SPTBN2 Promotes the Progression of Thyroid Cancer by Accelerating G1/S Transition and Inhibiting Apoptosis

Background Thyroid carcinoma (TC) is an increasingly common malignancy of endocrine organs, and its most frequently encountered histotype is papillary thyroid cancer (PTC). Identifying new potential gene alterations is important for completely elucidating the mechanism of PTC initiation and progression. Thus, we performed whole transcriptome sequence analysis (RNA-seq) on 79 PTC tissue samples and paired adjacent nontumor tissue samples to study the molecular mechanism of TC tumorigenesis and progression further. The results of RNA-seq analysis showed that spectrin beta, nonerythrocytic 2 (SPTBN2), was markedly overexpressed in PTC tissues relative to that in the paired nontumor tissues. Additionally, the analysis results for 502 PTC samples and 58 nontumor thyroid samples from The Cancer Genome Atlas dataset were consistent with our RNA-seq results. However, the molecular mechanisms and function of SPTBN2 in TC progression remain unknown. Methods We examined SPTBN2 gene expression in 48 papillary thyroid tumor tissues and paired adjacent normal thyroid tissues by using qRT-PCR. SPTBN2 expression in the TC cell lines was silenced by small interfering RNA. Then, the transfected TC cells were used to investigate the in vitro function of SPTBN2. Result The expression of SPTBN2 was significantly upregulated in our RNA-seq cohort, our local validated cohort, and TCGA RNA-seq cohort. The results of the in vitro experiment revealed that in TC cell lines, SPTBN2 downregulation considerably suppressed tumor cell proliferation, the cell cycle, migration, colony formation, and invasion and induced cell apoptosis. Furthermore, the protein levels of CCNE2, CDK2, CDK4, and Bcl-2 were downregulated, and those of P21, Bax, cleaved caspase-8, and cleaved caspase-3 had increased in transfected TC cells relative to in control TC cells. Conclusion The downregulation of SPTBN2 caused apoptosis and retarded G1/S cell cycle transition in TC cells. Thus, SPTBN2 may be a good candidate gene for TC diagnosis and therapy.


Introduction
Thyroid carcinoma (TC) is a highly common malignancy of endocrine organs; this disease accounted for approximately 2.9% of all new cases of malignancies in the United States of America in 2019 [1]. In the United States of America, the morbidity of thyroid cancer is now 2 times greater than that 30 years ago, and thyroid cancer is now the sixth most prevalent cancer in women [2,3]. In the majority population, papillary thyroid cancer (PTC) is the most common subtype of TC and accounts for 85% of newly diagnosed TC cases [4]. Although the morbidity of thyroid cancer is high, PTC has a good prognosis with a >90% 10-year sur-vival rate [5,6]. However, a large number of patients with PTC experience metastasis and recurrence after conventional therapeutic strategies [7]. Therefore, further studies on the probable molecular mechanism of thyroid tumors are needed to provide early accurate diagnosis and appropriate treatment to patients with TC.
With the advent of whole transcriptome sequencing (RNA-seq) technology, next-generation sequencing has become widely utilized to identify changes in genes between tumors and normal tissues. Although numerous studies have been carried out to explore the probable mechanisms of thyroid tumors, the precise biomechanisms underlying the progression and occurrence of thyroid tumors remain to be elucidated fully. A growing number of reports have shown that genetic alterations, such as gene mutations, epigenetic modifications, and copy number variations, play an important role in the initiation of TC [8,9]. For example, the point mutation of B-type Raf kinase can promote PTC tumorigenesis and progression via the initiation of the mitogenactivated protein kinase pathway [10,11]. Furthermore, PAX8/PPARγ rearrangements, RET/PTC rearrangements, and RAS mutations are important for the initiation and progression of differentiated TC [12][13][14]. Several biomarkers have been identified as appropriate diagnostic markers and potential therapeutic targets for TC. However, the identification of new potential gene alterations remains important for completely elucidating the mechanism of TC initiation and progression.
We performed RNA-seq on 79 PTC tissue samples and paired adjacent nontumor tissue samples to further study the underlying molecular mechanisms of TC. Through bioinformatics analysis, we discovered that spectrin beta, nonerythrocytic 2 (SPTBN2), a gene located on chromosome 11q13 in humans and encoding the membrane scaffold protein beta-III spectrin, is a potential powerful candidate gene that is associated with TC initiation and progression. Beta-III spectrin maintains dendritic architecture and stabilizes several membrane proteins [15]. A mutation in SPTBN2 causes spinocerebellar ataxias type-5, a human neurodegenerative disorder that is characterized by progressive locomotor incoordination, slurred speech, and uncoordinated eye movements [16,17]. Another study reported that the mutation of SPTBN2 can disrupt Hippo signaling activity in follicle cells [18]. However, the previous study mainly focused on the effect of SPTBN2 mutation on spinocerebellar ataxias type-5. The effect of the copy number variation of SPTBN2 on the tumorigenesis and metastasis of malignancies has never been studied. Here, we were the first to prove that SPTBN2 acts as a potential oncogene in the tumorigenesis and metastasis of thyroid malignancies by inhibiting apoptosis and promoting the G1/S cell cycle transition in malignant thyroid cells.

