LncRNA NEAT1 antagonizes the inhibition of melanoma proliferation, migration, invasion and EMT by Polyphyllin B

Polyphyllin B (PPB) is a compound with anti-tumor effects. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long-stranded noncoding RNA that induces epithelial-mesenchymal transition (EMT) of tumor cells and promotes tumor growth and metastasis. However, the role and mechanism of PPB on melanoma and the correlation between them remain unclear. In this study we screened NEAT1 by using LncRNA transcriptomic sequencing, and then transfected B16F10 cells using OVER-NEAT1 lentivirus. Next, we found that PPB had significant proliferation inhibition of melanoma and B16F10 cells through MTT assay and establishment of mouse subcutaneous transplantation tumor model; in addition, through wound healing assay, transwell assay and establishment of mouse melanoma lung metastasis model, we found that PPB significantly inhibited the invasion and migration of B16F10 cells in vitro, and inhibited the metastasis of melanoma to lung, bone and liver in vivo. Finally, changes in the expression levels of EMT-related proteins were assessed by western blot (WB) and immunohistochemistry, and PPB significantly downregulated the expression levels of MMP-9, N-cadherin, etc., and upregulated E-cadherin. While overexpressed NEAT1 showed the ability to promote melanoma proliferation, migration and invasion, in addition to partially reversed the inhibition of proliferation, migration and invasion of melanoma by PPB mentioned above.


Introduction
Melanoma, a tumor arising from the malignant transformation of melanocytes, occurs mostly in the skin and has a high mortality rate, accounting for nearly 73% of skin cancer-related deaths, with more than 320,000 new cases and 57,000 new deaths each year (Gershenwald and Guy 2016;Allgöwer et al. 2020;Sung et al. 2021). Melanoma can be treated surgically at the local stage with a 5-year relative survival rate of 98%, and although most primary melanomas can be cured by local excision, the prognosis for metastatic melanoma is poor, with a median survival of 9 months and a dramatically reduced long-term survival rate of only 10% (Turner et al. 2018). Therefore it is necessary to find new drugs or active ingredients for the treatment of metastatic melanoma.
EMT is a process by which epithelial cells acquire mesenchymal features. During EMT, cells display fibroblastlike morphology and cytoarchitecture, as well as enhanced migratory capacity, and in addition, these migrating cells often have invasive properties (Pastushenko and Blanpain 2019). In cancer, EMT is associated with tumor initiation, invasion, metastasis and resistance to therapy, with the ability to promote tumor cell migration and invasion (Yang et al. 2020). There is growing evidence that non-epithelial tumors, including melanomas, can also acquire mesenchymal-like properties that increase their propensity to metastasize and decrease their susceptibility to treatment (Pedri et al. 2022 Long non-coding RNAs (LncRNAs) can regulate a variety of biological functions in the body. Among the nuclear transcription factors, LncRNA NEAT1 has been reported to be involved in the invasion, migration and EMT of a variety of tumor cells, including osteosarcoma cells (Chen et al. 2021), lens epithelial cells (Dong 2020), endometrioid adenocarcinoma cells (Li et al. 2016) and retinoblastoma cells (Luan et al. 2021). These studies demonstrate that NEAT1 promotes cancer progression, migration, invasion and EMT.
Paris polyphylla is a herb widely distributed in southwest China, with a wide range of biological activities, including hemostatic, antibacterial and antivenom activities, and has been used for thousands of years (Shen et al. 2018;Guo et al. 2018;Qin et al. 2018). Traditionally used as an anti-tumor drug in China, including lung cancer, osteosarcoma, brain tumors and other cancers (Wang et al. 2018). Polyphyllin B (PPB) is an active ingredient extracted and isolated from the Chinese herb Paris polyphylla, with molecular formula C 51 H 82 O 20 . Polyphyllin B has been found to promote Ros-mediated Bax/Cyt-c apoptosis leading to suppress growth and aggression of glioma cells (Cheng et al. 2021). In addition, Polyphyllin B can inhibit migration and invasion of human bladder cancer by regulating EMT-associated factors and MMPs (Niu et al. 2020). However, the role of PPB on melanoma and the mechanisms remain unclear.
In this study we determined the effect of PPB on the proliferation, migration and invasion of melanoma. In addition we found that NEAT1 is involved in this process and explored the role of overexpressed NEAT1 in.
All C57BL/6 mice (male,5-6 weeks old) were acquired from Hangzhou Ziyuan Experimental Animal Technology Co,.Ltd. (Hangzhou, China, SCXK2019-0004). All procedures for animal experiments were performed following the "Principles of Laboratory Animal Care" and guidelines of the laboratory animal care committee of Anhui University of Chinese Medicine (Animal Ethics Number: AHUCM-mouse-2021062).

