It is now known that RCC1 is involved in the regulation of cell cycle processes and can be involved in the repair of DNA damage. Also, there have been many reports indicating that SNHG3 and SNHG12 are aberrantly expressed in many tumor tissues and can be involved in the development of a variety of tumors and diseases. We evaluated the expression of RCC1/SNHG3/SNHG12 in 33 different cancer types using several databases such as GEPIA, UALCAN, and TIMER2.0, and found that they were significantly differentially expressed in tumor tissues and normal tissues. The results showed that, overall, they were all upregulated in BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, and STAD tumor tissues, while downregulated in KICH. In addition, RCC1 and SNHG12 expression were upregulated in THCA, and RCC1 and SNHG3 expression were upregulated in UCEC. However, RCC1 and SNHG3 expression were downregulated in PCPG tumors, while SNHG12 showed upregulation. However, it is noteworthy that the number of normal samples relative to PCPG was only three and the expression differences were not significant.
Regarding the reason for the downregulation of RCC1/SNHG3/SNHG12 expression in KICH, the methylation of RCC1/SNHG3/SNHG12 genes was first analyzed by MEXPRESS tool (https://mexpress.be/). The results showed no statistical significance (Supplementary Fig. 3A). Next, we analyzed the tumor mutation landscape of RCC1/SNHG3/SNHG12 expression in KICH using the SangerBox tool. However, limited by the sample size and other factors, there was no significant difference in the mutation landscape of each gene under high or low expression of RCC1/SNHG3/SNHG12 (Supplementary Fig. 3B). Subsequently, we analyzed the overall differential genes in KICH tumors and their chromosomal distribution using the GEPIA2 tool. It was found that the differential genes were mostly altered with downregulated expression (Supplementary Fig. 3C). We then pooled all differential genes in BLCA, BRCA, CESC, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, and KICH. The genes were divided into two groups according to whether they were downregulated in KICH and upregulated in the rest of the cancers or upregulated in KICH and downregulated in the rest of the cancers. In each group, the intersection of the differential genes in each cancer was taken. However, neither group was finally enriched for the key genes (Supplementary Fig. 3D, E). Since KICH belongs to a rare type of kidney cancer, there are not many studies and analyses about it. Here, we speculate that it may be the result of the synergistic regulation of multiple genes.
In terms of genetic variation, since RCC1/SNHG3/SNHG12 are located adjacent to each other on the chromosome, their CNV and methylation levels are more similar. Especially, in OV, BRCA, LUSC, SARC, LGG and other tumors, their CNV and mRNA expression showed a very significant positive correlation. The effect of methylation level on the mRNA expression of SNHG3 and SNHG12 was greater than that of RCC1. This result may be due to the different lengths of the genes.
RCC1/SNHG3/SNHG12 are not only highly expressed in a variety of cancers, but also are risk factors for poor prognosis. Overall, RCC1/SNHG3/SNHG12 can be used as a marker of poor prognosis in the early stages of pan-cancer. High expression of RCC1/SNHG3/SNHG12 in patients with a variety of cancers including ACC, KIRP, LAML, LGG, LIHC, and PRAD predicted lower overall survival and disease free survival. We additionally analyzed the OS and DFS of RCC1/SNHG3/SNHG12 in KICH. The results showed a trend towards lower survival in patients with high expression of RCC1/SNHG3/SNHG12. This result was statistically different in RCC1, but not statistically significant in SNHG3 and SNHG12 (Supplementary Fig. 4).
In the next immune correlation analysis, we found some very interesting phenomena. Based on the results of the TIMER 2.0 tool, it appears that RCC1/SNHG3/SNHG12 were positively correlated with immune cell infiltration in MDSC and negatively correlated with hematopoietic stem cell and eosinophil. In addition, RCC1 was positively correlated with the degree of infiltration of common lymphoid progenitor. MDSC can significantly suppress the immune cell response, protect cancer from the immune system, and makes the tumor resistant to immunotherapy[41, 42]. T cell follicular helper (Tfh) is a specific subpopulation of CD4 + T cells that plays a key role in protective immunity. Tfh function is dysregulated in several diseases where antibody production is excessive or insufficient. The amount of circulating Tfh is increased in the blood of patients with autoimmune diseases[43–46]. We think that SNHG3/SNHG12 is positively correlated with immune infiltration in MDSC on the one hand, and with Tfh on the other hand, suggesting that the immune regulatory processes involved are complex. Meanwhile, combined with the results of survival analysis, the role of RCC1/SNHG3/SNHG12 in some cancers is dominated by suppression of immune response.
In contrast, for individual cancers, SNHG3 behaved more similarly to RCC1, especially in LGG, LIHC, PCPG, THYM, etc. In THYM, RCC1/SNHG3 was positively correlated with immune infiltration of CD8 + and CD4 + T cells and negatively correlated with immune infiltration of macrophage, NK cells, and cancer associated fibroblast (CAF). CAF is an important component of the tumor microenvironment and has multiple functions including matrix remodeling. Current studies suggest that CAF drives cancer growth and progression by remodeling the tumor microenvironment and contributes to increased tumor drug resistance[47–50]. We additionally analyzed the OS and DFS of RCC1/SNHG3/SNHG12 in THYM patients. The results showed that the expression of RCC1/SNHG3 correlated with OS in THYM patients, and the OS was higher in patients with high expression of RCC1/SNHG3. In contrast, this phenomenon was not mentioned in SNHG12 (Supplementary Fig. 5).