Materials and Methods
2.1. Tissues. The fresh malignant thyroid samples and paired nontumor thyroid samples that were used in this study were obtained from patients with PTC who underwent thyroid surgery at the First Affiliated Hospital of Wenzhou Medical University (China) from May 2017 to April 2019. The patients did not undergo chemo-or radiotherapy for pretreatment. All of the samples were snap-frozen in liquid nitrogen and stored at −80°C before RNA extraction. All of the tumor tissues used in the present study were examined blindly by three independent senior thyroid pathologists. Written consent/assent for the usage of the thyroid samples was given by the patients and their relatives. The procedures that were performed in our study were approved by and conducted in accordance with the ethical standards of the Institutional Review Board of the First Affiliated Hospital of Wenzhou Medical University (approval no. 2012-57).  (Table 1). KTC-1 (8 × 10 4 cells per well) and BCPAP (7 × 10 4 cells per well) were plated on six well-plates and incubated for 24 h before transfection. siRNA was transfected into the TC cell lines by using Lipofectamine RNAiMAX Reagent (13778, Invitrogen, Carlsbad, CA, US). The transfected TC cells were harvested from the six-well plates at 48 h posttransfection, and knockdown efficacy was determined via qRT-PCR.

The Cancer
2.5. RNA Extraction and qRT-PCR Analysis. Total RNA was extracted from thyroid tissues or cell lines with TRIzol reagent (15596026, Invitrogen, US). Afterward, the A260/ A280 ratio was used to evaluate RNA quality. By using a ReverTra Ace qPCR RT Kit (TOYOBO Life Science, Cat. No: FSQ-101, Japan), RNA was converted into cDNA. RNA expression levels were detected via qRT-PCR with an AB Prism 7500 fast system (Applied Biosystems, US) and SYBR qPCR Mix (QPS-201; TOYOBO Life Science, Japan). The primer pair details are shown in Table 2 was used for the in vitro Transwell migration assay. A total of 3:5 × 10 4 transfected TC cells were suspended in 300 μL of medium (RPMI-1640, free serum) and placed in the upper insert. The lower outer well contained 650 μL of 10% serum RPMI-1640 medium. After 20 h of incubation, the nonmigrated cells on the upper side of each membrane were removed by using sterile cotton swabs, and the migrated cells on the lower surface were fixed in 4% paraformaldehyde solution and stained with crystal violet (0.01%). Cell invasion capacity was evaluated by using an invasion chamber (354480, Corning, NY, US). All of the steps in this procedure were the same as those followed in the Transwell migration assay.
2.9. Apoptosis Analysis. Cell apoptosis was measured by applying an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (556547, BD, US). TC cells (5-6 × 10 cells per well) were cultured in six-well plates for 1 day and transfected for 2 days. The cells were collected and centrifuged at 1100 rpm for 5 min. Then, the supernatant was decanted, and the cells were washed with PBS (1 mL). These processes were repeated three times. Afterward, each tube was added with 4 μL of Annexin V-FITC and 4 μL of PI and then placed for 20 min in the dark. Apoptosis was studied with a BD FACSCalibur flow cytometer (Becton-Dickinson, CA, US).

SPTBN2 Was Highly
Overexpressed in PTC. RNA-seq was performed on 79 PTC tissue samples and paired adjacent nontumor tissues to examine the potential mechanism of PTC. We found that the expression of SPTBN2 in the PTC samples was highly upregulated compared with that in the adjacent nontumor thyroid tissues (Figure 1(a), Supplementary Table 1). Tissues from patients with PTC and normal thyroid tissues in TCGA dataset were also investigated ( Figure 1(c)). The mRNA expression level of SPTBN2 was also upregulated in our local validated cohort (48 PTC patients) as revealed by qRT-PCR (Figure 1(e)). Both results indicated that SPTBN2 was highly upregulated in the PTC samples relative to that in the nontumor thyroid samples. The diagnostic potential of SPTBN2 was evaluated on the basis of a ROC curve. ROC curve analysis was performed on all of the above cohorts (our RNA-seq cohort, our local validated cohort, and TCGA RNA-seq cohort) (Figures 1(b), 1(d), and 1(f)). The results indicated that SPTBN2 is a potential gene for the diagnosis of PTC.