Cell culture
The melanoma cell lines B16F10 was kindly provided by the Stem Cell Bank, Chinese Academy of Sciences (Beijing, China), and cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS, Gibco, CA, U.S.A.), 100 units/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO 2 .
Transcriptomic sequencing of LncRNA 6 C57BL/6 mice (male, 18-20 g, 5-6 weeks old) were housed under specific pathogen-free conditions at a temperature of 24 ± 1˚C and humidity of 55 ± 5% in a laminar airflow room with a 12 h light and 12 h dark circle. After acclimatization for 1 week, 6 mice were subcutaneously inoculated at the right axilla with 1 × 10 6 B16F10 cells per 100 μL PBS to produce subcutaneous graft tumor model. After 1 day of tumor inoculation, B16F10 cells injected-mice were randomly divided into two groups: 3 mice + 100 μL distilled water/2d (the control group); 3 mice + 100 μL PPB 1 mg/kg/2d (the PPB group). After 7 days of tumor inoculation, one intraperitoneal injection was given every other morning for 14 days. 14 days after starting the administration, mice were anesthetized by intraperitoneal injection of 100 μL of 1% sodium pentobarbital solution, cervically dislocated and executed, and the tumors were immediately surgically removed. Three replicates of each group were set up, trizol was added, total RNA was extracted, and the samples were sent to Tsingke Biotechnology Co., Ltd (Beijing, China). After passing the quality control, the whole transcriptome library was constructed for the tested samples, and PE150 sequencing was performed through Illumina platform.

Construction of overexpressed plasmid and lentivirus transfection
Incubate 2 × 10 5 cells/well in 6-well plates, after reaching 60-70% confluency, over-NC (NC) cells and over-NEAT1 (OE) cells were obtained by transfecting B16F10 cells using lentivirus (Hanbio Biotechnology, Shanghai, China) at 37 °C, 5% CO 2 according to the manufacturer's instructions. The B16F10 cells used in the follow-up experiments were B16F10 NC cells and OE cells. The sequence of NEAT1 over-RNA is shown in Supplementary Material. The mapping of the overexpression vector LV-m-NEAT1 is shown in Supplementary Fig. 1. Micrographs of NC cells and OE cells under fluorescence microscopy and results of verification of overexpression levels using QRT-PCR assays are shown in Supplementary Fig. 2.

Cell viability assay
Cell viability was assessed using MTT. NC cells and OE cells were inoculated into 96-well plates at a density of 6 × 10 3 cells/ well and cultured overnight. Cells were treated with different concentrations of PPB (DMSO < 0.1%) in each well for 24 h and 48 h, and then incubated with 20 μL of MTT (50 μg/mL final concentration) for 4 h in each well. Following solubilization with DMSO, the absorbance values were then measured at 570 nm. Cell viability = (absorbance value of PPB-treated group/absorbance value of control group) × 100%, and the IC 50 for 24 h and 48 h was calculated by entering the cell viability of each group into Graphpad 8.0.2.

Wound healing assay
The effect of PPB on cell migration was assessed using a wound healing assay. NC and OE cells were inoculated at a density of 5 × 10 5 cells/well and cultured for 24 h. After scraping the cell monolayer with a sterile micropipette tip, the wells were washed with PBS and treated with medium or PPB (1.39 μM) (DMSO < 0.1%) for 24 h. After drug treatment, pictures were taken under the microscope (200 ×) at 0 h and 24 h, and then the healing area was measured using Image J 6.0 software. The wound healing rate was calculated as: Healing rate = (wound area at 0 h-wound area at 24 h)/ wound area at 0 h × 100%.

