Analysis of the Mechanism of Maslinic Acid on Papillary Thyroid Carcinoma Based on RNA-Seq Technology

Objective This study analyzed gene sequence changes in the thyroid papillary carcinoma (PTC) cell line TPC-1 treated with the natural compound maslinic acid (MA) through RNA-sequencing (RNA-seq) and identified the necessary genes to provide a basis for the study of the molecular mechanism of action of MA in PTC treatment. Methods RNA-seq technology was used to detect genetic differences between the normal cell group (Nthy-ori 3-1) and the TPC-1 cell group (N vs T). Then, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Venn diagram analysis of shared genes, and protein–protein interaction (PPI) network analysis were used to analyze the therapeutic effect of the MA on TPC-1 cells. Real-time quantitative PCR (qRT-PCR) was used to verify six key genes. Results GO and KEGG analyses showed that four crucial signaling pathways are related to TPC development: cytoplasmic molecule (cell adhesion molecules), neuroactive ligand–receptor interaction, tumor transcriptional disorder, and cytokine–cytokine interaction. The Venn diagram revealed 434 genes were shared between the MA vs T-group and 387 genes were shared between the MATH vs T and N vs T groups. PPI and ClueGO showed that NLRP3, SERPINE1, CD74, EDN1, HMOX1, and CXCL1 genes were significantly associated with PTC, while CXCL1, HMOX1, and other factors were mainly involved in the cytokine–cytokine interaction. The qRT-PCR results showed that the expression of NLRP3, EDN1, HMOX1, and CXCL1 genes was significantly upregulated in the TPC-1 group but significantly downregulated after MA treatment (p < 0.01). SERPINE1 and CD74 genes were not expressed in TPC-1 cells, whereas they were significantly upregulated after MA treatment (p < 0.01). Conclusions This present study proves for the first time that MA can treat PTC, and the preliminary identification of key genes and rich signal transduction pathways provides potential biomarkers. It also provides potential biomarkers for the treatment of PTC with the natural compound MA and preliminarily discusses the therapeutic mechanism of action of MA against PTC, which is helpful for the further diagnosis and treatment of PTC patients.


Introduction
yroid carcinoma is the most common thyroid malignancy. According to the pathological type, it is mainly divided into five types: follicular thyroid carcinoma, papillary thyroid carcinoma (PTC), thyroid medullary carcinoma, anaplastic thyroid carcinoma, and undifferentiated carcinoma. PTC is the most common thyroid cancer that belongs to differentiated thyroid cancer, accounting for approximately 88% of thyroid cancer. e incidence rate of PTC is increasing year by year, mostly accompanied by lymph node metastasis [1]. Modern related research shows that the PTC is involves multiple genes and targets. Researchers have usually found that the TPC-1 pathogenesis is associated with sseveral classical pathways, including BRAF mutation, RET/ PTC rearrangement [2], PI3K/AKT/mTOR [3,4], and MAPK/ERK [5].
MA is a common pentacyclic triterpene compound mainly found in olive fruits and leaves [6,7], pomegranate flowers [8], and other natural plants. MA has various pharmacological activities, including antitumor [9], anti-AIDS [10], antioxidant [11], and antidiabetes [12]. rough the PI3K/AKT/NF-κB signaling pathway, MA plays an antiinflammatory role [13]. MA inhibits the growth of neuroblastoma cells by inducing apoptosis, activating caspase, inhibiting cell migration and invasion, and targeting the MAPK/ERK signaling pathway [14]. It exhibits an inhibitory effect on precancerous colon lesions in rats [15]. MA can significantly inhibit tumor formation in azomethane/dextran sodium sulfate mice and xenotransplantation mice and regulate the AMPK/mTOR pathway, thereby inhibiting the growth of colon tumors [16]. In addition, MA can induce apoptosis of pancreatic cancer cells [17] and act against breast cancer [18].
Gene sequencing technology can obtain the gene information of each sample through sample RNA extraction, primer design, sequencing, and bioinformatics sequence analysis, and can reflect the expression and regulation of genes in cells as a whole. Gene sequencing has become the most commonly used technology for studying the mechanisms of modern molecular pharmacology. It can widely and deeply mine the molecular genes of diseases to identify key target genes. Next-generation gene sequencing showed that the polycystic kidney disease and liver disease like protein 1 (PKHD1L1) may be a tumor suppressor gene in thyroid cancer [19]. Genomic DNA sequencing and mutation detection of PTC tumors showed that VEGF expression in PTC tissues was significantly lower than that in adjacent normal thyroid tissues, which was significantly related to invasive tumor behaviors such as lymph node metastasis and mutation [20]. Numerous studies on PTC have been conducted using next-generation gene sequencing. Zheng et al. [19] found significant differences in the expression of the PKHD1L1 gene between thyroid carcinoma and adjacent healthy thyroid tissues through next-generation sequencing. Yin et al. [20] detected BRAF mutations in PTC tumor tissues through genomic DNA sequencing. e results showed that CCDC67 expression was downregulated in PTC, and this expression was negatively correlated with PTC invasiveness and BRAF mutations.
Currently, no study has been reported on the role of the MA in PTC treatment. In this study, RNA-seq was used to detect TPC-1 cells and MA-treated TPC-1 cells to analyze the changes in the molecular function and signal transduction pathway and thus find target genes, offer broader information for the inhibition of TPC-1 proliferation, and provide more evidence for MA to treat PTC.

