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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (2): 217-227. doi: 10.19723/j.issn.1671-167X.2023.02.004

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Read-through circular RNA rt-circ-HS promotes hypoxia inducible factor 1α expression and renal carcinoma cell proliferation, migration and invasiveness

Yun-yi XU1,Zheng-zheng SU1,Lin-mao ZHENG1,Meng-ni ZHANG1,Jun-ya TAN1,2,Ya-lan YANG1,Meng-xin ZHANG1,Miao XU1,Ni CHEN1,2,Xue-qin CHEN1,2,Qiao ZHOU1,2,*()   

  1. 1. Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China
    2. Research Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China
  • Received:2022-11-16 Online:2023-04-18 Published:2023-04-12
  • Contact: Qiao ZHOU E-mail:zhou_qiao@hotmail.com

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Abstract:

Objective: To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α. Methods: Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3′ untranslated region (3′ UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. Results: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. Conclusion: The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.

Key words: Read-through circular RNA HIF1α-SNAPC1, Renal cell carcinoma, Hypoxia inducible factor 1α, Small nuclear RNA activating complex polypeptide 1, microRNA 539

CLC Number: 

  • R365

Table 1

Primer sequence of RT-PCR"

mRNA name Primer sequence(5′-3′) Length/bp
rt-circ-HS
(divergent primer)		 FP：TCACTTTACAGCAATGCCCA
RP：TCCTCACACGCAAATAGCTGA		 306
rt-circ-HS
(convergent primer)		 FP：GTGAACCCATTCCTCACCCA
RP：AGCACCAACTCTGATCTGGA		 209
rt-circ-HS full length
(divergent primer)		 FP：CACTTTACAGCAATGCCCA
RP：TCAGCACCAAGCAGGTCATAGG		 761
rt-circ-HS full length
(convergent primer)		 FP：GGTATAAGAAACCACCTATGAC
RP：CACACGATCACTTGGGTCC		 518
HIF1α FP：GATGTAATGCTCCCCTCACC
RP：ATTCATTGACCATATCACTATCCAC		 295
SNAPC1 FP：CAGGAGACGGACAGTGTACG
RP：TCGCCAAGCCAAAGCTAAAGC		 138
BNIP3 FP：ACCAACAGGGCTTCTGAAAC
RP：GAGGGTGGCCGTGCGC		 202
VEGF FP：AGGAGGGCAGAATCATCACG
RP：GCACACAGGATGGCTTGAAGA		 140
β-actin FP：CTGGCACCACACCTTCTACAATG
RP：CCTCGTAGATGGGCACAGTGTG		 248
U6 FP: TGGAACGATACAGAGAAGATTAGCA
RP：AACGCTTCACGAATTTGCGT
TaqMan probe：FAM-CCCCTGCGCAAGGA-MGB		 75
miR-539 FP: CGGCGGGAGAAATTATCCT
RP：GTGCAGGGTCCGAGGT
TaqMan probe：FAM-ACTGGATACGACACTCCACACCA-MGB		 74

Figure 1

Discovery and characterization of rt-circ-HS A, B, bioinformatics and sequencing analysis identified three novel read-through RNAs derived from HIF1α and SNAPC1, with respective genomic sequence structures; C, structure and sequencing of the novel rt-circ-HS derived from HIF1α-SNAPC1 read-through. The junction site of rt-circ-HS was verified by Sanger sequencing; D, rt-circ-HS full length convergent primers and divergent primers were designed for RT-PCR to characterize the rt-circ-HS derived from HIF1α exon 6-SNAPC1 exon 2 read-through RNA; E, the read-through RNAs of HIF1α exon 10-SNAPC1 exon 2 and HIF1α exon 9-SNAPC1 exon 2 did not form circRNAs. rt-circ-HS, read-through circular RNA HIFα-SNAPC1; HIF1α, hypoxia inducible factor 1α; SNAPC1, small nuclear RNA activating complex polypeptide 1; RT-PCR, reverse transcription-polymerase chain reaction; circRNA, circular RNA."

Figure 2

Expression of rt-circ-HS in different cell lines and renal cell carcinoma tissues A, RT-PCR showed that HIF1α exon 6-SNAPC1 exon 2 read-through RNA was present in different renal cancer cells, including A498, ACHN, 786-O, 769-P, OS-RC-2, normal renal epithelial cell HK-2, and human embryonic kidney cell HEK-293. The circular rt-circ-HS was highly expressed in renal cancer cell 786-O, weakly expressed in human embryonic kidney cell HEK-293, and was very low in other cells; B, HIF1α exon 6-SNAPC1 exon 2 read-through RNA, but not the circular rt-circ-HS was found in various cells lines, including prostate cancer cells (PC3, DU45, 22RV1), prostate epithelial cells (RWPE-1), bladder cancer cells (T24), cervical cancer cells (HeLa), glioma cells (U251, U87), astrocytes (HA) and microglia cells (HMC3); C, chromogenic ISH showed high expression of rt-circ-HS in different renal cell carcinoma tissue samples in tissue microarrays, the blue-purple ISH signals indicated the positive signal, and the nuclei were stained green. For each RCC type, low power fields (×40) were shown with insets of high power fields (×400). RT-PCR, reverse transcription-polymerase chain reaction; rt-circ-HS, read-through circular RNA HIFα-SNAPC1; HIF1α, hypoxia inducible factor 1α; SNAPC1, small nuclear RNA activating complex polypeptide 1; ISH, in situ hybridization; ccRCC, clear cell renal cell carcinoma; pRCC, papillary renal cell carcinoma; chRCC, chromophobe renal cell carcinoma; TFE3-RCC, TFE3-translocation renal cell carcinoma; FH-RCC, FH-deficient renal cell carcinoma."

