Scavenger receptor class A, member 5 is associated with thyroid cancer cell lines progression via epithelial‐mesenchymal transition

Thyroid cancer (TC) has become one of most common endocrine malignancies in recent decades. Due to gene background polymorphism, it's outcome goes quite differently in each patient. For exploring the mechanism, we performed whole transcriptome sequencing of paired papillary thyroid carcinoma (PTC) and adjacent thyroid tissues. As a result, scavenger receptor class A member 5 (SCARA5) might be a crucial anti‐oncogene associated with PTC. By RT‐qPCR, we first detected the expression of SCARA5 in PTC tissue and three type of TC cell lines. Besides, The Cancer Genome Atlas (TCGA) data were gathered to analysis the relationship between SCARA5 and clinical feature. A series of loss‐function experiments in TC cell lines (KTC‐1 and BCPAP) to investigate the function of SCARA5 in PTC. The results showed that SCARA5 expression in PTC was lower than adjacent normal tissue. And, it's consistent with the TCGA database. After analyse the correlation between SCARA5 expression and clinicopathological features in TCGA database, we discovered that downregulated SCARA5 is significantly connected age (P = .04) and tumour size (P = .032). Knockdown of SCARA5 in TC cell line could significantly increase the function of cells proliferation, colony formation, migration, and invasion. Furthermore, we also proved that SCARA5 could modulate the expression of epithelial‐mesenchymal transition‐related proteins, which influence invasion and migration. To best of our knowledge, SCARA5 is a suppressor gene which was associated with PTC and might be a potential therapeutic target in the future. Significance of the study Thyroid cancer (TC) has become one of most common endocrine malignancies in recent decades. By whole transcriptome sequencing of paired papillary thyroid carcinoma (PTC) and adjacent thyroid tissues, author discovered that scavenger receptor class A member 5 (SCARA5) might be crucial anti‐oncogene associated with PTC. Furthermore, knocking‐down of SCARA5 in TC cell line can increase the function of cells proliferation, colony formation, migration, and invasion. Author also proved that SCARA5 could modulate the expression of epithelial‐mesenchymal transition‐related proteins.