Cell Lines and Culture Conditions.
e human PTC cell line TPC-1 and human normal thyroid cells (Nthy-ori3-1) cells were purchased from the American Type Culture Bank (USA). Both cells were cultured in RPMI/1640 medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin at 37°C in a 5% CO 2 incubator. e medium was changed regularly. e cells were divided into Nthy-ori3-1, TPC-1, and MA + TPC-1 cell groups. MA (purity>98%) was purchased from Sichuan Weikeqi Biological Technology Co., Ltd., China.

RNA Isolation and Sequencing.
Nthy-ori3-1 and TPC-1 cells were cultured in RPMI/1640 medium for 24 h, and TPC-1 cells (66,700 cells/mL) were cultured in the medium containing 12, 24, and 48 μMMA for 48 h. e cells were washed with PBS and lysed using the TRIzol reagent (MRC, Germany). e RNA concentration (OD260/280 ratio) was detected using a nano-photometer spectrophotometer, and the RNA integrity was accurately detected using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). After passing the quality inspection, different libraries were pooled according to the effective concentration and target offline data volume and then sequenced using Illumina, and a 150bp paired-end read was generated. Four fluorescently labeled dNTPs, DNA polymerase, and adaptor primers were added to the sequenced flow cell for amplification. When each sequencing cluster extends the complementary strand, each fluorescently labeled dNTP releases the corresponding fluorescence. e sequencer then captures the fluorescent light signal and converts it into a sequencing peak through computer software, thereby obtaining the sequence information of the fragment to be tested.

Differential Gene Expression analysis.
Differentially expressed genes (DEGs) are genes whose expression levels have changed significantly. e gene expression differences between the T vs N, MATL vs T, MATM vs T, and MATH vs T groups were calculated. e DEGs were filtered and analyzed according to the following criteria: condition setting (|Fold change| > 2, p < 0.05).

Gene Ontology and Kyoto Encyclopedia of Genes and
Genomes Analyses. Volcano map and heat map analyses were performed on the four groups of DEGs to determine the changes in genes and enrichment of upregulated and downregulated genes. In the enrichment analysis of T vs N and MA vs T groups, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for DEG analysis. e first 20 enrichment pathways and the first 10 meaningful functional genes were selected, and their similarities were analyzed.

