SSRP1 Promotes Lung Cancer Progression by Blocking the WNT Pathway and is Negatively Regulated by miRNA-28-5p

Background: Structure-specic recognition protein 1 (SSRP1) plays important roles in the development of various tumors. Numerous reports have described the effects of microRNAs (miRNAs) on lung cancer apoptosis, metastasis and proliferation. However, the relationship between SSRP1 and miRNAs in the development of lung cancer remains unclear. Therefore, the purpose of our study was to explore the functions of SSRP1 in the occurrence and development of lung cancer. Methods: First, we analyzed the expression of SSRP1 in human lung cancer tissues and normal tissues, and the relationship between SSRP1 gene expression and overall survival through the UALCAN and GEPIA browsers. Second, we conducted experiments in vivo and vitro to demonstrate the roles and mechanisms of SSRP1 in the occurrence and development of lung cancer and its relationship with miR-28-5p. Results: Our study demonstrated over-expression of SSRP1 in tissue sections from patients with lung cancer and in lung cancer cell lines, as validated by bioinformatics analysis. The over-expression of SSRP1 was found to be associated with lung cancer development and low overall survival rates. Silencing of SSRP1 by siRNA inhibited lung cancer proliferation, migration and invasion by blocking the WNT signaling pathway in vitro and vivo. We also veried SSRP1 was negatively regulated by miR-28-5p (cid:0) as predicted by a variety of miRNA-related databases. Further studies showed that miR-28-5p mediated suppression of SSRP1 inhibited lung cancer cell proliferation. Conclusion: Therefore, our data suggested that SSRP1 promotes lung cancer progression by blocking the WNT pathway and is negatively regulated by miRNA-28-5p. SSRP1: Structure-specic recognition protein 1; FACT: Facilitates chromatin transcription; miRNAs: MicroRNAs ; UTRs: Untranslated regions; miR-28-5p: MicroRNA-28-5p; DMEM: Dulbecco's modied Eagle's medium ; NC: Negative control; LUSC: Human lung squamous cell carcinoma ; LUAD: Lung adenocarcinoma; PCNA: Proliferating cell nuclear antigen.

signi cantly inhibits proliferation, invasion and migration, and promotes apoptosis in colorectal tumors [13]. Josephine et al. have reported that inhibition of SSRP1 decreases resistance to Erlotinib in non-small cell lung cancer [7]. The crucial role of SSRP1 in tumors prompted us to determine whether SSRP1 might participate in the development of lung cancer.
MiRNAs participate in cellular processes including proliferation, apoptosis and tissue morphogenesis [15,16]. Many miRNAs have been shown to regulate the expression of genes with tumor-associated functions, and to play important roles in promoting or inhibiting tumorigenesis [17]. MicroRNA-28-5p (miR-28-5p) has been veri ed to have tumor suppressor activity in several types of tumors, such as hepatocellular carcinoma, prostate cancer and natural killer/T-cell lymphoma [18][19][20]. Moreover, Cheng Wang et al. have found that miR-28-5p suppresses human renal carcinoma cell lines' proliferation and migration [21]. Liang Ma et al. have shown that miR-28-5p inhibits the migration of breast cancer cells [22]. Studies have shown that the miR-28-5p/CAMTA2 axis regulates colorectal cancer progression through the WNT/β-catenin signaling pathway [23], and SSRP1 is regulated negatively by miR-28-5p [24]. However, the relationship and role of miR-28-5p, SSRP1 and WNT/β-catenin signaling in the development of lung cancer were not elucidated. In this study, we aimed to explore the expression and the detailed functions and channels of SSRP1 in the proliferation, migration and invasion of lung cancer, and to identify the upstream miRNA regulating SSRP1 in lung cancer, to assess whether SSRP1 might be used as a criterion for the diagnosis and prognosis of lung cancer patients.

Bioinformatics
The analysis of bioinformatic data on the expression level of SSRP1 in human lung cancer and corresponding normal tissues and the relationship between SSRP1 gene expression and overall survival rate in patients with lung cancer were assessed through the UALCAN (http://ualcan.path.uab.edu/index.html) and GEPIA (http://gepia.cancer-pku.cn/index.html) browsers.

