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Objective:To explore whether BHLHE40 can affect the sensitivity of thyroid cancer (TC) cells to cisplatin by activating oxidative phosphorylation (OXPHOS) pathway by targeting high mobility group A2 (HMGA2) .Methods:The mRNA expression of HMGA2 and its upstream transcription factor BHLHE40 in TC tissues was analyzed by TCGA-THCA and hTFtarget online databases. The si-HMGA2, oe-HMGA2, oe-BHLHE40, negative control si-NC and oe-NC were transfected into TC cells (K1 and SW579) by liposome transfection method. The mRNA expression levels of BHLHE40 and HMGA2 in TC cells (SW579, FTC-133, and K1) and normal thyroid cells (Nthy ori3-1) were detected by real-time quantitative PCR (qRT-PCR). The cell viability was detected by MTT assay, the half inhibitory concentration (IC 50) value of cisplatin was calculated by CCK-8 assay, the apoptosis level was detected by flow cytometry, and the expression of OXPHOS complex was detected by Western blotting. Seahorse XFe 96 was used to analyze the oxygen consumption rate of the TC cells. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the binding relationship between BHLHE40 and HMGA2. Results:TCGA database results showed that the mRNA expression levels of HMGA2 and BHLHE40 in TC tissues (10.57±2.58, 13.89±1.13) were higher than those in normal thyroid tissues (4.82±1.69, 12.28±1.01), with statistically significant differences ( t=16.69, P<0.001; t=10.43, P<0.001). The results of qRT-PCR showed that the relative mRNA expression levels of HMGA2 in normal thyroid cells (Nthy ori3-1) and TC cells (SW579, FTC-133, and K1) were 1.00±0.13, 2.94±0.23, 4.71±0.41 and 6.29±0.49, while those of BHLHE40 were 1.00±0.12, 2.60±0.23, 3.39±0.35 and 6.18±0.51 respectively, both with statistically significant differences ( F=130.50, P<0.001; F=125.20, P<0.001). Further pairwise comparison showed that mRNA expression levels of HMGA2 and BHLHE40 in TC cells were significantly higher than those in normal thyroid cells (all P<0.001). According to MTT experimental results, si-HMGA2 treatment significantly reduced the cell viability of K1 cells compared to the si-NC group (all P<0.05). In addition, compared to the oe-NC group, oe-HMGA2 treatment significantly increased the cell viability of SW579 cells (all P<0.05). Compared to the oe-NC+DMSO group, the oe-HMGA2+DMSO group showed enhanced cell viability of SW579 cells, while the OXPHOS pathway inhibitor Gboxin was able to reverse the effect of overexpressing HMGA2 on cell viability (all P<0.05). The results of flow cytometry and CCK-8 experiments showed that compared to the si-NC group (apoptosis level: 6.19%±0.28%; cisplatin IC 50 value: 17.47 μmol/L), knocking down HMGA2 could increase the apoptosis level (11.96%±0.32%; t=19.17, P<0.001) and cisplatin sensitivity (IC 50 value: 1.49 μmol/L) of K1 cells. In addition, compared to the oe-NC group (apoptosis level: 9.98%±0.32%; cisplatin IC 50 value: 8.17 μmol/L), overexpressing HMGA2 significantly decreased the apoptosis level (4.32%±0.25%; t=19.65, P<0.001) and cisplatin sensitivity (IC 50 value: 34.95 μmol/L) of SW579 cells. The results of dual-luciferase assay showed that compared with the si-NC group, knocking down the expression of BHLHE40 in human kidney epithelial 293T cells significantly reduced the luciferase activity of wild-type HMGA2 (0.31±0.02 vs. 1.00±0.11; t=10.69, P=0.004). However, there was no significant effect on the luciferase activity of mutant-type HMGA2 (1.06±0.11 vs. 1.00±0.07; t=0.80, P=0.470). ChIP results showed that the mRNA expression level of HMGA2 in K1 cells was significantly increased in the anti-BHLHE40 group (6.57±0.62) compared with the IgG group (1.00±0.10; t=15.36, P<0.001). Compared to the oe-NC+DMSO group, the oe-HMGA2+DMSO group showed decreased apoptosis level ( P<0.05) and cisplatin sensitivity of SW579 cells, with a significant increase in the expression of OXPHOS complexes Ⅰ-Ⅴ and cellular oxygen consumption rates (all P<0.05). The effect of overexpressing HMGA2 was reversed by treatment with oe-HMGA2+Gboxin (all P<0.05). The recovery experiment showed that compared to the oe-NC+si-NC group, overexpression of BHLHE40 in SW579 cells increased cell viability and the expression of OXPHOS complexes Ⅰ-Ⅴ, while decreasing apoptosis levels and increasing cellular oxygen consumption rates and cisplatin IC 50 values (all P<0.05). However, simultaneous knockdown of HMGA2 reversed the effect of overexpressing BHLHE40 (all P<0.05) . Conclusion:BHLHE40 can activate the OXPHOS pathway by targeting and regulating the expression of HMGA2, thereby affecting the sensitivity of TC cells to cisplatin.
