[Effect of REG3A on proliferation and invasion of glioma cells by regulating PI3K/Akt signaling pathway].

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.目的： 探讨胰岛再生源蛋白3A(REG3A)对胶质瘤细胞增殖和迁移的影响及其分子作用机制。 方法： 收集2017年10月17日至2018年10月18日于临沂市人民医院行手术治疗的10例胶质瘤患者的癌及癌旁组织，低级别和高级别胶质瘤各5例。体外培养不同胶质瘤细胞株(SF295、U251、TG905、A172和CRT)及原代胶质瘤细胞株PT1，采用实时荧光定量聚合酶链反应和Western blot检测组织标本及各细胞株中REG3A的mRNA和蛋白表达水平。通过脂质体转染si－REG3A至TG905细胞敲低REG3A的表达，通过慢病毒包装REG3A质粒感染SF295细胞上调REG3A的表达，使用REG3A重组蛋白和Akt抑制剂单独或联合处理胶质瘤细胞，采用细胞计数试剂盒8法检测细胞增殖能力，细胞划痕实验检测细胞迁移能力，Western blot法检测细胞中N－cadherin、vimentin和磷酸化Akt(p－Akt)的表达情况。 结果： 实时荧光定量聚合酶链反应(RT－qPCR)结果显示，U251、TG905、CRT、A172和PT－1细胞中REG3AmRNA的表达水平分别为2.129±0.13、2.22±0.59、5.02±0.31、6.62±1.34和9.18±0.61，高于SF295细胞(1.00±0.18，均P<0.001)。高级别胶质瘤组织中REG3AmRNA的表达水平为3.18±2.92，高于癌旁组织(1.00±1.14，P＝0.031)和低级别胶质瘤组织(0.90±0.67，P＝0.014)。Western blot和免疫组化检测结果与RT－qPCR结果一致。si－REG3A组TG905细胞的迁移率为(60.57±5.30)%，低于空白对照组[(79.65±12.09)%，P＝0.076]和si－NC组[(84.18±13.63)%，P＝0.038]。REG3A质粒转染组SF295细胞的迁移率为(96.05±6.41)%，高于空载转染组[(74.47±8.23)%，P＝0.021]。REG3A重组蛋白能够上调SF295细胞中N－cadherin、vimentin和p－Akt的表达。与对照组[(100.00±2.53)%]相比，REG3A重组蛋白组的增殖率[(117.70±10.24)%]明显提高，REG3A重组蛋白+Akt抑制剂组的增殖率[(98.31±3.64)%]明显低于REG3A重组蛋白组(P＝0.017)。REG3A重组蛋白+Akt抑制剂组的迁移率为(63.35±4.06)%，低于REG3A重组蛋白组[(89.26±11.07)%，P＝0.019]。 结论： REG3A能够通过激活磷酯酰肌醇－3－激酶/Akt信号通路促进人胶质瘤细胞的增殖和迁移。.