Pin1相互作用的长非编码RNA lncPIL-1调控乳腺癌恶性表型的研究

doi:10.3969/j.issn.1009-976X.2022.02.003

摘要/Abstract

摘要： 目的探究与Pin1蛋白结合的长非编码RNA lncPIL-1对乳腺癌细胞增殖、迁移和干性表型的影响。方法采用RNA免疫沉淀联合深度测序（RIP-seq）得到与Pin1特异性结合的长非编码RNA,并通过RNA结合蛋白免疫沉淀（RIP）和紫外交联免疫沉淀（CLIP）进行验证筛选;利用小干扰RNA（siRNA）敲降乳腺癌细胞lncPIL-1的表达,利用慢病毒感染得到lncPIL-1过表达乳腺癌细胞株,并通过qRT-PCR实验检测敲降及过表达效率;采用平板克隆方法检测lncPIL-1对乳腺癌细胞增殖能力的影响;采用Transwell实验检测lncPIL-1对乳腺癌细胞迁移侵袭能力的影响;采用ALDEFLUOR法检测lncPIL-1对乳腺癌细胞干性表型的影响。结果 RIP-seq及验证实验显示,lncPIL-1在Pin1免疫沉淀复合物中富集。乳腺癌患者高表达lncPIL-1与总生存率低密切相关。平板克隆实验显示lncPIL-1不影响乳腺癌细胞生长。Transwell实验中过表达lncPIL-1会加强乳腺癌细胞的迁移能力,而敲降lncPIL-1会降低乳腺癌细胞的迁移能力;ALDEFLUOR检测结果显示敲降lncPIL-1明显抑制乳腺癌细胞干性表型。结论 lncPIL-1与Pin1特异性结合并与乳腺癌患者预后不良高度相关,具有促进乳腺癌细胞运动迁移及干性表型的能力,提示lncPIL-1有望成为新的乳腺癌治疗靶点。

关键词: 长非编码RNA, Pin1, 乳腺癌, 迁移, 肿瘤干细胞

Abstract: Objective To explore the effect of Pin1-interacting long non-coding RNA lncPIL-1 on the proliferation, migration and stem cell phenotype of breast cancer cells. Methods RNA immunoprecipitation combined with high-throughput sequencing （RIP-seq） was used to obtain lncRNAs specifically bound to Pin1. RNA immunoprecipitation （RIP） and crosslinking-immunoprecipitation （CLIP） were used for validation. The expression of lncPIL-1 was knocked down by small interfering RNA （siRNA）, and the lncPIL-1 overexpressed breast cancer cell lines were obtained by lentivirus infection. The knockdown and overexpression efficiency were detected by qRT-PCR. The effect of lncPIL-1 on the proliferation of breast cancer cells was detected by plate cloning assay. Transwell assay was used to detect the effect of lncPIL-1 on the migration of breast cancer cells. ALDEFLUOR assay was used to detect the effect of lncPIL-1 on the stem cell phenotype of breast cancer cells. Results RIP-seq and validation experiments showed that lncPIL-1 was enriched in Pin1 immunoprecipitates. LncPIL-1 overexpression in breast cancer patients correlated with worse overall survival. Colony formation assay showed that lncPIL-1 didn′t affect breast cancer cell growth. Transwell assay showed that overexpression of lncPIL-1 enhanced the migration capabilities of breast cancer cells, and knockdown of lncPIL-1 reduced the capabilities. ALDEFLUOR assay showed that knockdown of lncPIL-1 significantly inhibited the stem cell phenotype of breast cancer cells. Conclusion lncPIL-1 specifically binds to Pin1 and is highly associated with poor prognosis of breast cancer patients. LncPIL-1 promotes the migration and cancer stem cell phenotypes of breast cancer cells, which suggests that lncPIL-1 may be a new therapeutic target for breast cancer.

Key words: long noncoding RNA, PIN1, breast cancer, migration, cancer stem cells

中图分类号:

R737.9

赵炜, 罗曼莉. Pin1相互作用的长非编码RNA lncPIL-1调控乳腺癌恶性表型的研究[J]. 岭南现代临床外科, 2022, 22(02): 126-133.

ZHAO Wei, LUO Man-Li. Studying the effect of Pin1-interacting lncPIL-1 on the malignant phenotypes of breast cancer cells[J]. Lingnan Modern Clinics In Surgery, 2022, 22(02): 126-133.

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