Integrated bioinformatics analysis of apolipoprotein M in thyroid cancer

Background Throid cancer is one of the most common cancer worldwide and its mechanism of development remains elusive.Apolipoprotein M (ApoM) is associated with lipid metabolism, inammation and atherosclerosis, but the prognostic value of apoM in thyroid cancer has not been well studied. Methods In this study, transcriptional expression, survival, gene ontology and networks of apoM in patients with thyroid cancer were analyzed using integrated bioinformatics tools including UALCAN, GEO, LinkedOmics, GeneMANIA, STRING, CircNET and KOBAS. Results Results indicated that ApoM is decreased in thyroid cancer tissues and is therefore negatively associated with malignant clinicopathological parameters. However, Kaplan-Meier analyses showed that ApoM expression is not an independent and signicant prognostic factor for overall survival in thyroid cancer. LinkedOmics Conclusions These results indicate that integrated bioinformatics analysis provide valuable information on apoM expression and potential regulatory networks in thyroid cancer. This information will be crucial in understanding the role of apoM in thyroid carcinogenesis. This study investigated ApoM involvement in development of thyroid cancer using integrated bioinformatics tools. Integrated bioinformatics is mainly used for determining differential expression of target molecules and generating survival analyses. We report here downregulation of ApoM in thyroid cancer tissues compared to that of normal thyroid tissues. The expression level of ApoM mRNA was signicantly associated with clinicopathological parameters form the TCGA database. There was no signicant association between ApoM expression levels and survival of thyroid cancer patients. Using the GEO database, we further validated tha ApoM mRNA levels were downregulated in the thyroid cancer tissues compared with the normal thyroid tissues. ApoM is predominately located in the cytocol Golgi and plays an active role in lipoprotein metabolism and HDL assembly. Low levels of ApoM mRNA were observed in thyroid cancer tissues at late disease stage, nodal metastasis and histologic subtype. This implies that ApoM expression in normal tissues could affect normal physiological cellular function and inhibit malignant transformation.


Background
Thyroid cancer is the most common endocrine malignancy and presents in four differentiation types; anaplastic carcinoma, medullary carcinoma, follicular carcinoma and papillary carcinoma [1]. It is responsible for 567,233 cases worldwide, ranking in ninth place cancer incidences, or 1 in 20 cancer diagnoses in 2018 [2]. The estimated percentage of people who live at least ve years after thyroid cancer diagnosis (5-year survival rate) is 98%. However, this rate varies depending on speci c type, location, spread and stage of the disease. For instance, based on location and spread, the 5-year survival rate of localized, regional and metastatic thyroid cancer is 99%, 96% and 56%, respectively [3]. With the prevalence of unhealthy lifestyle and improvements in disease awareness and detection services, the incidence of thyroid cancer increased by 4.73 times from 2.40 per 100,000 in 2003 to 13.75 per 100,000 in 2012 with an average annual increase of 20%, while thyroid cancer mortality only increased slightly around 0.32 per 100,000 from 0.26 per 100,000 to 0.36 per 100,000 [4]. Therefore, it is necessary to identify reliable and effective biomarkers and unravel the genetic elements involved in thyroid carcinogenesis.
Apolipoprotein M (ApoM) is mainly associated with high-density lipoprotein (HDL), with only a small proportion located in low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) [5]. Studies also indicate that ApoM plays a role in formation of preβ-HDL and in the antioxidative properties of HDL [6]. In addition, ApoM has been shown to play a role in stimulating HDL cholesterol e ux capacity and increasing its antioxidant activity [7,8]. ApoM is a carrier and a regulator of sphingosine 1-phosphate (S1P), an important multi-functional bioactive lipid. ApoM also exerts its anti-oxidation and antiin ammatory of HDL by delivering S1P to S1P receptors on endothelial cells [8][9][10]. Despite having atheroprotective capacity, apoM have been shown to play a role in development of some cancers. Our group has previously explored the biological effects of apoM in liver cancer [11], colorectal cancer [12] and lung cancer [13]. We have reported the role of ApoM in promoting proliferation and invasion in nonsmall cell lung cancers via upregulating sphingosine 1-phosphate receptor 1 (S1PR1) and activating the ERK1/2 and PI3K/AKT signaling pathways [13]. However, the expression and role of ApoM in thyroid cancer developmen and progression is unclear.
In this study, we explored the role of ApoM in thyroid cancer, deducing biological insights from The Cancer Genome Atlas (TCGA) datasets and Gene Expression Omnibus (GEO) datasets. Using integrated bioinformatics, we analyzed the correlation, survival, Gene Ontology (GO) and relative gene expression of apoM in patients with thyroid cancer. So, we can obtain overall information regarding the function of apoM to uncover the underlying mechanisms of thyroid cancer occurrence and development.

Statistical analysis
Graphpad Prism 7.0 software (Graphpad Software Inc) was used for statistical analysis. Differential expression analysis of ApoM was conducted using LIMMA software package hosted by Bioconductor 3.1 at p<0.05.

