RCL1 is abnormally expressed in various tumor tissues, and associated with prognosis and tumor progression.
We first explored the general expression of Rcl1 in multiple human cancers using the GEPIA2 website portal (Figure 1A). The analyses of the RNA-seq data of 23 malignancies in TCGA suggested that the expression of RCL1 was significantly lower in Cholangiocarcinoma and LIHC compared to the adjacent normal tissues. However, RCL1 expression of Colon and Rectum adenocarcinomas were significantly higher than the one in normal tissues.
The TISIDB web portal was used (Figure 1B) to further evaluate the correlation of RCL1 expression and survival prognosis. Notably, RCL1 expression had a significant impact in the prognosis of 8 cancers, including brain lower-grade glioma (LGG), glioblastoma multiforme, kidney renal clear cell carcinoma (KIRC), LIHC, ovarian serous cystadenocarcinoma(OV), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS), and uveal melanoma (UVM). Low RCL1 expression was remarkably associated with poor prognosis in all these cancer types except for UCEC and UCS.
Moreover, the association between RCL1 expression and tumor progression across human cancers was identified. It was revealed that RCL1 expression was positively correlated to tumor stage in KIRC, LIHC, and Stomach adenocarcinoma, as well as in UVM ((Figure 1C). Similarly, the expression of RCL1 in KIRC and LIHC was also positively associated with histological grade ((Figure 1D). However, the RCL1 downregulation was notably correlated with higher grade of Cervical squamous cell carcinoma and endocervical adenocarcinoma and UCEC (Figure 1D).
In summary, these results confirmed that RCL1 could be a potential tumor-associated gene in several melignancies. Notably, it was in LIHC that RCL1 expression was not only significantly down-regulated but also associated with prognosis, tumor progression across many human cancers.
Low RCL1 expression is correlated with poor clinicpathological outcomes in HCC patients.
Eleven HCC datasets were downloaded and analyzed (Figure 1E) to further verify RCL1 expression in HCC tumor tissue. For 9 HCC datasets including GSE22058, GSE25097, GSE36376, GSE14520, TCGA-LIHC, GSE76427, GSE54236, GSE63898, and ICGC-LIR-JP, the expression levels of Rcl1 in HCC tissues were generally lower than the ones in adjacent tissues (p < 0.001).
Univariate and multiple survival analyses were performed using R programming environment on the TCGA-LIHC dataset to promote understanding of the association between the RCL1 expression and the prognosis of HCC . Univariate analysis indicated that patients with the high RCL1 expression was associated with better overall survival (OS, HR = 0.607 (0.416 - 0.886)) and progression-free survival (PFS, HR = 0.661 (0.476 - 0.917)) (Figure 2A-2B). Univariate and multivariate analyses further confirmed that RCL1 expression is an independent factor for OS (HR = 0.616 (0.420 – 0.905)) and PFS (HR = 0.701 (0.502 – 0.98)) of HCC patients (Figure 2C, D, Table 1).
In addition, we studied the connection between RCL1 expression and the clinicopathological characteristics. It was found that low RCL1 expression was significantly correlated with female, advanced primary tumor (T classification) and TNM stage, higher AFP level, as well as vascular invasion in TCGA-LIHC cohort (Figure 3A-E). Besides, it was found that a decrease in the RCL1 expression was associated with increasing T classification, HBV infection, portal vein and hepatic vein invasion in ICJC-LIRI-JP corhort (Figure 3F-I). Meanwhile, a remarkable connection between RCL1 expression was lower in the patients with BCLC C stage, proliferation class, high AFP level, vascular invasion, as well as phosphorylation level of Akt, RPS6, and IGFR1 in GSE9843 corhort (Figure 3J-P). No significant relationship between the RCL1 mRNA expression and age, cirrhosis, as well as gender was found in HCC cohorts (Figure S1).
RCL1 expression significantly correlates with infiltrating levels of various immune cells in HCC, especially CD4+ T cells.