Transwell migration and invasion assay
The migration and invasion of NC and OE cells was assessed in transwell chambers containing 24 mm diameter polycarbonate membranes. The NC and OE cells (2 × 10 5 cells/well) were seeded in the upper chamber without matrigel coating for the migration assay and in the upper chamber precoated with matrigel for the invasion assay. After cells were treated with medium or PPB (1.39 μM) (DMSO < 0.1%) for 24 h, cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet, and then the wells were washed with PBS. Counted and photographed with a microscope.

Establishment of mouse subcutaneous transplantation tumor model and mouse melanoma lung metastasis model
24 C57BL/6 mice (male, 18-20 g, 5-6 weeks old) were housed under the same conditions as in described above. After 1 week of domestication, 12 mice were each injected with 100 μLPBS containing 1 × 10 6 B16F10 NC cells mixed in the right axilla, and another 12 mice were each injected with 100 μLPBS containing 1 × 10 6 B16F10 OE cells mixed in the right axilla to establish a mouse melanoma subcutaneous transplantation tumor model, and D1 was recorded on the day of inoculation. After 1 d of tumor cell inoculation, the mice were randomly divided into 4 groups of 6 mice each: 6 mice inoculated with NC cells + 100 μL saline/2d (NC control group); 6 mice inoculated with NC cells + 100 μL PPB 1 mg/kg/2d (NC administration group), 6 mice inoculated with OE cells + 100 μL saline/2d (OE control group), and 6 mice inoculated with OE cells + 100 μL PPB 1 mg/kg/2d (OE administration group). All mice were weighed every other day, and PPB solution prepared with saline was injected intraperitoneally once every other morning for 14 days after 7 days of tumor inoculation.14 d after initiation of drug administration, all mice were sacrificed by cervical dislocation. The tumor samples were immediately surgically removed, then weighed and photographed, and stored in -80℃ refrigerator and 4% paraformaldehyde.
A lung metastasis model of melanoma was established using 100 μL PBS containing 1 × 10 6 B16F10 NC cells or OE cells mixed in the tail vein of 12 C57BL/6 mice. 3 mice/ group,the grouping, administration and anesthesia were the same as described above. Mice are executed and bone, liver and lung tissue are immediately surgically removed and the lungs are photographed with a camera.

Flow cytometry analysis of B16F10 cells metastasis in vivo
Detection of Lungs and Liver metastases by B16F10 cells: fresh lung and liver tissues were ground, filtered, lysed of red blood cells, and then washed and centrifuged to obtain cell homogenates for f low detection of GFP fluorescence intensity. Detection of B16F10 cells on bone metastasis: remove the femur and tibia of one of the legs of each group of mice, then rinse the bone marrow cavity with a syringe (replaced with a 1 mL needle) aspirated with 2 mL of 1640 medium, f lush out the bone marrow, centrifuge the lysed red blood cells and wash again to obtain whole bone marrow to detect GFP f luorescence intensity.

Western blot analysis
Cells were collected after treatment with medium or PPB (1.39 μM) (DMSO < 0.1%) for 24 h; the tumor samples were removed from the -80 °C refrigerator, ground under an ice bath to obtain a homogenate. The protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 2 h and then reacted with the primary antibody (1:1000) overnight at 4 °C. After washing with TBST, the membranes were incubated with the goat anti-rabbit lgG or goat anti-mouse lgG (1:10,000) for 2 h at 4℃.

