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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 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: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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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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The high mobility group A2 (HMGA2), one ofnon-histone chromatin proteins, may alter chromatin structure and thereby regulate the transcription of several genes by either enhancing or suppressing transcription factors, and leading to malignant neoplasm formation. This paper focuses on the role of the HMGA proteins in human neoplastic diseases, and discusses the mechanisms by which they contribute to carcinogenesis, and diagnosis strategies based on targeting HMGA proteins.
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The high mobility group A2 (HMGA2) is a non-histone chromatin protein. It has no transcriptional activity of itself but could alter chromatin structure to regulate the transcription of other genes. HMGA2 gene is weakly expressed or does not express in normal tissues. The expression of HMGA2 is up-regulated during embryogenesis and malignant neoplasias. In several malignant tumors, HMGA2 is used as a diagnostic molecular marker or an independent prognostic indicator. Furthermore, HMGA2 not only elicits epithelial-mesenchymal transition but also maintains the differentiation potential and self-regeneration capability of stem cells. It plays an important role in initiation, proliferation, and metastasis of malignant neoplasias.