Cell culture and human tissue specimens
The human lung cancer cell lines A549, NCI-H446, H1299 and PC9, murine cells (LLC) and the normal human lung epithelial cell B2B were obtained from the Department of Respiratory Medicine, First Hospital of Jilin University (Changchun, Jilin, China) and Basic Medical School of Jilin University (Changchun, Jilin, China). Cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) containing 5.5 mmol/l D-glucose and supplemented with 10% FBS (BI, Israel), 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were cultured in a 37 °C incubator with 5% CO 2 . Five tissue samples from lung cancer patients (mean age, 58.2±6.85 years, ranging from 49 to 68; three female patients and two male patients) were collected at the First Hospital of Jilin University from September 2018 to May 2019. Patients receiving radiotherapy or chemotherapy were excluded from our study. Patients whose tissue samples were used in this research had provided written informed consent under an IRB-approved protocol. This study was approved by the local institutional review board of the First Hospital of Jilin University. The study methodologies conformed to the standards set by the Declaration of Helsinki.

Cell migration and invasion assays
Cell migration assays were conducted with Transwell chambers (Corning Costar, Cambridge, MA, USA) with an 8-mm pore size. The lower chamber was lled with DMEM containing 20% FBS, and the upper chamber contained cells resuspended in serum-free DMEM, at a concentration of 5 × 10 4 cells per well.
After cultivation under 5% CO 2 for 48 h at 37 °C, the bottom surfaces of the polycarbonate membranes in the upper chamber were wiped with cotton swabs to remove residual cells, and cells were counted visually under an IX71 inverted uorescence microscope (Olympus, Shinjuku, Tokyo, Japan) after staining with 0.1% crystal violet dye. Matrigel (BD Biosciences, San Jose, CA, USA) was also used in the Transwell chambers before cells were cultured in the invasion assays. Cell migration and invasion were determined by counting ve random elds under an optical microscope. The data are presented as mean ± standard deviation.

Western blot analyses
Cells and tissues were harvested and lysed in RIPA buffer for 30 min at 4-8 °C. Proteins were detected and quanti ed with a BCA protein assay kit (Thermo Fisher Scienti c, Inc., MA, USA). Protein samples (30-50 μg) were separated with 12% SDS-PAGE and transferred to polyvinylidene uoride membranes.
Nonspeci c binding was blocked with 5% low-fat milk with Tween-20 for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with the following primary antibodies . Lamin B1, encoded by the LMNB1 gene, is composed of a two-dimensional matrix of proteins located next to the inner nuclear membrane, which has been highly conserved during evolution. Therefore, Lamin B1 is often used as a nuclear protein internal control. The membranes were scanned for statistical analysis by enhanced chemiluminescence with a gel image processing system (Tanon, Shanghai, China).

Nuclear extraction
A nuclear and cytoplasmic protein extraction kit was purchased from Beyotime (Beyotime Biotechnology Inc., Nantong, China). Speci c experimental steps were performed according to the manufacturer's protocol.

Animal experiments
The study was approved by the Animal Research Ethics Committee of Jilin University. The siRNA used in vivo was synthesized by Genepharma (Suzhou, China) and dissolved in PBS buffer. The dose of siRNA in nude mice was 0.5 mg/kg.
Ten BALB/c male nude mice (18-22 g) were purchased from Beijing Vital River Laboratory Animal Technology and housed under a 12/12 h light/dark cycle in an air-conditioned room at 22 ± 2 °C, and were given free access to food and water. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scienti c Investigation Board of the College of Basic Medicine, Jilin University. Ten nude mice were randomly divided into two groups equally ( ve mice per group). A 100 µl subcutaneous injection containing 1 × 10 6 NCI-H446 cells was delivered into the upper right back in mice in each group. When the tumor size reached 3-5 mm, siSSRP1 or negative control (NC, used with isodose PBS) was inoculated into the xenograft tumor by multi-point injection every 3 days. Tumor volume was calculated with the following formula: V = (length) × (width) 2 /2. After 28 days, mice were killed by intraperitoneal administration of barbiturates (240 mg/kg) followed by cervical dislocation. Tumor tissue was removed and frozen at −80 °C for protein analysis or xed in 4% paraformaldehyde for hematoxylin-eosin (H&E) staining and immuno uorescence staining.

