [(59±7)% vs (27±3)%, t=13.200, P<0.01], and invasions number [(99±9)% vs (54±5)%, t=13.414, P<0.01] of RASFs were significantly increased after miR-584-3p and MINCR were inhibited simultaneously. Conclusion:Interfering lncRNA MINCR can inhibit the proliferation, migration and invasion of RASFs by targeting and up-regulating miR-584-3p.
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Objective:To investigate the differential expression of long non-coding RNA (lncRNA) in placental tissues of women with preeclampsia (PE) and the effect of MIR210HG on the biological function of HTR8/SVneo cells.Methods:A total of 39 cases of PE women (PE group) and 39 cases of normal pregnant women (CTL group) admitted to the Affiliated Hospital of Qingdao University from July 2018 to July 2019 were collected. (1) Transcriptome sequencing (RNA-seq) was used to analyze the differentially expressed lncRNAs in the placental tissues of the two groups. (2) The expression level of MIR210HG, one of the differentially expressed lncRNAs, in the placental tissues of the two groups was detected by real-time quantitative PCR. And the correlations between the expression level of MIR210HG and systolic blood pressure, diastolic blood pressure and neonatal birth weight were analyzed. (3) The constructed small interfering RNA and negative control (NC) RNA were transfected into the HTR8/SVneo cells. The cells were divided into MIR210HG knockdown (KD) group and NC group. The effects of living cell counting (CCK-8) and transwell assay on the proliferation and migration of HTR8/SVneo cells were detected. (4) RNA interacting with MIR210HG was predicted using the Encyclopedia of RNA Interactomes (ENCORI) database. Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Gene and Genomes (KEGG) and BioCarta pathway enrichment analysis were performed.Results:(1) A total of 26 significantly differentially expressed lncRNAs were found by RNA-seq, among which 21 lncRNAs were up-regulated and 5 lncRNAs were down-regulated. (2) The relative expression level of MIR210HG in the PE group was significantly higher than that in the CTL group (9.30±1.90 and 1.10±0.20, respectively; t=4.425, P<0.01). The relative expression level of MIR210HG had positive linear correlation with systolic blood pressure ( r2=0.234, P<0.05) and diastolic blood pressure ( r2=0.190, P<0.05), but had a negative linear correlation with newborn birth weight ( r2=0.157, P<0.05). (3) Compared with the NC group, the proliferation and migration ability of HTR8/SVneo cells in the KD group were increased (all P<0.05). (4) A total of 38 RNAs that might interact with MIR210HG were predicted by ENCORI database. GO functional annotation analysis showed that MIR210HG might be involved in the functions of 27 pathways, including the regulation of production of molecular mediator of immune response, etc; KEGG pathway analysis showed that MIR210HG might be involved in the function of 8 pathways including allograft rejection, etc; Biocarta pathway analysis showed that MIR210HG may be involved in the functions of 8 pathways, including the eukaryotic initiation factor (eIF) pathway, etc. Conclusion:The expression of MIR210HG is up-regulated in the placental tissue of PE women, and MIR210HG might be a regulator of the biological behavior of trophoblast cells.
