Nucleolar Protein 6 Promotes Cell Proliferation and Acts as a Potential Novel Prognostic Marker for Hepatocellular Carcinoma
Hepatocellular carcinoma (HCC) is the most common type of primary liver malignancy. Early-stage HCC is typically treated with resection or liver transplantation, while late-stage patients are limited to kinase inhibitors such as sorafenib and regorafenib, and immune checkpoint inhibitors like pembrolizumab and nivolumab. The lack of early diagnostic markers and limited treatment options contribute to the high lethality and poor prognosis associated with HCC. Therefore, a comprehensive analysis of HCC is essential to develop effective therapeutic strategies.
Nucleolar protein 6 (NOL6), also known as NRAP, utp22, and baa311h10.1, is encoded by the NOL6 gene on human chromosome 9. NOL6 is a nucleolar RNA-associated protein highly conserved among species. Its molecular functions include the establishment of RNA binding and localization, and it is associated with ribosome biogenesis through interaction with pre-rRNA primary transcripts. Alternative splicing at this locus has identified two splice variants encoding different subtypes. The Human Protein Atlas indicates that NOL6 is related to HCC prognosis, with high expression protecting against liver cancer occurrence. However, the specific mechanism of how NOL6 affects liver cancer prognosis remains unclear.
This study aimed to investigate the functions of NOL6 in human liver cancer cell lines, BEL-7404 and SMMC-7721. We constructed an NOL6-short hairpin RNA (shRNA)-expressing lentivirus and evaluated the effects of shRNA-mediated NOL6 knockdown on cell proliferation, colony formation, and apoptosis. Furthermore, we constructed a gene expression profile chip containing samples with NOL6 knockdown and studied potential downstream genes altered by changes in NOL6 expression, examining their functional pathways.
NOL6 Expression in HCC Tissues and Cells
We first validated the expression of NOL6 in The Cancer Genome Atlas database (368 liver cancer tissues vs. 160 normal liver tissues). NOL6 expression was higher in HCC tissues than in normal liver tissues, particularly in patients with higher tumor stage, grade, or lymph node positivity. These results were also verified in the TIMER database (Tumor Immune Estimation Resource). RNA sequencing data, including multiple tumor types, showed relatively high NOL6 expression in HCC and many other malignant tumors.
To investigate the role of NOL6 in HCC, we tested its expression in liver cancer cell lines by real-time quantitative PCR. NOL6 was highly expressed in various liver cancer cell lines compared to normal liver cells. Based on these findings, we selected BEL-7404 and SMMC-7721 cell lines for subsequent experiments.
Survival Rate of HCC with High NOL6 Expression
To determine if NOL6 gene expression is related to prognostic characteristics, we analyzed gene expression profiles in the Kaplan-Meier plotter database, which contains 364 HCC patients, using the univariate Cox proportional hazards regression model. High NOL6 expression (at the mRNA level) was significantly negatively related to overall survival (hazard ratio: 1.71, 95% confidence interval: 1.43–2.06, P < 0.001). Similar results were found in 341 HCC samples in the Human Protein Atlas, where high NOL6 expression was associated with poor prognosis, especially in patients with stage I–II HCC.
Knockdown of NOL6 Inhibits HCC Cell Growth
We constructed shRNAs containing NOL6-targeting or non-silencing sequences cloned into GV115 plasmid vectors. NOL6 shRNA lentivirus or non-silencing lentivirus (negative control) expressing GFP was generated and applied to BEL-7404 and SMMC-7721 cells. Western blot analysis showed that NOL6 protein expression was downregulated in cells infected with NOL6-shRNA lentivirus compared to those infected with non-silencing lentivirus. Quantitative real-time PCR (qRT-PCR) assays indicated that NOL6 mRNA levels were reduced by approximately 60% in both cell lines treated with NOL6 shRNA lentivirus.
