OPN Promotes Cell Proliferation and Invasion through NF-κB in Human Esophageal Squamous Cell Carcinoma

Background Osteopontin (OPN) is a phosphorylated glycoprotein. There is increasing evidence that the OPN gene played a major role in the progression of solid organ tumors. However, few studies have clarified how OPN regulated the functional role of human esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the effect of OPN in esophageal squamous cell carcinoma. Methods First, we screened Eca-109 and KYSE-510 cells to construct OPN silencing and overexpression models. Endogenous OPN of Eca-109 and KYSE-510 were knocked down or overexpressed using small interfering RNAs. QRT-PCR, Western blot, flow cytometry, and CCK-8 were used to detect the function of Eca-109 and KYSE-510 cells. Tumor formation in nude mice was used to measure tumor growth after OPN inhibition. Results Eca-109 and KYSE-510 cells contain the si-OPN arrest cell cycle in the S-phase and increase apoptosis. These changes were OPN downregulation of the NF-κB pathway that significantly reduced the protein levels of TNF-α, IL-1β, and p-p65. However, the activity of Eca-109 and KYSE-510 cells was enhanced in OPN overexpressing cells. Then, the in vivo tumor formation experiment in nude mice showed that the tumor volume and weight of nude mice after silencing OPN were significantly reduced. Conclusion This study contributed to understanding the vital role of OPN in ESCC development and progression. This could be a promising molecular target for developing new ESCC diagnostic and therapeutic strategies.


Introduction
Esophageal cancer, also known as esophageal cancer, is a common digestive tract tumor, and its incidence and mortality rates vary widely from country to country. China is one of the regions with a high incidence of esophageal cancer in the world, and the incidence is mostly in the middle-aged and elderly population [1,2]. Te typical esophageal cancer symptom is progressive difculty swallowing, frst dry food, then semiliquid food, and fnally water and saliva [3]. Most patients with esophageal cancer are already in the middle to the late stage when diagnosed with cancer, making the treatment process more passive. Terefore, early detection of esophageal cancer risk factors and prevention to reduce the occurrence of esophageal cancer are the key tasks in the prevention and treatment of esophageal cancer nowadays [4,5]. Terefore, the search for efective molecular targets has been a research hotspot and difculty in the feld of esophageal cancer.
Osteopontin (OPN) is a phosphorylated glycoprotein [6]. OPN molecule is 41.5 KD and is an acidic secreted glycoprotein [7]. OPN can promote cellular chemotaxis, invasion, and metastasis. It was involved in immune response, promotes blood vessel growth, and inhibits apoptosis [8]. Tere is growing evidence that OPN genes play a regulatory role in a variety of solid cancerous tumors [9,10]. Moreover, elevated OPN levels may be linked to poor patient prognosis [11,12]. Highly phosphorylated human breast OPN promotes cell migration in human placental choriocarcinoma cell lines, whereas less phosphorylated OPN does not stimulate cell migration [13]. Tese past studies used purifed OPN or recombinant OPN from noncancerous tissues for functional assays [14]. Although OPN expression has been extensively studied, the role and the exact mechanism of OPN in esophageal carcinogenesis remain unclear. Terefore, the role of OPN secreted by cancer cells in the behavior of cancer cells is not yet clear. Terefore, we attempted to examine the relationship between OPN and cancer cell proliferation and apoptosis.
OPN can activate nuclear factor-κB (NF-κB) to promote the secretion of ECM in chondrocytes [15]. NF-κB can activate genes that maintain cell proliferation consisting of two subunits, p65 and p50 [16]. Te NF-κB pathway is primarily an infammatory oncogenic signaling pathway that contributes to angiogenesis and proliferation and was constitutively activated in various human cancers [17,18]. NF-κB is a transcription factor that plays a signifcant regulatory role in cellular life processes, including immunity, stress response, adaptive immunity, and lymphoid organ formation [19,20]. As an early transcription factor, activation of NF-κB does not require newly translated proteins in regulation [21]. Te activated NF-κB is transported from the cytoplasm to the nucleus and then binds to specifc DNA sequences to form DNA/NF-κB complexes, which initiate transcription of downstream DNA sequences and synthesis of proteins, which in turn perform diferent biological functions [22]. Terefore, we attempted to explore the relationship between OPN and NF-κB pathways in esophageal cancer cells.
In this study, we examined the expression of OPN in esophageal squamous carcinoma cell lines. Small interfering RNA (siRNA) knocked down the expression of endogenous OPN in esophageal squamous carcinoma cell lines. A silent OPN nude mouse tumorigenic model was constructed to elucidate the role of OPN in regulating ESCC proliferation in vitro and in vivo. Te aim is to improve the understanding of esophageal cancer treatment.