UALCAN analysis
From the TCGA thyroid cancer dataset, ApoM had a signi cantly higher expression in normal thyroid tissues than in tumor tissues (Fig. 1). Moreover, the associations between the ApoM mRNA expression levels and some clinicopathological features, including different tumor stages, histological subtype and nodal metastasis status, were present ( Fig. 2 A-C). In survival analysis, the mRNA levels of apoM were not associated with overall survival in thyroid cancer (Fig. 2D). These ndings suggest ApoM could play a potential tumor-suppressive role in thyroid cancer development and progression.

GEO data validation
As shown in Fig.3, GEO data (GSE33630) analysis showed that apoM mRNA expression was signi cantly higher in normal tissues than in thyroid cancer tissues (P<0.001) (Fig. 3).

Correlation and functional enrichment analysis
LinkedOmics dataset was used to screen the genes that were associated with apoM. From the Pearson correlation analysis, 19,927 genes were correlated with ApoM (Fig. 4). To expound the potential biological functions of apoM, GO analysis was performed in KOBAS 3.0. The GO function analysis showed that apoM was mainly enriched in regulation of lipoprotein and HDL metabolism, reverse cholesterol transport and protein-lipid complex assembly (Fig. 5).

Discussion
This study investigated ApoM involvement in development of thyroid cancer using integrated bioinformatics tools. Integrated bioinformatics is mainly used for determining differential expression of target molecules and generating survival analyses. We report here downregulation of ApoM in thyroid cancer tissues compared to that of normal thyroid tissues. The expression level of ApoM mRNA was signi cantly associated with clinicopathological parameters form the TCGA database. There was no signi cant association between ApoM expression levels and survival of thyroid cancer patients. Using the GEO database, we further validated tha ApoM mRNA levels were downregulated in the thyroid cancer tissues compared with the normal thyroid tissues. ApoM is predominately located in the cytocol Golgi and plays an active role in lipoprotein metabolism and HDL assembly. Low levels of ApoM mRNA were observed in thyroid cancer tissues at late disease stage, nodal metastasis and histologic subtype. This implies that ApoM expression in normal tissues could affect normal physiological cellular function and inhibit malignant transformation.
ApoM mediates reverse cholesterol transport, plays an active role in formation of preβ-HDL and in the antioxidative properties of HDL [15]. However, the role of ApoM in cancer development and progression remains poorly understood. In our previous studies, ApoM enhanced proliferation and invasion in nonsmall cell lung cancers through upregulating S1PR1 and activating ERK1/2 and PI3K/AKT signaling pathways [13]. In another report, the levels of ApoM mRNA in colorectal cancer tissues were signi cantly increased in the patients with lymph node metastasis [12]. These ndings are however contradictory to the results of the present study, necessitating further investigation on the role of ApoM in carcinogenesis.
Protein-protein interaction (PPI) networks and correlations analysis identi ed potential interactions between ApoM and other genes and circular RNA.ApoM and other apolipoproteins including ApoA1,ApoB and ApoC3, are main components of HDL. It was demonstrated that apoA-I can inhibit intestinal tumor growth and metastasis by promoting cholesterol e ux [16]. Moreover, D-4F, an apoA-I mimetic peptide, inhibits proliferation and tumorigenicity of epithelial ovarian cancer cells by upregulating the antioxidant enzyme MnSOD [17]. This implies that ApoM and other members of the apolipoprotein family (ApoA-I, ApoB and ApoAC3) could be regulated by the transcriptional factors KLF6 and HNF4, long circRNAs to inhibit cell proliferation and cell cycle in cancer development and progression. Studies have shown that some transcriptional factors incuding HNF1, LXRα, ER and Foxa2 can regulate ApoM gene expression [18][19][20][21].
From the GO enrichment results, ApoM was enriched in regulation of lipoprotein and HDL metabolism, reverse cholesterol transport and protein-lipid complex assembly. A recentstudy demonstrated that lack of ApoM in mice increases the amount of brown adipose tissue (BAT), accelerates clearance of postprandial triglycerides, and protects against diet-induced obesity. Moreover, ApoM-de cient mice phenotype is S1Pdependent and re ects diminished S1P1 stimulation [22]. In genetically modi ed mice, changes in plasma ApoM concentration resulted in quantitative and qualitative changes in HDLs, while its overexpression reduced atherosclerosis [6]. Furthermore, ApoM concentration is strongly correlated to total cholesterol in healthy individuals [23]. Clinical and experimental data have shown that hypercholesterolemia and a high-fat, high-cholesterol diet can affect cancer development. External cholesterol can activate the oncogenic Hedgehog pathway, whereas internal cholesterol can induce mTORC1 signaling [24].
This study concludes that ApoM may serve as a diagnostic biomarker for patients with thyroid cancer.
Using integrated bioinformatics, molecular pathways and ApoM-related molecules involved in thyroid cancer were identi ed. Further studies are required to validate putative ApoM functions and mechanisms of its involvement in thyroid cancer. Downregulation of ApoM mRNA leves in thyroid cancer compared to normal thyroid tissues at ***p<0.001. downregulation of apoM in thyroid cancer was con rmed with UALCAN dataset. *** P<0.001 vs normal thyroid tissues