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Objective:To investigate the effects of long non-coding RNA (lncRNA) FBXL19-AS1 on the proliferation, migration and invasion of pancreatic cancer cells, and to determine the targeting relationship of lncRNA FBXL19-AS1 and microRNA-339-3p (miR-339-3p).Methods:From January 2017 to August 2019, 73 cancer tissues and matched normal pancreatic tissues adjacent to cancer from patients pathologically diagnosed as pancreatic cancer who underwent surgical resection in Yantai Hospital of Yantai were collected. Normal pancreatic epithelial cells (hTERT-HPNE) and 3 pancreatic cancer cell lines (Capan-1, SW1990, PaTu8988) were cultured in vitro. The real-time fluorescent quantitative PCR was used to detect the expression of lncRNA FBXL19-AS1 and miR-339-3p in pancreatic cancer tissues and cell lines. The Capan-1 cells were divided into the NC group (normal control group), si-NC group (transfected with meaningless negative sequence), si-FBXL19-AS1 group (transfected with FBXL19-AS1 small interfering RNA), miR-NC group (transfected with empty plasmid control), miR-339-3p group (transfected with miR-339-3p overexpression lentiviral vector), si-FBXL19-AS1+ anti-miR-339-3p NC group (cotransfected with FBXL19-AS1siRNA and miR-339-3p inhibitor negative control sequence) and si-FBXL19-AS1+ anti-miR-339-3p group (cotransfected with FBXL19-AS1siRNA and miR-339-3p inhibitor). CCK-8 method was used to detect cell proliferation activity. Transwell chamber was used to detect cell migration and invasion ability, and western blotting method was used to detect cell cyclinD1, matrix metalloproteinase 2 (MMP2) and MMP9 protein expression. Bioinformatics and dual luciferase reporter gene experiments were used to analyze the targeting relationship between lncRNA FBXL19-AS1 and miR-339-3p.Results:The expression of lncRNA FBXL19-AS1 in pancreatic cancer tissue was significantly higher than that in normal pancreatic tissue adjacent to cancer (2.96±0.21 vs 1.00±0.13, P<0.05), and the expression of miR-339-3p was significantly lower than that in normal pancreatic tissue adjacent to cancer (0.37±0.05 vs 1.00±0.11, P<0.05). The expression of lncRNA FBXL19-AS1 in Capan-1, SW1990 and PaTu8988 cells were 2.43±0.18, 1.97±0.13 and 1.73±0.14, respectively, which were significantly higher than that of hTERT-HPNE cells 1.00±0.07. The expression of miR-339-3p were 0.42±0.03, 0.54±0.03 and 0.57±0.04, respectively, which were all significantly lower than 1.00±0.05 of hTERT-HPNE cells. Among them, the expression of lncRNA FBXL 19-AS1 in Capan-1 cells was the highest, and the miR-339-3p was the lowest. Compared with the si-NC group, the absorbance value ( A450) of Capan-1 cells in the si-FBXL19-AS1 group, the number of migrating cells, and the number of transmembrane cells were significantly decreased (0.47±0.03 vs 0.94±0.06, 81.00±7.41 vs 187.00±16.13, 67.00±5.41 vs 141.00±9.24), the protein expression of cyclinD1, MMP2 and MMP9 was significantly reduced (0.44±0.03 vs 0.83±0.05, 0.48±0.03 vs 0.92±0.07, 0.38±0.02 vs 0.73±0.05). Compared with the miR-NC group, the A450, the number of migrating cells, and the number of transmembrane cells of Capan-1 cells in the miR-339-3p group were significantly decreased (0.54±0.04 vs 0.94±0.05, 98.00±6.53 vs 193.00±10.02, 83.00±6.58 vs 146.00±7.11, the protein expression of cyclinD1, MMP2 and MMP9 was significantly reduced (0.47±0.03 vs 0.81±0.07, 0.43±0.03 vs 0.94±0.06, 0.32±0.02 vs 0.71±0.06). Compared with the si-FBXL19-AS1+ anti-miR-NC group, the A450, the number of migrating cells and the number of transmembrane cells in the si-FBXL19-AS1+ anti-miR-339-3p group increased significantly (0.96±0.07 vs 0.48±0.03, 204.00±11.25 vs 83.00±5.11, 157.00±8.76 vs 64.00±4.12, P all <0.05), the protein expression of cyclinD1, MMP2 and MMP9 increased significantly (0.84±0.06 vs 0.42±0.03, 0.96±0.08 vs 0.47±0.08, 0.74±0.06 vs 0.37±0.02, P all <0.05). The luciferase activity of Capan-1 cells cotransfected with WT-FBXL19-AS1 and miR-339-3p mimics was significantly lower than that of the cotransfected with WT-FBXL19-AS1 and miR-NC (0.47±0.04 vs 1.00±0.10, P all <0.05). The difference of the luciferase of Capan-1 cells in the cotransfected MUT-FBXL19-AS1 and miR-339-3p mimics group and the cotransfected MUT-FBXL19-AS1 and miR-NC group was not statistically significant. Conclusions:LncRNA FBXL19-AS1 was highly expressed in pancreatic cancer tissues and Capan-1 pancreatic cancer cell lines. Knockdown of lncRNA FBXL19-AS1 can target miR-339-3p to regulate the proliferation, migration and invasion of pancreatic cancer cells, and promote the occurrence and development of pancreatic cancer.
