Circular RNA Circerbb2 Promotes Gallbladder Cancer Progression By
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Huang et al. Molecular Cancer (2019) 18:166 https://doi.org/10.1186/s12943-019-1098-8 RESEARCH Open Access Circular RNA circERBB2 promotes gallbladder cancer progression by regulating PA2G4-dependent rDNA transcription Xince Huang1,2†, Ming He1†, Shuai Huang1,2†, Ruirong Lin1,2†, Ming Zhan1†, Dong Yang1, Hui Shen1, Sunwang Xu1,2, Wei Cheng1, Jianxiu Yu3, Zilong Qiu4 and Jian Wang1* Abstract Background: CircRNAs are found to affect initiation and progression of several cancer types. However, whether circRNAs are implicated in gallbladder cancer (GBC) progression remains obscure. Methods: We perform RNA sequencing in 10 pairs of GBC and para-cancer tissues. CCK8 and clone formation assays are used to evaluate proliferation ability of GBC cells. qPCR and Western blot are used to determine expression of RNAs and proteins, respectively. CircRNA-protein interaction is confirmed by RNA pulldown, RNA immunoprecipitation, and fluorescence in situ hybridization. Results: We find that circRNA expression pattern is tremendously changed during GBC development. Among dozens of significantly changed circRNAs, a circRNA generated from the oncogene ERBB2, named as circERBB2, is one of the most significant changes. CircERBB2 promotes GBC proliferation, in vitro and in vivo. Other than being a miRNA sponge, circERBB2 accumulates in the nucleoli and regulates ribosomal DNA transcription, which is one of the rate-limiting steps of ribosome synthesis and cellular proliferation. CircERBB2 regulates nucleolar localization of PA2G4, thereby forming a circERBB2-PA2G4-TIFIA regulatory axis to modulate ribosomal DNA transcription and GBC proliferation. Increased expression of circERBB2 is associated with worse prognosis of GBC patients. Conclusions: Our findings demonstrate that circERBB2 serves as an important regulator of cancer cell proliferation and shows the potential to be a new therapeutic target of GBC. Keywords: Circular RNA, circERBB2, Gallbladder cancer, rDNA, PA2G4 Background made in the diagnosis, operation, and adjuvant therapy, Gallbladder cancer (GBC) is the most common bile tract little impact has been made on overall survival. malignancy with high malignancy and late presentation The development of high throughput sequencing has [1]. Surgical resection can achieve radical cure in early verified that circular RNA (circRNA) is an entire class of stage patients, but when GBC patients are symptomatic, abundant, non-coding RNAs ubiquitous among eukary- there is no effective treatment [2]. Five-year survival rate otes [4–6]. CircRNAs are generated when the precursor is < 10% for stage-III patients and < 5% for stage-IV pa- mRNAs (pre-mRNA) back-splice to join a downstream tients [3]. Although obvious improvement has been splice donor to an upstream splice acceptor. Recent studies show that circRNAs play critical roles in cellular * Correspondence: [email protected] differentiation, neural development, and disease occur- † Xince Huang, Ming He, Shuai Huang, Ruirong Lin and Ming Zhan rence [7–9]. Though most functional circRNAs are re- contributed equally to this work. 1Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, ported to work as miRNA sponges, only few circRNAs Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, People’s contain multiple binding sites to trap one specific Republic of China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. Molecular Cancer (2019) 18:166 Page 2 of 19 miRNA [5]. This indicates that some circRNAs may sequencing, 29 pairs were selected for RNA and protein function in ways beyond miRNA sponge. extraction (and only 28 pairs were succeeded in protein Many circRNAs have been implicated in cancer initi- extraction due to the limitation of tumor size), and 149 ation and progression. For examples, circITCH and cancer samples were embedded with paraffin to make circZNF292 affect cancer growth by targeting Wnt/β-ca- the tissue microarray. tenin signaling [10, 11]. CircFoxo3 has been implicated in inhibition of cell cycle progression by forming a Cell culture and transfection circFoxo3-p21-CDK2 ternary complex, which prevents GBC cell lines SGC-996 and GBC-SD and human em- CDK2 from promoting cell cycle progression [12]. Ele- bryonic kidney 293 T cells were cultured in high glucose vated circCCDC66 in colon polyps and colon cancer DMEM medium with 10% fetal bovine serum, 100 U/ml controls invasion and migration of colorectal cancer cells penicillin, and 100 μg/ml streptomycin. [13]. Moreover, some other circRNAs, like circNRIP1, Lipofectamine 2000 and polyethyleneimine were used circCSNK1G3, and circFAT1, affect several of the hall- for transfection of GBC cells and 293 T cells respectively, marks of cancer [14–16]. On the other hand, due