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Objective:To investigate the effects of miR-219a-5p on proliferation, invasion and migration of osteosarcoma U2OS cells by regulating high mobility group A2 (HMGA2) .Methods:Real-time quantitative PCR was used to detect miR-219a-5p mRNA expression levels in osteosarcoma U2OS cells and normal osteoblasts hFOB1.19. The U2OS cells were transfected with miR-219a-5p mimic (miR-219a-5p mimic group) and negative control mimic (mimic NC group) by liposome transfection. The expression levels of miR-219a-5p and HMGA2 mRNA in transfected cells were detected by real-time quantitative PCR. The level of HMGA2 protein was detected by Western blotting, cell proliferation ability was detected by CCK-8 assay and clonogenesis assay, cell migration ability was detected by scratching assay, cell invasion ability was detected by Transwell chamber assay, and the relationship between miR-219a-5p and HMGA2 was verified by double luciferase reporter gene assay.Results:Real-time quantitative PCR showed that the expression level of miR-219a-5p in osteosarcoma U2OS cells (0.11±0.01) was significantly lower than that in normal osteoblasts (1.00±0.06) , with a statistically significant difference ( t=26.83, P<0.001) . The results of CCK-8 showed that the cell absorbance values of the mimic NC group and miR-219a-5p mimic group were 0.52±0.02 and 0.42±0.02 after 24 h, 0.85±0.03 and 0.60±0.03 after 48 h, and 1.12±0.02 and 0.72±0.02 after 72 h respectively. The proliferation activity of the miR-219a-5p mimic group was significantly lower than that of the mimic NC group, with statistically significant differences ( t=6.97, P<0.001; t=16.65, P<0.001; t=26.78, P<0.001) . The results of clonogenesis assay showed that the number of clones in the miR-219a-5p mimic group was 157.00±15.39, which was significantly lower than that in the mimic NC group (294.00±15.51) , with a statistically significant difference ( t=9.70, P<0.001) . The results of scratch experiment showed that the percentage of scratch area in the miR-219a-5p mimic group was (40.53±2.92) % after 24 h culture, which was significantly higher than that in the mimic NC group [ (21.71±3.11) %], with a statistically significant difference ( t=7.26, P=0.002) . The results of Transwell chamber assay showed that the number of cells penetrating the membrane in the miR-219a-5p mimic group was 128.67±18.67, which was significantly lower than that in the mimic NC group (317.67±14.33) , with a statistically significant difference ( t=15.65, P<0.001) . The results of double luciferase reporter gene assay showed that in MUT-HMGA2 cells, transfection with miR-219a-5p mimic (4.30±0.26) had no significant effect on luciferase activity compared with the mimic NC group (4.40±0.28) , with a statistically significant difference ( t=0.85, P=0.690) . In WT-HMGA2 cells, compared with the mimic NC group (4.50±0.25) , the lucifase activity of the miR-219a-5p mimic group (2.88±0.16) was significantly decreased, with a statistically significant difference ( t=19.15, P<0.001) . After miR-219a-5p was overexpressed, HMGA2 mRNA and protein expressions in osteosarcoma U2OS cells (0.77±0.01; 0.37±0.01) were downregulated compared with the mimic NC group (1.00±0.02; 1.00±0.01) , with statistically significant differences ( t=16.38, P<0.001; t=42.02, P<0.001) . Conclusion:In osteosarcoma cells, miR-219a-5p can inhibit the proliferation, migration and invasion of osteosarcoma cells by down regulating the expression of HMGA2.
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Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.
