Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma

Abstract

Although expression of non-protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.

Fig. 1.

ucRNAs are aberrantly expressed in malignant hepatocytes. (A) Genome-wide expression profiling was performed in HepG2 malignant cells and normal human hepatocytes (HH). Fifty-six ucRNAs were aberrantly expressed in malignant hepatocytes with P < 0.05, with 12 ucRNAs increased and 7 decreased by greater than twofold. The ratio of expression of these ucRNAs in malignant cells relative to nonmalignant cells is plotted against the P value. Selected ucRNAs with a greater than threefold change in expression are annotated. (B) The genomic locations of the ucRNA as exonic, nonexonic, or possibly exonic relative to protein-coding genes is depicted for all ucRNAs and for the group of ucRNAs that are aberrantly expressed in malignant hepatocytes. Selective enrichment of a specific group of ucRNA based on their genomic relationship to known protein-coding genes was not observed.

Fig. 2.

uc.338 is overexpressed in HCC cells lines. RNA was extracted from different cell lines and uc.338 expression evaluated by quantitative real-time-PCR. The expression of uc.338 was normalized to that of RNU6. Bars represent the mean and SEM of four samples. uc.338 expression was increased in all HCC cell lines compared with normal human hepatocytes (HH). *P < 0.05 relative to human hepatocytes. Nonmalignant epithelial cells (hatched bars): HE, hepatocytes; BE, biliary epithelia; PE, prostatic epithelia. Malignant cells (solid bars): CCA, cholangiocarcinoma; PaC, pancreatic cancer; CRC, colorectal cancer; PC, prostate cancer; BC, breast cancer.

Fig. 3.

uc.338 is overexpressed in human HCC tissues. uc.338 expression was evaluated in a total of 221 HCCs, 72 cases of noncirrhotic liver tissues, and 97 cases of cirrhotic adjacent liver tissues. Paraffin-embedded, formalin-fixed liver tissues were incubated with LNA–anti-uc.338. (A) uc.338 expression was classified as negative, weak, moderate, or strong based on the percentage of cells with detectable staining for uc.338. The proportion of cases of HCC, cirrhotic liver, or noncirrhotic liver within each class is depicted in the columns. (B) The mean and 95% confidence intervals of uc.338 expression in noncirrhotic liver, cirrhotic liver, and HCC tissues is shown. *P < 0.05. (C) uc.338 expression was compared between 156 HCCs and their corresponding adjacent liver tissues. Picture of representative cases are shown. (D) An expression score was derived as the ratio of the difference in expression between HCC and adjacent liver tissues to the SD of uc338 in all tissues and plotted with the size of the bubble representing the number of cases.

Fig. 4.

Nuclear expression of uc.338 in HCC cells. (A) Paraffin-embedded, formalin-fixed liver tissues were incubated with LNA–anti-uc.338. uc.338 was frequently detected in nuclei of HCC in situ. (B) RNA was extracted from the nuclear and the cytoplasmic fraction of liver cells and uc.338 expression evaluated by real-time-PCR. Bars represent the mean and SEM of relative expression of uc.338 from two experiments performed in four replicates. *P < 0.05.

Fig. 5.

uc.338 and PCBP2 are independently regulated. (A) Schematic representation of the partial exonic location of uc.338 within the PCBP2 gene. The exons of PCBP2 are indicated by dark gray boxes, and uc.338 is depicted as the light gray box. The location of the uc.338 forward (F) and reverse (R) primers used for real-time-PCR and probe used for in situ hybridization are shown. With the exception of the forward primer, these primers are located within the PCBP2 intronic region. Of the siRNAs targeting uc.338, siRNA-1 is entirely intronic, whereas siRNA-2 overlaps a few nucleotides of the coding sequence of PCBP2. F′ and R′ indicate primers used for detection of PCBP2. (B) Expression of uc.338 is plotted against PCBP2 mRNA expression in samples of normal and HCC cell lines. There is no linear correlation between uc.338 and PCBP2 gene expression (P = 0.08). (C) HepG2 cells were transfected with siRNA against PCBP2 or siRNA control. Bars represent the mean of two independent experiments performed in four replicates. *P < 0.05 compared with control siRNA. (D) The expression of uc.338 was decreased in HepG2 and Huh-7 cells by using two different siRNAs against uc.338. After 48 h, RNA was collected and uc.338 and PCBP2 expression were evaluated by real-time PCR. Bars represent the mean and SEM of two experiments performed in four replicates. *P < 0.05 relative to siRNA control. These data indicate that uc.338 does not regulate the expression of PCBP2 and that the expression of uc.338 is independent of PCBP2.

