Long Non-coding RNA PTCSC3 Promotes Apoptosis of Human Papillary Thyroid Carcinomas by Regulating S100A4

Objective: We investigated the effect of papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) on the apoptosis of human papillary thyroid carcinomas cells by regulating S100A4. Methods: Normal cells Nthy-ori 3-1, cancer cells SW579 and TPC-1 were used to study. Proliferation of human papillary thyroid carcinomas cells was detected by CCK8, clone formation and transwell. Tube formation assay was used to observe the formation of in vitro tubes. Western blot was applied to detect the expressions of S100A4. After S100A4 overexpressed or down expressed, the above experiments were repeated. Tumor volume and weight was measured. Ki67 expression and tumor microvascular density (MVD) were detected by immunohistochemistry. Apoptosis of tissues was detected by TUNEL. Expression of apoptosis-related proteins, VEGF and MMP-9 were detected. Results: PTCSC3 overexpression and S100A4 lowexpression had signicantly decreased proliferation, invasion, migration and tube formation of cancer cells. Meanwhil, the expression of Ki67 and MVD in tumor tissues were signicantly decreased (p<0.05). Conclusion: Up-regulation of PTCSC3 can inhibit the expression of S10044, and promoted cell apoptosis and tube formation of papillary thyroid carcinomas cells.


Introduction
In the past few decades, patients with papillary thyroid carcinomas have increased rapidly (1) and papillary thyroid carcinomas (PTC) account for about 80% -85% of all thyroid carcinomas (2) . The survival rate of PTC can decrease drastically due to distant metastases. Long non-coding RNA (lncRNA) is a non-protein-encoding RNA molecule of more than 200 nucleotides in length. Studies have shown that lncRNA is related to many biological processes, including the development and immunity of cell, cell proliferation and differentiation, modulation of apoptosis, function as markers of genomic imprinting, and act as competing endogenous RNAs (ceRNAs) that share micro RNAs (miRNA) biding sites (3)(4)(5) Dysregulation of lncRNA expression among various cancers has been implicated to have a potential effect on tumorigenesis (6,7) Increasing number of lncRNAs have been characterized in different malignancies and they are mainly involved in the modulation of carcinogenesis and cancer progression (8) .
Papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) is an intergenic long noncoding RNA. It was reported to be involved in tumor suppression in thyroid carcinoma (9) . The PTCSC3 gene is exclusively signi cantly suppressed in PTC tumor tissues (10) . Studies have shown that lncRNA PTCSC3 downregulates the expression of S100A4 gene, leading to reduced cell motility and invasiveness (11) . S100A4 is a Ca 2+ -binding protein that are associated with diverse cellular processes, there aer cell invasion, proliferation and cell-cell interaction (12)(13)(14) . S100A4 was reported to be overexpressed in human PTC tissues and lymph node metastases (15,16) , and knockdown of S100A4 inhibited the growth and metastasis in anaplastic papillary thyroid carcinomas cells (17) . VEGF and MMP-9 are downstream targets of S100A4 and the suppression of S100A4 also results in suppressed expressions about VEGF and MMP-9, which in turn suppresses the invasion of thyroid carcinoma cells (18,19) VEGF is an essential endothelial cell mitogen and is an important factor in angiogenesis (20) . Studies have shown that increased expression of VEGF promotes thyroid carcinoma cell growth and migration, while the decreased expression of VEGF inhibits tumor growth (21) . MMP-9 has also been shown to associated with the migration and survival of endothelial cells (22,23) , Caspase-3 and Caspase-9 are two essential caspases that play critical roles in apoptosis, which is the process of programmed cell death (24,25) .
Thus, it is signi cant to research the effects and potential mechanisms of the lncRNA PTCSC3 on the angiogenesis and growth of thyroid carcinoma xenografts, which might provide a direction for treating papillary thyroid carcinomas. overexpression group (PTCSC3), PTCSC3 inhibitor negative control group (NC1) and PTCSC3 inhibitor group (si-P). The lentivirus plasmids were constructed by Shanghai Genechem Co., Ltd.