Data Visualization Analysis to Identify Essential Gene
Targets.
e Venn diagram was used to determine the shared genes of MATH, MATM, MATL vs T, and T vs N groups, and the Java plug-in ClueGo in Cytoscape 3.8.0 (http://www.cytoscape.org) was used to compare the shared genes between MATH vs T, MATM vs T, MATL vs T, and MA administration groups and the T vs N group for enrichment analysis. In addition, the protein interaction (PPI) network analysis was performed using string (https://stringdb.org/) to enrich the genes. e protein node map was imported into Cytoscape 3.8.0, data were visualized, key target genes were identified, degree values were combined, and gene enrichment was performed in ClueGO (showed genes that are enriched) and then the key genes were verified.

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Verification of the Expression of Related Factors through qRT-PCR.
After sequencing the same batch of RNA samples, total RNA quantification and reverse transcription were performed to synthesize cDNA, and the 2 × Power Up SYBR Green Master Mix kit was used to verify key genes according to the primer sequences listed in Table 1.

Data
Analysis. e SPSS 23.0 software was used for repeated measurement analysis of variance (ANOVA). Differences between groups were analyzed using the LSD test and one-way ANOVA. If p < 0.05, the difference was considered significant. In addition, GraphPad Prism 8 (GraphPad Software, Inc.) was used to draw each group of bar graphs.

N vs T and MA vs T differential expression analysis.
According to the conditions of |Fold change| > 2 and p < 0.05 for the screening of genes, N vs T gene volcano map exhibited 4381 differential genes. Of these genes, 2202 were upregulated and 2179 were downregulated. e MATH vs T group exhibited 823 different genes, including 357 upregulated and 466 downregulated genes. e MATM vs T group exhibited 86 different genes including 27 upregulated and 59 downregulated genes e MATL vs T group had 65 different genes. Of them, 24 genes were upregulated and 41 were downregulated (Figure 1(a)). In addition, the MATH group was more similar to the T group (Figure 1(b)).

GO and KEGG Analyses in the Tvs N and MA vs T Groups.
GO analysis found that the selected DEGs in the T vs N group were mainly involved in biological processes such as extracellular matrix organization, extracellular structure organization, and neuron projection guidance, and cell composition, including extracellular matrix, proteinaceous extracellular matrix, and receptor complex. In biological processes, cell composition and molecular function are the most common functions. According to KEGG analysis, genes and genomic pathways related to the cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, transcriptional misregulation in cancer, cell adhesion molecules, etc. were significantly enriched in the T vs N group (Figures 2(a) and 2(b)). e MA vs T group mainly participated in biological processes, such as responding to a molecule of bacterial origin, a response to a lipopolysaccharide, and negative regulation of cytokine production; cell composition, including the receptor complex, side of the membrane, and external side of the plasma membrane; and molecular functions such as cytokine activity, receptor ligand activity, and cytokine receptor binding. According to the KEGG analysis, pathways mainly enriched in the MA vs T group were the cytokine-cytokine receptor interaction, the IL-17 signaling pathway, and the TNF signaling pathway (Figures 2(c) and 2(d)). e cytokine receptor interaction pathway was significantly enriched in the T vs N and MA vs T groups.

Analysis of DEGs shared by the MA vs T and T vs N
Groups. Venn diagram analysis of MATH, M, L vs T, and T vs N groups revealed that 434 genes were shared between the MA vs T and T vs N groups. In total, 15 genes were shared between the MATL vs T and N vs T groups, 32 genes were shared between the MATM vs T and N vs T groups, and 387 genes were shared between the MATH vs Tand N vs Tgroups (Figure 3(a)); the total gene PPI (TSV format) was calculated.
e degree values are shown in Table 2. In addition, the analysis of MATL vs T, MATM vs T, and MATH vs T groups by using ClueGO revealed that the MATH vs T group was most correlated with the N vs T group (Figure 3(b)). An analysis of the shared genes of the MA vs T and T vs N groups showed that their main molecular functions were related to cytokine secretion; this result is basically the same as that of the previous analysis (Figure 3(c)).We then visually analyzed the data, combined with the calculation of the shared gene degree values, and found that the genes with the highest correlation were mainly SERPINE1, NLRP3, CD74, and EDN1 (Figure 3(d); Table 2). Table 2. e PPI analysis results were imported into the Cytoscape to calculate the degree value, and the genes with a degree value of ≥4 were selected. MMP9, matrix metallopeptidase 9; CCL5, C-C motif chemokine ligand 5; CXCL1, C-X-C motif chemokine ligand 1; SERPINE1, serpin family E Member 1; NLRP3, NLR family pyrin domain containing

