脂肪酸结合蛋白5促进宫颈癌细胞增殖侵袭和转移

FABP5 promotes cell growth, invasion and metastasis in cervical cancer

目的探讨脂肪酸结合蛋白5(FABP5)在宫颈癌发生和发展中的作用机制。方法选取人宫颈癌细胞株C33A、Siha、Caski、HeLa和HCC94,206例Ⅰa2期~Ⅱa2期宫颈癌组织蜡块以及40例正常宫颈组织,采用实时荧光定量PCR和Western blot法检测5种宫颈癌细胞和正常宫颈组织中FABP5 mRNA和蛋白的表达。构建慢病毒载体,采用细胞计数盒8法、平板克隆形成实验、划痕实验、Transwell侵袭实验检测干扰FABP5表达对Siha细胞增殖、迁移和侵袭的影响。以酶联免疫吸附法检测干扰FABP5表达后Siha细胞中基质金属蛋白酶2(MMP-2)和MMP-9的表达。建立裸鼠移植瘤和肺转移瘤模型,观察移植瘤生长曲线和转移瘤数目,分别采用实时荧光定量PCR法、Western blot法检测移植瘤组织中MMP-2和MMP-9 mRNA和蛋白的表达情况。结果与正常宫颈组织的细胞比较,FABP5 mRNA和蛋白在宫颈癌细胞中均呈高表达(P<0.05)。体外实验结果显示,与空白对照组和阴性对照组比较,Siha-FABP5-RNAi组Siha细胞中FABP5 mRNA和蛋白的表达明显受到抑制,增殖能力和克隆形成能力明显减弱,损伤修复能力和侵袭能力明显减弱。空白对照组、阴性对照组和Siha-FABP5-RNAi组Siha细胞的克隆形成率分别为(84.6±4.5)%、(84.6±5.1)%和(21.2±2.6)%,差异有统计学意义(P<0.05)。空白对照组、阴性对照组和Siha-FABP5-RNAi组的穿膜细胞数分别为(72.8±4.7)个/高倍视野、(72.6±3.3)个/高倍视野和(21.4±2.3)个/高倍视野,差异有统计学意义(P<0.05)。体内实验结果显示,空白对照组、阴性对照组和Siha-FABP5-RNAi组裸鼠皮下移植瘤体积分别为(921.4±63.0)mm^3、(1 021.4±56.0)mm^3和(139.6±36.0)mm^3,与空白对照组和阴性对照组比较,Siha-FABP5-RNAi组的裸鼠成瘤能力明显降低(P<0.001),且每侧肺叶肿瘤转移灶明显减少(P<0.001)。实时荧光定量PCR法检测结果显示,空白对照组、阴性对照组和Siha-FABP5-RNAi组裸鼠移植瘤组织中MMP-2 mRNA的相对表达量分别为1.00±0.10、1.00±0.10和0.34±0.13;MMP-9 mRNA的相对表达量分别为1.00±0.10、1.00±0.10和0.38±0.17。Siha-FABP5-RNAi组MMP-2和MMP-9 mRNA的表达较空白对照组和阴性对照组明显降低(均P<0.05)。MMP-2和MMP-9蛋白的表达与mRNA的表达趋势一致。结论 FABP5可促进宫颈癌细胞的增殖、迁移、侵袭和转移,可能通过调节MMP-2和MMP-9的表达发挥作用。

Objective To investigate the functions of FABP5 in the carcinogenesis and development of cervical cancer. Methods The expression of FABP5 was detected in several cervical cancer cell lines (C33A, Siha, Caski, HeLa and HCC94), 206 cases of cervical cancer tissues with stage Ⅰa2-Ⅱa2 and 40 cases of normal cervical tissues by real-time PCR and Western blotting. Then, the cells were infected with lentivirus-mediated siRNA-targeting FABP5. CCK-8 cell proliferation, colony formation, wound healing and transwell assays were used to investigate the effects of FABP5 on in vitro cell proliferation, migration and invasion. And in vivo xenograft model and lung metastasis model were used to observe the transplanted tumor growth and metastasis in female athymic nude mice. Furthermore, the total protein and RNA were extracted from the primary xenografts to determine the expression levels of FABP5, metalloproteinase-2 and metalloproteinase-9 using Enzyme linked immunosorbent assay (ELISA), real-time PCR and Western blotting. Results FABP5 expression was found to be significantly unregulated in cervical cancer tissues than that in normal cervical tissues (P<0.05). Compared with the Siha-NC group and uninfected group, the expression of FABP5 mRNA and protein in Siha-FABP5-RNAi group was significantly inhibited along with the decrease of cell proliferation, colony formation, wound healing and invasion ability. The clone formation rates of Siha cells in uninfected group, Siha-NC group and Siha-FABP5-RNAi group were (84.6±4.5)%,(84.6±5.1)% and (21.2±2.6)%, respectively. Moreover, the transwell assay showed that invasive cells in three groups were (72.8±4.7)/HPF,(72.6±3.3)/HPF and (21.4±2.3)/HPF, respectively. All of the difference was statistically significant (P<0.05). Furthermore, FABP5 silencing significantly reduced tumor growth and lung metastases in nude mice in vivo (P<0.001). The subcutaneously xenografted volume in uninfected group, Siha-NC group and Siha-FABP5-RNAi group was (921.4±63.0) mm^3,(1 021.4±56.0) mm^3 and (139.6±36.0) mm^3, respectively. The real-time quantitative PCR results showed that the relative expression levels of MMP-2 and MMP-9 mRNA were 1.00±0.10 and 1.00±0.10, 1.00±0.10 and 1.00±0.10 as well as 0.34±0.13 and 0.38±0.17 in xenografted tumor tissues of uninfected group, Siha-NC group and Siha-FABP5-RNAi group, respectively. MMP-2 and MMP-9 was significantly downregulated after FABP5 inhibition(P<0.05). Additionally, the protein expression trend of MMP-2 and MMP-9 in three groups was consistent with the mRNA levels. Conclusion FABP5 might promote the carcinogenesis and metastasis of cervical cancer via up-regulating MMP-2 and MMP-9.