H&E staining and Immunohistochemistry
Tissues were xed in 4% paraformaldehyde solution for at least 4 h at room temperature, and this was followed by dehydration, dipping in wax, para n embedding and cutting into sections. The sections were then H&E stained. For immunohistochemistry, sections were incubated with serum or BSA for 30 min at room temperature, and then were incubated with diluted primary antibody for 2 h and then with secondary antibody. The samples were observed under a BX53 uorescence microscope (Olympus, Shinjuku, Tokyo, Japan).

Online searches of miRNA-related databases
The miRNA-related databases used in this research were as follows: TargetScan

Statistical analyses
Data analysis was performed on at least three independent experimental groups, and the results are expressed as mean ± standard deviation. Statistical analyses were performed in SPSS version 17.0. Comparison of the two sets of data was performed with unpaired Student's t-test. One-way ANOVA was followed by Dunnett's test to compare more than two sets. Differences were considered signi cant at *p< 0.05 and **p< 0.01, vs. the NC group.

Results
SSRP1 expression is upregulated in lung cancer and is associated with overall survival rate To determine the role of SSRP1 in lung cancer, we analyzed the expression of SSRP1 in human tumor tissues from the online UALCAN browser and found that SSRP1 expression was upregulated in both human lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) samples ( Fig. 1A and 1B, p < 0.01), compared with normal samples. The GEPIA browser also showed that the mean expression level of SSRP1 was higher in LUAD and LUSC than in the corresponding normal tissues (Fig. 1C, p < 0.01).
Overall survival rate analysis showed that lung cancer patients with high expression of SSRP1 showed a low overall survival rate, as compared with patients with low expression of SSRP1 (Fig. 1D, p < 0.05).
Immunohistochemistry results showed that the SSRP1 levels were higher in grade I-IV lung cancer than normal lung tissues. Furthermore, the SSRP1 levels in high-grade lung cancer were signi cantly higher than those in lower grade lung cancer (Fig. 1E) was signi cantly inhibited after SSRP1 siRNA transfection. These data revealed that blocking the expression of SSRP1 represses the proliferation of lung cancer cells.

SSRP1 affects the cell cycle and apoptosis in lung cells
To further explore the regulatory role of SSRP1 in lung cancer cell proliferation, we examined cell cycle and apoptosis in lung cancer cells by ow cytometry. NCI-H446 and H1299 cells treated with SSRP1 siRNA for 48 hours showed signi cant G0/G1 phase arrest, as compared with cells treated with NC ( Fig. 3A). Simultaneously, the percentage of apoptotic cells in NCI-H446 and H1299 cells transfected with SSRP1 siRNA was markedly higher than that in the NC group (Figs. 3B and 3C). Thus, knockdown of SSRP1 inhibited lung cancer cell cycle progression from G0/G1 phase to S phase and induced lung cancer cell apoptosis.

Downregulation of SSRP1 inhibits migration and invasion of lung cancer cells
We additionally explored the effects of SSRP1 on migration and invasion in lung cancer cells. Transwell assays showed that downregulation of SSRP1 with SSRP1 siRNA transfection signi cantly impaired migration (Figs. 4A and 4B) and invasion (Figs. 4C and 4D) in both NCI-H446 and H1299 cells. Therefore, SSRP1 silencing inhibited migration and invasion of lung cancer cells.

