Objective To observe the effect of forkhead box Q1(FOXQ1) short hairpin RNA (shRNA) on sensitivity of oxaliplatin chemotherapy in hepatpcellular carcinoma cell line SMMC-7721. Methods ① Complementary shRNA oligonucleotides targeting the FOXQ1 gene and negative control-shRNA were designed and inserted into lentiviral vector. shRNA lentivirus vectors were transfected into SMMC-7721 cells and the lentivirus vector with the best silencing effect was screened. ② SMMC-7721 cells were divided into interference group (SMMC-7721 cells were transfected with FOXQ1-shRNA-1), negative control group (SMMC-7721 cells were transfected with negative control-shRNA), and blank control group (SMMC-7721 cells did not received any treatment), and the expressions of FOXQ1 mRNA and its protein in SMMC-7721 cells were detected at 72 hours after transfection. ③ SMMC-7721 cells were divided into interference group, negative control group, blank control group, interference+oxaliplatin group, negative control+oxaliplatin group, and blank control+oxaliplatin group, apoptosis rates and viability of SMMC-7721 cells were detected at 48 hours after transfection. Results ① The expressions of FOXQ1 mRNA and its protein in SMMC-7721 cells of the FOXQ1-shRNA-1 group were both lower than those of the FOXQ1-shRNA-2 group and FOXQ1-shRNA-3 group (P<0.05), so the FOXQ1-shRNA-1 was the best lentiviral vector. ② Compared with the negative control group and the blank control group, the expressions of FOXQ1 mRNA and its protein of the interference group were both lower (P<0.05), but there was no significant difference between the negative control group and the blank control group (P>0.05). ③ Whether added oxaliplatin or not, compared with the negative control group and the blank control group, the apoptosis rates of the interference group were higher (P<0.05), but the viability of the interference group was lower (P<0.05), and there was no significant difference between the negative control group and the blank control group under the same condition (P>0.05). The apoptosis rates of groups (including the interference group, the negative control group, and the blank control group) which added oxaliplatin were higher than those groups didn’t add oxaliplatin (P<0.05), but viability of groups (including the interference group, the negative control group, and the blank control group) which added oxaliplatin was lower than those groups didn’t add oxaliplatin (P<0.05). Conclusion Down-regulation of expression of FOXQ1 by shRNA in hepatocellular carcinoma cell line SMMC-7721 can effectively induce apoptosis and increase sensitivity of SMMC-7721 cells to oxaliplatin.
Objective To investigate whether miRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colon cancer cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway. Methods miR-34a expression levels were detected in colon cancer tissues and colon cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search, functional luciferase assay, and Western blotting method were used to demonstrate the downstream target of miR-34a in colon cancer cells. Cell viability was measured with cell counting kit-8. Apoptosis and macroautophagy of colon cancer cells were analyzed by flow cytometry and transmission electron microscopy, and expressions of Beclin1 and LC3Ⅱ protein were detected by Western blotting method. Results Expression of miR-34a was significantly reduced while expressions of TGF-β and Smad4 mRNA were increased in colon cancer patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a expression levels and increased TGF-β and Smad4 expression levels in both parental cells and the OXA-resistant colon cancer cells. Activation of macroautophagy contributed to OXA resistance in colon cancer cells. Expression levels of Smad4 and miR-34a in colon cancer patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in colon cancer cells. Conclusion miR-34a mediates OXA resistance of colon cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.
ObjectiveTo observe the effects of different concentrations of trastuzumab alone or in combination with oxaliplatin on proliferation, apoptosis, and cell cycle of SW-620 human colon cancer cell, and to explore its mechanism. MethodsSW-620 human colon cancer cells were cultured in vitro. ① Cell proliferation experiment: the cells were divided into two large groups: trastuzumab group and trastuzumab combined with oxaliplatin group. There were eight concentration groups in each large group (five holes for each group). The concentration of the trastuzumab group was 0, 0.001, 0.01, 0.1, 1, 10, 100, and 1 000 μg/mL, corresponding to the trastuzumab combined with oxaliplatin group. The concentration of the antibiotic was the same as before, except that oxaliplatin (10 μmol/L) was added. The absorbance (A) value of each group was measured by CCK-8 method. ② Apoptosis experiment: the same proliferation experiment was performed in the group, except that the concentrations of trastuzumab only included 0, 0.1, 1 and 10 μg/mL. Flow cytometry was used to detect the proportion of apoptotic cells and cell cycle distribution in each group. ③ Determination of human epidermalgrowth factor receptor-2 (her-2). The SW-620 cells were divided into two large groups, the concentration of trastuzumab group concluded 0, 100, and 1 000 μg/mL, as well as the concentration of trastuzumab in the trastuzumab combined with oxaliplatin group concluded 0, 1, and 10 μg/mL. Expressions of her-2 protein in SW-620 cells were detected by Western blot method. Results① Cell proliferation assay: the A values at100 μg/mL and 1 000 μg/mL were significantly lower than that at 0 μg/mL (P<0.05). At the same concentration, the A value of the trastuzumab combined with oxaliplatin group was lower than that of the trastuzumab group (P<0.05 ), and the A value gradually decreased with the increase of the concentration of trastuzumab. ② Apoptosis experiment: the proportion of apoptotic cells in the trastuzumab combined with oxaliplatin group was higher than that in the trastuzumab group (P<0.05) at the same concentration of trastuzumab. Flow cytometry: after treatment with different concentrations of trastuzumab combined with oxaliplatin, cells in G1 phase showed a downward trend, and cells in S phase showed an upward trend in a dose-dependent manner. At 1 and 10 μg/mL concentration of trastuzumab, the trastuzumab combined with oxaliplatin group significantly reduced the proportion of cells in the G1 phase of SW-620 cell cycle compared with the trastuzumab group, but S phase ratio was higher (P<0.05). The proportion of G2 phase cells was significantly higher in the trastuzumab combined with oxaliplatin group than the trastuzumab group at 0.1, 1 and 10 μg/mL concentrations of trastuzumab (P<0.01). ③ Expressions of her-2 protein: the expression level of her-2 protein gradually decreased at 1, 100, and 1 000 μg/mL trastuzumab group (P<0.05). The expression levels of her-2 protein in 0, 1 and 10 μg/mL trastuzumab combined with oxaliplatin group also gradually decreased (P<0.01). ConclusionsHigh concentration of trastuzumab can inhibit the proliferation of SW-620 human colon cancer cells and induce apoptosis. Trastuzumab combined with oxaliplatin has synergistic effect on inhibiting cell proliferation and promoting apoptosis.