低氧诱导的长链非编码RNA 68在肝癌中的功能及其作用机制

doi:10.3969/j.issn.1674-8115.2024.06.005

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (6): 702-712.doi: 10.3969/j.issn.1674-8115.2024.06.005

低氧诱导的长链非编码RNA 68在肝癌中的功能及其作用机制

谭露¹(), 沈少明², 何平³()

^1.上海交通大学基础医学院病理生理学系，上海 200025
^2.上海交通大学医学院附属仁济医院衰老与组织修复研究院，上海 200240
^3.上海交通大学医学院附属仁济医院上海市肿瘤研究所，上海 200240

收稿日期:2024-02-26 接受日期:2024-03-15 出版日期:2024-06-28 发布日期:2024-06-28
通讯作者: 何平 E-mail:tanlu98@sjtu.edu.cn;phe@shsci.org
作者简介:谭露（1998—），女，硕士生；电子信箱：tanlu98@sjtu.edu.cn。
基金资助:
国家自然科学基金(82103233);上海市高水平地方高校创新团队(SHSMU-ZDCX20211800)

Function and mechanism study of hypoxia-induced long non-coding RNA 68 in hepatocellular carcinoma

TAN Lu¹(), SHEN Shaoming², HE Ping³()

^1.Department of Pathophysiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Institute of Aging and Tissue Repair, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
^3.Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China

Received:2024-02-26 Accepted:2024-03-15 Online:2024-06-28 Published:2024-06-28
Contact: HE Ping E-mail:tanlu98@sjtu.edu.cn;phe@shsci.org
Supported by:
National Natural Science Foundation of China(82103233);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20211800)

摘要/Abstract

摘要：

目的·探究肝癌细胞系中受低氧诱导的长链非编码RNA 68（hypoxia-induced long non-coding RNA 68，HILRNA68）的功能以及其相关机制。方法·利用长链非编码RNA（long non-coding RNA，lncRNA）芯片研究低氧（hypoxia）与常氧（normoxia）分别处理12 h的肝癌细胞系SMMC-7721中表达变化的lncRNA，通过R语言DEseq2程序包分析低氧下表达显著变化的lncRNA，并利用实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）验证差异lncRNA。通过短发夹RNA（short hairpin RNA，shRNA）稳定敲除细胞内的低氧诱导因子（hypoxia inducible factors，HIFs）用以研究低氧下HILRNA68转录是否受HIFs的调控。通过细胞核浆分离，结合qRT-PCR检测以及RNA荧光原位杂交技术（RNA fluorescence in situ hybridization，RNA-FISH）实验研究HILRNA68的亚细胞定位。通过小干扰RNA（small interfering RNA，siRNA）分别在SMMC-7721和MHCC-97H细胞中敲低HILRNA68以研究其在低氧下的细胞功能。通过细胞生长计数实验以及Transwell细胞侵袭实验分别探究低氧下HILRNA68对肝癌细胞的生长增殖以及侵袭转移能力的影响。此外，通过双荧光素酶报告基因实验研究敲低HILRNA68后是否抑制HIF1α的转录活性。结果·通过对lncRNA芯片的结果进行差异基因分析，共得到247个升高和17个降低的lncRNA［定义为倍数变化（fold change）≥4，伪发现率（false discovery rate，FDR）≤0.05］。在差异基因中发现HILRNA68在多个经低氧处理的细胞系中均稳定升高约10倍。低氧下敲低HIF1α、HIF2α、HIF1β均显著抑制HILRNA68的升高（均P<0.05），荧光素酶报告基因实验表明其转录受到HIFs的调控。亚细胞定位研究表明HILRNA68主要定位于细胞核。细胞功能实验结果表明敲低HILRNA68显著抑制肝癌细胞SMMC-7721与MHCC-97H在低氧下的增殖以及侵袭能力（均P<0.05）。机制研究表明，敲低HILRNA68显著抑制低氧下HIF1α的转录活性（P<0.05）；HIF1α靶基因在低氧下的升高在敲低HILRNA68后被显著抑制（P<0.05）。结论·研究鉴定出一批低氧下表达显著变化的lncRNA，并功能注释了其中升高的HILRNA68。HILRNA68受HIFs调控而升高，升高后促进细胞低氧下的增殖以及侵袭转移能力；机制上，HILRNA68低氧下表达升高后促进HIF1α的转录活性。

关键词: 低氧, 低氧诱导因子, 长链非编码RNA, 细胞增殖, 细胞侵袭, TCONS_00027424

Abstract:

Objective ·To investigate the biological roles and associated mechanisms of the hypoxia-induced long non-coding RNA 68 (HILRNA68) in hepatocellular carcinoma (HCC) cell lines. Methods ·Long non-coding RNA (lncRNA) microarray analysis was conducted to study the differential expression of lncRNAs in the HCC cell lines cultured under hypoxia treatment and normoxia treatment separately for 12 h, and DEseq2 R package was used for the analysis of differentially expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the differential lncRNAs. Short hairpin RNAs (shRNAs) were used to knock down hypoxia-inducible factors (HIFs) to investigate whether HILRNA68 transcription was regulated by HIFs under hypoxia. Nucleus-cytoplasmic isolation combined with qRT-PCR and RNA fluorescence in situ hybridization (RNA-FISH) experiments were used to investigate the subcellular localization of HILRNA68. HILRNA68 was knocked down in SMMC-7721 and MHCC-97H cells by small interfering RNA (siRNA) to investigate its cellular function under hypoxia. The impact of HILRNA68 on the cell proliferation and invasion capabilities of HCC cells under hypoxia was examined by cell counting and Transwell assays. Dual-luciferase reporter assay was employed to identify how HILRNA68 regulated the transcriptional activity of HIFs under hypoxia. Results ·By differential expression analysis of lncRNAs, a total of 247 and 17 significantly (defined as fold change≥4, FDR≤0.05) up- and down-regulated lncRNAs, respectively, were identified. Among these differentially expressed genes, lncRNA HILRNA68 was up-regulated about 10-fold in multiple HCC cell lines when cultured under hypoxia for 12 h. Knockdown of HIF1α, HIF2α, and HIF1β significantly suppressed (all P<0.05) the upregulation of HILRNA68 under hypoxia. Luciferase reporter assay suggested that the transcription of HILRNA68 was regulated by HIFs. Subcellular localization studies revealed that HILRNA68 was mainly localized in the nucleus. Biological function experiments showed that silencing of HILRNA68 significantly inhibited the proliferation and invasion of HCC cells under hypoxia (all P<0.05). Mechanistic studies demonstrated that knock-down of HILRNA68 significantly suppressed the transcriptional activity of HIF1α under hypoxia (P<0.05) and the up-regulation of these canonical HIFs targets under hypoxia was also significantly inhibited after HILRNA68 knockdown (P<0.05). Conclusion ·The current study identifies a series of differential hypoxia-regulated lncRNAs and functionally annotates the upregulated HILRNA68. HILRNA68 is directly up-regulated by HIFs which promotes cell proliferation and invasion under hypoxia. Mechanistically, the upregulation of HILRNA68 under hypoxia enhances the transcriptional activity of HIF1α.

Key words: hypoxia, hypoxia inducible factor (HIF), long non-coding RNA (lncRNA), cell proliferation, cell invasion, TCONS_00027424

中图分类号:

R735.7

谭露, 沈少明, 何平. 低氧诱导的长链非编码RNA 68在肝癌中的功能及其作用机制[J]. 上海交通大学学报（医学版）, 2024, 44(6): 702-712.

TAN Lu, SHEN Shaoming, HE Ping. Function and mechanism study of hypoxia-induced long non-coding RNA 68 in hepatocellular carcinoma[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(6): 702-712.

图/表 9

表1 qRT-PCR引物序列

Tab 1 Primer sequences for qRT-PCR

Primer	Forword (5'→3')	Reverse (5'→3')
HILRNA44	GGAACGAACTCTTGCGTGTG	AGGAGCGCACTTCCTCATTC
HILRNA68	AAGCGGAGCCTGAAGATGTG	CCAACCCGGCTGCTACTTTAT
GLUT1	CTGTCGTGTCGCTGTTTGTG	AAAGATGGCCACGATGCTCA
PDK1	CTGGCTGTGGCTTCTCTAGC	CCGAAGTCCAGGAACTGCTT
PGK1	TGTGGTCCTGAAAGCAGCAA	AGTTGACTTAGGGGCTGTGC
BNIP3L	GGACTCGGCTTGTTGTGTTG	TCGACTAGGTGGGACGACAT
VEGF	TAAGTCCTGGAGCGTTCCCT	ACGCGAGTCTGTGTTTTTGC
18S rRNA	GTAACCCGTTGAACCCCATT	CCATCCAATCGGTAGTAGCG

表2 shRNA靶向序列

Tab 2 shRNA targeting sequences

shRNA	Sequence (5'→3')
shHIF1α#1	CCGCTGGAGACACAATCATAT
shHIF1α#2	GTGATGAAAGAATTACCGAAT
shHIF1β#1	GGCTCAAGGAGATCGTTTATT
shHIF1β#2	GCCTACACTCTCCAACACAAT
shHIF2α	CAGTACCCAGACGGATTTCAA