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Objective:To investigate the effects of micro RNA(miR)-296-3p on cell proliferation, migration and invasion of tongue squamous cell carcinoma and its underlying mechanism.Methods:Normal oral epithelial mucosal keratinocytes HOK was used as the control. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of miR-296-3p and nucleoprotein 1(NUPR1) mRNA in tongue squamous cell carcinoma cell lines CAL27, SCC15 and SCC9, and Western blot was used to detect the expression of NUPR1 protein. CAL27 cells were divided into NC group, miR-con group, miR-296-3p group, si-con group, si-NUPR1 group, miR-296-3p+pcDNA group and miR-296-3p+pcDNA-NUPR1 group, then CCK8 method was used to detect the cell activity. Transwell was used to detect the cell migration and invasion. Western blot was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), matrixmetallo proteinase-2(MMP-2) and matrixmetallo proteinase-9(MMP-9). Dual-luciferase reporter assay system was implemented to verify the relationship between miR-296-3pand NUPR1.Results:Compared with the HOK cells, the content of miR-296-3p in the tongue squamous cell line CAL27, SCC15 and SCC9 groups was significantly reduced (0.54 ± 0.08, 0.38 ± 0.05, 0.59 ± 0.07 vs. 1.04 ± 0.12, t = 10.401, 15.231 , 9.718, P<0.05), while the expression levels of NUPR1mRNA (5.94 ± 0.40, 4.48 ± 0.45, 5.19 ± 0.48 vs.0.94 ± 0.12, t = 35.918, 22.803, 25.769) and protein (0.79 ± 0.09, 0.54 ± 0.05, 0.62 ± 0.08 vs. 0.28 ± 0.04, t = 15.535, 12.182, 11.404) were significantly increased (all P<0.05). Compared with those in the NC group or miR-con group, CAL27 cell activity, migration number, invasion number and the protein expression of PCNA, MMP-2 and MMP-9 in the miR-296-3p group were decreased (all P<0.05). Compared with those of the NC group or the si-con group, the CAL27 cell activity, migration number, number of invasions and the protein expression of PCNA, MMP-2 and MMP-9 in the si-NUPR1 group were decreased (all P<0.05). miR-296-3p negatively regulatedthe expression of NUPR1 in CAL27 cells. Compared with the miR-296-3p+pcDNA group, CAL27 cell viability (138.34 ± 5.73 vs. 98.54 ± 5.89, t = 14.530), migration number (95.28 ± 7.56 vs. 67.92 ± 5.23, t = 8.929), invasion number (184.53 ± 17.57 to 101.26 ± 10.64, t = 12.162) and the protein expression of PCNA (0.68 ± 0.07 to 0.35 ± 0.06, t = 10.738), MMP-2 (0.43 ± 0.05 to 0.29 ± 0.04, t = 6.559) and MMP-9 (0.58 ± 0.06 vs. 0.33 ± 0.08, t = 7.500) in the miR-296-3p+pcDNA-NUPR1 group were increased (all P<0.05). Conclusions:miR-296-3p may inhibitthe proliferation, migration and invasion of tongue squamous cell carcinoma cells by negatively regulating NUPR1.