MTT Analysis.
10 μL of 12 mmol/L MTT (Honorgene, AWC0118b) stock solution was added to each well. Another negative control was made by adding 10 μL of MTT stock solution (AWC0118a, Abiowell) to 100 μL of medium alone. Te plates were incubated at 37°C for 4 h. When the cell density was higher (more than 1 × 10 5 cells per well), the incubation time could be shortened to 2 h. 50 μLL of DMSO (AWC0147a, Abiowell) was added to each well and mixed thoroughly with a pipette. Te culture plate was incubated at 37°C for 10 min. Te absorbance (OD) value at 490 nm was analyzed with the enzyme labeling instrument (MB-530, Huisong).

Flow Cytometry Analysis.
Cells were collected by digestion with EDTA-free trypsin. Cells were washed twice with PBS (AWC0409, Abiowell) and centrifuged at 2000 rpm for 5 minutes to collect approximately 3.2 × 10 5 cells. 500 μL of Binding bufer was in addition to suspend the cells. After adding 5 μL of Annexin V-APC, 5 μL of Propidium Iodide (Solarbio, CA1020) was added, mixed at room temperature, protected from light, and the reaction was performed for 10 min. Within 1 h, and observed and detected by fow cytometry.
Cells were digested and the cell suspension was plated into a Petri dish containing a coverslip. Cells were grown on coverslips for 48 hours in a CO 2 incubator. Take out the coverslip and operate in the following order: rinse with PBS for 3 minutes, fx in methanol: glacial acetic acid � 3 : 1 fxative for 30 minutes, then stain with Giemsa solution for 10 minutes, and fnally, rinse with tap water. After drying, the coverslips were fipped on the glass slides and examined microscopically (DSZ2000X, Cnmicro).

Statistical Analysis.
All data were analyzed using GraphPad Prism 9.0 software (GraphPad Software, La Jolla, CA, USA). Te results were expressed as mean ± standard deviation (SD). An unpaired t-test was used to compare the two groups with a normal distribution. Comparisons among multiple groups were conducted using a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Diferences were considered statistically signifcant at P < 0.05.

Te Expression of OPN in the Esophageal Carcinoma and Normal Esophageal Epithelial Cell Lines.
To observe the expression of OPN in esophageal cancer cell lines, we selected Eca-109, KYSE-450, KYSE-510, and normal esophageal epithelial cell line HEEC for experiments. QRT-PCR and Western blot were used to detect the expression of OPN. Te data results showed that the mRNA and protein of OPN are highly expressed in the three esophageal cancer cells (Figures 1(a) and 1(b)), and there is a statistical signifcance (P < 0.05). We selected Eca-109 and KYSE-510 cells for subsequent experiments.

OPN Silenced Cell Model was Constructed.
Te above results showed that OPN was highly expressed in Eca-109, KYSE-450, and KYSE-510 cell lines, and there was no signifcant diference. We randomly selected Eca-109 and KYSE-510 cell lines for subsequent experiments. To investigate the efect of OPN on Eca-109 and KYSE-510 cells, we constructed a silent OPN cell model to inhibit the expression of OPN. As displayed in Figures 2(a)-2(d), compared to the si-NC, cells transfected with si-OPN had signifcantly decreased OPN mRNA or protein levels. As shown in Figure S1, OPN mRNA or protein levels were signifcantly higher in Eca-109 and KYSE-510 cells transfected with oe-OPN compared to oe-NC. Te above data indicated that the cell model was successfully constructed.

Te Impact of Knockdown OPN on the Proliferation of Esophageal Squamous Carcinoma Cells.
To study the efect of OPN silencing on the proliferation of esophageal cancer cells, the proliferation efciency of Eca-109 and KYSE-510 cells was observed by MTT assay. Compared with the si-NC group, the proliferation ability of Eca-109 and KYSE-510 cells in the si-OPN group was considerably decreased (Figure 3(a)). Cell proliferation and cell cycle were detected by fow cytometry. Te data showed that Eca-109 and KYSE-510 cells containing si-OPN blocked the S-phase cell cycle and enhanced apoptosis (Figures 3(b) and 3(c)). Te result of overexpression of OPN was the opposite ( Figure S2). In conclusion, silencing OPN efectively inhibited the malignant progression of cells.

