2023 Volume 46 Issue 10 Pages 1385-1393
Cutaneous melanoma is an aggressive cancer, which is the most common type of melanoma. In our previous studies, gambogenic acid (GNA) inhibited the proliferation and migration of melanoma cells. Maternally expressed gene 3 (MEG3) is a long noncoding RNA (lncRNA) that has been shown to have inhibitory effects in a variety of cancers. However, the mechanisms in melanoma progression need to be further investigated. In the current study, we investigated the inhibitory effect of GNA on melanoma and its molecular mechanism through a series of cell and animal experiments. We found that GNA could improve epithelial mesenchymal transition by up-regulating the expression of the lncRNA MEG3 gene, thereby inhibiting melanoma metastasis in vitro and in vivo.
Melanoma is the third most common type of skin cancer, originating from the uncontrolled proliferation of melanocytes in the skin, which has a high clinical impact due to its high risk of early metastasis and poor prognosis.1,2) Patients with early-stage melanoma can easily be cured by surgical excision. However, metastatic melanoma patients have fewer treatment options. Although advanced checkpoint immunotherapy and BRAF-targeted therapy have improved the treatment and survival of melanoma patients,3) the toxic effects and innate and acquired drug resistance of these therapies are unavoidable problems currently. In addition, there is currently no pharmacological intervention that has demonstrated both statistically significant improvement and favorable prognosis in the treatment of malignant melanoma. Therefore, urgent efforts are needed to identify more efficacious and less toxic therapeutic agents.
As an important regulatory factor, long noncoding RNAs (lncRNAs) are involved in the regulation of a variety of normal cellular physiological processes.4) Previous studies have shown that some lncRNAs may play a role in tumor development by affecting cell proliferation, invasion and metastasis, such as HOTAIR, GAS5, p21, H19, MEG3, NEAT1 and PTENP1.5–7) Of the genes, maternally expressed gene 3 (MEG3) is located on human chromosome 14q32.3 within the DLK1-MEG3 locus. There are 10 exons in this gene and 35kb in length, encoding about 1.6 kb of lncRNA.8,9) MEG3 is abundantly expressed in many tissues and has a key role in development and growth.10) Uncontrolled expression of this gene could lead to moderate or severe developmental disabilities in humans.11) Furthermore, uncontrolled MEG3 expression has been observed in a variety of primary human cancers. Many human tumors and tumor-derived cell lines often have abnormal expression of MEG3, which has carcinogenic function.12–14) Overexpression of MEG3 inhibits the tumorigenic effect of nasopharyngeal carcinoma cells in vivo.15) In conclusion, these results indicate that MEG3 is one of the lncRNAs with tumor inhibitory activity.16) Further studies have shown that MEG3 can regulate tumorigenesis by regulating the interaction of tumor-related genes such as p53, RB, MYC and transforming growth factor β (TGF-β).9,17)
Natural product compounds play a pivotal role in the discovery and development of anticancer drugs.18,19) Gambogenic acid (GNA), the major bioactive ingredient of Gamboge,20) was found in the dry resin of Garcinia hanburyi HOOK. f. (Guttiferae). As previously reported, gambogenic acid inhibits a range of tumor cells, including human liver cancer cells,21,22) lung cancer cells,23,24) nasopharyngeal carcinoma cells25) and melanoma cells.26) However, the mechanism by which gambogenic acid inhibits melanoma metastasis is not yet known. Therefore, this study investigated the inhibitory effect and mechanism of gambogenic acid on melanoma metastasis in vivo and in vitro, and the mechanism of action was determined to be related to the regulation of MEG3.
GNA (>99.0%) was purified by HPLC by Dr. X. Wang of Anhui University of Chinese Medicine.27) Dacarbazine (DTIC) was purchased from Sinopharm Yixin Pharmaceutical Co. (Changchun, China).
Cell CultureMelanoma cell lines B16F10, B16F10-GFP-LUC and B16 (The American Type Culture Collection) were cultured in 25 cm2 culture flask with 1640 medium (containing 1% penicillin–streptomycin and 10% fetal bovine serum) in 5% CO2 at 37 °C. The culture medium was changed every other day. The logarithmic growth phase cells were selected in the experiment.