to lack according to the manufacturer’s protocols. of the open end, circRNAs are resistant to exoribonu- clease treatment and can stably exist in some body fluids Silencing of gene expression by siRNAs like serum, saliva, and even urine [17]. This makes cir- CircERBB2, PA2G4 and TIFIA were silenced by at least cRNAs novel biomarkers for cancer diagnosis and prog- 2 siRNAs. Sequences of siRNAs were listed in Additional nosis from non-invasive clinical samples [17–19]. file 1: Table S1. For siRNA transfection, siRNAs were The active ribosomal DNA (rDNA) is transcribed by mixed with Lipofectamine 2000 with final concentration RNA polymerase I (Pol I) to produce pre-ribosomal of 50 μM. Total protein or RNA was collected 48 h later RNA (rRNA), which is named as 45S and processed to to determine effects of siRNAs. yield 5.8S, 18S and 28S rRNA. Cells undergoing malig- nant proliferation are dependent on a concomitant in- Plasmids creased rate of protein synthesis, which in turn, is pLenti-CMV-circERBB2 dependent on synthesis of ribosome. Thus, ribosome Full length gDNA from intron 2 (− 791 bp from back- synthesis is a rate-limiting step for malignant cellular splicing site) to intron 7 (+ 1342 bp from back-splicing proliferation [20]. Cancer cells, which are hallmarked by site) of ERBB2 gene was PCR amplified from the gDNA enlarged nucleoli, often have abnormal rDNA transcrip- of SGC-996 cells. pLenti-CMV-EGFP-3Flag vector was tion by Pol I [21, 22]. The rDNA transcription and Pol I treated with EcoRI and XbaI restriction endonuclease to activity are regulated by many factors including some remove EGFP-3Flag. Then intron 2 to intron 7 gDNA of proteins, non-coding RNAs and epigenetic alterations ERBB2 was cloned into the vector with T4 DNA-ligase. [23–26]. Many factors associated with rDNA transcrip- Back-splicing between exon 3 and exon 7 was mediated tion are related with malignant transformation and can- by sequence of flanking introns. cer progression, and rRNA synthesis has been recognized as a potential target for cancer therapeutic pLenti-CMV-PA2G4-Flag intervention [21, 23]. Full length PA2G4 cDNA was amplified from cDNA of Here, we report the function and clinical implication 293 T cells and cloned into pLenti-CMV-EGFP-3Flag of a circRNA derived from ERBB2 gene locus, referred vector using EcoRI and BamHI. to circERBB2. Differing from general circRNAs that are located in cytoplasm and function as miRNA sponges, pLenti-CMV-PA2G4-mCherry-Flag circERBB2 is enriched in nucleoli, up-regulates Pol I ac- pLenti-CMV-mCherry-3Flag vector was treated with tivity and rDNA transcription, finally promotes GBC EcoRI for linearization. Full length PA2G4 cDNA was proliferation and progression. CircERBB2 has important amplified from cDNA of 293 T cells and cloned into the clinical implications for GBC patients. linearized vector using seamless clone kit (Beyotime). Then PA2G4 cDNA with mutant C-terminal polybasic Methods region was obtained by overlap extension PCR and Patients and samples cloned into the linearized vector using seamless clone All surgically removed primary GBC and para-cancer kit. specimens were obtained from Biliary-Pancreatic Sur- gery Department of Shanghai Renji Hospital, with in- pLenti-CMV-TIFIA-Flag formed consent. Specimens were collected in accordance Full length TIFIA cDNA was amplified from cDNA of with institutional protocols. Ten pairs of cancer and cor- 293 T cells and cloned into pLenti-CMV-EGFP-3Flag responding para-cancer samples were selected for RNA vector using EcoRI and BamHI. Huang et al. Molecular Cancer (2019) 18:166 Page 3 of 19 pGL3-rDNA-IRES days. Then cells were fixed with methanol and stained The rDNA promoter luciferase reporter vector was gen- with crystal violet. erously given by Chen Ling-Ling group of Shanghai In- stitute of Biochemistry and Cell Biology, Chinese RNA pulldown Academy of Sciences [25]. CircRNA pulldown was performed using Magnetic RNA-protein Pull-down Kit (Thermo), with some modi- RNA isolation, reverse transcription PCR (rt-PCR) and fications of manufacturer’s protocol. We designed qPCR desthiobiotin-labeld DNA probe (Additional file 1: Table Total RNA from cells or tissue samples were extracted S1) targeting back-splicing sequence of circERBB2 to ef- with Trizol Reagent (Sigma) according to the manufac- fectively and specifically capture circERBB2 from total turer’s protocol. First strand cDNA was synthesized with RNA. 107 293 T cells were transfected with circERBB2 RevertAid First Strand cDNA Synthesis Kit (Thermo) over-expression (OE) vector or control vector. 48 h later, according to the manufacturer’s protocol. qPCR was total RNA from the two groups was extracted and incu- done with SybrGreen PCR Master Mix (Yeasen) and bated with 100 nmol DNA probe respectively at 70 °C Quantstudio™ DX Real-Time PCR Instrument (ABI).