OPN Afected the Expression of NF-kB in Esophageal
Squamous Carcinoma Cells. In view of the efect of the NF-kB pathway on cell proliferation, we investigated whether inhibition or overexpression of OPN afected the expression of the NF-kB pathway in Eca-109 and KYSE-510 cells. Te total cellular levels of p65 and p-p65 were analyzed by Western blot. Te data showed that downregulation of endogenous OPN reduced TNF-α, IL-1β, and p-p65 protein levels ( Figure 4). Up-regulation of endogenous OPN can promote the protein expression of TNF-α, IL-1β, and p-p65 ( Figure S3). In conclusion, OPN had an efect on the NF-κB pathway.

OPN Knockdown Could Inhibit Tumor Proliferation In
Vivo. Te above results indicated that inhibition of OPN in vitro could afect the proliferation of cells. We continued to explore whether the downregulation of OPN could inhibit tumor proliferation in vivo. Cells were transfected with OPN silencing plasmids and injected into nude mice under the armpit. Figures 5(a) and 5(b) displayed tumor growth in nude mice within 40 days. Compared with the si-NC group, the tumors in the si-OPN group appeared later and grew slowly. OPN knockout tumors were found to have signifcantly lighter fnal weight than the Control ( Figure 5(c)). Collectively, these data demonstrate that OPN could encourage the development of tumor growth in vivo.

Discussion
Esophageal cancer is one of the most common GI tumors with a high mortality rate in EC patients. Tis article explored that silencing OPN inhibits the proliferation of Eca-109 and KYSE-510 cells. Te NF-κB pathway was inhibited and apoptosis was increased in Eca-109 and KYSE-510 cells with down-regulated OPN gene expression. Tis showed that modulation of OPN can mediate NF-κB to inhibit the progression of esophageal cancer cells.
It is pointed out that the level of OPN is related to the tumor grade and prognosis of patients with bladder cancer, breast cancer, prostate cancer, and colon cancer [23][24][25]. Tis suggests that this protein was involved in tumor formation and was closely associated with tumor progression. High OPN levels are associated with lymph node metastasis.

Genetics Research
Patients with high OPN levels have smaller overall survival than those with low OPN levels [26]. In the present study, we found that OPN plays a major role in regulating ESCC cell proliferation and NF-κB p65 expression. Previous studies have shown that OPN was highly expressed in ovarian cancer tissues, and overexpression of OPN promotes ovarian cancer cell proliferation and is an unfavorable factor for the survival and prognosis of ovarian cancer patients.
Targeting OPN has potential implications for providing new therapeutic opportunities for ovarian cancer patients [27]. Tis study used Eca-109 and SKYE-510 cells as ESCC tumor cell models and HEEC cells as normal esophageal epithelial cell    It has been noted that the mechanism by which OPN enhances tumor aggressiveness may be related to nuclear factor kappa B (NF-lB) activation [28]. NF-κB activation was associated with several tumorigenic processes [29,30]. Te NF-lB pathway was an important pro-infammatory signaling pathway that plays an important role in carcinogenesis. OPN activates the NF-lB signaling pathway mainly by binding to integrin αvβ3, ultimately leading to tumor progression [31,32]. OPN promotes tumor growth in breast cancer by activating the CD44/NF-kappa B pathway in cells with low integrin β3 levels [33]. Interestingly, the knockdown of endogenous OPN reduced the proliferation of Eca-109 and KYSE-510 cells. Tese changes suggest that OPN downregulation signifcantly reduces NF-lB p65 and p-p65 protein levels. Tese results indicated that OPN could mediate the proliferation and apoptosis of Eca-109 and KYSE-510 by regulating the expression of NF-κB p65. Te clinical study of its in-depth mechanism needs to be further explored.
In conclusion, our data demonstrated that OPN can regulate ESCC cell proliferation through NF-κB. Knockdown of OPN expression in Eca-109 and KYSE-510 cells inhibited cell proliferation and promoted apoptosis. Overexpression of OPN in Eca-109 and KYSE-510 cells promoted cell proliferation and inhibited apoptosis. Tis fnding could serve as a basis for a potential target for treating esophageal cancer.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
Te Ethical Institutional Animal Care and Use Committees of Hunan Cancer Hospital approved the protocol of this work (Number: 2021-098). Te treatment of animals during the experiment conforms to the standards of "Guiding Opinions on Being kind to Experimental Animals" issued by the Ministry of Science and Technology in 2006.

Conflicts of Interest
Te authors declare that they have no conficts of interest.