Materials And
The transfection e ciency was tested by RT-PCR after 24h.

CCK8
Logarithmic cells were seeded into 96-well plates with 2×10 4 /ml with 100 μl of medium added to each well. After culture for 24h, 48h, 72h and 96h at 37 ℃ and 5% CO 2 . Then 10 μl CCK-8 solution was added to each well (tonghua institute of chemistry, Japan) and mixed well for further culture for 4h. The absorbance (OD) of each well at 450 nm was measured by the microplate reader.

Clone formation assay
The density was modulated to 250 cells/ml, and 2ml cells were added to each well of the six-well plate. The cells were incubated at 37℃ and 5%CO2 for 2-3 weeks, then fresh medium was changed every three days. The cells were xed with methanol, 1ml gemsa solution was added to each well, and the cells were stained for 30min. The cells were washed with ultrapure water for 2 times, and the water around the dish was sucked off with a lter. Take pictures with a camera.

Tube formation assay
Firstly, 50 µL Matrigel (BD Bioscience, USA) was applied to 96-well plates and cured at 37℃ for 1h. Then, HUVECs were digested and centrifuged at 1000rpm for 5min. The cell supernatants (4×104 cells/wells) were resuspended and inoculated on the matrix glue, placed in the medium, and incubated at 37℃ in an incubator with 5% CO2 (Thermo, USA) for 48h. The digital images were obtained by counting under an inverted microscope using the digital Camera system on the microscope (Jenoptik ProgRes Camera, Germany).

Flow cytometry
After treatment, the cells were cultured for 24 hours then collected. The cells were resuscitated by precooling with 1×PBS (4℃), centrifuged at 1000rpm for 5-10 minutes, and washed. Then 300 μl of 1×Binding Buffer was added to suspend the cells. Annexin V-APC of 5 μl was added and mixed, then incubated for 15 minutes at room temperature without light. Five minutes before loading, 5 μl of PI was added for dyeing, and 200 μl of 1×Binding Buffer was added before loading. The samples were nally detected by ow cytometry (Beckman Coulter, Brea, CA, USA) and analyzed by CellQuest software (BD Bioscience, San Diego, CA).
The other steps are the same as above.

Construction and grouping of nude mice with xenograft tumors
Twenty-four SPF Balb/c female nude mice, 16-18g, 4 weeks old, purchased from Charles River. License number SCXK (Beijing) was 20160006. The nude mice were fed in the aseptic, independent and air supply isolation cage at air laminar ow puri cation room, where kept the constant temperature (26-28℃) and humidity (relative humidity 40%-60%). Feed, drinking water and bedding material were sterilized. Animal experiments followed the guidelines of NIH (NIH pub.no.85-23, revised 1996), which have been approved by the animal protection and use committee of the 960 th Hospital of the PLA Joint Logistics Support Force. Cells in the logarithmic growth phase were digested with 0.25% trypsin. Cells were collected and counted. The concentration was adjusted to 2.5×10 6 /ml. And 0.2 ml was inoculated on the right forelimb close to soft skin on the back of the nude mice. Twenty-four nude mice were randomly divided into (1) Control group.

Tumor volume calculation
Vernier calipers were used to measure tumor length (L) and short diameter (W) every 7 days to calculate tumor volume. Tumor volume (V) = (long diameter × short diameter 2) /2. After 35 days, the nude mice were anesthetized by intraperitoneal injection of 0.6% pentobarbital sodium (40mg/kg). The mice were sacri ced by neck dissection. The tumor tissue was collected and weighed. The tumor tissue was xed in 4% paraformaldehyde and embedded in para n for 24 hours. Part of the tissues was placed in the freezer tube and stored in the refrigerator at -80℃.

