IDH1-R132H突變體通過調(diào)節(jié)HIF-1α通路抑制膠質(zhì)瘤U87細胞惡性進展

【摘要】 目的 探究異檸檬酸脫氫酶1（IDH1）-R132H突變體通過調(diào)節(jié)低氧誘導因子-1α（HIF-1α）通路抑制膠質(zhì)瘤U87細胞惡性進展。方法 實時熒光定量聚合酶鏈式反應（qRT-PCR）和蛋白免疫印跡法檢測膠質(zhì)瘤組織和細胞IDH1 mRNA和蛋白表達水平。將U87細胞分為對照組、Vector組、IDH1 wt組、IDH1-R132H組，qRT-PCR和Western Blot檢測IDH1 wt、IDH1-R132H轉(zhuǎn)染效率，噻唑藍（MTT）和克隆形成實驗檢測細胞增殖，Transwell檢測細胞遷移，流式細胞儀檢測細胞凋亡，Western Blot檢測IDH1、PCNA、基質(zhì)金屬蛋白酶-2（MMP-2）、Bcl-2相關(guān)X蛋白（Bax）、B細胞淋巴瘤/白血病-2（Bcl-2）、血管內(nèi)皮生長因子（VEGF）、HIF-1α、HIF-2α蛋白表達。結(jié)果 與癌旁組織和正常膠質(zhì)細胞NHAs相比，膠質(zhì)瘤組織和人神經(jīng)膠質(zhì)母細胞瘤細胞T98G、U138、U87中IDH1 mRNA和蛋白表達水平明顯降低（P＜0.05）。qRT-PCR和Western Blot結(jié)果表明，IDH1 wt、IDH1-R132H 轉(zhuǎn)染成功；與對照組或Vector組比較，IDH1-R132H組細胞活性、PCNA、MMP-2、Bcl-2蛋白表達明顯降低，VEGF、HIF-1α、HIF-2α蛋白表達明顯升高，且 IDH1-R132H 組克隆細胞數(shù)和遷移細胞數(shù)減少，細胞凋亡率、Bax蛋白表達明顯增加（P＜0.05）；但對照組、Vector組、IDH1 wt組上述指標兩兩比較，差異均無統(tǒng)計學意義（P＞0.05）。結(jié)論 IDH1-R132H突變體可能通過調(diào)節(jié)HIF-1α通路抑制膠質(zhì)瘤U87細胞增殖和遷移，并誘導細胞凋亡。

【關(guān)鍵詞】 IDH1-R132H突變體；HIF-1α通路；膠質(zhì)瘤；細胞增殖；細胞遷移；細胞凋亡

【中圖分類號】 R739.41" 【文獻標志碼】 A" 【文章編號】 1672-7770（2024）06-0654-06

The IDH1-R132H mutant inhibits malignant progression of glioma U87 cells by regulating the HIF-1α pathway FANG Song， TANG Guoqiang， YE Youzhong， LI Mingsong， CHEN Jiabei.Department of Neurosurgery， Chenzhou First Peoples Hospital， Chenzhou 423000， China

Corresponding Author： CHEN Jiabei

Abstract：" Objective To investigate the inhibition of malignant progression of glioma U87 cells by the regulation of hypoxic-induced-factor-1α（HIF-1α） pathway by mutant isocitrate dehydrogenase 1 R132H（IDH1-R132H）. Methods Real-time quantitative fluorescent polymerase chain reaction（qRT-PCR） and Western Blot were used to detect IDH1 mRNA and protein expression levels in glioma tissues and cells. U87 cells were divided into control group， Vector group， IDH1-wt group and IDH1-R132H group. The transfection efficiency of IDH1-wt and IDH1-R132H were detected by qRT-PCR and Western Blot， and cell proliferation was detected by MTT and clonal formation experiments. Cell migration was detected by Transwell， apoptosis was detected by flow cytometry， Western Blot analysis of IDH1， PCNA， matrix metalloproteinase-2（MMP-2）， Bcl-2 associated X protein（Bax）， B-cell lymphoma/leukemia-2（Bcl-2）， vascular endothelial growth factor（VEGF）， HIF-1α， HIF-2α protein expression. Results The expression levels of IDH1 mRNA and protein in glioma tissues and human glioblastoma cells T98G， U138， U87 were significantly lower than those in para-cancerous tissues and normal glioblastoma cells（Plt;0.05）. qRT-PCR and Western Blot proved successful transfection of IDH1-wt and IDH1-R132H. Compared with control group or Vector group， IDH1-R132H group significantly decreased cell activity， PCNA，"MMP-2 and Bcl-2 protein expression， significantly increased VEGF， HIF-1α and HIF-2α protein expression， decreased the number of cloned cells and migrated cells， and significantly increased apoptosis rate and Bax protein expression， and the number of cloned cells and migrated cells in IDH1-R132H group were decreased， while the apoptosis rate and Bax protein expression were significantly increased（Plt;0.05）. However， pairwise comparison of the above indicators in control group， Vector group and IDH1-wt group showed no statistical significance（Pgt;0.05）. Conclusions The IDH1-R132H mutant may inhibit the proliferation and migration of glioma U87 cells and induce apoptosis by regulating the HIF-1α pathway.

Key words： IDH1-R132H mutant; HIF-1α pathway; glioma; cell proliferation; cell migration; apoptosis

膠質(zhì)瘤是神經(jīng)外科常見顱內(nèi)腫瘤，始于神經(jīng)膠質(zhì)細胞，占原發(fā)腦腫瘤60%以上，可嚴重影響患者神經(jīng)系統(tǒng)癥狀，多數(shù)患者術(shù)后易復發(fā)，膠質(zhì)母細胞瘤5年生存率未超過10%^［1］。近幾年雖然膠質(zhì)瘤綜合治療已獲得重大突破，但治療效果不甚理想。2008年，有研究通過高通量測序首次發(fā)現(xiàn)，膠質(zhì)瘤中存在異檸檬酸脫氫酶1（isocitrate dehydrogenase 1，IDH1）基因點突變，多項研究顯示，約80%～90%世界衛(wèi)生組織Ⅱ/Ⅲ級膠質(zhì)瘤患者、繼發(fā)膠質(zhì)瘤患者存在IDH1/2基因突變，這一項新發(fā)現(xiàn)為膠質(zhì)瘤診療帶來新的希望^［24］。……