Funding information
Natural Science Foundation of Zhejiang Province, Grant/Award Numbers: LGF18H160032, LY13H160034; Science and Technology Project of Wenzhou, Grant/Award Number: Y20180855 Thyroid cancer (TC) has become one of most common endocrine malignancies in recent decades. Due to gene background polymorphism, it's outcome goes quite differently in each patient. For exploring the mechanism, we performed whole transcriptome sequencing of paired papillary thyroid carcinoma (PTC) and adjacent thyroid tissues. As a result, scavenger receptor class A member 5 (SCARA5) might be a crucial anti-oncogene associated with PTC. By RT-qPCR, we first detected the expression of SCARA5 in PTC tissue and three type of TC cell lines. Besides, The Cancer Genome Atlas (TCGA) data were gathered to analysis the relationship between SCARA5 and clinical feature. A series of loss-function experiments in TC cell lines (KTC-1 and BCPAP) to investigate the function of SCARA5 in PTC. The results showed that SCARA5 expression in PTC was lower than adjacent normal tissue. And, it's consistent with the TCGA database. After analyse the correlation between SCARA5 expression and clinicopathological features in TCGA database, we discovered that downregulated SCARA5 is significantly connected age (P = .04) and tumour size (P = .032). Knockdown of SCARA5 in TC cell line could significantly increase the function of cells proliferation, colony formation, migration, and invasion. Furthermore, we also proved that SCARA5 could modulate the expression of epithelial-mesenchymal transition-related proteins, which influence invasion and migration. To best of our knowledge, SCARA5 is a suppressor gene which was associated with PTC and might be a potential therapeutic target in the future.
Significance of the study: Thyroid cancer (TC) has become one of most common endocrine malignancies in recent decades. By whole transcriptome sequencing of paired papillary thyroid carcinoma (PTC) and adjacent thyroid tissues, author discovered that scavenger receptor class A member 5 (SCARA5) might be crucial antioncogene associated with PTC. Furthermore, knocking-down of SCARA5 in TC cell line can increase the function of cells proliferation, colony formation, migration, and invasion. Author also proved that SCARA5 could modulate the expression of epithelialmesenchymal transition-related proteins.
Ru-Tian Hao and Ou-Chen Wang contributed equally to this study.  6 Accumulating studies had published that the genomic mutations such as stimulation of oncogene or silencing of tumour suppressor genes is a crucial step in the development and tumorigenesis of TC. [7][8][9] In particular, B-type Raf kinase V600E, a famous gene mutation, can accelerate PTC tumorigenesis and progression via abnormally triggering the mitogenactivated pathway kinase pathway. 10 Besides, another notable mutations such as RAS mutation, 11 TERT mutation, 12 PTEN mutation, 13,14 PIK3CA mutation 15,16 and TP53 mutation 17,18 also play a significant role in thyroid carcinoma. Whereas numerous scholars had made remarkable progress in TC research in decades, but there are still many underlying molecular mechanisms yet remain unclear.
Scavanger receptors (SRs) are a type of receptor superfamily which contains one or several repeats of highly conserved SRCR domain protein module. 19 Many biomolecules such as modified lipoproteins, lipids, polyribonucleotide and polysaccharides are able to combine this membrane receptor. 20 In the previous study, SRs had been divided into eight discrete classes based on their ability to identify different type of low density lipoprotein. 21 In 2006, SCARA5 had been discovered and identified as the new member of this superfamily. 22 Unlike the other family members mainly detected in macrophages, SCARA5 was widely expressed in diverse human tissues such as bladder, ovary, kidney, testis, adrenal gland, skin and trachea. 23,24 Recently, an investigator have defined function of SCARA5 was that it can serve as nontransferrin iron delivery receptor to bound iron and help to the developing kidney. 25 More intriguingly, there are accumulating studies validated that SCARA5 act as a tumour suppressor in many human cancer. In human hepatocellular carcinoma (HCC), Huang et al found that SCARA5 expression have been down-regulated because of promoter hypermethylation and loss of heterozygosity; suppression of SCARA5 was also correlates with cellular invasion and overall progression. 26 A considerable report noted that up-regulation of SCARA5 could inhibit lung cancer lines tumorigenesis and progression in vitro. 27 Furthermore, Ying Liu et al testified that inhibition of SCARA5 could promote tumorigenicity, colony formation, cell invasion and metastasis in oral squamous cell carcinoma. 28 Emerging shreds of evidence have manifested that SCARA5 act as effective anti-tumour gene in many tumour, however, whether the SCARA5 gene have essential role in PTC still remains unclear.
With the help of sequencing technology, we have been revealed whole transcriptome bioinformation of 19 pairs PTC samples and its adjacent normal thyroid tissues. 29 The results showed that SCARA5 might act as a vital anti-tumour gene in the PTC. By RT-qPCR, we performed 57 pairs PTC samples to further validated this sequencing result.
Besides, the connection between SCARA5 expression and the clinico- To best our knowledge, SCARA5 is a suppressor gene which was associated with PTC and might be a potential biomarker in the future.

| The Cancer Genome Atlas database
TC RNA-seq data and corresponding clinical information were downloaded from the TCGA database (https://tcgadata.nci.nih.gov/tcga/).
In result, total of 502 TCs with complete clinicopathologic features such as age, gender, lymph node metastasis, tumour size, clinical stage (ACJJ7), and histological type were collected.

| RNA extraction and RT-qPCR
For RNA isolation, TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) was used to isolate 60 pairs of tissue specimens and TC cell line samples according to manufacturer's protocol. The A260/A280 ratio and spectrophotometric value were used to assess the RNA quality and quantity, respectively. As for cDNA synthesizing, we were used ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) at 16 C for 5 minutes, 42 C for 30 minutes and 98 C for 5 minutes. Real-time PCR analysis was performed in triplicate on an ABI Prism 7500 sequence detection system (Thermo Fisher Scientific, Inc.) by using the THUNDER-BIRD SYBR qPCR Mix (TOYOBO) following the manufacturer's protocol.
The thermocycling conditions were: 95 C for 2 minutes, followed by 40 cycles of 95 C for 15 seconds and 60 C for 60 seconds, and a final step of 72 C for 5 minutes. The relative expression of mRNA was calculated using the 2 −ΔΔCq method. 30 The primer sequences for PCR are as

| CCK-8 proliferation assay
Firstly, BCPAP and KTC-1 cells (about 1.5 × 10 3 cells) were plated into 96-well plates. After 24 hours, we use above methods to transfect cells with siRNA. Subsequently, when we observed cell adherence, we added 10 μL of CCK-8 solution into each well and incubated at 37 C for 2 to 4 hours. All cell plates were continuously incubated for 5 days. Final, we measured the absorbance of each wells at 450 nm and drawn cell proliferation curves. All experiments were performed in triplicate.