Immunochemistry
After routine sections of the tumor tissue, the toasted slides were dewaxed with xylene and hydrated with gradient ethanol solution. And 3% H2O2 methanol solution was used to inactivat for 20min, followed by

TUNEL
The thickness of the section was 4 μm. After conventional dewaxing of xybenzene and dehydrating with gradient ethanol, apoptosis detection kit (No.: ZK-8005, ZSGB-BIO, China) was used for quantitative detection of apoptosis by TUNEL. Five elds were randomly selected under a 400-x optical microscope (BX50 /Olympus, Japan). Cell lines were identi ed as apoptotic cells with brown or tan granules and apoptotic cell morphological characteristics. Apoptotic index (AI) was calculated to re ect the degree of apoptosis. Apoptotic index = (number of apoptotic positive cells /total number of cells) ×100% 2.15 Statistical analysis SPSS 19.0 was used for data processing. Data analysis results were presented as mean±SD. T test was used for data analysis between two groups. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis. The relationship between the expression difference in PTCSC3 and S100A4 and the clinicopathological characteristics of papillary thyroid carcinomas patients was analyzed by chi-square test. The correlation between PTCSC3 and S100A4 expression was analyzed by Pearson correlation analysis. P <0.05 indicated that the difference was statistically signi cant.

The effect of PTCSC3 on the proliferation of human papillary thyroid carcinomas cells
As shown in Figure 1, compared with Nthy-ori 3-1, the expression of PTCSC3 mRNA in human papillary thyroid carcinomas cells was obviously decreased (p<0.05), while the expression of S100A4 mRNA was visibly increased (p<0.05). The differential expression was most pronounced in TPC-1 cells. TPC-1 cells were selected for subsequent experiments. Compared to the control group, there was no obvious difference in PTCSC3 mRNA expression, cell proliferation, invasion, migration and tube formation ability between the NC1 and NC2 groups (p>0.05). The level of PTCSC3 mRNA was obviously increased in PTCSC3 group, and the cell proliferation, invasion, migration and tube formation ability were signi cantly decreased (p<0.05). The expression of PTCSC3 mRNA was visibly decreased in the si-P group, and cell proliferation, invasion, migration and tube formation capacity were signi cantly increased (p<0.05).In comparation with the PTCSC3 group, the expression of PTCSC3 mRNA was signi cantly decreased in the si-P group, and the cell proliferation, invasion, migration and tube formation ability were signi cantly increased (p<0.05).

The effect about PTCSC3 on apoptosis of human papillary thyroid carcinomas cells
As shown in Figure 2, in comparation with the control group, the apoptosis rate and the expression of apoptosis-related protein Bax, Cleaved caspase-3 of the PTCSC3 group was visibly increased, while the level of Bcl-2 protein was decreased. However, the results of si-P group were opposite (p<0.05). Compared to PTCSC3 group, the apoptosis rate and the expression of apoptosis-related protein of si-P group were signi cantly decreased, while Bcl-2 protein level was markedly increased (p < 0.05).
3.3 PTCSC3 regulates the effect of S100A4 on the proliferation of human papillary thyroid carcinomas cells As shown in Figure 3, the expression of S100A4 gene and protein was signi cantly decreased in the PTCSC3 group compared with the Control group, whereas that in the si-P group was increased (p<0.05). Compared to the PTCSC3 group, the expression of S100A4 gene and protein in the si-P group was markedly increased (p<0.05). Compared to the control group, the expression of S100A4 mRNA was signi cantly decreased in the PTCSC3, si-S, P+NC4, and P+S groups (p<0.05). In comparation with the PTCSC3 and si-S groups, the expression of S100A4 mRNA was obviously increased in the P+S group (p<0.05). Compared to the control group, the proliferation, invasion, migration, and tube formation ability of the PTCSC3, si-S, P+NC4, and P+S groups were visibly lower (p<0.05). Compared with the PTCSC3 and si-S groups, the proliferation, invasion, migration and tube formation ability of P+S group were signi cantly increased (p<0.05).

