Validating the role of PTGIS gene in colorectal cancer by bioinformatics analysis and in vitro experiments – Scientific Reports

Prostacyclin synthase (PTGIS), also known as CYP8, PGIS, PTGI and CYP8A1, is a member of the cytochrome P450 superfamily. It can catalyze the production of arachidonic acid to PGI2 and be involved in various physiological and pathological processes such as adenylate cyclase activation, cAMP elevation and protein kinase A activation, thereby playing the role of vasodilation, platelet aggregation and proliferation vascular smooth muscle cells and inhibits migration, etc^10,11. It can regulate many physiological functions of the cardiovascular system, such as angiogenesis. Studies have shown that differential expression of PTGIS-encoding genes in many diseases is closely related to the occurrence and development of diseases such as pulmonary hypertension (PAH), hepatic fibrosis, etc. As far as we know, there have not been many studies on the functional relationship between PTGIS and tumors. Therefore, whether PTGIS plays an important role in tumors remains to be fully elucidated. Based on the online bioinformatics database, this study comprehensively demonstrated the PTGIS gene expression profiles of 31 different tumors, including gene expression and survival analysis. These results showed that PTGIS expression was significantly downregulated in 17 cancer types, ACC, BLCA, BRCA, CESC, COAD, ESCA, KICH, KIRP, LIHC, LUAD, LUSC, OV, PRAD, READ, THCA, UCEC, UCS. Among the above cancers, only five cancers prognosis were significantly correlated with the expression of PTGIS. Analysis of the survival prognosis plots revealed that high expression of ACC, BLCA, COAD, KIRP, LUSC, and PTGIS was significantly correlated with poor prognosis. It is precisely because of the discovery that the high expression of PTGIS gene is associated with poor prognosis of patients, in this study, we used colorectal cancer cells with significantly lower expression of PTGIS gene than normal colorectal tissue as a cell model to explore its pathophysiological processes of occurrence and development by overexpressing PTGIS gene. It is reported that mutations play an important role in the formation and development of tumors and are considered important targets for tumor diagnosis and treatment¹². Through the analysis of this study, we found that PTGIS mutations are also abundant in different tumors. In particular, the mutation rate of colorectal cancer patients is the highest at over 8%. Among them, PTGIS has the highest frequency of gain type changes. Therefore, this study focuses on the relationship between PTGIS and colorectal cancer.

Currently, colorectal cancer has become a common cancer with the highest incidence and mortality in the world, and the onset is becoming younger in middle age¹³. Early treatment of colorectal cancer patients is mainly limited to surgery, radiotherapy and chemotherapy with obvious side effects and low quality of life after surgery. In recent years, many targeted drugs have emerged for the treatment of advanced colorectal cancer, including antibodies that inhibit EGFR receptor activity, such as cetuximab, panitumumab, kinase inhibitors: regorafenib, etc. However, since cancer, including colorectal cancer, is often a case Due to this very heterogeneous disease, these targeted therapies must inevitably be confronted with the problem of group benefit selection. Therefore, it is very important to further research the mechanism of occurrence and development of colorectal cancer and explore more comprehensive, accurate and effective early detection and treatment methods. Most of the factors that lead to the occurrence of colorectal cancer can be modified and the occurrence monitored, so the prediction of colorectal cancer is of great importance¹⁴.

In this work, we used a bioinformatics database to analyze the correlation between the PTGIS gene and the occurrence and development of colorectal cancer. It was found that the expression of the PTGIS gene was different in normal colorectal tissues and colorectal cancer tissues. We found that expression of the PTGIS gene in colorectal cancer tissue was lower than that in normal colorectal cancer tissue. The q-PCR and WB results confirmed the above results. The expression of the PTGIS gene has an impact on the prognosis of colorectal cancer, the overall survival rate of patients with low expression of the PTGIS gene was higher than that of patients with high expression of the PTGIS gene, the analysis results of the linkedOmics database match those of the GEPIA2 database. In this study, while observing the relationship between PTGIS and the proliferation and invasion of colorectal cancer cells, we found that PTGIS was normally weakly expressed in cancer cells, and SW480 cells overexpressing PTGIS had stronger proliferative ability and higher activity, decreased apoptosis and increased Invasiveness was demonstrated compared to the control group, suggesting that PTGIS gene expression differs from common tumor genes and low PTGIS gene expression leads to better prognosis. The mechanism of the PTGIS gene involved in colorectal cancer progression needs further investigation.