The results of immunoassays based on the GSCA tool showed that SNHG3 behaved more similarly to RCC1, while there were some differences in SNHG12. Similarly, RCC1/SNHG3/SNHG12 showed a very significant correlation with various immune cell infiltration in THYM. When RCC1/SNHG3/SNHG12 was analyzed as a gene set, its overall performance was very similar to that of RCC1. We suggest that the gene encoding the protein plays a primary role in the process of immune regulation, while the lncRNA may play a secondary supporting role. Taken together, RCC1/SNHG3/SNHG12 are involved in the process of immune cell infiltration, and the process is more complex. In particular, RCC1 and SNHG3 can be used as immune detection markers for THYM.
The phenomenon of RCC1/SNHG3/SNHG12 in immune regulation is also reflected in the regulation of cellular processes. In particular, the correlation of RCC1 was more significant in apoptosis and cell cycle. The correlation of SNHG3/SNHG12 was not as significant as that of RCC1, but the regulatory trend remained consistent with RCC1. In addition, the effects of RCC1/SNHG3/SNHG12 on each cellular pathway in BRCA, PRAD, THCA, and THYM were significantly correlated, especially in THYM. Notably, in addition to the involvement of RCC1 in DNA Damage Response, Cell Cycle, and Apoptosis pathways that are currently known, RCC1/SNHG3/SNHG12 also positively correlated with hormone AR pathway and negatively correlated with hormone ER in THYM. Androgen receptor (AR) belongs to the steroid hormone family and is involved in the regulation of normal growth and development of various target organs. The current research and application of AR are mainly in prostate cancer and breast cancer[51, 52]. Several studies have been reported on the involvement of LncRNA in the regulation of AR[53, 54]. In Philling et al. study, activation of AR increased cell viability and survival and attenuated G2/M arrest. AR negatively regulated spindle checkpoint signaling, leading to premature mitotic progression and apoptotic cell death evasion[55]. Estrogen receptors (ER) belong to protein molecules, including nuclear and membranous receptors[56]. Most reports on ER have focused on breast cancer diagnosis and treatment[57, 58]. In bladder cancer, ERα activation is thought to have an inhibitory role in tumor growth, as its knockdown promotes the growth of cancer cells and xenograft tumors[59]. And in a study of gastric cancer, the authors found that ERα overexpression significantly inhibited cell growth and proliferation, promoted apoptosis, and blocked cell entry into the G1/ G0 phase[60, 61]. Thus, it seems reasonable that RCC1/SNHG3/SNHG12 are positively correlated with the AR pathway and negatively correlated with the ER in THYM. On the one hand, they positively regulate the AR pathway to attenuate G2/M arrest and promote the mitotic process. On the other hand, they negatively regulate the ER pathway to ensure that cells enter the G1/G0 phase and ensure the stability of the mitotic process. The overall trend is consistent with the regulation of cell cycle and DNA damage repair by RCC1.
After GO and KEGG analysis of the proteins that can interact with RCC1/SNHG3/SNHG12, we found that the cellular components and biological processes they are involved in are very similar to the localization and function of RCC1. GO analysis showed that they are mostly located on chromatin in the nucleus and their molecular functions are mainly involved in chromatin binding, DNA binding, p53 binding, etc. Remarkably, in addition to the positive regulation of cell cycle and mitosis, they are also involved in the regulation of a variety of cells and their differentiation in terms of biological processes, including hemopoiesis, myeloid cell, stem cell, myeloid leukocyte, etc. The positive correlation of RCC1/SNHG3/SNHG12 with immune cell infiltration in MDSC seems to be more convincing in the previous immune correlation analysis. In a subset of tumors, high expression of RCC1/SNHG3/SNHG12 caused enhanced mitotic and DNA damage repair processes in tumor cells, thereby enhancing cell viability and promoting cell proliferation. At the same time, RCC1/SNHG3/SNHG12 may in turn influence the regulation of immune cell infiltration in this tumor by directly or indirectly regulating the proliferation and differentiation of some immune cells.
In conclusion, RCC1/SNHG3/SNHG12 may play a cancer-promoting role in many tumors, while it seems to play a cancer-suppressing role in THYM. We conjecture that there seems to be a phenomenon, which we refer to as "homodomain effect", where coding and non-coding genes at the same or very adjacent positions on the chromosome have similar and possibly synergistic effects on cellular function. This effect is dominated by coding genes, and non-coding RNAs located on their sequences, such as lncRNAs, can produce the same or similar effects. Of course, this conjecture needs to be verified by more experiments and studies. Meanwhile, the aberrant expression and role of RCC1/SNHG3/SNHG12 in different tumors and its potential clinical application value also deserve further exploration.