A novel biomarker Linc00974 interacting with KRT19 promotes proliferation and metastasis in hepatocellular carcinoma

Abstract

Location-associated long noncoding RNA (lncRNA) was reported to interact with target protein via a cis-regulatory process especially for the Flank10kb class lncRNA. Based on this theory, we aimed to explore the regulatory mechanisms of Linc00974 and KRT19 (an lncRNA beyond the Flank10kb class with protein) when we first confirmed the aberrant expression in hepatocellular carcinoma in a previous study. Knockdown of Linc00974 resulted in an inhibition of cell proliferation and invasion with an activation of apoptosis and cell cycle arrest in vitro, which was also validated by a subcutaneous and tail vein/intraperitoneal injection xenotransplantation model in vivo. We further investigated the interaction pattern of Linc00974 and KRT19. MiR-642 was identified, by acting as the competing endogenous RNA in regulating Linc00974 and KRT19. Linc00974 was increased owing to an abnormal hypomethylation promoter, which induced the upregulation of KRT19 via ceRNA interaction, resulting in the activation of the Notch and TGF-β pathways as detected by cDNA microarray. We also discovered Linc00974F-1 stably expressed in the plasma. By the combined analysis of Linc00974F-1 with CYFRA21-1, we found that these joint indicators predicted growth and metastasis of tumor in HCC patients. In conclusion, the combination of Linc00974 and KRT19 may be novel indices for clinical diagnosis of tumor growth and metastasis in HCC, while Linc00974 may become a potential therapeutic target for the prevention of HCC progression.

Figure 1

Linc00974 was upregulated with a high correlation with KRT19 in HCC. (a) A relatively increased level of Linc00974 was detected in HCC tissues compared with the corresponding adjacent tissues (n=150). Data was log-transformed as presented with mean±S.D. (b) Subcellular localization investigation indicated that the transcript for Linc00974 was located mainly in the cytoplasm of Huh7 and HepG2 cell lines, according to the results of RT-PCR amplified with separated cytoplasm RNA and nucleus RNA. HPRT was used as the control for cytoplasmic expression and U2 for cytonuclear expression. (c) The bisulfite sequencing method was used to detect methylation of CpG island predicted in Linc00974. The methylation level of the Linc00974 promoter was downregulated in tumor tissues compared with the corresponding adjacent tissues. (d) Paraffin sections from the above 150 patients were used to examine KRT19 expression; the left indicated a negative stain of KRT19 while the right presented the positive stain. (e) Pearson correlation showed a positive correlation between expression levels of KRT19 and Linc00974 with a P<0.0001, R²=0.7753. (f) Relative expression level of KRT19 mRNA and protein in HCC samples. Data was log-transformed as presented with mean±S.D. (*P<0.01)

Figure 2

Linc00974 promote cell proliferation in KRT19-positive cells. (a) Immunohistochemical analysis was applied to detect KRT19 in HCC cell lines. We selected Huh7 (left panel) as a 100% positive stain while Hep3B (right panel) indicated an absence of KRT19 with a magnification of × 200. (b) The CCK8 assay presented showed that a decreased level of Linc00974 inhibited the growth of Huh7, and by contrast, no difference was observed in Hep3B. Absorbance at 450 nm was presented as the mean±SEM. (c) The EDU assay confirmed the functional role of Linc00974 in cell proliferation. Stable knockdown of KRT19 reduced the proliferation of Huh-7 after 24 h (P<0.01) compared with the negative control while no significant differences in proliferation were found in Hep3B with a magnification of × 200. The IOD value of cells treated with control plasmids was normalized to 100%. (d) Cells treated with lncRNA shRNA and KRT19 shRNA followed. Forty-eight hours after treatment, cells were stained and analyzed by flow cytometry. LR, early apoptotic cells; UR, terminal apoptotic cells. (e) At 48 h after treatment, cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G1–G0, S, or G2–M phase. All experiments were performed in triplicate and presented as the mean±S.E.M. (*P<0.05, **P<0.01)