HE, melanin and immunohistochemistry staining
HE staining: tumor samples were dehydrated and embedded, followed by deparaffinization and hematoxylin staining for 15 min. Next we treated the tissue with hydrochloric acid and alcohol for 10 s and placed it in an ammonia solution. Finally, the tissues were stained with eosin for 5 min.
Melanin staining using fontana-masson stain kit. Tumor samples were incubated in warm Amino Silver Solution, Gold Chloride Solution (0.2%), Sodium Thiosulfate Solution (5%), and Nuclear Fast Red Solution, respectively, for melanin staining according to the manufacturer's instructions.
Immunohistochemistry staining: the tumor samples were removed from 4% paraformaldehyde, embedded in paraffin, cut into thin slices and dewaxed. The slices were soaked in the order of 100%-95%-80% concentration of ethanol for 5 min each time and washed with distilled water. Slices were incubated with 3% H 2 O 2 for 20 min at room temperature to remove endogenous peroxidase activity, and then the slices were washed with PBS. The slices were placed into primary antibodies and incubated overnight at 4 °C. The primary antibodies (1:300) comprised MMP2, MMP9, Vimentin, E-cadherin, N-cadherin and ki-67. Subsequently, the slices were washed with PBS and incubated with secondary antibodies (1:3000) for 30 min at 37 °C. Finally, the slices were stained with aminobenzidine (DAB), rinsed with distilled water and stained with hematoxylin for 1 min.
The slices were photographed under a light microscope in three random high-power fields. The Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze the images. The black color developed in the melanin staining and brown color developed in the immunohistochemistry test was set as the unified standard for judging the positive reaction of all photographs, and each photograph was analyzed to obtain the integral optical density (IOD) and the pixel area of the tissues (AREA) for each photograph. Then, the average optical density values (AOD) were calculated using the formula AOD = IOD/AREA.

Statistical analysis
Quantitative study results were plotted using GraphPad Prism 8.0.2 software. All experiments were repeated at least three times. Data were compared using one-way analysis of variance (ANOVA) and Dunnett's test, and P < 0.05 was considered a statistically significant difference.

Chemical structure of PPB and its effect on the LncRNA expression profile of melanoma
The chemical structure of PPB is shown in Fig. 1A. To investigate the changes in LncRNA expression profiles before and after PPB treatment, melanoma samples were (B) Volcano map of LncRNA differential expression in mouse melanoma after administration of 100 μL saline/2d or PPB 1 mg/kg/2d for 14d: the horizontal coordinate is the fold change of expression levels in different administration groups/different control groups, the vertical coordinate is the statistical significance of the change in expression, and different colors represent different classifications sequenced for LncRNA transcriptomics after 14 d of PPB administration. DEseq was used for differential expression analysis to compare the control group with the PPB group. And genes with |log2Ratio|≥ 1 and q < 0.05 were selected as the screening conditions for significant differential expression. The analysis showed that a total of 3321 genes were changed after 14 d of PPB administration, and the volcano map of differentially expressed genes showed 2241 genes upregulated and 1080 genes downregulated (Fig. 1B). For differentially expressed LncRNAs, then annotated according to known databases (Ensembl, NCBI, GENCODE, HGCN), the top 6 most significantly differentially expressed LncRNAs are listed in Table 1, showing that NEAT1 is downregulated and most significantly differentially expressed.