SPTBN2 Expression Was Closely Related to
Clinicopathological Characteristics in PTC. We examined the relationship between clinicopathological features and SPTBN2 expression in our local validated cohort and TCGA cohort to gain insights into the mechanism of SPTBN2 in the progression of PTC. The PTC tissues in TCGA cohort (502 patients with PTC) and our local validated cohort (48 patients with PTC) were classified individually into the high SPTBN2 expression group or the low SPTBN2 expression group in accordance with their median expression level value of SPTBN2. The results of the analyses showed that in TCGA cohort, histological type, LNM, tumor size, and disease stage were related to SPTBN2 upregulation (Table 3). Similar results were found for our validated cohort. Specifically, the upregulation of SPTBN2 was correlated with tumor size (P = 0:004), disease stage (P < 0:001), and LNM (P = 0:002) ( Table 4). We omit the nonstatistically   (Table 3) in Table 4. All analytical results provided support that SPTBN2 may act as an oncogene in PTC.

SPTBN2 Overexpression Was Correlated with Increased
Tumor Size. Logistic regression studies were applied to evaluate the relationship between SPTBN2 and tumor size in PTC. In the univariate logistic regression analysis, age, gen-der, SPTBN2 expression, LNM, and disease stage were significant risk factors for increased tumor size (Table 5). Subsequently, multivariate logistic regression analysis was conducted on the variables that were found to be statistically significant in the univariate logistic regression analysis. The results indicated that age, gender, SPTBN2 expression, and disease stage were significant independent risk factors for increased tumor size (Table 6).

Knockdown of SPTBN2 Inhibited TC Cell Migration and
Invasion In Vitro. By subjecting TCGA cohort and our validated cohort to the χ 2 test, we found that LNM was significantly correlated with SPTBN2 expression (Tables 3 and 4). Therefore, we studied the function of SPTBN2 in the migration/invasion capacities of TC. Migration capacity was assessed by using migration assays. The results demonstrated that in malignant thyroid cell lines, migration capacity was significantly suppressed by the knockdown of SPTBN2 (Figures 3(a) and 3(b)). The invasion capacity of the transfected TC cell lines was detected via invasion assays. The results showed that the invasion capability of the TC cell lines was also inhibited by the knockdown of SPTBN2 (Figures 3(c) and 3(d)).

Knockdown of SPTBN2 Induced Apoptosis and Retarded G1/S Cell Cycle Transition in TC Cell Lines In Vitro.
The GSEA results revealed that in TCGA cohort, the cell cycle/ apoptosis pathway was closely associated with SPTBN2 expression in thyroid cancer ( Figure 4). Given that cell cycle regulation is closely associated with cell proliferation, we analyzed the function of SPTBN2 in the TC cell cycle. The proportion of G1-phase cells increased with the knockdown of SPTBN2, whereas that of S-phase KTC-1 and BCPAP cells decreased (Figures 5(a) and 5(b)). Hence, the G1/S transition was significantly suppressed with the knockdown of SPTBN2. Cell apoptosis is another cause of cell growth inhibition. Therefore, the percentage of apoptotic cells after siRNA transfection was assessed. The ratio of apoptotic cells after transfection with SPTBN2-siRNA significantly increased relative to that after transfection with the negative control (Figures 5(c) and 5(d)). These results indicated that SPTBN2 markedly promoted cell cycle transition and suppressed TC cell apoptosis.

