长链非编码RNA B3GALT5-AS1靶向miR-361-3p调节大鼠胰腺腺泡细胞增殖及凋亡的分子机制

Molecular Mechanism of Long Noncoding RNA B3GALT5-AS1 Targets miR-361-3p in Regulatng Proliferation and Apoptosis of Rat Pancreatic Scinar Cells

探讨长链非编码RNA(lncRNA)1,3-半乳糖基转移酶–多肽5反义RNA(B3GALT5-AS1)对雨蛙素诱导的大鼠胰腺腺泡细胞AR42J增殖、凋亡的调控机制。用雨蛙素诱导AR42J细胞构建急性胰腺炎细胞损伤模型;用脂质体将pcDNA-B3GALT5-AS1组(转染pcDNA-B3GALT5-AS1)、si-B3GALT5-AS1组(转染si-B3GALT5-AS1)、miR-361-3p组(转染miR-361-3p mimics)、anti-miR-361-3p组(转染anti-miR-361-3p)、pcDNA-B3GALT5-AS1+miR-361-3p组(共转染pcDNA-B3GALT5-AS1和miR-361-3p mimics)、si-B3GALT5-AS1+anti-miR-361-3p组(共转染si-B3GALT5-AS1和anti-miR-361-3p)转染至AR42J细胞,再用雨蛙素诱导细胞损伤。细胞计数试剂盒-8(cell counting kit-8,CCK-8)、流式细胞术、酶联免疫吸附实验(ELISA)分别检测细胞增殖率,细胞凋亡率和肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)含量;免疫印迹(Western blot)、实时荧光定量逆转录聚合酶链反应(qRT-PCR)分别检测细胞中细胞周期蛋白D1(cyclin D1)、存活蛋白(survivin)、前体半胱氨酸天冬氨酸蛋白酶3(procaspase-3)、前体半胱氨酸天冬氨酸蛋白酶-9(procaspase-9)蛋白水平及B3GALT5-AS1、miR-361-3p的表达;双荧光素酶报告实验检测B3GALT5-AS1与miR-361-3p的结合力。与对照组相比,雨蛙素诱导的AR42J细胞的增殖率显著降低,凋亡率明显升高,TNF-α、IL-6的含量显著升高(P<0.05)。模型细胞中B3GALT5-AS1表达异常降低,miR-361-3p表达异常升高,且过表达B3GALT5-AS1和抑制miR-361-3p可促进受损细胞的增殖,抑制凋亡,上调cyclin D1、survivin、procaspase-3、procaspase-9蛋白表达水平;并且抑制B3GALT5-AS1和过表达miR-361-3p可以抑制受损细胞增殖,促进细胞凋亡,下调cyclin D1、survivin、procaspase-3、procaspase-9蛋白表达水平。B3GALT5-AS1能够结合miR-361-3p。miR-361-3p可恢复B3GALT5-AS1对受损细胞增殖、凋亡的调节作用。lncRNA B3GALT5-AS1可促进雨蛙素诱导的AR42J细胞增殖,抑制凋亡,其机制与靶向miR-361-3p有关。

This study was aimed to investigate the regulation mechanism of lncRNA(long noncoding RNA)B3GALT5-AS1(1,3-galactosyltransferase-polypeptide 5 antisense RNA)on the proliferation and apoptosis of rat pancreatic acinar cells induced by caerulein.AR42J cells were induced by caerulein to construct a model of acute pancreatitis cell injury;AR42J cells were transfected pcDNA-B3GALT5-AS1 group(transfected pcDNA-B3GALT5-AS1),si-B3GALT5-AS1 group(transfected si-B3GALT5-AS1),miR-361-3p group(transfected miR-361-3p mimics),anti-miR-361-3p group(transfected anti-miR-361-3p),pcDNA-B3GALT5-AS1+miR-361-3p group(co-transfected pcDNA-B3GALT5-AS1 and miR-361-3p mimics),si-B3GALT5-AS1+anti-miR-361-3p group(co-transfected si-B3GALT5-AS1 and anti-miR-361-3p)with lipidosome,and then induced by cellulin.CCK-8(cell counting kit-8),flow cytometry,ELISA(enzyme-linked immunosorbent assay)seperately were used to detect cell proliferation rate,apoptosis rate,TNF-α(tumor necrosis factor-α)and IL-6(interleukin-6)contents;Western blot and qRT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain reaction)were used to detect cyclin D1,survivin,procaspase-3(precursor cysteine expression of acid aspartic protease-3),procaspase-9(precursor cysteine aspartic protease-9)protein levels and B3GALT5-AS1,miR-361-3p expression;dual luciferase reporting experiment was used to detect the binding ability of B3GALT5-AS1 to miR-361-3p.Compared with the control group,the proliferation rate was significantly decreased;the apoptosis rate was significantly increased;the levels of TNF-αand IL-6 were significantly increased in AR42J cells induced by caerulein(P<0.05).B3GALT5-AS1 expression was abnormally reduced and miR-361-3p expression was abnormally increased in model cells.B3GALT5-AS1 overexpression or miR-361-3p inhibition could promote the proliferation;inhibit apoptosis,and up-regulate cyclin D1 and survivin.Procaspase-3,procaspase-9,inhibition of B3GALT5-AS1 and overexpression of miR-361-3p could inhibit the proliferation of damaged cells;promote cell apoptosis,and down-regulate the protein expression levels of cyclin D1,survivin,procaspase-3,and procaspase-9.B3GALT5-AS1 was capable of binding miR-361-3p.miR-361-3p could reverse the regulation of B3GALT5-AS1 on the proliferation and apoptosis of injured cells.lncRNA B3GALT5-AS1 could promote the proliferation and inhibit apoptosis of AR42J cells induced by caerulein,and the mechanism was related to targeting miR-361-3p.