In this study, we identified a 5-gene-based prognostic model by comparing normal tissues with rectal cancer and further validated the predictive power of the model in two independent rectal cancer datasets. Our findings indicated that the OS rates increased with the risk-score in the TCGA and GSE133057 rectal cancer datasets. Strikingly, both the high- and low-risk groups showed a significant association with the TME, especially non-regulatory CD4+ T cells and CD8+T cells. Moreover, the data showed that the risk-score was significantly associated with vascular invasion (Table I). Based on these results, we propose the use of the 5-gene-based classification model as a novel molecular-based prognostic tool to evaluate the survival of rectal cancer patients. The model provides a starting place for further research regarding the role of genes in the development of rectal cancer.
Rectal cancer is a tumour with a relatively high prevalence but without convincing prognostic and predictive molecular markers. It has been established that biomarkers can classify rectal cancer patients with certain subtypes, some of which may benefit from tailored therapy. Several important factors have been linked to rectal cancer survival, and these molecules participate in key processes involved in cancer development, including cell migration and invasion[19–23]. Although these factors were considered to be significant in colorectal cancer, the findings were not verified in rectal cancer via validation in an independent dataset. Our results were validated in a separate cohort with rectal cancer patients. These data supported the findings that the 5-gene-based signature well served as a predictor for survival in rectal cancer patients.
Our study showed a strong correlation between the 5-gene-based tumour markers and clinical outcomes of rectal cancer. The molecular function of CLIC5, ENTPD8, PACSIN3, HGD, and GNG7 remains unclear, partly due to the lack of original research in rectal cancer. Chloride intracellular channel 5 (CLIC5) belongs to the family of chloride (Cl−) channels which are responsible for encoding chloride intracellular channel (CLIC) proteins. To date, there are six known members in the CLIC family (CLIC1-6), and accumulating evidence supports their role in tumour biology, especially gastrointestinal cancer. A previous study identified upregulated changes in CLIC1 in colorectal cancer (CRC), which was shown to be associated with poor prognosis in CRC patients. CLIC4 was also found to be overexpressed in CRC, and its upregulation was correlated with unfavourable 5-year clinical outcomes. To date, there have been no reports of a relationship between CLIC5 and rectal cancer. Ectonucleoside triphosphate diphosphohydrolase 8 (ENTPD8) is a member of the ectonucleoside triphosphate diphosphohydrolases (E-NTPDase) family, plays an essential role in ATP metabolism and is mainly expressed in the intestine. Although ENTPD8 is still poorly understood, there is evidence that it plays a crucial role in pancreatic cancer and exhibits metabolic activity toward gene-metabolite networks. PACSIN3 was identified as an intracellular adapter protein that regulates endocytosis, vesicle transport, membrane internalization, and actin reorganization. As reported, PACSIN3 is one of the mobility-related genes that is downregulated in ING5-overexpressing SGC-7901 gastric cancer cells. Another study also found that PACSIN3 was decreased in prostate cancer. To our knowledge, there is no report of a study about PACSIN3 in rectal cancer. The HGD gene encodes one of the enzymes called homogentisate 1,2 dioxygenase, which is required for the catabolism of the amino acids tyrosine and phenylalanine and is generally active in the kidneys and liver to catalyze oxidation-reduction reactions. The current study showed that high HGD mRNA expression (≥ 3-fold) was associated with poorer survival of, histological grade, advanced stage, and metastasis of cholangiocarcinoma patients. Previous results have shown that HGD is a potential key factor in the regulatory mechanism of BRAFV600E-mediated PTC. However, there was no significant discrepancy in overall survival. G protein γ subunit 7 (GNG7), which is a component of the large G γ family, was first identified to be a downregulated differentially expressed gene in pancreatic cancer and was then found in gastrointestinal tract cancer (including oesophagal, gastric, and colorectal cancer). In a previous study, it was shown that GNG7 acts as a potential tumour suppressor both in vitro and in vivo. A similar study also demonstrated that it was a tumour suppressor gene in clear cell renal cell carcinoma and lung adenocarcinoma. Taken together, our research is the first work to evaluate the survival value of a 5-gene-based tumour marker in rectal cancer and might aid in the improvement in molecular prognosis.