| Colony formation assay
At the beginning, BCPAP and KTC-1 cells (about 1.5 × 10 3 cells) were seeded into 96-well plates and consecutively incubated for 8 to 14 days. When we observed 50~70 cell in one colony formation at inverted light microscope (Olympus Corporation, Japan), the magnification is ×10. Then we use 4% paraformaldehyde (Sigma) to fix cell with for 30 minutes. Subsequently, 0.01% crystal violet stained cell for 30 minutes. All assays were performed in triplicate. BioCoat Matrigel Invasion Chambers (Corning, New York) was use for invasion assays. The procedure was similar with above protocol described for the migration assay.

| Protein extraction and western blot analysis
The samples were cleaved in RIPA lysis buffer (Beyotime, Shanghai, China). By using SDS-PAGE on a 10% gel and electro transferred to PVDF membranes total proteins (20 μg) in the lysate were removed.   SCARA5 both in our validated cohort and TCGA cohort. Based on the median value, the TCGA cohort was divided into two parts which are low expression and high expression groups respectively. As showed in Table 1, result showed that SCARA5 expression was correlated with Age (P = .04), Tumour size (P = .032) and disease stage (P = .003) (based on the seventh edition of the American Joint Committee on Cancer Staging Manual 31 ) in the TCGA cohort. But remained factors such as gender, lymph node metastasis and extrathyroidal invasion and we could not find their association with the expression of SCARA5. In local cohort, we found same trend was consistent with the results of TCGA cohort ( intensified the risk of tumour growth (Table 4).

| SCARA5 influence in tumorigenesis via modulating epithelial-mesenchymal transition in KTC-1 and BCPAP cell lines
Emerging researches have pointed that epithelial-mesenchymal transition (EMT) was an indispensable process during tumour progression and metastasis. [32][33][34] Furthermore, we aimed to found the potential mechanism by which this gene contributes to PTC tumour progression. Though a series of Western blotting, we detected EMT-related protein expression. As shown in Figure 5, 37 However, the function of SCARA5 in TC still remain to be fully elucidated.
In this study, we examined 57 matched PTC tumour tissue and adjacent normal tissues to testified the function of SCARA5 in PTC.
The mRNA expression level of SCARA5 was evaluated in local cohort and different PTC cell lines via RT-qPCR. in vitro experiments illustrated that downregulation of SCARA5 induced PTC cell proliferation and the abilities of migration and invasion. Finally, we measured the protein expression of EMT-related molecules via immunoblotting and found low E-cadherin and high N-cadherin and vimentins expression in the Si-SCARA5 cell lines.
There are some limitations still exist in current study. First, the relationship between SCARA5 and the prognosis of PTC in large samples needs to be investigated. Second, the exact molecules involved in the role of SCARA5 to modulate EMT pathway in PTC tumorigenesis need further study. In addition, animal experiments should be performed to verify the anti-tumour function of SCARA5.
In a summary, the present study investigated the association between SCARA5 and tumour growth in TCGA cohort and local cohort. And, downregulation of SCARA5 induce TC cell proliferation, colony formation, migration, and invasion though EMT pathway.
These fascinating findings provide potential molecular markers for the diagnosis and treatment of PTC.

ACKNOWLEDGEMENTS
Chen Zheng wrote the manuscript. Er-jie Xia performed the main experiment. Chen Zheng and Rui-Da Quan collected and processed the data. The authors thank Adheesh Bhandari for assistance. We would also like to express appreciation for Ru-Tian Hao and Ou-Chen wang helped with intellectual matters and material resources. They designed this work together and supported our team.