SRPX2 promotes cancer cell proliferation and migration of papillary thyroid cancer

Thyroid cancer is the endocrine tumor with the highest incidence at present. It originates from the thyroid follicular epithelium or follicular paraepithelial cells. There is an increasing incidence of thyroid cancer all over the world. We found that SRPX2 expression level was higher in papillary thyroid tumors than in normal thyroid tissues, and SRPX2 expression was closely related to tumor grade and clinical prognosis. Previous reports showed that SRPX2 could function by activating PI3K/AKT signaling pathway. In addition, in vitro experiments showed that SRPX2 promoted the proliferation and migration of papillary thyroid cancer (PTC). In conclusion, SRPX2 could promote the malignant development of PTC. This may be a potential treatment target for PTC. Supplementary Information The online version contains supplementary material available at 10.1007/s10238-023-01113-1.


Introduction
Thyroid cancer is one of the most common cancers, and it ranks seventh among cancers diagnosed in women [1,2].Its incidence has significantly increased over the past few decades.According to the difference in tumor origin and differentiation, thyroid cancer can be further divided into PTC, follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) [3].PTC is the most common type of thyroid cancer, accounting for about 80% of all thyroid malignancies [4].Thyroid cancer therapy includes surgery, chemotherapy, radiotherapy, and targeted therapy.Although most thyroid cancers are curable, advanced thyroid cancers show increased incidence and mortality rates [5].The development of systematic treatments for advanced thyroid cancer using molecular landscapes is an emerging field [6].Fortunately, it is reported that the genomics of thyroid cancer has promoted new therapeutic targets.
SRPX2, also known as sushi repeat-containing protein X-linked 2, is located on Xq22.1.SRPX2 was first identified as the downstream target gene of E2A-HLA in leukemia in 1999 [7].The SRPX2 protein is related to nerve development and cell growth and widely expressed in normal tissues [8,9].Besides, SRPX2 is highly expressed in cancer tissues and cell lines, including glioma, mesothelioma, osteosarcoma, esophageal, gastric, lung, and colorectal cancers [8,[10][11][12][13][14][15].Recent studies have revealed that a variety of signaling pathways are involved in the upregulation of SRPX2 in human diseases, such as the binding of transcription factors [16,17].In addition, inflammation can also promote the expression levels of SRPX2 [18].Moreover, SRPX2 is regulated by its upstream molecules including TGFβR1/ SMAD3, NFATc3/c-JUN, MAN1 (LEM) domain containing 1 (LEMD1), miR-149, and FOXP2 [17,[19][20][21][22].The effects Haiwei Guo and Ruiqi Liu have contributed equally to this study.of these mechanisms are not only limited to the upregulation of SRPX2 but also regulate the biological behaviors of tumor cell proliferation, migration, and invasion.Therefore, the upregulation of SRPX2 may be an important promoting factor for tumor development and metastasis.
SRPX2 promotes cell migration in gastric cancer, but not cellular growth.SRPX2 can increase the interaction between endothelial cells and tumors, regulating tumor progression and metastasis [8].SRPX2 overexpression plays a malignant role in colorectal cancer by regulating cell proliferation, adhesion, migration, and invasion [11].Moreover, SRPX2 regulates glycolytic metabolism in colon cancer cells through the PI3K-Akt pathway [11].SRPX2 also increases osteosarcoma cell proliferation by activating the Hippo signaling pathway [23].The role of SRPX2 in thyroid cancer, however, is unclear.
We searched for thyroid cancer biomarkers and found that SRPX2 was upregulated in PTC.In vitro experiments also show that SRPX2 plays a vital role in the proliferation and migration of PTC.Our study aimed to clarify the biological functions and regulatory mechanisms of SRPX2 in PTC.

Tissue samples and clinical data collection
The Zhejiang Provincial People's Hospital provided six pairs of formalin-fixed, paraffin-embedded thyroid cancer specimens and adjacent non-tumor specimens.We provide the basic information of the patient in Supplementary Table 1.No samples were collected from patients undergoing chemotherapy or radiotherapy.Ethical approval was obtained from the Institutional Ethical Review Board of Zhejiang Provincial People's Hospital (Institutional Review Board number QT2022435) before commencing sample analysis.

RNA sequencing
Trizol (Invitrogen) was used to isolate RNA from three biologically repeated siControl and three biologically repeated siSRPX2-1-transfected TPC1 cells after 36 h.Transcriptome expression profiling was analyzed by RNA sequencing using NovaSeq 6000 platform (Illumina) by Shanghai Bioegene Co., Ltd.After the final transcriptome was generated, String-Tie (http:// ccb.jhu.edu/ softw are/ strin gtie/ ,versi on: strin gtie-2.1.6) and ballgown (http:// www.bioco nduct or.org/ packa ges/ relea se/ bioc/ html/ ballg own.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples).The genes with the false discovery rate (FDR) parameter below 0.05 and absolute fold change > 2 were considered differentially expressed genes.Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.