表3 siRNA靶向序列

Tab 3 siRNA targeting sequences

siRNA	Sequence (5'→3')
siNC	TTGTTACTCGCTCACATTTAA
si#1	CGTACCTGGCCTTAGTAATTT
si#2	GAGCCTGAAGATGTGATTAAA

图1 低氧下HILRNA68在肝癌细胞中表达显著升高Note：A. LncRNA microarray of SMMC-7721 exposed to hypoxia for 12 h. B. Differential lncRNA analysis of SMMC-7721 under hypoxia and normoxia. Red: up-regulated lncRNA; blue: down-regulated lncRNA. Fold change≥4, FDR≤0.05. C. Heatmap analysis of the indicated differentially expressed genes (DEGs), with data normalized using Z-score methods. N—normoxia; H—hypoxia. D. qRT-PCR analysis of indicated lncRNA in SMMC-7721 cells under normoxia and hypoxia. E. qRT-PCR analysis of HILRNA68 in human HCC cell lines (PLC/PRF/5, MHCC-97H, MHCC-97L, and Bel-7402) under normoxia and hypoxia.

Fig 1 HILRNA68 was up-regulated in HCC cells under hypoxia

图2 低氧下HILRNA68在肝癌细胞中的表达受HIFs调控Note： A. Western blotting analysis of SMMC-7721 cells indicated the knockdown efficiency of HIF1α/HIF2α/HIF1β. B. qRT-PCR analysis of indicated RNAs of SMMC-7721 cells with or without knockdown HIF1α/HIF2α/HIF1β under hypoxia. The P values indicate the differences between each group and the control group (shNC). C. Western blotting analysis of PLC/PRF/5 cells indicated the knockdown efficiency of HIF1α/HIF2α/HIF1β. D. qRT-PCR analysis of indicated RNA of PLC/PRF/5 cells with or without knockdown HIF1α/HIF2α/HIF1β under hypoxia. The P values indicate the differences between each group and the control group (shNC). E. Reference sequence diagram of HILRNA68. TSS—transcription start site. F. Western blotting analysis of HEK-293T cells transfected with HIF1α over-expression plasmid. G. Dual-luciferase assay results of HEK-293T transfected with HIF1α over-expression plasmid. H/N—hypoxia/normoxia.

Fig 2 Expression of HILRNA68 in HCC under hypoxia was regulated by HIFs

图3 HILRNA68主要定位于细胞核Note： A. Histogram about subcellular distribution of indicated RNAs in SMMC-7721 cells. U1 as a nucleus marker, GAPDH as a cytoplasm marker. B. Fluorescence of RNA-FISH results of SMMC-7721 hybridized with HILRNA68. DAPI indicates nucleus.

Fig 3 HILRNA68 was mainly located in the nucleus

表3 HILRNA68的编码潜力分析

Tab 3 Coding potential analysis of HILRNA68

RNA	Coding probability	Label
HILRNA68	0.009 4	Noncoding
HIF1α	1.000 0	Coding
XIST	0.219 1	Noncoding

图4 HILRNA68低氧下抑制肝癌细胞的生长增殖与侵袭转移Note： A. qRT-PCR analysis of HILRNA68 knockdown efficiency in SMMC-7721. B. Cell proliferation results of SMMC-7721 cells with or without HILRNA68 knock-down. C. Histogram of Transwell cell invasion analysis of SMMC-7721 cells with or without HILRNA68 knock-down. D. qRT-PCR analysis of HILRNA68 knockdown efficiency in MHCC-97H. E. Cell proliferation results of MHCC-97H cells with or without HILRNA68 knock-down. F. Histogram of Transwell cell invasion analysis results of MHCC-97H cells with or without HILRNA68 knock-down. The P value indicates the differences between each group and the control group (siNC).

Fig 4 HILRNA68 inhibited the tumor proliferation, invasion and metastasis of hepatocellular carcinoma cells under hypoxia

图5 HILRNA68调控HIFs的转录活性Note： A. Luciferase activity of SMMC-7721 cells with or without HILRNA68 knock-down under hypoxia. B. qRT-PCR analysis of indicated mRNAs in SMMC-7721 under hypoxia. C. Luciferase activity of MHCC-97H cells with or without HILRNA68 knock-down under hypoxia. D. qRT-PCR analysis of indicated mRNAs in MHCC-97H cells under hypoxia. The P values indicate the differences between each group and the control group (siNC).

Fig 5 HILRNA68 regulated the transcriptional activity of HIFs

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低氧诱导的长链非编码RNA 68在肝癌中的功能及其作用机制

Function and mechanism study of hypoxia-induced long non-coding RNA 68 in hepatocellular carcinoma

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