| Objective: Breast cancer is one of the most common malignant tumors in women.In 2018,breast cancer was the most common type of cancer among 8.6 million new female cancer patients(24.2%,one fourth of all new female cancer cases worldwide were breast cancer),and the cancer was the most common in 154 countries and regions.Breast cancer accounts for 15% of the 4.2 million female cancer deaths,which is the main cause of cancer deaths.Triple-negative breast cancer(TNBC)is the most aggressive subtype of breast cancer,with the highest incidence of metastatic diseases and the lowest overall survival rate.Therefore,there is an urgent need to develop targeted therapy for tri-negative breast cancer.Many studies have shown that MTDH(Metadherin)expression in breast cancer tissues is significantly higher than that in normal tissues,and is closely related to adverse prognosis such as chemotherapy resistance and cancer recurrence and metastasis.In this study,we constructed a model of MTDH overexpression and knockdown in breast cancer cells by using lentivirus packaging vectors to study the effects of MTDH on proliferation,apoptosis,cell cycle and chemoresistance of cancer cells,so as to provide experimental theoretical basis for reversing chemoresistance.Methods:1.The MTDH overexpression vector,knockdown vector and empty vector were constructed by lentiviral packaging,and infected with triple-negative breast cancer cells MDA-MB-231(cells named 231-OE,231-KD,231-NC).High expression and knockdown efficiency were detected by q-PCR and Western blot.2.CCK8 method was used to detect the effect of MTDH overexpression or knockdown on the proliferation and the sensitivity to paclitaxel of cells aboye.Flow cytometry was used to detect the apoptosis and cell cycle changes of MTDH overexpression and knockdown in the medicated and untreated groups,and the expression of AKT/GSK3β pathway-related proteins before and after MTDH overexpression and knockdown were detected by Western blot.Happening.3.In each group of breast cancer cell lines,AKT inhibitor MK2206 and paclitaxel were used separately or in combination to observe cell proliferation,apoptosis and cyclical changes.Western blot was used to detect the expression of related proteins in each pathway.Results:1.Construction and validation of MTDH overexpression and knockdown stable cell linesWe successfully constructed stable MTDH overexpression and knockdown cell lines(231-OE,231-KD,231-NC)in breast cancer cell line MDA-MB-231.The relative quantity of MTDH at the level of gene and protein was detected by q-PCR and Western blot.The level of expression of 231-OE was significantly higher than that of 231-NC and 231 groups.The level of expression of 231-KD was significantly higher than that of 231-NC and 231 groups.There were statistical differences between the groups below 231-NC and 231.(P< 0.01).2.Effects of MTDH overexpression/knockdown on cell proliferation,apoptosis and cell cycle in different groupsThe CCK8 method was used to determine the OD450 nm values of the four groups of cells 231-OE,231-KD,231-NC and 231 at 0h,24 h,48h and 72 h,and then the proliferation of each group was detected.The results showed that the proliferation of 231-OE group was significantly higher than that of control group 231-NC and 231(P < 0.01),proliferation of 231-KD group was significantly lower than that of control group 231-NC and 231(P < 0.05).The apoptotic rate of 231-OE in the experimental group was lower than that in the 231-NC group(5.367 ±0.569)and 231(6.607 ±0.666)groups(P < 0.01).The apoptotic rate of 231-KD(13.900+1.350)group was significantly higher than that of the control group(231-NC and 231),with significant statistical difference(P<0.01).The results of cell cycle assay showed that the proportion of S phase and G2/M phase in 231-OE group increased significantly,and the proportion of G0/G1 phase decreased significantly.In 231-KD group,the proportion of G0/G1 phase and G2/M phase increased significantly.,the proportion of S period is reduced.3.The effect of MTDH overexpression and knockdown on Taxol sensitivityIn CCK8 experiment,the inhibition rate of paclitaxel(0.1 ug/)on 231-OE cell