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Objective:To explore the anti-hepatoma effect of compound <italic>Phylanthus urinaria</italic> Ⅱ （ CPU Ⅱ） by inhibiting the expression of the long non-coding RNA （lncRNA） colon cancer associated transcript-1 （CCAT1） and restoring the expression of microRNA let-7a. Method:Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the expression of lncRNA CCAT1 in normal liver cells （LO2 cells） and hepatocellular carcinoma HepG2 cells， and the differences in expression between these two types of cells were compared. The methylthiazolyl tetrazolium（MTT） assay was used to detect the proliferation of HepG2 cells after treatment with different concentrations of CPU Ⅱ and 5-fluorouracil（5-FU） for 24， 48 and 72 h. Hepatocellular carcinoma HepG2 cells were cultured <italic>in vitro </italic>and set into three gropes： cell control group， CPU Ⅱ low-dose group （0.8 g·L<sup>-1</sup>） and high-dose group （1.6 g·L<sup>-1</sup>）. Real-time PCR was used to detect the mRNA expression of lncRNA CCAT1， microRNA let-7a and its target genes high mobility group protein A2（HMGA2）， and N-RAS in each grope. Western blot was used to detect the protein expression of HMGA2， and Cyclin D<sub>1</sub> in each grope. Result:As compared with LO2 cells， expression of lncRNA CCAT1 in HepG2 cells was significantly up-regulated （<italic>P</italic><0.05）. Results of MTT assay showed that the 50% inhibiting concentration（IC<sub>50</sub>）<sub> </sub>of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649， 0.044 648 g·L<sup>-1 </sup>respectively. As compared with the control group， CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L<sup>-1</sup>） significantly inhibited the proliferation of HepG2 cells （<italic>P</italic><0.05）， and the effect was most remarkable in CPU Ⅱ high-dose group （<italic>P</italic><0.05）. The results of Real-time PCR showed that as compared with control group， the expression of lncRNA CCAT1 mRNA was significantly inhibited in CPU Ⅱ high-and low-dose groups （<italic>P</italic><0.05）， and the expression of microRNA let-7a mRNA was obviously up-regulated in high-dose group （<italic>P</italic><0.05）， but the expression of HMGA2 mRNA in CPU Ⅱ high-and low-dose groups as well as the expression of N-RAS mRNA in CPU Ⅱ low-dose group were down-regulated （<italic>P</italic><0.05）. Western blot results showed that as compared with the cell control group， the protein expression of HMGA2 and Cyclin D<sub>1</sub> in CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L<sup>-1</sup>） was significantly down-regulated （<italic>P</italic><0.05）. Conclusion:CPU Ⅱ can inhibit the expression of lncRNA CCAT1， recover the expression of microRNA let-7a， and suppress the mRNA and protein expression of related downstream target genes in hepatoma cells line HepG2， thereby inhibiting the proliferation of hepatocellular carcinoma cells and exerting anti-hepatocellular carcinoma effect.
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Objective:To investigate the effects of miRNA-26a (miR-26a) on the target gene HMGA2 on the proliferation and migration of hepatoma cells and the underlying mechanism. Methods:Liver cancer tissue samples ( n = 30) and adjacent normal tissue samples ( n = 30) pathologically confirmed by Wenzhou Hospital of Traditional Chinese Medicine between September 2018 and September 2019 were collected. MiR-26a mimics, control mimics (miR-Control), high-mobility group A2 protein (HMGA2) siRNA or negative control siRNA (Control) were transfected into human hepatoma cell lines HepG2 or Huh-7 cells. The expression of miR-26a in hepatocellular carcinoma tissue was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). MTT assay and scratch test were performed to determine the ability of cell proliferation and migration. RT-qPCR and western blotting were performed to detect miR-26a and HMGA2 mRNA expression. The relationship between miR-26a and HMGA2 mRNA was analyzed using Bioinformatics and luciferase reporter gene assay. Results:RT-qPCR results showed that the expression level of miR-26a in hepatocellular carcinoma tissue was 0.11 ± 0.02, which was significantly lower than that in normal tissues (0.25 ± 0.03, t = 21.268, P < 0.05). The expression level of miR-26a in stage III + IV was 0.05 ± 0.01, which was significantly lower than that in stage I + II (0.09 ± 0.01, t = 15.491, P < 0.05). Cell experiment showed that in the miR-26a group, the proliferation ability of Huh-7 cells was (3.10 ± 0.30) and (4.10 ± 0.40), and the proliferation ability of HepG2 cells was (3.08 ± 0.31) and (4.11 ± 0.40), which was significantly lower than that in the control group [(3.90 ± 0.40), (5.50 ± 0.60), (3.92 ± 0.41), (5.49 ± 0.58), t = 8.764, 10.634, 11.148, 10.728, all P < 0.05]. In the miR-26a group, the migration ability was (0.50 ± 0.06), (0.65 ± 0.07), which was significantly lower than that in the control group [(1.00 ± 0.10), (0.96 ± 0.10), t = 23.483, 13.910, both P < 0.05]. Bioinformatics and in vitro experiments showed that HMGA2 was a direct target of miR-26a. Restoring the expression of HMGA2 in miR-26a mimics-transfected cells, compared with that in the miR-26a group [(0.24 ± 0.02), (0.31 ± 0.03);(0.45 ± 0.05)], could significantly reverse the inhibitory effect of miR-26a on tumor cell proliferation and migration [(0.31 ± 0.03), (0.40 ± 0.04);(0.93 ± 0.08), t = 10.634, 9.859, 27.868, all P < 0.05). Conclusion:miR-26a inhibits the proliferation and migration of hepatoma cells by directly targeting HMGA2. The abnormal decrease of miR-26a and the increase of HMGA2 may be the important factors that participate in the occurrence and development of liver cancer.