Overexpression of NEAT1 reverses the inhibition of PPB on B16F10 cell proliferation and melanoma growth in mice
To evaluate the proliferation inhibition of B16F10 cells and melanoma by PPB and and the effect of NEAT1 in it. We used the establishment of MTT assay and a C57BL/6 mice subcutaneous transplantation tumor model to conduct the study, respectively.
B16F10 NC cells and OE cells were treated with different concentrations of PPB (0, 0.5, 1, 2, 4, 8, 16 μM) for 24 h and 48 h, and cell viability was measured by MTT assay. PPB significantly reduced the cell viability of B16F10 cells in a time-and dose-dependent manner ( Fig. 2A-2B). OE cells were significantly more proliferative than NC cells ( ** P < 0.01, ## P < 0.01) (Fig. 2C). Graphpad 8.0.2 was used to plot and calculate the IC 50 , and the results are shown in Table 2.
We established a subcutaneous transplantation tumor model in C57BL/6 mice. There was no differential increase in body weight in any group of mice 2 weeks after cell inoculation (Fig. 2E). The tumor is displayed in Fig. 2D. The tumor weight and tumor-to-body weight ratio of mice in the OE administration group were significantly less than those in the control group ( ** P < 0.01, ## P < 0.01); while those in the OE control group were significantly larger than those in the NC control group ( * P < 0.05); in addition, those in the OE administration group were significantly larger than those in the NC control group ( + P < 0.05) (Fig. 2F-2G).
For further histological analysis of melanoma tissue, we performed HE staining, ki-67 staining and melanin staining of the tumors. We found that the tumor cells in the control group had vigorous growth, regular morphological arrangement, tight structure, large and deeply stained nuclei visible under the microscope, melanin formation in the cytoplasm, and ki-67 staining showing mostly yellow granules in the nuclei, and high AOD values (high level of positive expression) for melanin and ki-67 staining results; moreover, these phenomena were more obvious in the tumors of OE controls ( Fig. 2H-J, * P < 0.05, ** P < 0.01). However, after PPB treatment, most of the cells in the administered group were seen to be wrinkled and sparsely arranged with irregular morphology, and the nuclei were significantly deformed and necrotic, and ki-67 staining showed mostly blue granules in the nuclei, and low AOD values (low level of positive expression) for melanin and ki-67 staining results ( Fig. 2H-J, * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01). These phenomena and results were reversed in the OE-PPB group compared to the NC-PPB group ( Fig. 2H-J, + P < 0.05, ++ P < 0.01).
These results suggest that overexpression of NEAT1 promotes B16F10 cell and melanoma growth in vitro and in vivo, and is involved in the reversal of the inhibition of their growth by PPB.

Overexpression of NEAT1 reverses the inhibition of migration and invasion of B16F10 cells and melanoma in mice by PPB
We investigated the effects of PPB and overexpression of NEAT1 on the migration and invasion of B16F10 cells in vitro using wound healing assay and transwell assay, respectively. We found that compared with the control group, after PPB treated B16F10 cells for 24 h, wound healing rate was decreased and fewer cells crossing the upper chamber without or with matrigel ( ** P < 0.01 vs. NC, ## P < 0.01 vs. OE); in addition, the OE control group had an increased wound healing rate and more cells crossed the upper chamber compared to the NC control cells ( * P < 0.05 vs. NC); interestingly, the cells in the OE-administered group also showed stronger migration and invasion ability than those in the NC-administered group ( + P < 0.05 vs. NC-PP) (Fig. 3A-3E).
To investigate the effects of PPB and overexpression of NEAT1 on lung, liver and bone metastases of melanoma in vivo, we established a mouse melanoma lung metastasis model. We found that after 14 d of PPB treatment, the number of pulmonary metastatic nodules was significantly lower in the dosing group; moreover, the number of pulmonary metastatic nodules was higher in the OE control group than in the NC control group; we also found that compared with the NC dosing group, OE administration group had a higher  number of pulmonary metastatic nodules compared with the NC administration group (Fig. 3F). The cells were obtained by grinding the pulmonary, hepatic and bone marrow of different groups of mice, and the GFP fluorescence intensity was detected by flow cytometry (Becman culter, FC500). Observing the experimental results we found that, compared with the control group, the pulmonary, hepatic and bone marrow of the administered group of mice after 14 d of PPB treatment had different degrees of melanoma metastasis inhibition; in addition, melanoma metastasis was more significant in the OE control group compared to the NC control group (Fig. 3G).