RNA preparation and real-time quantitative PCR
Twenty-four hours after transfection, total RNA was isolated by RNA-Quick Purification Kit (ES Science, China) and quantified by absorbance at OD 260 nm.Total RNA was reverse-transcribed into cDNA by using the PrimeScript RT Reagent Kit (Takara Biotechnology, China).All the PCRs were carried out using qPCR SYBR Green Master Mix (Yeasen, China).The LightCycler 480 (Roche Diagnostics) was used for the real-time PCR assays.In order to normalize the expression of mRNA, GAPDH was used as a reference.The primers used for the related genes are listed in Table 1.

Proliferation assays
Cells transfected with the specified siRNAs were inoculated into six-well plates (1 × 10 4 cells each well).The cells were digested with trypsin/EDTA in suspension every other day to determine the number of cells.

Wound healing assay
For wound healing experiments, TPC1 and IHH4 cells were incubated in six-well plates until the cell density reached 90%.In the center of the hole, a straight line was drawn with a sterile pipette tip.The wound was subsequently washed with PBS, and the cells were cultured in serum-free 1640 for 12 h.The wound area was captured by a microscope and measured by ImageJ software.

Transwell Assay
Transwell assays were performed in Transwell plates (LAB-SELECT, China, 6.5 mm).Equal amounts of TPC1 and IHH4 cells were inoculated into the upper chamber containing 200 µL serum-free 1640 (5 × 10 4 cells).The lower chamber contained 700 µL of 1640 containing 10% FBS for induction of cell migration.The cells were cultured for 24 h.The migrated cells were photographed and counted under a microscope and then analyzed by ImageJ software.

Statistical analysis
The means and standard deviation are calculated from at least three independent experiments performed in duplicate.For statistical significance, we used GraphPad Prism 8.0 software to perform t-tests (and nonparametric tests) and one-way ANOVA (and nonparametric or mixed).Statistical methods are selected based on the sample size and sample distribution characteristics.The Kaplan-Meier method was used for the survival analysis.The significance of a statistical test was defined as *P < 0.05, **P < 0.01, ***P < 0.001.

SRPX2 expression levels are upregulated in PTC
Six pairs of tumor tissues and adjacent non-tumor specimens were sequenced (Fig. 1A), and high-expression genes were selected for further study.We assessed the SRPX2 levels based on RNA sequencing data from Time2.0 (http:// timer.comp-genom ics.org).In cancer tissues, SRPX2 expression was higher than in normal tissues, especially in CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), LUAD (lung adenocarcinoma), LUAC (lung adenocarcinoma), GBM (glioblastoma multiforme), READ (rectum adenocarcinoma), STAD (stomach adenocarcinoma), and THCA (thyroid carcinoma) (Fig. 1B).To verify SRPX2 expression levels in thyroid cancer, we first analyzed the data from UALCAN (http:// ualcan.path.uab.edu/ index.html), which included 505 samples of thyroid cancer tissues and 59 thyroid normal tissues.SRPX2 mRNA expression levels were higher in thyroid cancer tissues than in normal tissues according to UALCAN results (Fig. 1C,  Supplementary Fig. 1).In addition, in the presence of the most common BRAF mutation in thyroid cancer, the expression of SRPX2 was significantly higher than that in WT group (Supplementary Fig. 2).We further analyzed SRPX2 mRNA levels in three human PTC cell lines (TPC1, IHH4, and BCPAP) (Fig. 1D).As shown in Fig. 1D, SRPX2 mRNA levels were significantly increased compared with NTHY3.Besides, we extracted the total RNA from PTC tissues and paracancerous tissues of the six-paired samples, and SRPX2 mRNA levels have shown to have a significant difference (Fig. 1E).To further confirm whether SRPX2 expression is indeed upregulated in PTC, we performed immunohistochemical (IHC) analysis of SRPX2 expression in cancer and paracancerous tissues.IHC also showed that SRPX2 was highly expressed in PTC tissue compared with normal paracancerous tissue (Fig. 1F, G).Database analysis together with PCR and IHC results revealed that SRPX2 expression is upregulated in PTC.

High expression of SRPX2 predicts a poor survival prognosis
To further evaluate the correlation between the expression level of SRPX2 and the clinical significance in patients with thyroid cancer, we found higher SRPX2 expression in all cancer stages and lymph node metastasis grades of thyroid cancer tissues than in normal tissues by using the UALCAN and GEPIA databases (http:// gepia.cancer-pku.cn/ index.html) (Fig. 2A-C).The GEPIA database was used to investigate the correlation between SRPX2 expression and thyroid cancer prognosis.As a result, SRPX2 expression significantly affected the prognosis of thyroid cancer patients (Fig. 2D).The Human Protein Atlas (https:// www.prote inatl as.org) also revealed that patients with thyroid cancer who exhibited higher SRPX2 expression (n = 400) have shorter survival time than those with lower expression patients (n = 101) (Fig. 2E).These results reveal that SRPX2 is an important prognostic factor and has important clinical value in thyroid cancer.