Fig. 6.

Cloning of the transcript including uc.338. (A) Schematic representation of the transcript including uc.338 (TUC338) in relation to PCBP2 gene. By performing 5′ and 3′ RACE, we identified 237 nt upstream and 130 nt downstream of the ultraconserved region that was reported by Bejerano et al. (23). The complete sequence of the TUC338 transcript is reported. (B) Northern blotting analysis for TUC338 and PCBP2 was performed as described in Materials and Methods. TUC338 was expected to be 590 nt long, and PCBP2 was ∼1,200 nt long.

Fig. 7.

TUC338 modulates cell growth in HCC cells. (A) HepG2 and Huh-7 cells were transfected with siRNAs against TUC338 or control siRNA for 48 h and then plated. After 24, 48, and 72 h, cells were counted by trypan blue staining. Mean values of three independent experiments with SEM are represented. *P < 0.05 compared with control. Cell viability after transfection with siRNA-1 was not significantly different from that with siRNA-2 (P > 0.05 at each time point in each of the cell lines). (B) HepG2 cells were transfected with siRNA-2 anti-TUC338 or control siRNA for 48 h, and analysis of cell-cycle distribution was performed by flow cytometry. Compared with controls, there was a reduction of 30% in S phase and of 12% in G2/M phase for cells transfected with siRNA against TUC338. Bars represent the mean and SEM of three experiments. *P < 0.05 compared with controls. (C) HepG2 cells were transfected with siRNA anti-TUC338 or control siRNA by nuclear transfection for 48 h and then plated in agar in 96-well plates. Anchorage-independent growth was assessed fluorometrically after 7 d. Bars represent the mean and SEM of two experiments performed in seven replicates. *P < 0.05.

Fig. 8.

TUC338 modulates cell growth in mouse hepatocytes. BNL-CL.2 are nonmalignant embryonic mouse hepatocytes. BNL-SVA.8 cells are derived from BNL-CL.2 after SV40 transformation, and they exhibit transformed cell growth. (A) TUC338 expression was assessed by real-time-PCR and normalized to that of RNU6. Bars represent the mean and SEM of three experiments. (B) BNL-CL.2 and BNL-SVA.8 were plated in soft agar, and colonies were counted after 4 wk. Representative pictures are shown along with the mean and SEM of three independent experiments. *P < 0.05. (C) BNL-CL.2 and BNL-SVA.8 cells were plated in 6-well plates, and cell number was counted by trypan blue staining at different time points. Mean and SEM derived from three independent experiments are shown. *P < 0.05 compared with BNL-CL.2. (D) BNL-SVA.8 cells were transfected with siRNA-1 anti-TUC338 or siRNA control for 48 h. Bars represent mean and SEM of four replicates. At the indicated times, cells were counted after trypan blue staining. Mean and SEM from three independent experiments are represented. *P < 0.05 compared with siRNA control.

Fig. 9.

TUC338 expression modulates expression of cell-cycle regulatory proteins. HepG2 and Huh7 cells were serum-starved for 48 h before transfection with either control siRNA or anti-TUC338 siRNA. Cells were collected 48 h after transfection, and protein lysates were obtained. Lysates were also obtained from untransfected cells and normal human hepatocytes (HH). Western blotting was performed for the indicated cell-cycle–associated proteins, and their expression was quantitated by densitometry and normalized to that of vinculin. The expression relative to cells transfected with a control siRNA is reported along with representative immunoblots. PCNA, proliferating cell nuclear antigen.