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BACKGROUND: Cyclic RNA plasmacytoma variant translocation 1 (circPVT1) is involved in the senescence of fibroblasts, but the relationship of circPVT1 with nucleus pulposus senescence and its mechanism are still unclear. OBJECTIVE: To investigate the expression of circPVT1 in nucleus pulposus cell senescence and to explore its possible mechanism. METHODS: Human nucleus pulposus cells were cultured in vitro, and the senescence of nucleus pulposus cells was induced by ionizing radiation (5 Gy, 6 days). The expression of circPVT1 and let-7 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). CircPVT1 siRNA and anti-let-7 were transfected into normal nucleus pulposus cells, which were divided into control group, si-NC+anti-NC group, si-circPVT1+anti-NC group, si-NC+anti-let-7 group, and si-circPVT1+anti-let-7 group. The expressions of circPVT1 and let-7 mRNA were detected by qRT-PCR. Cell counting kit-8 assay was used to detect the inhibition of cell proliferation. Plate cell clone formation assay was used to detect colony formation. Cell senescence was detected by SA-β-gal staining. The expressions of p21, p27, let-7 target high mobility group protein A2 (HMGA2) and KRAS were detected by western blot assay. Double luciferase activity assay was used to verify the relationship between let-7 and target regulation of HMGA2 and KRAS. RESULTS AND CONCLUSION: (1) Compared with normal nucleus pulposus cells, the expression of circPVT1 was decreased, while let-7 expression and the positive rate of SA-β-gal staining were increased in the irradiated cells (P < 0.05). (2) Compared with the control group and si-NC+anti-NC group, the si-circPVT1+anti-NC group appeared to have decreased expression of circPVT1 mRNA, HMGA2 and KRAS proteins and number of clones formed as well as increased let-7 mRNA expression, p21, p27 protein expression, cell inhibition rate and positive rate of SA-β-gal staining (P < 0.05). However, opposite changes were found in the si-NC+anti-let-7 group in relative to the control group (P < 0.05). (3) The expression of circPVT1 mRNA, clone formation, and expressions of HMGA2 and KRAS proteins in the si-circPVT1+anti-let-7 group were higher than those in the si-circPVT1+anti-NC group, and lower than those in the si-NC+anti-let-7 group. Let-7 mRNA expression, cell inhibition rate, positive rate of SA-β-gal staining, and expressions of p21 and p27 proteins in the si-circPVT1+anti-let-7 group were lower than those in the si-circPVT1+anti-NC group, and higher than those in the si-NC+anti-let-7 group (P < 0.05). Double luciferase activity assay showed that HMGA2 and KRAS were the targets of let-7. These findings indicate that inhibition of circPVT1 can inhibit the aging of nucleus pulposus cells. The mechanism may be through binding let-7 to inhibit the targeting of HMGA2 and KRAS proteins.
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@#[Abstract] Objective: To investigate the effect of apatinib (APA) combined with cisplatin (DDP) on the proliferation, invasion and migration capacity of gastric carcinoma (GC) cells and its molecular mechanism. Methods: Cancer and para-cancerous tissue samples resected from 50 GC patients, who were surgically treated in Wuwei People's Hospital from January 2016 to June 2019, were collected for this study; in addition, GC cell lines MGC803 and SGC7901 were also collected. qPCR was used to detect the HMGA2 expression in tissues and mRNA expressions of molecules related to cell proliferation, migration and invasion in GC cell lines. MGC803 and SGC7901 cells were transfected with pcHMGA2 by liposome transfection technology. After treatment with DDP and APA at different concentrations, the cells were divided into NC, pcHMGA2, pcHMGA2+DDP and pcHMGA2+DDP+APA groups. Protein expression of HMGA2 in GC cells was detected by Western blotting, and proliferation, migration and invasion of the cells were detected by MTT and Transwell assay, respectively. Results: The mRNA expression of HMGA2 in GC tissues was higher than that in para-cancerous tissues (P<0.05), and the survival rate of GC patients in the high expression group was significantly reduced (P<0.01). DDP significantly inhibited the proliferation, invasion and migration of MGC803 and SGC7901 cells (all P<0.01); the proliferation, invasion and migration of MGC803 and SGC7901 cells in DDP+APA group significantly decreased (all P<0.01) as compared with DDP group; APA significantly enhanced the inhibitory effect of DDP on HMGA2 expression in GC cells (P<0.01); APA enhanced the anticancer activity of DDP against GC by down-regulating HMGA2 expression. Conclusion: APA promotes the anticancer activity of DDP against GC, and its molecular mechanism is the promotion of the inhibitory effect of DDP on HMGA2 expression.
