长链非编码RNA PRMT5-AS1调控电离辐射诱导肝癌细胞铁死亡机制的研究

Mechanism of lncRNA PRMT5-AS1 regulating radiation-induced ferroptosis in hepatocellular carcinoma cells

目的探讨长链非编码RNA PRMT5-AS1对电离辐射诱导的肝癌细胞铁死亡的影响。方法在MHCC-97H细胞中构建PRMT5-AS1过表达模型,在HepG2细胞中构建PRMT5-AS1敲低模型。使用X射线照射,吸收剂量为10 Gy,剂量率为3 Gy/min。采用Western blot和qRTPCR实验检测基因表达水平。采用台盼蓝染色流式细胞术检测PRMT5-AS1表达对受照肝癌细胞脂质过氧化以及铁死亡的影响。采用CCK-8实验检测PRMT5-AS1表达水平对电离辐射照射后肝癌细胞死亡的影响。双荧光素酶报告实验检测let-7c-5p与PRMT5-AS1和SLC7A11之间结合作用。结果MHCC-97H细胞中过表达PRMT5-AS1能够显著降低电离辐射引起的细胞死亡(对照组vs.PRMT5-AS1过表达组:27.57%vs.18.30%,t=14.94,P<0.05)。HepG2细胞中敲低PRMT5-AS1可显著增加电离辐射引起的细胞死亡(对照组vs.PRMT5-AS1敲低组:17.26%vs.28.26%,t=13.63,P<0.05)。过表达PRMT5-AS1能够明显抑制由电离辐射诱导的细胞内脂质活性氧(ROS)水平增加(对照组vs.PRMT5-AS1过表达组:17.01%vs.12.52%,t=12.80,P<0.05),敲低PRMT5-AS1可显著增加电离辐射诱导的脂质ROS水平增加(对照组vs.PRMT5-AS1敲低组:14.54%vs.17.72%,t=5.93,P<0.05)。CCK-8实验结果表明,过表达PRMT5-AS1能够显著抑制Erastin诱导的细胞活性降低(对照组vs.PRMT5-AS1过表达组:87.92%vs.109.06%,t=2.87,P<0.05),敲低PRMT5-AS1则促进Erastin抑制细胞活性(对照组vs.PRMT5-AS1敲低组:82.56%vs.60.58%,t=38.35,P<0.05)。Western blot和荧光定量PCR结果表明,过表达PRMT5-AS1能够明显提高SLC7A11的蛋白和mRNA水平(t=26.24,P<0.05),敲低PRMT5-AS1后SLC7A11的蛋白和mRNA水平均显著降低(t=5.60,P<0.05)。荧光素酶报告基因实验表明PRMT5-AS1与let-7c-5p之间存在相互作用(t=9.74,P<0.05)。PRMT5-AS1可以与let-7c-5p形成ceRNA网络,靶向调节SLC7A11。let-7c-5p能够逆转由过表达PRMT5-AS1引起的SLC7A11表达水平增加、脂质ROS水平和细胞死亡减少(t=3.01、4.11,P<0.05),而敲低SLC7A11能够逆转PRMT5-AS1引起的脂质ROS抑制和细胞死亡减少(t=21.35、7.15,P<0.05)。结论长链非编码RNA PRMT5-AS1通过PRMT5-AS1/let-7c-5p/SLC7A11轴抑制电离辐射诱导肝癌细胞铁死亡的发生。

Objective To evaluate the effect of long non-coding RNA PRMT5-AS1 on ferroptosis of hepatocellular carcinoma cell(HCC)after ionizing irradiation.Methods The PRMT5-AS1 overexpression model was constructed in MHCC-97H cells and the PRMT5-AS1 knockdown model was constructed in HepG2 cells.X-ray irradiation(IR)was performed with an absorbed dose of 10 Gy and a dose rate of 3 Gy/min.Western blot and qRT-PCR were used to detect gene expression.The effect of PRMT5-AS1 expression on lipid peroxidation and ferroptosis of HCC after IR was detected by Trypan blue staining flow cytometry.The effect of PRMT5-AS1 expression on the death of HCC after IR was detected by CCK-8 assay.Dual luciferase assay to detect the binding of let-7c-5p to PRMT5-AS1 and SLC7A11.Results Overexpression of PRMT5-AS1 in MHCC-97H cells could significantly reduce cell death induced by IR(Vector vs.PRMT5-AS1:27.57%vs.18.30%,t=14.94,P<0.05).Knockdown of PRMT5-AS1 in HepG2 cells significantly increased cell death induced by IR(siNC vs.siPRMT5-AS1:17.26%vs.28.26%,t=13.63,P<0.05).Flow cytometry result show that overexpression of PRMT5-AS1 can significantly inhibit the increase of intracellular lipid ROS level induced by IR(Vector vs.PRMT5-AS1:17.01%vs.12.52%,t=12.80,P<0.05),and knockdown of PRMT5-AS1 significantly increases the lipid ROS level induced by IR(siNC vs.siPRMT5-AS1:14.54%vs.17.72%,t=5.93,P<0.05).The result of CCK-8 experiment showed that overexpression of PRMT5-AS1 could significantly inhibit Erastin induced cell activity reduction(Vector vs.PRMT5-AS1:87.92%vs.109.06%,t=2.87,P<0.05),and knockdown of PRMT5-AS1 could promote Erastin′s inhibitory effect on cell activity(siNC vs.siPRMT5-AS1:82.56%vs.60.58%,t=38.35,P<0.05).Western blot and fluorescent quantitative PCR result showed that the protein and mRNA levels of SLC7A11 were significantly increased after overexpression of PRMT5-AS1(t=26.24,P<0.05),and the protein and mRNA levels of SLC7A11 were significantly decreased after knockdown of PRMT5-AS1(t=5.60,P<0.05).The correlation between PRMT5-AS1 and let-7c-5p was confirmed by luciferase report gene experiment(t=9.74,P<0.05).The result of luciferase reporter gene experiment showed that PRMT5-AS1 could form ceRNA network with let-7c-5p to regulate SLC7A11.Let-7c-5p was able to reverse the increase in SLC7A11 expression levels,decrease in Lipid-ROS levels and cell death induced by overexpression of PRMT5-AS1(t=3.01,4.11,P<0.05).And knockdown of SLC7A11 reversed Lipid-ROS inhibition and reduced cell death caused by PRMT5-AS1(t=21.35,7.15,P<0.05).Conclusions LncRNA PRMT5-AS1 inhibits IR-induced ferroptosis in HCC through the PRMT5-AS1/let-7c-5p/SLC7A11 axis.