To assess the role of NOL6 in HCC cell proliferation, cells were infected with shRNA lentivirus and counted continuously for 5 days using Cellomics Arrayscan. The number of BEL-7404 cells increased approximately five-fold in the control group and two-fold in the NOL6-shRNA group from day 0 to day 5, indicating that NOL6 knockdown inhibits BEL-7404 cell proliferation. Similar results were observed in SMMC-7721 cells. MTT assays also showed lower optical density at 490 nm (OD490) in the NOL6 downregulated group, representing reduced cell proliferation activity.
Knockdown of NOL6 Inhibits Colony Formation of HCC Cells
We studied the colony formation ability of HCC cells treated with NOL6 shRNA lentivirus. Cells were allowed to grow for 12 days to form colonies. NOL6 knockdown resulted in a significant reduction in the number of colonies in both HCC cell lines (P < 0.01), suggesting that NOL6 is associated with the colony-forming ability of HCC cells.
Knockdown of NOL6 Increases HCC Cell Apoptosis
We detected the effect of NOL6 shRNA on HCC cell apoptosis using annexin V-APC staining by fluorescence-activated cell sorting (FACS) 72 hours after infection. The percentage of apoptotic cells in the NOL6 knockdown group increased several times, indicating that NOL6 knockdown promotes HCC cell apoptosis.
Potential Downstream Effectors of NOL6: MAPK8, CEBPA, and FOSL1
After applying NOL6 shRNA lentivirus or non-silencing lentivirus to BEL-7404 cells, we divided the cells into two groups and used human gene expression chips to detect differentially expressed genes. Ingenuity Pathway Analysis (IPA) revealed genes related to cell proliferation and apoptosis. Genes such as MAPK8, CEBPA, and FOSL1 were at the center of the gene relationship network. Western blot analysis showed that MAPK8, CEBPA, and FOSL1 expression changed in HCC cells with differential NOL6 expression, suggesting that NOL6 affects HCC cell proliferation and apoptosis through these genes.
Pathway enrichment analysis of differentially expressed genes showed that the top four downregulated functional pathways included cancer, organismal injury and abnormalities, gastrointestinal disease, and hepatic system disease.
Discussion
NOL6 is a nucleolar protein highly conserved throughout evolution. Previous studies have linked its nucleolar localization to ribosomal biogenesis. Li et al. showed that loss of NOL6 expression resulted in G1 phase arrest and cell death induction, demonstrating its involvement in rRNA processing and cell cycle progression.
This study proved that NOL6 is highly expressed in HCC cells and associated with poor prognosis in HCC patients. Knockdown of NOL6 significantly inhibited cell proliferation and colony formation and promoted apoptosis in HCC cells, suggesting its role in tumor progression. NOL6 may become a target for anti-HCC treatment.
Through GeneChip and IPA analysis, MAPK8, CEBPA, and FOSL1 were identified as potential downstream genes of NOL6. These genes are closely related to cell apoptosis. FRA1, encoded by the FOSL1 gene, can directly bind to CEBPA’s promoter. NOL6 can upregulate FRA1 and JNK1 (protein of MAPK8), both of which can downregulate CEBPA and affect apoptosis. CEBPA, a tumor suppressor, prevents uncontrolled cell growth and is involved in blood cell maturation and HCC development.
Targeted molecular therapy, including RNA interference, has shown great potential in cancer treatment by specifically reducing target gene expression. Lentivirus-mediated NOL6 knockdown targets proliferating cells without genetic toxicity, making it a useful target in anti-HCC therapy.
In summary, NOL6 is involved in cell proliferation and apoptosis in HCC cell lines, and its expression is linked to HCC patient prognosis. NOL6 may regulate cell proliferation and apoptosis through MAPK8, CEBPA, and FOSL1 genes, playing a role in HCC occurrence and development. Further research is needed to elucidate the specific and detailed functions of NOL6.
doi.org/10.1097/CM9.0000000000001655
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