SRPX2 knockdown inhibits the proliferation of PTC cells
To explore the cellular function of SRPX2 in PTC, we designed two different siRNAs for SRPX2 to carry out our experiments.RT-qPCR was used to detect the SRPX2 mRNA level 24 h after transfection of TPC1 and IHH4 cells.
The expression level of SRPX2 has decreased (Fig. 3A).WB (Western blot) experiments also showed that SRPX2 was effectively knocked down by two different siRNAs in the TPC1 and IHH4 cell lines (Fig. 3B).By using siSRPX2-1 and siSRPX2-2 as interference, we successfully generated PTC cell lines with low SRPX2 expression.Next, we assessed the growth and proliferation of PTC cells in the siSRPX2-1 and siSRPX2-2 groups.We found that SRPX2 inhibition resulted in growth inhibition of TPC1 and IHH4 cells (Fig. 3C, D).

SRPX2 inhibits PTC cell migration
To identify the function of SRPX2 in the migration of PTC cells, we used siRNAs to knockdown SRPX2 in TPC1 and IHH4 cells.After that, scratch assay showed that the downregulation of SRPX2 inhibited the migration of TPC1 and IHH4 cells compared with control transfection (Fig. 4A, B).In addition, the Transwell assay also showed that silenced expression of SRPX2 reduced the migration ability of TPC1 and IHH4 (Fig. 4C, D).Collectively, these results suggest that SRPX2 promotes PTC tumor migration.

SRPX2-related downstream molecules and signaling pathways
To identify potential downstream genes of SRPX2, we screened target genes using sequenced mRNAs.By comparing the gene expression changes between the control and SRPX2 knockout groups, heatmap clustering analysis revealed that the expression of some genes decreased after the knockdown of SRPX2.Through literature review, we identified several SRPX2 downstream genes with potential clinical value, including PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2 (Fig. 5A).Based on gene sequencing, we generated a volcanic map of all SRPX2associated genes and found a positive correlation between the levels of PTGDS, GREM1, LAMA4, S100A14, PREX2, GLS2, and SRPX2 (Fig. 5B).We then performed KEGG analysis of the SRPX2 gene and identified the top 20 KEGGenriched terms (Fig. 5C).The KEGG analysis revealed that SRPX2 was highly correlated with the PI3K/AKT signaling pathway, calcium signaling pathway, amphetamine signaling pathway, and ECM receptor interaction.Taken together, these findings suggest that SRPX2 mediates a range of malignant biological functions in PTC cell lines by activating the PI3K/AKT pathway and downstream target genes.
In order to verify our hypothesis that SRPX2 is directly proportional to the expression of downstream target genes, we performed SRPX2 knockdown in the TPC1 and IHH4 cell line and verified it by RT-qPCR.As expected, the mRNA levels of PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2 decreased when SRPX2 was effectively knocked down (Fig. 5D, E).Previous studies have suggested that SRPX2 is involved in PI3K/AKT pathway, suggesting that SRPX2 may be involved in tumor metastasis.The results of the Transwell experiment also showed that after the SRPX2 gene interfered, the metastatic ability of PTC cells was decreased.This suggests that the SRPX2 protein may be one of the key regulatory factors for PTC cell metastasis.We next examined the protein level of vimentin, N-cadherin, E-cadherin, and FN1, which are typical epithelial-mesenchymal transition (EMT) regulating proteins.We found that SRPX2 knockdown resulted in decreased levels of N-cadherin and vimentin in the TPC1 and IHH4 cell lines, whereas the protein levels of E-cadherin and FN1 did not change significantly (Fig. 5F).At the same time, WB results showed that P-PI3K levels were decreased after SRPX2 knockdown, whereas PI3K protein levels were not significantly changed (Fig. 5F).These results suggest that SRPX2 regulates PTC via the PI3K/AKT pathway and promotes EMT via N-cadherin and vimentin.We also identified several SRPX2 downstream genes, including PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2, which require further experiments to identify the detailed mechanisms (Fig. 6).