Collectively, several studies have shown the clinical importance of immune infiltrates in colorectal cancer. Galon and his colleagues examined tumour-infiltrating lymphocytes (TILs) in approximately 400 colorectal cancer samples and found that CD8+ and CD45RO+ T cells in the tumour were superior predictors to the histopathological staging methods. Previously, studies have also shown that CD4+ and CD8+ T cells were promised survival predictors in colorectal cancer patients. A significant correlation was observed between the density of CD8+ T cells in the peritumoral region and a longer disease-free interval (P = 0.009), and Kaplan–Meier analysis later suggested that the percentage of CD8+ T cells might have clinical application in the stratification of the relapse risk of patients (P = 0.006). Yasuda K et al. reported that tumour-infiltrating lymphocytes (TILs), especially density CD4+ T cells and CD8+ TILs, were strongly associated with the tumour treatment response of rectal cancer after neoadjuvant chemoradiotherapy (nCRT). As mentioned earlier, our study found that the 5-gene signature was strongly correlated with tumour-specific CD4+ and CD8+ type T cells. Obviously, these data were consistent with studies of TILs in colorectal cancer.
Another interesting finding in our study was that the 5-gene signature was predicted to be intersected in hsa-miR-6887-3p, while only GNG7 correlated with hsa-miR-6887-3p. MicroRNAs (miRNAs) are critical tumorigenesis mediators in many human cancers. The role of miRNAs as clinical biomarkers in colorectal cancer research is promising . Many studies have documented aberrant miRNA levels as biomarkers for colorectal cancer and have reported the evaluation of their potential roles as diagnostic and prognostic indicators. Li H demonstrated that hsa-miR-6887-3p inhibited the tumorigenesis of colorectal cancer by downregulating Mex3a expression and functioned as an important regulator in the hsa-miR-6887-3p/Mex3a/RAP1GAP signalling axis. However, the clinical application of miRNAs as predictive biomarkers in rectal cancer remains to be seen. Our data also suggested the need to further studies to investigate the biological role of hsa-miR-6887-3p and GNG7 in rectal cancer.
It is noteworthy that the 5-gene signature was correlated with the vascular invasion (P = 0.038), but not the lymphatic invasion. The vascular invasion has been associated with an increased risk of regional and distant metastasis in colorectal cancer patients. Some authors have reported that vascular invasion was a strong prognosticator for rectal cancer that influenced disease progression and survival[37–39]. Thus, a few analyses have failed to indicate the prognostic value of vascular invasion in colorectal, colon, and rectal cancer survival[40, 41]. It is therefore important to identify vascular invasion-related biomarkers to better diagnose and classify rectal cancer patients. However, until now, no validated tumour markers have been identified[41–43]. Our findings suggested that the vascular invasion is critical for the progression of colorectal cancer which warrants further investigation. The lymphatic invasion has also been reported to be linked with an adverse prognosis of colorectal cancer. However, our data did not show any statistically significant association between lymphatic invasion and prognosis. Based on our results, further research into the function of the 5-gene signature in vascular invasion is warranted.
One potential limitation of our research was that it was predominantly based on bioinformatics results. Our study was designed to decrease bias and increase the repeatability of the analytic results based on the use of two independent large-sample datasets. Nevertheless, our findings needed to be validated in a more prospective study, and the correlation between gene markers and other factors requires further investigation. Another key question is whether the 5 genes indeed affect the progression of rectal cancer. Consequently, future research will be essential to understand the biological association between the expression of these 5 genes and rectal cancer.
To summarize, as highlighted in this paper, the 5-gene-based signature was a robust prognosticator for rectal cancer patients. Moreover, the risk-score generated by the signature was shown to have the ability to further classify the patients into low- and high-risk groups. In addition, the prognosis of the two groups was significantly different, and the high-risk group had a more unfavourable OS. Our findings were confirmed in an independent validation set and subsequently evaluated by time-ROC curves and nomograms. Further analyses demonstrated that the two groups of patients differed significantly with respect to nonregulatory CD4+ T cells and CD8+ T cells. Our results also suggested that there were clear differences in vascular invasion (P = 0.038) between the high- and low-risk groups, and the high-risk group exhibiting a higher risk of vascular invasion.
More importantly, this was an independent prognostic factor based on the Cox regression model. Our research aimed to uncover the complex interaction between 5 genes and clinical outcomes in rectal cancer through the use of microRNA and single-cell analysis tools. In summary, our results indicated that the 5-gene-based signature could contribute to the prognostic evaluation of rectal cancer and might pave the way for new therapeutic strategies in the foreseeable future.