In the last two decades, about 20% of colorectal cancer patients have already formed metastases at the time of diagnosis¹⁵. Some patients classified as low-risk colorectal cancer also have some chance of recurrence or metastasis after treatment¹⁶. The survival rate of patients with metastatic colorectal cancer is less than 15%, while the survival rate of patients with non-metastatic colorectal cancer is over 75%¹⁷. It can be seen that colorectal cancer metastasis is the main factor leading to death. Tumor invasion is a key step in tumor growth and metastasis, which is of great importance for tumor treatment and prognosis. In cancer, EMT is considered to be one of the main mechanisms leading to metastasis and metastatic cancer¹⁸. When EMT is activated, cancer cells gain the ability to invade and metastasize^19,20. In this study, Spearman was used to analyze the correlation between a single gene and the pathway score. It has been found that PTGIS may be related to the occurrence of the EMT mechanism in colorectal cancer. It provides ideas for further research on PTGIS and colorectal cancer.

From the results of the immune infiltration, we can find that the PTGIS gene is positively correlated with mast cell-activated macrophages M2 and macrophages M0 in COAD, which can promote the development of a tumor. In addition, it was negatively correlated with follicular helper T cells, CD8+ T cells, memory-activated T cells CD4+, activated NK cells, resting mast cells, B-cells, plasma cells that have the function of tumor killing or to inhibit. Therefore, it shows that the PTGIS gene can promote the occurrence of cancer in COAD. Furthermore, the PTGIS gene in READ was positively correlated with macrophage M2 and macrophage M0, which could promote tumor development. In addition, it negatively correlated with follicular helper T cells, CD8+ T cells, resting T cell CD4+, resting NK cells, and B cell plasma cells that had the function of inhibiting tumors. Therefore, this shows that the PTGIS gene can promote the occurrence of cancer in READ. In this study, PTGIS was positively correlated with M2 macrophages, which may indicate that PTGIS may be activated in macrophage polarization, and the expression of PTGIS will affect the activation of T cells, which is negatively correlated, indicating that PTGIS inhibits T cell function in colorectal cancer.

Finally, the associated protein network diagram and major biological processes involved in PTGIS gene were retrieved from STRING website, which confirmed that the major enriched biological processes including metabolic pathways, arachidonic acid (AA) metabolism, steroid biosynthesis, pathways in cancer etc^21,22. The mechanism of the PTGIS gene involved in the progression of colorectal cancer needs further. PGI2 is the main metabolite of AA, and PGI2 can activate PPAR to promote the occurrence of cancer²³. PTGIS is a key factor in the increase of PCI2 in tumors, and may affect the release of inflammatory factors through the synergistic effect of AA pathway, thus affecting the recruitment of immune cells. The above results indicate that PTGIS may play an immunosuppressive role by affecting tumor immune infiltrating cells.

However, there are some limitations in this study. We used bioinformatics methods to analyze the differences of PTGIS expression between tumor tissues and normal tissues and their effects on tumor biological characteristics, and predicted the possible downstream mechanisms of PTGIS and the effect on immune infiltration, at the same time, the in vitro experiments of cell biological function were done. Nevertheless, in order to investigate the role of PTGIS genes in the development of colorectal cancer and detailed mechanisms by which PTGIS influences immune infiltration, we need to further research the molecular mechanisms of PTGIS in colorectal cancer in vitro, and, all above results should also be verified in vivo and clinical trials (Supplementary file).

In conclusion, the PTGIS gene may play an important role in the development, invasive and prognosis of colorectal cancer, and may also play a special role in immune infiltration. At present, there are few studies on the role of the PTGIS gene in the pathogenesis of colorectal cancer. Further study on the relationship between PTGIS Gene and colorectal cancer is of great significance for finding highly specific markers for early detection of colorectal cancer and providing new targets for subsequent treatment of colorectal cancer.