The correlation of RCL1 expression in HCC samples with immune infiltration levels were investigated using TIMER2.0 website portal. The results showed that RCL1 expression significantly correlated with the infiltrating levels of myeloid-derived suppressor cell (MDSC, r = -0.395, p = 2.32e-14), endothelial cell (r = 0.336, p = 1.57e-10), hematopoietic stem cell (r = 0.296, p = 2.21e-08), Tregs (r = 0.279, p = 1.34e-07), monocyte (r = 0.261, p = 9.2e-07), granulocyte-monocyte progenitor (r = 0.255, p = 1.53e-06), CD4+ T cells (r = -0.31, p = 3.92e-09), DCs (r = -0.204, p = 1.35e-04), macrophages (r = -0.197, p = 2.35e-04), CD8+ T cells (r = 0.16, p = 2.81e-03), B cells (r = -0.125, p = 2.01e-02), and neutrophils (r = -0.133, p = 1.36e-02) in LIHC, although no significant correlation with tumor purity was found (Figure 4). Furthermore, the RCL1 expression was negatively associated with the infiltration level of naïve CD4+ T cell (r = -0.131, p = 1.51e-02), Th1 cell (r = -0.182, p = 6.78e-04), Th2 cell (r = -0.245, p = 3.98e-06), M0 macrophages (r = -0.266, p = 5.44e-07), but not with M1 macrophages (r = 0.025, p = 6.40e-01)and M2 macrophages (r = -0.081, p = 1.33e-01) (Figure 4).
Moreover, RCL1 expression was found to be significantly different between molecular and immune subtypes by exploring the TISIDB web portal (p < 0.001, Figure 5A-B). Furthermore, the expression levels of RCL1 in the patients with TP53 and IDH1 mutation were lower than the ones in the patients with TP53 and IDH1 wild-type (Figure 5C, 5D).The expression levels of RCL1 in the patients with CTNNB1 mutation were higher than the ones in the patients with CTNNB1 wild-type (Figure 5E) and no significant difference was observed between the expression levels of RCL1 in TERT mutation and wild-type patients (Figure 5F).
Rcl1 is down expression in HCC cells and suppresses HCC cell growth and metastasis in vitro.
The endogenous Rcl1 expression levels were detected in a collection of liver cancer cell lines and L-02 cells. Both mRNA and protein expression levels of Rcl1 were generally lower in all liver cancer lines in comparison with L-02 cells (Figure 6A, 6B). The Rcl1 expression of high-invasive HCC cell lines was substantially lower than the one in the low-invasive cell lines. These findings were further supported by immunofluorescence staining (Figure 6C). Interestingly, it was also revealed that there was a marked difference in the distribution of Rcl1 protein between the liver cells and cancer cells. In particular, Rcl1 was mostly located in the nucleus in the HCC cell lines, while it was uniformly distributed in nucleus and cytoplasm in the liver cell lines (Figure 6C).
Then a recombinant plasmid vector encoding Rcl1 (FLAG-Rcl1) was conducted and an empty vector was used as control (Ctrl). The overexpression of Rcl1 in Huh-7 cells was validated with RT-PCR and western blot analyses (Figure 7A, 7C). Forced Rcl1 expression could markedly inhibit cell growth as supported by cell viability assay in Huh-7 cell (Figure 7E). Moreover, transwell assays indicated that the overexpression of Rcl1 significantly impaired Huh-7 cell’s ability to migrate and invade (Figurre 7G). Furthermore, Rcl1 was knockdown by transfecting the shRcl1 vector or empty vector into Hep-3B cell line. The efficiency of knockdown was confirmed by RT-PCR and western blot analyses (Figure 7B, 7D). Consistently, knockdown of Rcl1 in Hep-3B cell strikingly enhanced cell viability, migration, and invasion (Figure 7F, 7H).
Rcl1 could potentially participate in regulating cell cycle and metabolism-associated signal pathways.
Notably, the Rcl1positive group of genes were enriched in multiple cellular metabolic processes such as xenobiotic, fatty acid, bile acid, adipogenesis, and oxidative phosphorylation, while the Rcl1negative subgroup of genes were enriched in cell cycle regulation including G2M checkpoint, E2F targets and mitotic spindle (Figure 8A, C). Moreover, gene ontology (GO) analysis revealed that Rcl1 could potentially promote the activation of protein-binding and transmembrane transport, while simultaneously inhibiting microtubule and protein kinase activity (Figure 8B). Mitochondria and chromosomes were the main cellular organelles of the Rcl1positive and Rcl1negative groups, respectively (Figure 8D).
The effects of Rcl1 on cell cycle distribution was evaluated using flow cytometry analysis. We found that the overexpression of Rcl1 resulted in a significant increase of cells in G2M phase (35.10% vs 22.06%, p = 0.002) and a concomitantly significant decrease of cells in S phase (25.62% vs 39.53%, p = 0.0002) in Huh-7 cell (Figure 8E). As expected, upon Rcl1 knockdown, the ratio of cells in G2M phase was decreased (18.89% VS 26.58%, p = 0.003), the ratio in S phase was increased (32.33% VS 17.59%, p = 0.0001), and the ratio in the G0/1 phase was decreased (50.78% VS 55.84%, p = 0.040) (Figure 7F).