Ssrp1 Inhibition Blocks Lung Cancer Growth In Vivo
Next, we used the NCI-H446 cells to construct a lung cancer xenograft model to explore the role of SSRP1 in lung cancer in vivo. The xenograft model construction and administration method were as shown in Fig. 5A. Animal experiment results revealed that the tumor size and volume in siSSRP1 group were much smaller than those in the NC group (Figs. 5B and 5C). However, the body weight did not show a signi cant difference between the groups (Fig. 5D), thus indicating that SSRP1 siRNA did not cause any damage to the bodies of the mice.
Immunohistochemistry showed that the expression of SSRP1 in the siSSRP1 group was markedly downregulated (Fig. 5E). As an index of cell proliferation, proliferating cell nuclear antigen (PCNA) expression was inhibited in the siSSRP1 group (Fig. 5E), compared with the NC group. Bcl-2 in the siSSRP1 group was markedly downregulated (Fig. 5E), and the expression of Bax in the siSSRP1 group was clearly upregulated (Fig. 5E), as compared with that in the NC group. MMPs play a key role in matrix remodeling [25] and are associated with tumor invasion [26]. We found that the expression of matrix metalloproteinase (MMP)-2 and MMP-9 was inhibited in the siSSRP1 group. However, no organ toxicity was observed by H&E staining (Fig. 5F).
Furthermore, SSRP1 silencing increased the expression of the cell cycle regulator p21 and the proapoptotic factor Bax, but decreased the expression of Cyclin D1 and Bcl2 in NCI-446 and H1299 cells (Figs. 5G and 5H). The results above demonstrated that SSRP1 inhibition blocked lung cancer growth both in vitro and in vivo.

SSRP1 affects lung cancer cell activity by inhibiting the WNT signaling pathway
The WNT pathway is considered the classical signaling pathway regulating tumor proliferation and metastasis [27]. To determine whether the WNT signaling pathway participates in SSRP1 regulation of lung cancer growth, we used western blotting to detect alterations of critical proteins in the WNT signaling pathway. Western blotting showed that SSRP1 knockdown inhibited expression of p-GSK3β and β-catenin, which are critical proteins in the WNT signaling pathway (Figs. 6A and 6B), in total protein extract from NCI-446 and H1299 cells, and p-GSK3β and β-catenin were also downregulated in nucleoprotein (Figs. 6C and 6D) from NCI-446 and H1299 cells. In addition, protein expression of p-GSK3β and β-catenin was inhibited in the siSSRP1 group in the xenograft model of lung cancer (Figs. 6E and 6F). Therefore, SSRP1 silencing may block proliferation and promote apoptosis by blocking the WNT signaling pathway.
Upregulation of SSRP1 in lung cancer is associated with miR-28-5p MiRNAs are associated with the initiation and progression of cancer [28]. We previously con rmed that SSRP1 expression affects lung cancer progression. To investigate whether miRNAs regulate SSRP1 and whether SSRP1 affects the development of lung cancer, we used four miRNA-related databases to predict miRNAs that might regulate SSRP1 (Fig. 7A). The predictive results revealed that SSRP1 miRNA contains a potential miR-28-5p seed sequence within its 3′UTR (Fig. 7B). To further con rm whether SSRP1 is the target gene of miR-28-5p, we generated a luciferase expression plasmid containing the wild-type or mutant SSRP1 3′UTR (report-SSRP1-wt and report-SSRP1-mut, respectively). Luciferase activity results indicated that miR-28-5p overexpression suppressed report-SSRP1-wt-expressed luciferase, whereas report-SSRP1-mut was unaffected under the same conditions (Fig. 7C). To determine whether miR-28-5p negatively regulates SSRP1, we constructed miR-28-5p mimics and inhibitors (Figs. 7D and E) and used them to transfect NCl-446 and H1299 cells. When the expression of miR-28-5p was upregulated, the expression of SSRP1 was inhibited in both cell lines (Figs. 7F and G). In addition, the expression of miR-28-5p was downregulated, and the expression of SSRP1 was upregulated in both cell lines (Figs. 7H and I). In conclusion, SSRP1 is negatively regulated by miR-28-5p in lung cancer.

