LAMC3低表达对卵巢癌细胞紫杉醇耐药的影响

Effect of low expression of LAMC3 on paclitaxel resistance in ovarian cancer cells

目的探讨层粘连蛋白γ3亚基(LAMC3)低表达对卵巢癌细胞紫杉醇耐药的影响。方法采用Western blot检测卵巢癌紫杉醇耐药细胞HeyA8-R(H-R)及其亲本细胞HeyA8(H)中LAMC3的表达水平。通过慢病毒转染H-R细胞构建LAMC3低表达的稳定细胞株H-R-C3 shRNA3及其对照细胞株H-R-C3 Scramble。通过CCK-8法检测细胞对紫杉醇的半数抑制浓度(IC_(50)),通过平板克隆形成实验验证细胞克隆形成能力。通过Western blot实验检测多药耐药、铁死亡、细胞周期、凋亡等关键通路蛋白的表达情况。结果与卵巢癌亲本细胞H和对照细胞H-R-C3 Scramble相比,LAMC3在卵巢癌紫杉醇耐药细胞H-R和H-R-C3 shRNA3中的表达显著降低。敲减LAMC3表达可降低卵巢癌紫杉醇耐药细胞对紫杉醇的耐药性及其平板克隆形成能力。Western blot实验结果显示,在梯度浓度紫杉醇药物处理下,与H-R-C3 Scramble细胞相比,H-R-C3 shRNA3细胞的多耐药蛋白多重耐药1/ATP结合盒亚族B1(MDR1/ABCB1)表达水平降低,铁死亡蛋白溶质载体家族7成员11(SLC7A11)表达水平升高,周期蛋白p21 Waf1/Cip1、细胞周期蛋白E2(CCNE2)表达水平升高,周期素依赖性激酶2(CDK2)、周期素依赖性激酶4(CDK4)表达水平降低,凋亡过程中剪切产生的亚基蛋白cleaved半胱氨酸天冬氨酸蛋白酶-9(caspase-9)、cleaved半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、cleaved聚ADP-核糖聚合酶(PARP)表达水平升高,差异有统计学意义(P<0.05)。结论LAMC3低表达可能通过影响多药耐药、细胞周期、细胞凋亡等方式来降低卵巢癌紫杉醇耐药细胞对紫杉醇的耐药性,提示LAMC3有望成为一个应用于卵巢癌耐药治疗的潜在新靶点。

Objective To investigate the effect of low expression ofγ3 subunit of laminins(LAMC3)on paclitaxel(PTX)resistance in ovarian cancer(OC)cells.Methods Western blot was used to detect the expression of LAMC3[JP4]in the PTX-resistant OC cells HeyA8-R(H-R)and its parent cells HeyA8(H).Stable transfected cell line H-R-C3 shRNA3 with low expression of LAMC3 and its control cell line H-R-C3 Scramble was constructed by transfection of lentivirus into H-R cells.CCK-8 assay was used to detect the half maximal inhibitory concentration of cells to PTX(IC_(50)).The cell clone formation ability was measured by plate clone formation assay.Western blot was also used to detect the expression levels of key pathway proteins such as multidrug resistance,ferroptosis,cell cycle and apoptosis.Results LAMC3 was significantly down-regulated in the PTX-resistant OC cells H-R and H-R-C3 shRNA3 compared with its parent cells H and control cells H-R-C3 Scramble.The knockdown of the expression of LAMC3 not only reduced the resistance to PTX but also decreased the clone formation ability of PTX-resistant OC cells.The results of Western blot showed that compared with those in the H-R-C3 Scramble cells,the protein expression levels of multidrug resistance 1/ATP-binding cassette subfamily B member 1(MDR1/ABCB1)decreased,and the solute carrier family 7 member 11(SLC7A11)increased,and the cyclin p21 Waf1/Cip1 and cyclin E2(CCNE2)increased,and the cyclin dependent kinase 2(CDK2)and cyclin[LM]dependent kinase 4(CDK4)decreased in the H-R-C3 shRNA3 cells under the concentration gradient treatment of PTX.Moreover,cleaved caspase-9,cleaved caspase-3,cleaved poly ADP-ribose polymerase(cleaved PARP)increased which belonged to subunit proteins when produced during apoptosis.All the differences were statistically significant(P<0.05).Conclusion Low expression of LAMC3 may reduce the resistance of PTX-resistant OC cells to PTX by affecting multidrug resistance,cell cycle and apoptosis,suggesting that LAMC3 may become a new potential target for drug resistance treatment in OC.