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@# Objective: To investigate the mechanism of lncRNA HCG18/miR-17-5p/HMGA2 axis regulating the proliferation and metastasis of non-small cell lung cancer (NSCLC) cells. Methods: Sixty-two pairs of NSCLC tissues and corresponding para-cancerous tissues collected at Central Hospital of Chengde City from June 2017 to June 2018 were used for this study; in addition, NSCLC cell lines (A549, NCI-H1299, H1650, NCI-H460) and human lung epithelial BEAS-B cells were also collected. mRNA expression levels of HCG18, miR-17-5p and high-mobility group AT-hook 2 (HMGA2) in NSCLC tissues and cell lines were measured by quantitative Real-time polymerase chain reaction (qPCR). Si-HCG18, miR-17-5p, miR-17-5p+HCG18 or pcDNA3.1-HMGA2 were transfected into A549 cells and NCI-H460 cells; CCK-8 assay was used to detect the proliferation of transfected cells, Transwell assay was used to detect the migration and invasion ability of cells, and Wb was used to analyze the expressions of HMGA2 and EMT associated proteins (E-cadherin, N-cadherin and vimentin). The target relationships between HCG18 and miR-17-5p, or between miR-17-5p and HMGA2 were confirmed by dual luciferase reporter gene assay. Mice A549 cell xenograft model with HCG18 knockdown was constructed, and the growth of transplanted tumor was observed. Results: lncRNA HCG18 was highly expressed in NSCLC tissues and cells (all P<0.01); HCG18 level was significantly increased in patients at late stage or with lymphnode metastasis; and high HCG18 level was correlated with poor prognosis and low survival rates of NSCLC patients (all P<0.01). Knockdown of HCG18 significantly inhibited NSCLC cell proliferation, migration and invasion (all P<0.01), up-regulated E-cadherin expression but suppressed N-cadherin and vimentin expression (all P<0.01), and the volume of xenograft was obviously decreased (P<0.05). Dual luciferase reporter gene assay confirmed the relationship between HCG18 and miR-17-5p as well as miR-17-5p and HMGA2. miR-17-5p transfection significantly inhibited NSCLC cell proliferation, migration and invasion (all P<0.01), and up-regulated E-cadherin expression, reversely suppressed N-cadherin and vimentin expression (all P<0.01); however, miR-17-5p + HCG18 transfection reversed the effect of miR-17-5p on NSCLCcells.Conclusion:HCG18promotes the proliferationandmigrationofNSCLCcellsthrough regulating miR-17-5p/HMGA2 axis.
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@# Objective: To observe the effects of miR-204 on the proliferation and invasion of retinoblastoma (RB) cells and to explore the potential regulatory mechanism. Methods: The expression level of miR-204 in RB cell lines (Y79, SO-RB50, and HXO-Rb44) as well as in normal human retinal pigment epithelial cell line hTERT RPE-1 was detected using qRT-PCR. The Y79 cells were divided into two groups (negative control group and miR-204 group) by respectively transfecting Y79 cells with NC-mimics and miR-204 mimics using liposome transfection method. The effects of miR-204 on Y79 cell proliferation was detected with CCK-8 assay; while the effect of miR-204 on migration and invasion of Y79cellsweredeterminedbycellscratchassayandTranswellassay,respectively.Besides, thepotentialtargetgeneofmiR-204waspredictedbybioinformatics;and the influence of miR-204 on the expression of high mobility group AT-hook 2 gene (HMGA2) at both mRNA and protein levels was detected using qRT-PCR and Western blotting, respectively. Results: miR-204 expression in RB cell lines Y79, SO-RB50 and HXO-Rb44 was remarkably lower than that in normal human retinal pigment epithelial cell line hTERT RPE-1 (P<0.01). miR-204 expression in Y79 cells was markedly up-regulated after transfection with miR-204 mimics (P<0.01) along with significantly reduced cell proliferation, migration and invasion capacities (all P<0.01), and mRNA and protein expressions of HMGA2 were also outstandingly reduced (P<0.01). Conclusion: miR-204 is lowly expressed in RB cell lines; in addition, miR-204 over-expression can suppress RB cell proliferation, migration and invasion, the mechanism of which might be related to down-regulation of the expression of HMGA2.
