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Objective To evaluate the clinical value of free glycoprotein non-metastatic melanoma protein B(GPNMB)as a drug resis-tance and prognostic marker for non-small cell lung cancer(NSCLC)patients with epidermal growth factor receptor(EGFR)amplifica-tion accompanied by mutations.Methods Fifty-five cases of NSCLC patients with EGFR amplification associated with mutations who received treatment from March 2018 to September 2019 were included as the observation group.All patients received an EGFR-tyrosine kinase inhibitor(EGFR-TKI)as the first-line treatment;67 blood samples from the physical examination center during the same period were randomly included as healthy control.We compared the expression levels of free GPNMB between the two groups,explored the correlation between GPNMB expression and the clinicopathological information in the observation group;and combined the clinical efficacy to evaluate its value as a drug resistance marker.Through follow-up,the progress free survival(PFS)of patients was statistically analyzed,and through multivariate Cox regression analysis,independent risk factors affecting the survival in the observation group were explored.Results Compared with that in the control group,the expression level of free GPNMB in the observation group was signi-ficantly up-regulated.The expression level of free GPNMB in the observation group is significantly related to the clinical efficacy of EGFR-TKI(P = 0.016).Patients with high GPNMB expression have significantly stronger drug resistance,and patients with high GPNMB expression have significantly shorter PFS duration(P = 0.032).A high free GPNMB expression(HR = 4.029,95%CI:1.942-8.358,P＜0.001)is also an independent risk factor affecting patient survival.Conclusion The expression level of free GPNMB in patients with EGFR amplification accompanied by mutant NSCLC is significantly up-regulated,and its high expression is significantly related to the enhancement of the patient's drug resistance.High GPNMB expression is significantly related to the poor prognosis of patients and is an independent risk factor affecting patient survival.
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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 explore the characteristics and biological functions of epigenetic treatment to DNMT1 expression in NSCLC cell lines. Methods Lung cancer cells were divided into 4 groups , and drugs with different concentrations. The effect of epigenetic treatment on NSCLC cell line was detected by CCK-8 and MTT. The effect of epigenetic treatment on the expression of DNMT1 in NSCLC cell line was detected by Western blot. Results CCK-8 and MTT assay showed that treatment with 5-Aza-CdR and MS-275 inhibited the proliferation of NSCLC cells. Western blot showed that treatment with 5-Aza-CdR and MS-275 inhibited the DNMT1 expression in NSCLC cells. Conclusion The expression of DNMT1 gene in the NSCLC cell lines may be suppressed effectively by epigenetic treatment , and the gene may inhibit the proliferation and growth of NSCLC cell lines.