lines was significantly lower than that of 231-NC(0.118 ±0.016)and 231(0.192 ±0.341)after 48 hours(P < 0.01),and the inhibition rate of paclitaxel on 231-KD cells(0.404 ±0.040)was significantly higher than that of the two control groups(231-NC and 231)after 48 hours(P < 0.01).The apoptotic rate of 231-OE group was significantly lower than that of 231-NC group(12.933+1.500)and 231-NC group(11.833+1.527)48 hours after paclitaxel treatment,while that of 231-KD group(22.063+1.839)was significantly higher than that of control group(231-NC and 231),with statistical difference(P < 0.01).The cell cycle results showed that after 48 hours of paclitaxel treatment,231-OE compared with the control group(231-NC and 231,G0/G1 phase ratio decreased,S phase and G2/M phase increased;231-KD compared with the control group(231-NC and 231)G0/G1 and S phases decreased,and G2/M phase increased.4.Effects of MTDH overexpression or knockdown on AKT/GSK3β pathwayWestern blot results showed that the overexpression of MTDH increased p-AKT and p-GSK3β,while after MTDH knocked down,p-AKT and p-GSK3β also decreased.It is suggested that the increase of MTDH can activate AKT/GSK3β signaling pathway,and MTDH knockdown can inhibit AKT/GSK3β signaling pathway.In the detection of cell cycle-related proteins,the expression of CyclinD1 and phosphorylated RB increased after MTDH overexpression,while the expression of CyclinD1 and phosphorylated RB decreased after MTDH knockdown.These results suggest that MTDH overexpression can promote the expression of cyclin and accelerate the process of cell cycle,while MTDH knockdown can inhibit the expression of cyclin and delay the process of cell cycle.5.MK2206 inhibits AKT/GSK3β pathway and reverses MTDH overexpression-induced drug resistanceThe results of CCK8 showed that the cell inhibition rate(0.499±0.066)of paclitaxel combined with MK2206 in 231-OE cells was significantly higher than that of the control group(231-OE-P(0.118±0.016)and 231-OE-M(0.202±0.087).),with significant difference(P<0.05);paclitaxel combined with MK2206 in 231-KD cells after 48 h inhibition rate(0.628±0.015)compared with two control groups(231-KD-P(0.404±0.040)and 231-KD-M(0.448±0.028))was significantly increased with significant difference(P<0.01).The results of apoptosis detection showed that the apoptosis rate of paclitaxel combined with MK2206 in 231-OE cells after 48 h was compared with the control group(231-OE-P(6.600±0.400)and 231-OE-M(7.800±).0.283)increased with significant difference(P<0.05);paclitaxel combined with MK2206 in 231-KD cells for 48 h after apoptotic rate(91.233±0.902)compared with two control groups(231-KD-P(22.063±1.839)and 231-KD-M(48.200 ± 1.868)were significantly elevated with significant differences(P < 0.05).The results of cell cycle assay by flow cytometry showed that 231-OE-P+M was compared with the control group(231-OE-P and 231-OE-M)G0/G1 phase and G2/M phase after 48 hours of paclitaxel combined with MK2206.Increased,S phase decreased;231-KD-P+M decreased compared with the control group(231-KD-P and 231-KD-M)G0/G1 phase and S phase,and G2/M phase increased.Conclusions:1.In human breast cancer cell line MDA-MB-231,MTDH overexp-ression and knockdown stable cell lines can be stably constructed.2.Breast cancer cells MTDH overexpressing showed proliferation increased,apoptosis decreased,cell cycle was blocked and resisted to paclitaxel.On the contrary,breast cancer cell lines MTDH knocked down showed proliferation slower,apoptosis increased and G0/G1 phase was prolonged,which were more sensitive to paclitaxel.3.Overexpression of MTDH can activate AKT/GSK3β pathway and induce paclitaxel resistance.When MTDH was knocked down,AKT/GSK3β pathway was inhibited,the sensitivity to paclitaxel was increased,and downstream signals such as CyclinD1 and p-RB were significantly downregulated.4.MK2206 can inhibit the activation of AKT/GSK3β pathway induced by MTDH overexpression and increase the sensitivity to taxol,thus reversing the taxol resistance induced by MTDH overexpression.MTDH knockdown can increase the antitumor synergism between taxol and MK2206. |
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