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Objective To investigate the role of high mobility group protein2(HMGA2)and E-Cadherin in the development of prostate cancer(PCa). Methods A total of 60 paraffln-embedded specimens of PCa were collected from patients who underwent operation in Dalian Central Hospital from 2008 to 2016.The expression lev-els of HMGA2 and E-Cadherin in the specimens were examined by immunohistochemical staining.The correlations between the expression of HMGA2 and E-Cadherin and clinical features such as age,Gleason score,invasion,me-tastasis and TNM stage were studied. Results The expression levels of HMGA2 in PCas were significantly higher than that in controls(χ2=51.818,P<0.01),but the positive expression levels of E-Cadherin in PCas were lower than that in controls(χ2=53.494,P<0.01).The positive expression of HMGA2 and the loss of E-Cadherin were confirmed in PCa accompanied by metastasis,seminal invasion,Gleason score>7 and stageⅢ~Ⅳ(P<0.05). Overexpression of HMGA2 was associated with down-regulation of E-cadherin(Spearman′s r=-0.569,P<0.01). Conclusions Up-regulation of HMGA2 and down-regulation of E-Cadherin are cooperatively correlated with the invasion and metastasis in PCa.
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BACKGROUND: Accumulating studies have demonstrated that high-mobility group A2 (HMGA2), an oncofetal protein, plays a role in tumor development and progression. However, the molecular role of HMGA2 in ovarian carcinoma is yet to be established. MicroRNAs (miRNAs), a group of small noncoding RNAs, negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. The aim of this study was to investigate the potential involvement of a specific miRNA, miR-219-5p, in HMGA2-induced ovarian cancer. METHODS: The ovarian cancer cell line, SKOV3, was employed, and miR-219-5p and HMGA2 overexpression vectors constructed. The CCK-8 kit was used to determine cell proliferation and the Transwell® assay used to measure cell invasion and migration. RT-PCR and western blot analyses were applied to analyze the expression of miR-219-5p and HMGA2, and the luciferase reporter assay used to examine the interactions between miR-219-5p and HMGA2. Nude mice were employed to characterize in vivo tumor growth regulation. RESULTS: Expression of miR-219-5p led to suppression of proliferation, invasion and migration of the ovarian cancer cell line, SKOV3, by targeting HMGA2. The inhibitory effects of miR-219-5p were reversed upon overexpression of HMGA2. Data from the luciferase reporter assay showed that miR-219-5p downregulates HMGA2 via direct integration with its 3'-UTR. Consistent with in vitro findings, expression of miR-219-5p led to significant inhibition of tumor growth in vivo. CONCLUSION: Our results collectively suggest that miR-219-5p inhibits tumor growth and metastasis by targeting HMGA2.
Subject(s)
Humans , Animals , Female , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , HMGA2 Protein/metabolism , MicroRNAs/physiology , Ovarian Neoplasms/genetics , Cell Movement/genetics , HMGA2 Protein/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Neoplasm Invasiveness , Neoplasm MetastasisABSTRACT
Background:More and more evidences suggest that microRNA plays an important role in the development of gastric cancer (GC).Expression of miR-129 in GC tissues is found abnormal, however, the mechanism of miR-129 in cell proliferation and invasion is still undefined.Aims:To investigate the expression of miR-129 in GC tissue and GC cell lines and the mechanism of miR-129 in proliferation and invasion of SGC-7901 cells.Methods:Eighty-two GC tissues and corresponding paracancerous tissues were collected, human gastric epithelial cell line and different GC cell lines were cultured, qPCR was conducted to assess miR-129 expression.SGC-7901 cells were transfected with miR-129 mimic or miR-NC, and then were transfected with overexpressed HMGA2 plasmid.Colony formation assay was used to detect cell proliferation ability, and Transwell chamber was used to assess cell invasion ability.Pearson correlation analysis was used to analyze the correlation between miR-129 and HMGA2 mRNA expression.Luciferase assay was performed to determine the activity of luciferase.mRNA and protein expressions of miR-129, HMGA2 were determined by qPCR and Western blotting, respectively.Results:Compared with paracancerous tissues, expression of miR-129 was significantly decreased in GC tissues (P<0.05);when compared with human gastric epithelial cells, expression of miR-129 was significantly decreased in GC cell lines (P<0.05).Compared with miR-NC group, proliferation and invasion abilities of SGC-7901 cells were inhibited in miR-129 mimic group (P<0.05).HMGA2 mRNA expression in GC tissues was significantly upregulated (P<0.05), and was negatively correlated with miR-129 expression (r=-0.543 9, P<0.01).Luciferase activity in wild-type miR-129 mimic group was significantly lower than that in miR-NC group (P<0.05);mRNA and protein expressions of HMGA2 were decreased after transfection with miR-129 mimic (P<0.05).Compared with miR-129+vector group, proliferation and invasion of SGC-7901 cells were significantly increased in miR-129 mimic+HMGA2 group (P<0.05).Conclusions:The expression of miR-129 is decreased in GC tissue and cells;miR-129 inhibits SGC-7901 cells proliferation and invasion by negatively regulating HMGA2.