MiR-28-5p inhibits lung cancer proliferation by regulating the expression of SSRP1
To further determine whether miR-28-5p participates in the progression of lung cancer by regulating SSRP1 expression, we used miR-28-5p mimic and inhibitor to detect the in uence of miR-28-5p on NCl-446 cell proliferation. Compared with that of cells transfected with NC miRNA, the cell viability of NCl-446 cells at 450 nm OD decreased after miR-28-5p mimic transfection for 48 and 72 h (Fig. 8A)-results opposite from those observed for the miR-28-5p inhibitor (Fig. 8B). In addition, the cell numbers and colony formation ability of NCl-446 cells decreased signi cantly after miR-28-5p mimic transfection (Figs. 8C, 8D and 8E) and increased after miR-28-5p inhibitor transfection (Figs. 8F, 8G and 8H). These results showed that miR-28-5p inhibits the progression of lung cancer by inhibiting the expression of SSRP1.

Discussion
FACT, a heterodimer consisting of two subunits, SSRP1 and suppressor of Ty16, plays a crucial role in transcription and DNA replication, recombination and repair [29,30]. SSRP1 is thought to be involved in the development of several cancers and to have different roles in different cancers. SSRP1 knockdown increases the sensitivity of HCC cells to doxorubicin, 5-Fluorouracil and cisplatin, and SSRP1 is negatively regulated by miR-497 in hepatic cancer [8]. Reports have indicated that SSRP1 silencing inhibits colorectal cancer proliferation, migration and invasion via the AKT pathway, and the lncRNA LOC101927746 acts as a competing endogenous RNA that suppresses miR-584-3p and then activates its target gene SSRP1, thus inhibiting the growth of colorectal cancer [13,31]. However, SSRP1 inhibition suppresses the activity of the phosphorylation of the MAPK signaling pathway and consequently blocks the proliferation and metastasis of glioma cells [10]. FACT is also both a marker and a target of aggressive breast cancer cells [32]. In non-small cell lung cancer, knockdown of SSRP1 decreases cancer cell growth and overcomes resistance to Erlotinib by modulating the nuclear factor-kappa B signaling pathway [7]. However, the detailed function of SSRP1 in the lung cancer proliferation, metastasis and invasion remains unclear.
Bioinformatics analysis showed that SSRP1 was over-expressed in both LUSC and LUAD. Moreover, SSRP1 expression was higher in patients with high-grade lung cancer than in patients with low-grade lung cancer and in normal samples. The overall survival rate of patients was negatively correlated with the expression of SSRP1. Meanwhile, the expression of SSRP1 was signi cantly higher in a series of lung cancer cells, especially in NCl-H446 and H1299 cells, than in normal human lung epithelial cells. These results suggest that SSRP1 might regulate the development of lung cancer, and that SSRP1 overexpression might predict a high clinical grade of lung cancer and low survival rate of lung cancer patients.
To further explore the function of SSRP1 in lung cancer, we silenced SSRP1 by using siRNA to detect the effects of SSRP1 inhibition on lung cancer in vivo and vitro. First, we used several methods, including CCK-8, colony formation assays and FACS ow cytometric analysis, to validate that SSRP1 knockdown inhibited cell viability and proliferation and induced apoptosis in NCl-H446 and H1299 cells. Decreased expression of SSRP1 weakened the growth and volume of lung cancer cell xenograft tumors. The immunohistochemistry results in lung cancer cell xenograft tumors showed that SSRP1 silencing inhibited the growth of lung cancer and induced apoptosis in vivo. These results indicated that the downregulation of SSRP1 inhibited lung cancer cell proliferation and stimulated anti-apoptosis function.
Furthermore, we explored the function of SSRP1 in lung cancer invasion and metastasis. NCl-H446 and H1299 cell migration and invasion ability were both inhibited after SSRP1 knockdown. Similarly, the immunohistochemistry results in lung cancer cell xenograft tumors showed that down-regulation of SSRP1 weakened migration and invasion ability in vivo. Furthermore, SSRP1 knockdown did not result in toxicity in organs including the heart, liver, spleen, lung and kidney.