Discussion
In recent years, the incidence rate of thyroid cancer has increased, and the new cases of thyroid carcinoma with a larger diameter and non-early cancer have shown an upward trend [25,26].In addition to the continuous increase in the prevalence rate, it is also a hot research topic that the prognosis of PTC is difficult to judge and there is no recognized optimal treatment [27].Presently, benign and malignant thyroid nodules are mainly differentiated using traditional ultrasound and needle aspiration biopsy [28,29].However, due to the varying experience of the operators and the location of sampling, there is a misdiagnosis rate in clinical practice.Currently, there are no clear markers for thyroid cancer.Therefore, identifying molecular markers of thyroid cancer is of great significance for the early diagnosis, treatment, and prevention of postoperative recurrence of thyroid cancer.
Our study identified a meaningful gene that could affect PTC prognosis through extensive bioinformatics analysis.We found that SRPX2 was overexpressed in PTC and positively correlated with higher histological grade, metastatic lymph node grade, and worse prognosis.Previous studies have found that SRPX2 takes part in human embryonic stem cell differentiation, cognitive impairment, and epileptic activity [19,30,31].Most published data show that SRPX2 has a carcinogenic effect and is abnormally expressed in various tumors [15,32,33].Our study also showed that the mRNA level of SRPX2 in PTC tissues was significantly higher than paracancerous tissues, which was confirmed by cell experiments and IHC.All these results suggest that SRPX2 is a prognostic biomarker of PTC.
Yu et al. found that SRPX2 targets the FAK/AKT pathway in hepatocellular carcinoma (HCC) to increase the mobility of HCC cells [34], and SRPX2 promotes EMT in small cell lung cancer [14].In addition, previous studies have shown that SRPX2 partially realizes its function through the FAK-dependent pathway, and SRPX2 targets FAK to exert malignant biological effects in thyroid cancer [35].However, studies on SRPX2 in thyroid cancer are rare and the mechanism of action of SRPX2 in thyroid cancer remains unclear.Our article is the first showing that SRPX2 enhances the cell proliferation and migration ability of PTC by regulating the PI3K/AKT signaling pathway, N-cadherin, and vimentin and additionally explores more potential regulating genes.Through KEGG analysis, we confirmed that the expression of SRPX2 was closely related to the PI3K/AKT signaling pathway, and WB verified that the level of P-PI3K decreased after knocking down SRPX2.Furthermore, the results of thermal cluster analysis and the volcanic map of SRPX2 showed that the expression of PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2 in thyroid carcinoma was positively correlated with SRPX2.In addition, the RT-qPCR results showed that their mRNA levels decreased to different degrees after SRPX2 knockdown.Other studies have also shown that PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2 have potential value in promoting cancer progression and predicting cancer prognosis [36][37][38][39][40][41].This is the first research to reveal a direct correction between SRPX2 and these oncogenes, which needs further study.
Our study had some limitations.The number of patients included in this study was limited, and it is difficult to explain the differences in age, race, and geographical distribution.Therefore, additional in vivo and in vitro experiments are required for verification.
In conclusion, our study shows that SRPX2 is highly expressed in PTC and is involved in tumor progression.An increase in SRPX2 expression is associated with a decrease in overall survival in patients with PTC and may serve as an independent prognostic factor.In addition, SRPX2 promotes the malignancy of PTC through the PI3K/AKT, N-cadherin, and vimentin pathways.We also identified potential downstream regulatory genes, including PTGDS, GREM1, LAMA4, S100A14, PREX2, and GLS2.Therefore, our study offers new insights into the role and mechanism of SRPX2 in thyroid cancer and its potential as a biomarker for PTC prognosis.

Fig. 1
Fig. 1 SRPX2 shows high expression in thyroid cancer.A Six pairs of thyroid tumor samples and adjacent paracancerous tissues were sequenced.B The levels of SRPX2 in different human tumor types were compared by Time2.0 database.C The significant increase in SRPX2 expression in thyroid cancer was further validated by using the UALCAN cancer database.D RNA analysis of SRPX2 expression in three human thyroid cancer cell lines (TPC1, IHH4, BCPAP) and a

Fig. 2 Fig. 3 SRPX2Fig. 4 SRPX2
Fig. 2 The expression level of SRPX2 is related to the prognosis of thyroid cancer.A-C Boxplot graphs show the relative expression of SRPX2 in all stages (A, C) and all lymph node metastasis grades B of thyroid cancer and normal tissues in the UALCAN (A, B) and GEPIA (C) database.D-E Kaplan-Meier survival curve shows that the overall survival rate of the patients with low SRPX2 expression is better than that of the patients with high SRPX2 expression.

Fig. 5
Fig. 5 Pathways and downstream molecules involved in SRPX2.A RNA-seq Heat map of the gene expression profile form siControl and siSRPX2-1 treated TPC1 cells.The differentially expressed genes shown in the heat map were marked with red line.B The volcanic map shows all the genes associated with SRPX2 in thyroid cancer.C KEGG analysis based on gene sequencing data was used to predict the potential function of SRPX2 and the signal pathways involved.

Table 1
The primers of the genes Gene symbol Forward/reverse primer