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Objective To study the expression and regulation of polycystic ovary syndrome ( PCOS) susceptibility gene Hmga2 in endometrial receptivity and decidualization. Methods Experiments including early pregnancy, delayed implantation and activation, artificial decidualization and ovarian steroid hormone treatment were performed, and Q-PCR and Western blot analyses were applied to explain the role of Hmga2 in endometrial receptivity. Results The Hmga2 ex-pression increased gradually with pregnancy. Its expression at the implantation site was significantly higher than that at the inter-implantation site,in the embryo activation group than in delayed implantation group, and in the artificial decidualiza-tion than the non-decidualization. The expression of Hmga2 was positively correlated with the expression of estrogen and progesterone, and was positively regulated by estrogen and progesterone in vivo. Conclusions Our findings indicate that the expression of Hmga2 is closely related to the embryo implantation process in early pregnancy in mice, is involved in the process of endometrial decidualization, and is influenced by the activated blastocyst and steroid hormones.
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Objective To study the expression and regulation of polycystic ovary syndrome ( PCOS) susceptibility gene Hmga2 in endometrial receptivity and decidualization. Methods Experiments including early pregnancy, delayed implantation and activation, artificial decidualization and ovarian steroid hormone treatment were performed, and Q-PCR and Western blot analyses were applied to explain the role of Hmga2 in endometrial receptivity. Results The Hmga2 ex-pression increased gradually with pregnancy. Its expression at the implantation site was significantly higher than that at the inter-implantation site,in the embryo activation group than in delayed implantation group, and in the artificial decidualiza-tion than the non-decidualization. The expression of Hmga2 was positively correlated with the expression of estrogen and progesterone, and was positively regulated by estrogen and progesterone in vivo. Conclusions Our findings indicate that the expression of Hmga2 is closely related to the embryo implantation process in early pregnancy in mice, is involved in the process of endometrial decidualization, and is influenced by the activated blastocyst and steroid hormones.
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The high mobility group protein A2 (HMGA2) has been demonstrated as an architectural transcription factor that is associated with pathogenesis of many malignant cancers; however, its role in prostate cancer cells remains largely unknown. To explore whether HMGA2 participates in the development and progression of prostate cancer, small interfering RNA (siRNA) targeted on human HMGA2 was transfected to suppress the HMGA2 expression in prostate cancer PC3 and DU145 cells, and then the cellular biology changes after decreased the expression of HMGA2 was examined. Our results showed that knockdown of HMGA2 markedly inhibited cell proliferation; this reduced cell proliferation was due to the promotion of cell apoptosis as the Bcl-xl was decreased, whereas Bax was up-regulated. In addition, we found that HMGA2 knockdown resulted in reduction of cell migration and invasion, as well as repressed the expression of matrix metalloproteinases (MMPs) and affected the occurrence of epithelial-mesenchymal transition (EMT) in both cell types. We further found that decreased HMGA2 expression inhibited the transforming growth factor-β (TGF-β)/Smad signalling pathway in cancer cells. In conclusion, our data indicated that HMGA2 was associated with apoptosis, migration and invasion of prostate cancer, which might be a promising therapeutic target for prostate cancer.
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The high mobility group A2 (HMGA2),one of non-histone chromatin protein,can specificly bind to AT-rich DNA sequences by its AT-hook and work as oncofetal gene and architectural transcription factor.HMGA2 expresses in almost all kinds of malignant neoplasms,which is closely related to the formation,development and poor prognosis of the neoplasms.HMGA2 plays a very important role in every biological process including cell proliferation,cell cycle,stem cell self-renewal,epithelial-mesenchymal transition and DNA damage repair.It is of great significance to study the effect and mechanism of HMGA2 in neoplasms.
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Objective To discuss the potential relationship between endometrial serous carcinoma (ESC) and tubal epithelial lesions by pathologic examination of fallopian tubes with ESC. Methods A total of 30 cases of typical ESC were reexamined and chosen by the pathologist. In each case, bilateral fallopian tubes were submitted to examination of pathologic morphology and immunostaining for p53, annexin Ⅳ(ANX-Ⅳ), human epidermal growth factor receptor 2(HER2)/neu, and high-mobility group protein A2 (HMGA2). Results Fallopian tubal epithelial lesions were found in 15 cases, including 9 cases tubal serous carcinoma, 2 cases serous tubal intraepithelial carcinoma (STIC) and 2 cases epithelial hyperplasia. Both sides of tubal serous carcinoma and STIC were found in 1 case. The results showed the positive expression for p53 in 26(87%)out of 30 endometrial malignant specimens tissues and 9(30%)tubal tissues samples (P>0.05). Twenty-five(83%)endometrial malignant specimens tissues and 6(20%)tubal tissues samples showed the positive expression of ANX-Ⅳ. Twenty-one(70%)endometrial malignant tissues and 7(23%) tubal tissues showed the positive expression of HER2/neu. Twenty-five(83%) endometrial malignant tissues and 6(20%)tubal tissues showed the positive expression of HMGA2. While, there were significant differences among the expression of three proteins between endometrium and the fallopian tube site (all P<0.05). Conclusions STIC may be associated with the occurrence of ESC. The expression of p53 was positively correlated between the fallopian tube and the endometrium. ANX-Ⅳ,HER2/neu and HMGA2 were extensively expressed in ESC.