Figure 3

Effects of rt-circ-HS on biological behavior of renal carcinoma cells A, CCK-8 assays showed that rt-circ-HS promoted the survival of renal cell carcinoma cells; B, EdU assays demonstrated rt-circ-HS promoted the proli-feration of renal cell carcinoma cells (immunofluorescence ×200), EdU positivily were shown by bar graph (* P < 0.001); C, Transwell cell migration assays demonstrating rt-circ-HS promoted migration of renal cell carcinoma cells (crystal violet staining ×200), the bar graph showed the number of transmembrane cells in each group(* P < 0.001); D, Transwell cell invasion assays showed that rt-circ-HS promoted invasion of renal cell carcinoma cells (crystal violet staining ×200), the bar graph showed the number of transmembrane cells in each group (* P < 0.001). CCK8, cell counting kit 8; EdU, 5-ethynyl-2'-deoxyuridine; rt-circ-HS, read-through circular RNA HIFα-SNAPC1; HIF1α, hypoxia inducible factor 1α; SNAPC1, small nuclear RNA activating complex polypeptide 1; si-NC, small interference RNA negative control; si-rt-circ-HS, small interference RNA of rt-circ-HS; rt-circ-HS-OE, overexpression plasmid of rt-circ-HS."

Figure 4

Regulation of parental genes HIF1α and SNAPC1 by interfering rt-circ-HS A, si-RNA were used to knock down rt-circ-HS in 786-O cells, which resulted in downregulation of mRNA expressions of HIF1α, SNAPC1, BNIP3 and VEGF, whereas artificial rt-circ-HS-OE in A498 cells promoted the mRNA expression of HIF1α, SNAPC1, BNIP3 and VEGF; B, Western blot showed that rt-circ-HS promoted the protein expression of HIF1α and SNAPC1. RT-PCR, reverse transcription-polymerase chain reaction; si-RNA, small interference RNA; si-NC, small interference RNA negative control; si-rt-circ-HS, small interference RNA of rt-circ-HS; rt-circ-HS-OE, overexpression of rt-circ-HS; rt-circ-HS: read-through circular RNA HIFα-SNAPC1; HIF1α, hypoxia inducible factor 1α; SNAPC1, small nuclear RNA activating complex polypeptide 1; BNIP3, BCL2 interacting protein 3; VEGF, vascular endothelial growth factor."

Figure 5

Promotion of HIF1α expression through competitive inhibition of miR-539 by rt-circ-HS A, miR-539 binding sequence was present in both the rt-circ-HS junction site and the HIF1α 3′UTR (703-715 nt) by bioinformatics analysis; B, expression of miR-539 (U6 as internal control) was increased and the mRNA of HIF1α was down-regulated (β-actin as internal control) by miR-539 mimic transfection in 786-O cells (** P < 0.01, *** P < 0.001); C, dual luciferase reporter gene assays showing that miR-539 mimics suppressed reporter gene activity of pGL3-circ-HS-WT, but not pGL3-circ-HS-MUT (+, plasmids or mimics added at transfection; -, plasmids or mimics were not added; *** P < 0.001); D, dual luciferase reporter gene assays demonstrating miR-539 mimics suppressed reporter gene activity of wild-type pGL3-HIF1α-WT, but not mutated pGL3-HIF1α-MUT. Simultaneous transfection with pGL3-circ-HS-WT, but not pGL3-circ-HS-MUT, reversed the inhibitory effects of miR-539 mimic on reporter gene activity of pGL3-HIF1α-WT, but not the pGL3-HIF1α-MUT (+, plasmids or mimics added at transfection; -, plasmids or mimics were not added; * P < 0.05, ** P < 0.01); E, schematic summary of the major findings of the present study. RT-PCR, reverse transcription-polymerase chain reaction; Q-PCR, real-time quantitative polymerase chain reaction; rt-circ-HS, read-through circular RNA HIFα-SNAPC1; miR-539, micro RNA 539; 3′ UTR, 3′ untranslated region; HIF1α, hypoxia inducible factor 1α; SNAPC1, small nuclear RNA activating complex polypeptide 1; BNIP3, BCL2 interacting protein 3; VEGF, vascular endothelial growth factor; U6, U6 small nuclear RNA; WT, wild type; MUT, mutation."

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