The expression levels of EMT-related proteins were detected by WB and immunohistochemistry
The results of WB showed that after 14 d of PPB administration, PPB down-regulated E-cadherin and up-regulated the expression of other proteins (MMP-9, N-cadherin, Vimentin, Twist, Snail, Zeb1) ( * P < 0.05, ** P < 0.01, # P < 0.05, Mice administered 100μL saline/2d or 1 mg/kg/2d PPB after 14d, (F)Representative photographs of lung tissues from different groups of mice. (G) The results obtained from lung, liver and bone marrow homogenates of different groups of mice were examined by flow cytometry for GFP fluorescence intensity, with a more rightward shift of the peak representing a stronger degree of melanoma metastasis ## P < 0.01); in addition, E-cadherin was down-regulated and other proteins were up-regulated in the OE control group compared to the NC control group ( * P < 0.05, ** P < 0.01). We also found that E-cadherin was down-regulated and other proteins were up-regulated in the OE administration group compared to the NC administration group ( + P < 0.05, ++ P < 0.01) (Fig. 4A-4B).
We further used immunohistochemical staining to detect the expression levels of EMT-related proteins. We found that after 14 d of PPB administration, an increase in the number of positive cells with brownishyellow nuclei and increased AOD values in the slices of E-cadherin, a decrease in the number of positive cells and AOD values in the other slices (MMP-2, MMP-9, N-cadherin, Vimentin) in the administration group compared to the control group ( Fig. 4C-D, * P < 0.05, ** P < 0.01, ## P < 0.01); in addition, the OE control group had fewer positive cells and lower AOD values in E-cadherin sections and more positive cells and higher AOD values in other sections compared to the NC control group; finally, we also found a reversal of these phenomena and results in the OE administration group compared to the NC control group (Fig. 4C-D, ** P < 0.01, + P < 0.05, ++ P < 0.01). These results are consistent with the results of WB.
EMT is an essential process in tumor progression and metastasis, and tumor cells that undergo EMT have a greater capacity for migration and invasion. PPB inhibited melanoma migration and invasion, and overexpression of NEAT1 promoted melanoma migration and invasion, and was resistant to PPB (Fig. 5).