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Objective To investigate the role of HMGA2 expression and epithelial to mesenchymal cell transformation in the regulation of TGF -β1 in human gastric cancer cells .Methods Human gastric cancer cell SGC-7901 was treated by TGF-β1 at different time points(0,3,6,24,48,72 h).We observed the changes in cell morphology of SGC-7901 through the microscope;HMGA2 assay,E-cadherin,Vimentin protein expression wereperformedbyWesternblot;cellinvasioncapabilitywasanalyzedbytranswellchamberassay.Results (1) With the prolonged duration of action of TGF -β1,SGC-7901 changes in cell morphology ,cells gradually be-came longer and larger.(2)In the role of TGF-β1,gastric cancer cells SGC -7901 are varying degrees of HM-GA2 expression and E -cadherin、Vimentin.HMGA2 protein begun to express from 0h to 6hrs.and HMGA2 pro-tein became the strongest at 6hrs and decreased slightly from 24hrs.E-cad protein expression at 0h begun at 3hrs highest expression ,and the reduction started from 3hrs.Vimentin protein was the highest at 48hrs then gradu-ally decreased.Conclusion In the TGF-β1 induced the process of epithelial to mesenchymal cell transforma-tion in gastric cancer cells SGC -7901 ,HMGA2 protein play an important role .HMGA2 can be used as snail pro-moter,combined with Smads ,promotes the process of epithelial -mesenchymal transformation .HMGA2 may also be regulated by Wnt /β-Catenin signaling pathway on growth and apoptosis of tumor cells .
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Purpose To detect high mobility group protein A2 (HMGA2) expression in breast cancer, and to analyze its relationship with clinicopathological features and the levels of HMGA2 in different molecular subtypes of breast carcinomas. Methods An immu-nohistochemical study was undertaken for measuring the levels of HMGA2 in 58 breast carcinomas. Results ( 1 ) The expression of HMGA2 was 0, 62. 5%, 60. 0%, 100. 0% and 80. 0% in Lum A, Lum B, HER-2-OE, basal-like breast carcinoma (BLBC) and un-classification phenotype respectively ( triple negative breast cancer, 92. 3%) . High expression of HMGA2 was associated with the tri-ple-negative breast cancer (TNBC) subtypes (P<0. 01). (2) An association was identified between high expression of HMGA2, and high tumor grade, lymph node metastasis (P<0. 05), positive Ki-67, CK5/6 and EGFR (P<0. 05), negative ER and PR (P<0. 01), but no association was observed for tumor size and patients’age. Conclusion An association is identified between high ex-pression, and high tumor grade, lymph node metastasis, positive Ki-67, EGFR and CK5/6, negative ER and PR, that means a high expression of HMGA2 is associated with an adverse outcome in breast cancer. High expression of HMGA2 are associated with the TNBC subtypes. Thus recognizing HMGA2 as a rational target in TNBC. The results of the study have implications for therapeutic target iden-tification and the design of future clinical trials for TNBC.
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BACKGROUND: Follicular thyroid carcinoma (FTC) is the second most common thyroid malignancy and its differential diagnosis includes follicular adenoma (FA) and adenomatous goiter (AG). Several ancillary markers have been suggested to aid in the diagnosis of FTC, but the successful use of these methods still needs to be validated. METHODS: In the present study, we verified the immunoexpression of HMGA2, CEACAM6, survivin, and SFN/14-3-3 delta in lesions including 41 AGs, 72 FAs, and 79 FTCs. We evaluated their diagnostic usefulness, combined with galectin 3, Hector Battifora mesothelial 1 (HBME1), cytokeratin 19, and cyclin D1, in diagnosing FTC. RESULTS: The expressions of HBME1 (65.8%) and HMGA2 (55.7%) were significantly higher in FTCs than in FAs and AGs (p<.001 and p=.005, respectively). HBME1 was the only marker that was more frequently expressed in FTCs than in FAs (p=.021) and it was more frequently expressed in follicular neoplasms than in AGs (p<.001). Among the novel markers, the combination of HMGA2 and HBME1 showed the highest sensitivity (72.2%) and specificity (76.1%) for diagnosing FTC. CEACAM6, survivin, and SFN/14-3-3 delta were barely expressed in most cases. CONCLUSIONS: Our present results show that only HMGA2 can be beneficial in differentiating FTC using the novel markers.