Figure 3

Decreased level of Linc00974 inhibited cell invasion with the downregulation of KRT19 in KRT19-positive cells. (a) Cell morphology graph of invasive cells in Huh7 and Hep3B cells after stable transfection of Linc00974 shRNA or negative control. (b) Cells were treated with KRT19 shRNA and control shRNA in Huh7. Magnification: × 400. The number of cells treated with control plasmid was normalized to 100%, and data are presented as the mean±S.E.M. based on at least three independent experiments. *P<0.05. (c) Immunohistochemical analysis was applied to detect the expression of KRT19 in cells treated with Linc00974 shRNA and control. IOD analysis indicated that Huh7 cells transfected with Linc00974 shRNA had a decreased level of KRT19 while no difference was obtained in Hep3B cells. Magnification: × 400. IOD value of cells treated with control plasmid was normalized to 100%. IOD value was presented as the mean±S.E.M. *Indicates a significant difference compared with the control group (P<0.05). **P<0.01

Figure 4

Linc00974 downregulation suppressed tumor growth and metastasis in vivo. (a) Bilateral axillary of BALB/C nude mice were subcutaneously transplanted with Huh7 or Hep3B stably expressed with Linc00974 shRNA (left axilla) or control (right axilla) (n=5). As was indicated by arrows, 4 weeks after implantation, Linc00974 decreased Huh7 attenuated tumor growth in nude mice. The volume of each tumor was calculated as the length × width² × 0.5. The tumor volume of cells treated with controls was normalized to 100%. Data were presented as the mean±S.E.M. *Indicates a significant difference compared with controls (P<0.05). (b) Representative figures of tail vein xenograft model indicated lung colonization, which was formed in Huh7 cells compared with controls (n=10). The number of mice with metastastic foci in the lung was calculated in each group, as presented in the table. Four out of the 10 mice in Huh7 treated with controls presented lung colonization while no metastatic foci were obtained in cells treated with Linc00974 shRNA. The chi-squared test was used to analyze the difference (P=0.025). (c) An intraperitoneal transplantation vaccination tumor model was applied to detect abdominal metastases. As presented in the left panel, fluorescence intensity was measured using the IVIS Lumina II system. Mice injected withHuh7 resulted in the suppressed metastasis of tumor cells compared with controls; however, no statistically significant difference was obtained in Hep3B. The value of fluorescence intensity in control cells was normalized to 100% data were presented as the mean±S.E.M. *Indicates a significant difference compared with the control group (P<0.05), **P<0.01

Figure 5

Linc00974 could regulate KRT19 via ceRNA interacting mechanism with the invasion of miR-642. (a) Relative RIP experiments were performed using an antibody against KRT19 on extracts from Huh7 with IgG as a negative control. The enrichment of the Linc00974 was normalized to the input. The purified RNA was used for RT-PCR analysis. The results showed that no bands were detected from the RNA in the group with anti-KRT19. (b) Relative expression level of miR-642 in clinical samples. Data was log-transformed as presented with mean±S.D. (c) Bioinformatics predicted the binding site between the miR-642 with Linc00974 and KRT19, and the mutation types were conducted into the pGL3 plasmid as presented. (d) Cells were co-transfected with miR-642a mimics or control, Renilla luciferase vector pRL-SV40 and Linc00974 full length or KRT19 3′UTR luciferase reporters for 48 h. Both firefly and Renilla luciferase activities were measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. Cells treated with controls of miRNA were normalized to 100%. (e) KRT19 protein expression level was detected by western blotting. Cells were treated with Linc00974 shRNA or miR-642a shRNA and the respective control plasmid. IOD was calculated for each band. All tests were performed in triplicate and presented as the mean±S.E.M. *P<0.05, **P<0.01

Figure 6

Linc00974 might act as a biomarker in HCC patients. (a) Five primers spaced every 500-bp across the complete Linc00974 transcript were designed. qRT-PCR was used to detect the expression of all fractions in HCC plasma samples. The results indicated that fraction1 was the highest expressed in plasma. (b) The PCR product was applied for agarose electrophoresis for validation. (c) Expression of Linc00974 was detected in patients in whom plasma was obtained from both preoperative and postoperative samples, by comparing with patients free of tumor. ROC curve analysis of merged Linc00974F-1 and CYFRA21-1 was employed to detect the diagnostic efficiency of HCC. Sensitivity and specificity are listed in the left of the curve. (d and e) Expression of Linc00974 was detected in subgroups grouped by tumor diameter (cutoff: 5 cm) and metastasis. Further ROC curve analysis was used for merged Linc00974F-1 and CYFRA21-1 to predict tumor growth and metastasis in HCC. All experiments are presented as the mean±S.E.M. *Indicates significant difference compared with the control group (P<0.05)