Discussion
Cancer becomes fatal when tumor cells spread from the primary site in the body to distant organs (a process called metastasis) (Finley 2019), cancer metastasis is the most common cause of death in cancer patients. Recently, tumor cell metastasis as a new target for cancer therapy has become a subject of interest. Melanoma originates as a result of malignant proliferation of melanocytes within the epidermis. Melanoma carries a high risk of death because of its ability to spread rapidly and invade new organs. Melanoma is a malignant tumor with high metastatic rate and high mortality. EMT has been reported to be associated with melanoma growth and metastasis, cellular metastasis involves a transition from an epithelial to a mesenchymal state. Despite some progress, metastatic melanoma is a key cause of treatment failure and high mortality (Sinha et al. 2021;Wang et al. Fig. 5 This figure aims to briefly illustrate that PPB regulates the expression of EMT-associated proteins to inhibit melanoma migration and invasion, and overexpression of NEAT1 is involved in this process and is resistant to PPB 2020). Tumor cell metastasis causes melanoma to have a high mortality burden in advanced stages, therefore, finding potential drugs to stop the metastasis of melanoma cells is increasingly important for the treatment of melanoma.
The herbal monomeric compound PPB has been found to have great potential in the treatment of cancer in recent years. PPB has been found to inhibit the growth, migration and invasion of a variety of tumors including bladder cancer (Niu et al. 2020) and glioma (Cheng et al. 2021). However, to date, no studies have reported the effect of PPB on melanoma. Our study revealed for the first time that PPB inhibits melanoma growth in vivo.
LncRNAs have been shown to play crucial roles in cancer biology. To screen which LncRNAs play a role in PPB inhibition of melanoma growth, we performed transcriptome sequencing of tumor samples, the results showed that NEAT1 was the most significantly differentially expressed LncRNA. Next, to investigate the role played by NEAT1 in PPB against melanoma, we used lentiviral to transfect melanoma cells B16F10 to overexpress NEAT1. NEAT1 drives tumor initiation and progression by modulating the expression of genes involved in the regulation of tumor cell growth, migration, invasion, metastasis, EMT, stem cell-like phenotype, chemoresistance and radioresistance, indicating the potential for NEAT1 to be a novel diagnostic biomarker and therapeutic target (Dong et al. 2018;Li et al. 2021). It has been demonstrated that NEAT1 is aberrantly expressed in many tumors and functions as a pro-oncogene, promoting tumor cell proliferation, migration, invasion and EMT, including laryngeal cancer cells , retinoblastoma cells (Luan et al. 2021), melanoma (Xia et al. 2019, p. 3).
In our study, we found that PPB significantly inhibited the proliferation, migration and invasion of B16F10 cells by MTT, wound healing and transwell assays. The results of in vivo experiments also showed, PPB significantly inhibited melanoma growth; this was further confirmed by the results of HE, ki-67 and melanin staining; flow cytometry also showed that PPB significantly inhibited the metastasis of B16F10 cells to lung, liver and bone in vivo. These results suggest that PPB inhibits the growth and metastasis of B16F10 cells and melanoma well in vitro and in vivo.
In addition, we found that NEAT1 overexpressing cells demonstrated enhanced migration and invasion abilities in wound healing and tranwell assays. The results of in vivo experiments also showed that overexpressed NEAT1 caused melanoma grow faster and had stronger metastatic ability.
The histological analysis of tumor tissues demonstrated that NEAT1 overexpression caused melanoma cells to grow more vigorously, with larger cross-sectional area and higher proliferation index in vivo. In addition, overexpression of NEAT1 resulted in stronger metastasis of melanoma to lung, liver and bone.
Normally, the cells that form epithelial sheets in various tissues of the body display apical-basal polarity and are held together laterally by tight junctions and adherens junctions, the latter of which are formed by cell surface epithelial cadherin (E-cadherin) molecules.This organization is crucial for the structural integrity of epithelia. Upon activation of EMT, E-cadherin expression is repressed, which leads to the loss of the typical polygonal,cobblestone morphology of epithelial cells.The cells acquire a spindle-shaped mesenchymal morphology and express markers that are associated with the mesenchymal cell state, notably neural cadherin (N-cadherin), Vimentin and Fibronectin (Kalluri and Weinberg 2009;Dongre and Weinberg 2019). In addition, transcription factors including Snail1/Snail, Snail2/Slug, Twist and Zeb1 are involved in the orchestration of EMT (Stemmler et al. 2019). During EMT, increased expression of the mesenchymal markers Vimentin and N-cadherin and downregulation of the epithelial marker E-cadherin, a potent suppressor of tumor cell invasion and metastasis, have been observed (Lin et al. 2010;Nakamura and Tokura 2011). MMP2 and MMP9 are important enzymes known to degrade the extracellular matrix, which is capable of degrading the extracellular matrix and basement membrane, which regulate the degradation of the extracellular matrix, but an imbalance in cellular regulatory mechanisms during EMT allows excessive degradation of the extracellular matrix and thus promotes invasion and metastasis (Ribatti et al. 2020). Our immunohistochemical and WB results showed that PPB inhibited the expression of EMT transcription factors Snail, Twist and Zeb1, prevented EMT, eventually downregulated mesenchymal markers Vimentin and N-cadherin, and upregulated epithelial marker E-cadherin, inhibiting tumor cell migration and invasion. The effect of overexpressed NEAT1 on the expression of EMT-related proteins was just the opposite of PPB, showing an enhanced ability of tumor cell migration and invasion.
To further investigate whether LncRNA NEAT1 is involved in the action of PPB on melanoma and B16F10 cells, and the role it plays in this, we also compared the NCadministered groups and OE-administered groups, and the results of all experiments showed that the OE-administered group had greater proliferation, migration and invasion abilities in vivo and in vitro. For the first time in the literature, we reveal that NEAT1 is involved in inhibition of melanoma growth, metastasis and EMT in mice by PPB, and overexpressed NEAT1 exhibited resistance to PPB in the treatment of melanoma.

Conclusion
In summary, PPB can inhibit the growth and metastasis of mouse melanoma cells and melanoma in vitro and in vivo, and in addition, overexpressed NEAT1 not only promotes their growth and metastasis but also shows resistance to PPB during the treatment of mouse melanoma. Therefore, using PPB and knocking down or knocking out NEAT1 to treat melanoma may be a new therapeutic strategy in the future.