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Additional Supporting Information may be found in the online version of this article.

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HEP_24563_sm_SuppFig1.tif2221KSupplementary Fig. 1. Expression levels of six lncRNAs in cirrhotic and non-cirrhotic HCC tissues (A) or non-tumor tissues (B) (Supplementary Table 3, Cohort 1). Normal liver tissues were used as controls (Supplementary Table 4). *plt;0.05.
HEP_24563_sm_SuppFig2.tif4800KSupplementary Fig. 2. Co-expression network in HCC tissues. The co-expression network includes 2698 connections among 1063 genes that are correlated at |r| > 0.50. A node with a cyan node line represents lncRNA, and a node without a node line represents a protein-coding gene. Red nodes denote up-regulated genes, and blue nodes denote the down-regulated genes. Real lines between two nodes indicate positively correlated interactions between genes, and dashed lines indicate negatively correlated interactions.
HEP_24563_sm_SuppFig3.tif2942KSupplementary Fig. 3. Co-expression network of paired non-tumor tissue. The co-expression network includes 2687 connections among 794 genes that are correlated at |r| > 0.50. A node with a cyan node line represents lncRNA, and a node without a node line represents a protein-coding gene. Red nodes denote up-regulated genes, and blue nodes denote the down-regulated genes. Real lines between two nodes indicate positively correlated interactions between genes, and dashed lines indicate negatively correlated interactions.
HEP_24563_sm_SuppFig4.tif7376KSupplementary Fig. 4. Full-length human lncRNA-HEIH gene cloning. (A) Left, Agarose gel electrophoresis of nested PCR products from the 5′-RACE procedure. Molecular weight markers (base pairs) are indicated on the right. The major PCR product is marked by an arrow on the left. Right, Sequencing of second-round PCR products revealed the boundary between the universal anchor primer and lncRNA-HEIH sequences. The adenine marked by a red arrow indicates a putative transcriptional start site. (B) Left, Agarose gel electrophoresis of nested PCR products from the 3′-RACE procedure. Molecular weight markers (base pairs) are indicated on the left. The major PCR product is marked by an arrow on the right. Right, Sequencing of second-round PCR products reveals the boundary between a universal anchor primer and lncRNA-HEIH sequences. The cytosine marked by a red arrow indicats a putative transcriptional end site. (C) Nucleotide sequence of the full-length human lncRNA-HEIH gene. The EZH2 binding region is indicated in red.
HEP_24563_sm_SuppFig5.tif3283KSupplementary Fig. 5. Characteristics of lncRNA-HEIH. (A) lncRNA-HEIH is a RNA Pol II transcript. ChIP analyses of Huh7 cells were conducted on the lncRNA-HEIH promoter region (up, primer set a-c) using anti-RNA Pol II antibodies. Enrichment is determined relative to input controls. The data are the mean ± s.d. of three independent biological replicates. (B) lncRNA-HEIH is polyadenylated. The level of lncRNA-HEIH in purified polyadenylated RNAs and total RNA was detected using RT-PCR. (C) Detection of lncRNA-HEIH by QD-FISH. HCC and non-tumor tissue sections were subjected to QD-FISH and observed under ultraviolet light excitation in a fluorescence microscope. Positive signals for lncRNA-HEIH were detected in HCC specimens. Negative signals were found in the hybridization mixture containing lncRNA-HEIH oligonucleotides without digoxin. Original magnifications: 400×.
HEP_24563_sm_SuppFig6.tif1924KSupplementary Fig. 6. Expression levels of lncRNA in HCC cell lines. (A) Expression of lncRNA in HCC cell lines was detected by RT-PCR. LncRNA-HEIH expression in Huh7 (B) and HepG2 (C) cells that were stably transfected with lentivirus encoding shRNA against lncRNA-HEIH. LncRNA-HEIH expression in stable Hep3B (D) and SMMC-7721 (C) cell clones that were stably transfected with pcDNA3.1 encoding lncRNA-HEIH cDNA.
HEP_24563_sm_SuppFig7.tif1981KSupplementary Fig. 7. LncRNA-HEIH promotes proliferation of HCC cells. (A) HepG2 cells stably transfected with lentivirus encoding shRNA against lncRNA-HEIH were seeded in 96-well plates, and cell proliferation was assessed daily for three days using the CCK-8 assay (up). Changes of the proliferation marker PCNA were shown by western blotting analysis and normalized to ß-actin (down). (B) SMMC-7721 cells stably transfected with pcDNA3.1 encoding lncRNA-HEIH cDNA were seeded in 96-well plates, and cell proliferation was assessed daily for three days using the CCK-8 assay. Changes in the proliferation marker PCNA were shown by western blotting analysis and normalized to ß-actin (down) (n=3).
HEP_24563_sm_SuppTab1.doc29KSupplementary Table 1. Clinical characteristics of 5 HCC patients used for lncRNA.
HEP_24563_sm_SuppTab2.doc87KSupplementary Table 2. Oligonucleotide Sequences used in this study.
HEP_24563_sm_SuppTab3.doc53KSupplementary Table 3. Clinical Characteristics of the HCC Patients.
HEP_24563_sm_SuppTab4.doc38KSupplementary Table 4. Clinical Characteristics of the Patients who provided cirrhosis and normal liver tissuse.
HEP_24563_sm_SuppTab5.doc43KSupplementary table 5. Functions of genes connected to lncRNA-HEIH in subnetwork.

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