SPTBN2 Knockdown Suppressed the Proliferation of PTC Cells by Arresting G1/S Transition and Promoting Cell
Apoptosis. After transfecting TC cells with siRNA, we performed WB analysis to detect the expression levels of signaling biomarker proteins that were correlated with the cell cycle/apoptosis in TC cell lines. The results illustrated that in TC cells, the knockdown of SPTBN2 significantly inhibited the expression levels of cell cycle/anti-apoptotic proteins, including CCNE2, CDK2, CDK4, and Bcl-2 ( Figure 6(a)). Furthermore, the downregulation of SPTBN2 significantly increased the expression levels of P21, cleaved caspase-8, cleaved caspase-3, and Bax in TC cells (Figures 6(a) and  6(b)). These results indicated that the knockdown of

Discussion
Thyroid cancer is a heavy burden on society, and its morbidity has continuously increased on an annual basis [17]. PTC, the most frequent thyroid cancer type (>85%), has been attracting growing attention [18]. Approximately 10% of patients with PTC cannot benefit from conventional thera-pies due to metastasis and recurrence [19]. Thus, exploring the fundamental molecular mechanisms of TC is essential for the identification and management of this malignancy.
A number of studies have reported that numerous genes play a vital role in the tumorigenesis of TC [20][21][22]. However, the current understanding of the molecular mechanism of TC is far from comprehensive. The widely used next-generation sequencing technology provides an opportunity for elucidating the molecular mechanisms of TC. Previously, we subjected 79 paired PTC samples and nontumor thyroid tissues to whole transcriptome sequencing. Through RNA-seq data analysis, we discovered that SPTBN2 expression in PTC samples was  The SPTBN2 gene is located on chromosome 11q13.2 [23]. It codes for a protein called SPTBN2 or beta-III spec-trin. Beta-III spectrin functions in diverse cellular functions, including the determination of cell form, the arrangement of transmembrane proteins, and the association of organelles. SPTBN2 mutation can result in spinocerebellar ataxias type-5. This disease is characterized by neural degeneration, uncoordinated eye movements, and locomotor incoordination. SPTBN1, which is an important paralog of SPTBN2, and other nonerythroid spectrins exert either oncogenic or   13 Disease Markers tumor suppressor activities by affecting various cancerrelated biological processes, including cell differentiation, DNA damage repair, and epithelial-to-mesenchymal transition [24]. A growing body of evidence has extensively proven that SPTBN2 plays a vital role in the diagnosis of cholangiocarcinoma. For example, by using a bioinformatics approach, Feng et al. demonstrated that SPTBN2 is highly associated with poor prognosis in ovarian cancer [25]. Zhang et al. and Yue et al. reported that SPTBN2 may play a vital role in the progression of colorectal cancer [26,27]. Wu et al. reported that SPTBN2 is positively correlated with the poor prognosis of lung adenocarcinoma [28]. However, the probable mechanisms of SPTBN2 in this malignancy has never been investigated before [29][30][31].
Although bioinformatics studies have revealed that SPTBN2 may be correlated with the initiation and progression of malignancies, the molecular mechanism of SPTBN2 in thyroid cancer has never been investigated. In this study, we discovered that SPTBN2 was exceedingly overexpressed in PTC tissues in our RNA-seq and TCGA datasets. Then, we evaluated the expression of SPTBN2 in 48 PTC samples and paired adjacent nontumor tissues via qRT-PCR and explored the biological function of SPTBN2 in vitro. The results of qRT-PCR were consistent with those of previous RNA-seq and TCGA analyses.
Previous in vitro studies on TC cell lines (BCPAP and KTC-1) knocked down SPTBN2 expression by using siRNA. The results of the cell experiments indicated that the knockdown of SPTBN2 could inhibit proliferation and metastasis capacity and induce apoptosis in the TC cell line in vitro. Furthermore, the signaling pathways that might be associated with SPTBN2 were analyzed via the GSEA. Since the GSEA showed downregulation of SPTBN2 related to the apoptosis and cell cycle signaling pathways in TCGA dataset, we focused on the expression level of the apoptosis relative protein (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9) and the cell-cycle relative protein (P21, CCNE2, CDK2, and CDK4) in the transfected TC cell line. Moreover, the downregulation of SPTBN2 led to a substantial increase in the protein levels of Bax, cleaved-caspase-8, and cleavedcaspase-3 and a reduction in CCNE2, CDK2, CDK4, and  Figure 6: Downregulation of SPTBN2 inhibited the tumorigenesis and progression of TC cell lines by arresting G1/S transition and promoting cell apoptosis. (a) Levels of CCNE2, CDK2, CDK4, Bcl-2, P21, caspase-3, cleaved-caspase3, caspase-8, cleaved-caspase8, and Bax were evaluated via WB analysis with β-actin as the loading control. (b) Quantitative results of WB analysis (normalized to β-actin). * P < 0:05, * * P < 0:01, * * * P < 0:001,and * * * * P < 0:0001. The data are presented as the mean ± standard deviation (three independent experiments).