Discussion
MA is a naturally occurring pentacyclic triterpene compound. Being an excellent pharmacological active product, MA has attracted people's interest because of its high-quality biological properties. MA has an inhibitory effect on various tumors. It targets the MAPK/ERK signaling pathway and inhibits cell migration and invasion. As the MA  Figure 1: Gene volcano map (a). Cluster analysis of differential genes (b). In the volcano map, blue dots indicate that genes have no significant differences, red origin indicates that genes are upregulated, and green origin indicates that genes are downregulated. In the MA vs T heatmap analysis, each row or column of the heatmap represents a module. e darker the color of the module, the more red or blue, the higher the correlation.    Evidence-Based Complementary and Alternative Medicine concentration increases, the inhibition rate becomes stronger [14]. MA can also inhibit the proliferation of renal cancer cell lines and the angiogenesis of endothelial cells, thereby exhibiting an antirenal cancer effect [21]. It can also prevent the formation of colon tumors by inhibiting the AMPK/mTOR signaling pathway [22]. MA exhibits numerous biological activities. However, researchers are yet to unravel how MA therapeutically targets oncogenic cell signaling cascades in different cancers [23]. Moreover, the effect of MA on PTC and its mechanism of action require further study. e sequencing results showed that TPC-1 DEGs were significantly higher in the presence of MA, with the number of downregulated genes being significantly higher than that of upregulated genes. is suggests that MA mainly acts as an inhibitor of TPC-1 cells. e results of GO analysis of MA action on TPC-1 cells showed that MA and T groups were mainly involved in biological processes such as bacterial-derived molecular reactions, lipopolysaccharide reactions, and negative regulation of cytokine production. Cell composition analysis revealed correlations between the receptor complex side of the membrane, the outer side of the plasma membrane, cytokine activity, receptor ligand activity, and cytokine receptor binding. According to the KEGG analysis, the pathways mainly enriched were the cytokine-cytokine receptor interaction, the IL-17 signaling pathway, and the TNF signaling pathway, with the cytokine-cytokine receptor interaction significantly enriched in the T vs N and MA vs T-groups. ClueGO analysis of MA action on TPC-1 revealed that the correlation between the MATH vs T, MATM vs T, and MATH vs T groups was the highest. Analysis of shared genes in the MAvs T and T vs N groups revealed that their main molecular functions were related to cytokine secretion. is result is essentially the same as that of the previous analysis (Figure 3(c)).
en, visual analysis of the data combined with calculation of the shared gene degree values, revealed that the SERPINE1, NLRP3, CD74, and EDN1 genes were closely related to these genes.
In the present study, the bioinformatics function of the MA on TPC-1 cells was analyzed through next-generation gene sequencing. MA induced TPC-1 cells by triggering cytokine receptor interaction signals and through regulation of various cytokine functions. At 48 μMMA, the most common genes were shared with TPC-1 cells. Moreover, SERPINE1, NLRP3, CD74, EDN1, HMOX1, and CXCL1 were verified as the key genes by qRT-PCR. e results were consistent with those of gene sequencing. Importantly, gene enrichment analysis revealed that HMOX1, CXCL1, NLRP3, and other genes were found to be involved in the cytokine signaling pathway and jointly promote the anticancer effect of MA. Gene sequencing enables us to better understand the molecular mechanisms of the anti-TPC-1 effect of MA and provides a new clue for the further study of its pharmacological activity.     Evidence-Based Complementary and Alternative Medicine 11