On the basis of preliminary results, SSRP1 may have potential value in the lung cancer diagnosis and therapy, and further studies on SSRP1 are urgently needed. Wnt/β-catenin signaling is an evolutionarily conserved pathway that plays crucial role in embryogenesis, development and homeostasis. When the Wnt pathway is active, Gsk3 is bound to β-catenin and translocates into cell nucleus as a transcription factor. Then the pathway stimulates the expression of downstream genes including cyclin D1 and cyclindependent kinase inhibitor CDKN1A (p21), alters cell cycle progression, and plays a role in tumorigenesis [27,33]. After wnt/β-catenin signaling is inhibited, the expression of Bcl2 increases and the expression of Bax decreases, and the changes in the expression of the aforementioned genes ultimately promote apoptosis. In this study, we found that blocking SSRP1 gene expression inhibited not only the expression of p-Gsk3β, β-catenin, cyclin D1 and p21 in total cell protein, but also the expression of p-Gsk3β and βcatenin in the nucleus in NCl-H446 and H1299 cells. These results showed that Wnt/β-catenin signaling might be activated in lung cancer cells and SSRP1 inhibition represses lung cancer growth by blocking the WNT signaling pathway, in agreement with ndings from a previous study on colorectal cancer [24].
MiRNAs negatively regulate target gene expression by binding the mRNA 3´UTR regions of target genes, which encode proteins and participate in a variety of cellular physiological process in tumors [28]. Numerous studies have suggested roles of miRNAs in anti-tumor therapy [34][35][36], thus prompting us to speculate that a certain miRNA may regulate the entire process of lung cancer progression by regulating the expression of SSRP1. Given the above ndings, we used four RNA-related databases to predict the possible binding of miRNAs to SSRP1 and identi ed miR-28-5p as a miRNA that might regulate SSRP1 expression. The potential target site of SSRP1 3'UTR binding to miR-28-5p is located at 335-342. MiR-28-5p has been reported to regulate colorectal cancer by inhibiting SSRP1 expression [24], and miR-28-5p inhibits the migration and invasion of gastric cancer cells by suppressing AKT phosphorylation [37]. MiR-28-5p was transfected to verify changes in target gene expression in luciferase reporter assays, which revealed that SSRP1 was a target gene of miR-28-5p and was negatively regulated by miR-28-5p. Transfection of miR-28-5p mimics resulted in the downregulation of SSRP1 and also inhibited the proliferation of lung cancer cells, thus re ecting the ndings of SSRP1 inhibition by siRNA. However, transfection of miR-28-5p inhibitor reversed this effect. Importantly, the diagnostic ability of miR-28-5p combined with SSRP1 was stronger than that of miR-28-5p or SSRP1 alone, and also stronger than that of the tumor markers used in the clinic. These data may provide new potential diagnostic and therapeutic targets for lung cancer in the future and highlight the clinical relevance of this study. However, further research to clarify the molecular mechanism of the interaction between miR-28-5p and SSRP1 is required. Our study demonstrates the roles of SSRP1, miR-28-5p and the WNT signaling pathway in lung cancer growth.

Conclusion
In conclusion, our study reported that silencing of SSRP1 by siRNA inhibited lung cancer proliferation, migration and invasion by blocking the WNT signaling pathway, however, miR-28-5p mediated suppression of SSRP1, thus in uencing lung cancer growth via the WNT signaling pathway.       Up-regulation of SSRP1 in lung cancer is associated with miR-28-5p. (A) MiR-28-5p may regulate SSRP1, according to a Venn diagram, as determined by bioinformatics analyses. (B) The sequence of miR28-5p and the region where the SSRP1 3'UTR may bind miR-28-5p. (C) NCl-H446 cells were co-transfected with luciferase reporter vectors containing wild type or mutant SSRP1 3'UTR (report-SSRP1-wt and report-SSRP1-mut, respectively) and miR-28-5p mimics for 48 h, and the luciferase activity was determined. (D-E) Relative miR-28-5p expression in NCl-H446 and H1299 after transfection with miR-28-5p mimics and inhibitor, determined by qPCR. (F-I) SSRP1 protein levels after transfection with miR-28-5p mimics and inhibitor in NCl-H446 and H1299 for 48 h, as determined by western blot analyses. *p < 0.05, **p < 0.01 vs. the NC group.