LAIR-1在卵巢癌的表达及其对卵巢癌细胞增殖和侵袭能力的影响

LAIR-1 is a collagen receptor which belongs to the immune inhibitory receptor family. In our recent study, we showed for the first time that LAIR-1 was expressed in ovarian carcinoma cells in a tissue microarray using immunohistochemical staining, and the expression of LAIR-1 was positively associated with higher grade tumor, which suggests that LAIR-1 maybe a carcinogenic factor. Besides, the expression of LAIR-1 was also detected in several ovarian carcinoma cell lines, but down-regulation of LAIR-1 expression by lentivirus-mediated shRNA in HO8910 cells showed that LAIR-1 reduced cell proliferation, colony formation and cell invasion of HO8910 cells. The role of LAIR-1 in the pathogenesis of ovarian cancer need to be further investigated. The project is intended to use shRNA gene knockdown strategy, CRISPR/Cas9 system, as well as nude-mouse transplanted tumor model to identify the function of LAIR-1 in the cell proliferation and invasion of ovarian carcinoma. We will use qRT-PCR, Western blot to identify in mRNA and protein level the expression of LAIR-1 and its clinical significance in ovarian carcinoma. Then, we will further investigate the effect of LAIR-1 on the signal pathways related to cell proliferation and invasion by using gene microarray, antibody microarray. . This study may be helpful for a better understanding of molecular pathogenesis of ovarian cancer, and may provide new idea for the development of early diagnostic reagents and targeted- therapy drugs.

LAIR-1是以胶原为配体的免疫抑制性受体。我们近期首次利用免疫组化的方法在卵巢癌组织芯片中发现LAIR-1在卵巢癌细胞表达，肿瘤分级越高LAIR-1的表达越高，提示LAIR-1是促癌蛋白；我们在多种卵巢癌细胞株也发现LAIR-1的表达；但是，shRNA干扰LAIR-1基因在卵巢癌细胞株HO8910的表达，发现LAIR-1抑制HO8910的增殖和侵袭。因此，LAIR-1在卵巢癌的作用需要深入研究。本项目拟在前期工作基础上，利用qRT-PCR、Western blot、RNA干扰、CRISPR/Cas9编辑等技术以及卵巢癌裸鼠原位移植瘤模型，明确LAIR-1在卵巢癌组织的表达、临床意义及在卵巢癌增殖和侵袭中的作用；采用基因芯片、抗体芯片分析LAIR-1在卵巢癌增殖和侵袭相关的重要信号通路中的作用。本项目的研究将进一步阐明卵巢癌致病机制、为早期诊断试剂和靶向治疗药物的开发提供新的依据。

近年来的研究发现LAIR-1在卵巢癌在内的一些实体癌中过度表达。LAIR-1在癌症进展中的作用不清楚。本项目主要分析LAIR-1对人卵巢癌细胞体外生物学功能及体内肿瘤形成能力的影响，探讨LAIR-1影响卵巢癌细胞生长的分子机制。实验选用LAIR-1低表达和中等程度表达的卵巢癌细胞株SKOV3和HO8910。首先克隆并明确了HO8910细胞株的LAIR-1基因的 cDNA序列，利用慢病毒转染技术构建了高表达LAIR-1的SKOV3细胞，利用 LAIR-1 慢病毒 RNAi 干扰载体构建下调 LAIR-1表达的卵巢癌 HO8910 细胞。通过CCK-8 实验、细胞克隆形成实验、细胞凋亡以及迁移实验，对LAIR-1过表达后对人卵巢癌SKOV3细胞增殖、迁移、凋亡的影响进行分析；采用裸鼠荷瘤实验，体内验证LAIR-1对人卵巢癌SKOV3细胞成瘤能力的影响；另外，采用Western Blot技术分析LAIR-1对卵巢癌相关信号通路PI3K-AKT-mTOR的影响；j结合免疫共沉淀技术（CO-IP），对潜在的LAIR-1结合蛋白进行质谱分析，探讨LAIR-1可能的作用机制；通过 Seahorse 生物能量分析仪，检测下调 LAIR-1 表达后对 HO8910 细胞生物能量代谢的影响。.本研究发现LAIR-1在体外可以抑制卵巢癌细胞SKOV3的生长和迁移，促进其凋亡；过表达LAIR-1的SKOV3细胞中，PI3K、AKT、mTOR以及p-AKT和p-mTOR表达均显著降低，LAIR-1干扰的HO8910细胞则表现为相反的趋势，体内实验结果与上述结果基本一致。共IP实验和质谱分析鉴定出高表达LAIR-1的SKOV3细胞中潜在的LAIR-1结合蛋白。Seahorse 生物能量分析结果显示LAIR-1 表达显著下调的 HO8910 细胞，ATP 产生明显改善，同时其基础呼吸和最大呼吸能力均明显提高。分析结果表明LAIR-1通过直接抑制PI3K-AKT-mTOR或在翻译水平上调节蛋白质合成来抑制卵巢癌细胞的生长，同时LAIR-1 分子通过影响线粒体生物能量代谢抑制人卵巢癌 HO8910 细胞的生长。本研究初步明确了LAIR-1在卵巢癌中的作用和分子机制，为卵巢癌的诊断和治疗提供了新思路。