PTCSC3 regulates the effect of S100A4 on apoptosis of human papillary thyroid carcinomas cells
In Figure 4, in comparation to control group, the apoptosis rate and the expression of apoptosis-related protein of PTCSC3, si-S, P+NC4 and P+S groups were effectively promoted, while the level of Bcl-2 was obviously inhibited (p<0.05). Compared to PTCSC3 and si-S group, the apoptosis rate and the expressions of Bax and Cleaved caspase-3 of P+S group were signi cantly down regulated. The Bcl-2 protein expression was signi cantly promoted (p<0.05).
3.5 PTCSC3 regulates the effect of S100A4 on tumor growth In Figure 5, compared to the control group, the tumor growth rate, volume and weight of the other groups were reduced, while the percentage of Ki67 positive cells, MVD values and expressions of VEGF and MMP-9 in the tumor tissues were obviously down-regulated(p<0.05). Compared to PTCSC3 and si-S group, the tumor growth rate of P+S group was signi cantly accelerated, the tumor volume and weight were visibly increased, while the percentage of Ki67 positive cells, MVD values, and protein expressions of VEGF and MMP-9 in tumor tissues were effectively increased (p<0.05).

PTCSC3 regulates the effect of S100A4 on tumor apoptosis
In the gure 6, compared to control group, the apoptotic index and the expressions of apoptosis-related protein in other groups were signi cantly increased. But expression of Bcl-2 was visibly prohibited (p<0.05). Compared to PTCSC3 and si-S group, the tumor apoptotic index and the expressions of Bax and Cleaved caspase-3 in P+S group was signi cantly down-regulated, and the expression of Bcl-2 protein was signi cantly promoted (p<0.05).

Discussions
LncRNA PTCSC3 has been shown to affect the development of papillary thyroid carcinoma (11) . To elucidate the biological functions of PTCSC3, thyroid carcinoma xenografts models in nude mice were constructed with gain-of-function experiments in thyroid carcinoma cell lines. The results indicated that overexpression of PTCSC3 inhibited the formation of thyroid carcinoma xenograft tumor and angiogenesis. The roles of dysregulated lncRNAs in carcinogenesis and cancer progression have been widely investigated in various studies (8) . Different lncRNAs have been continuously identi ed in different types of malignancies. For instance, increased expression of LncRNA H19 and resulting cell proliferation in pancreatic ductal adenocarcinoma (26) . LncRNA HULC was rst found to be highly expressed in liver cancer and was identi ed as an oncogene in glioma (27) . LncRNA BISPR regulates the microRNA miR-21-5p and promotes the progression of thyroid papillary carcinoma (28) . The studies suggested that the signaling network of lncRNAs regulated cancer development is complex and needs thorough investigations.
To elucidate the potential mechanisms by which PTCSC3 regulates the thyroid carcinoma cell invasion, the functions of S100A4 with gain-of-function experiments were also investigated. The data also illustrated that the abundant expression of PTCSC3 in the TPC-1 cell lines correlated with signi cantly suppressed expression of S100A4. Downstream target genes of S100A4 were also investigated in this study in order to elucidate how S100A4 functions in promoting thyroid carcinomas cell invasion and proliferation. VEGF and MMP-9 are two major genes that been characterized as downstream of S100A4 and depletion of S100A4 in thyroid carcinoma cells restrain their capabilities of proliferation, angiogenesis and invasion by down regulating the expression of VEGF and MMP-9. In this study, we detected the level of VEGF and MMP-9 and showed that they were signi cantly up regulated with abundant presence of S100A4, which con rmed that the underlying mechanism of PTCSC3 down regulates the expression of S100A4. Eventually, it leads to reduced expression of VEGF and MMP-9 and ultimately the suppression of thyroid carcinomas tumor development. The VEGF signaling pathway can affect EMT and tumor differentiation (29,30) . MMPs also have been shown to promote invasion of blood vessels and lymphatics by digesting extracellular substrates in the development of cancers (31) . Some have reported that MMPs facilitate the induction of the EMT pathway (32) . These emerging correlations also lead to us to reveal possible relationships between leading actors in the EMT developed in thyroid carcinomas cells.
Genes involved in the apoptosis caspase pathways were also investigated in this study to further understand the mechanisms of regulations between PTCSC3 and S100A4 in suppression of thyroid carcinomas tumor development. Studies have reported distinct roles of caspase family members during intrinsic apoptosis. Caspases involved in apoptosis have been classi ed by their actions into initiator caspases (Caspase-8 and -9) and inhibitor caspases (Caspase-3, -6 and -7) (33) . In the present study, the expression of Caspase-3 was detected and the results showed that they were signi cantly suppressed with abundant presence of S100A4. The results demonstrated that the potential mechanism of PTCSC3 down regulates the expression of S100A4, which in turn leads to abundance of Caspase-3 and ultimately promoting apoptosis of thyroid carcinomas tumor cells. Previous studies also reported other targeted gene for PTCSC3, for example the tumor suppressor gene neuregulin-1 (NRG1) involved in interactions with PTCSC3 in thyroid carcinoma (34,35) . A recent study of glioma revealed that PTCSC3 inhibits the proliferation and invasion of glioma cells and suppressed the epithelial-mesenchymal transition (EMT) by regulating the Wnt/β-catenin signaling pathway (36) . Therefore, besides the regulation through S100A4, the possible mechanisms that PTCSC3 regulates the proliferation and invasion of cancer cells could be complicated and involve multiple signaling pathways. Therefore, more investigations such as genome wide association studies are needed to reveal potential pathways involved in the regulations of PTCSC3 or S100A4.

Conclusion
In conclusion, the overexpression of PTCSC3 inhibits proliferation, invasion, metastasis and angiogenesis of thyroid carcinomas cells, and also promotes apoptosis of thyroid carcinomas cells in vivo. These functions of PTCSC3 are regulated by the suppression of S100A4 pathway through downstream targeting genes. Regulation of LncRNA PTCSC3 and S100A4 could be an effective potential therapeutic and preventive approach for thyroid carcinomas.

Declarations
Ethics approval and consent to participate Animal experiments were followed the NIH guidelines (NIH Pub. No. 85-23, revised 1996) and have been approved by the Animal Protection and Use Committee of Huai'an Second People's Hospoital.

Not applicable
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Q W and L Jcarried out the experimental work and the data collection and interpretation. Q W and Y Z participated in the design and coordination of experimental work, and acquisition of data. L Jnd J W participated in the study design, data collection, analysis of data and preparation of the manuscript. Q W and H D carried out the study design, the analysis and interpretation of data and drafted the manuscript.
All authors read and approved the nal manuscript. (F) Transwell assay for cell invasion (x400); (G) Transwell assay for cell migration (x400); (H) tube formation The experiment was conducted to observe the ability of tube formation in vitro (×200).
Compared with the Nthy-ori 3-1 group, $p<0.05; and compared to the control group, *p<0.05; compared to PTCSC3, # p<0.05. Data analysis results were repeated 3 times and presented as mean±SD. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis. Effect of PTCSC3 on apoptosis in human papillary thyroid carcinomas cells. (A) Apoptosis; (B) Western blot. compared to the control group , *p<0.05; compared to PTCSC3 group, #p<0.05. Data analysis results were repeated 3 times and presented as mean±SD. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis. PTCSC3 regulates the effects of S100A4 on apoptosis in human papillary thyroid carcinomas cells. (A) Apoptosis; (B) Western blot. Compared to the control group, *p<0.05; compared to PTCSC3, #p<0.05; compared to si-S group, ^ p<0.05. Data analysis results were repeated 3 times and presented as mean±SD. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis. the control group, *p<0.05; compared to PTCSC3, #p<0.05; compared to si-S group, ^ p<0.05. Data analysis results were repeated 3 times and presented as mean±SD. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis. compared to si-S group, ^ p<0.05. Data analysis results were repeated 3 times and presented as mean±SD. ANOVA was used for data analysis between multiple groups. And Dunnett-t test was used for subsequent analysis.