Transplanted and Metastatic Melanoma ModelsMouse transplanted melanoma model, B16F10 melanoma cells were taken at logarithmic growth stage and prepared into a cell suspension at a concentration of 2 × 107 cells/mL under aseptic conditions. Inoculating B16F10 melanoma cells in C57BL/6 mice’s skin. Mice inoculated with B16F10 cells were divided into 5 groups randomly. The negative control group was given the same amount of normal saline every three days for eight times, and the positive control group was given DTIC* (70 mg/kg) by intraperitoneal injection every 3 d for eight times. The GNA administration groups were divided into GNA group (8 mg/kg), GNA group (4 mg/kg) and GNA group (2 mg/kg) by intraperitoneal injection every three days for eight times. The doses of GNA used was based on our preliminary study,28) and the concentration of positive control drug was calculated from the dose used in clinic. [* DTIC: Dacarbazine, a clinical drug to treat malignant melanoma.]
One Mouse metastatic melanoma model, B16F10-GFP-LUC melanoma cells in logarithmic growth phase and fluorescently labelled with green fluorescent protein (GFP) were prepared as a cell suspension at a concentration of 2 × 107 cells/mL under aseptic conditions. Inoculating B16F10-GFP-LUC melanoma cells in C57BL/6 mice’s caudal vein. The administration mode, cycle and groups in mice with metastatic melanoma were the same as those of the transplanted melanoma mice. The weight was recorded every 3 d. Twenty-two days later, the mice were euthanized after anesthesia with 2% Pentobarbital Sodium, and pulmonary metastasis foci were photographed, weighed and recorded the longest diameter (a) and the maximum transverse diameter (b) in vertical direction were measured with vernier caliper. The tumor volume was calculated according to the formula V (mm3) = ab2/2. Then, pulmonary metastasis foci were collected for RT-quantitative PCR (RT-qPCR) and Western blotting, as well as hematoxylin–eosin (H&E) and immunohistochemistry staining. Fresh lung, liver tissues, the femur and tibia of one leg of each group from metastatic melanoma mice were collected for detection of GFP fluorescence intensity in metastases by flow cytometry.
The other mouse metastatic melanoma model was established by using melanoma cells B16F10-GFP-LUC with knockdown of MEG3. The negative control group and sh-MEG3 group were given the same amount of normal saline every 3 d for eight times, and GNA group and GNA + sh-MEG3 group were given GNA (4 mg/kg) by intraperitoneal injection every 3 d for eight times. The modeling methods and detection indexes were consistent with the metastatic tumor models mentioned above.
All animals are provided by Animal Research Center of Anhui University of Chinese Medicine, and conform to the Experimental Animal Ethics of Anhui University of Chinese Medicine, Approval Number: AHUCM-mouse-2021061.
Ethics StatementAll animal experiments comply with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The animal study was reviewed and approved by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine.
Colony Formation AssayB16F10 and B16 cells (1000 cells/well) in the logarithmic growth phase were seeded in plates with medium different concentrations of GNA. The culture medium was replaced every 2 d. After 10 d incubation, cells were fixed by 4% polyformaldehyde (500 µL/well) for 20 min. Staining cells with crystal violet for 30 min. Cells were observed and counted using a camera (Olympus, Japan).
Scratch Wound Healing AssayB16F10 and B16 cells (1 × 106 cells/well) were seeded in 6-well plates with 3 replica wells per group. Cells scratch wound with phosphate buffered saline (PBS) after each well and the migration of cells around the wound was observed by microscopy at 0 and 24 h after administration. Image J software was applied to calculate the healing rate.
Cell Migration AssayFor the transwell chamber migration assay, B16F10 and B16 cells were seeded in the chamber during logarithmic growth (placed in 24-well plates). 5 × 104 cells were added to each well in the chambers, and 800 µL of drug-containing medium containing 10% fetal bovine serum (FBS) was added to the lower 24-well plate. The cells in the lower chamber were immobilized with 4% polyformaldehyde after 24h and stained with crystal violet for 30 min, after which the number of migrating cells was counted.
Cell Invasion AssayAdd the Matrigel to the chambers (placed in a 24-well plates) and refrigerate at 4 °C overnight. When Matrigel solidified, B16F10 and B16 cells (5 × 104/wells) were added, and 800 µL of drug-containing medium containing 10% fetal bovine serum was added to the lower 24-well plate. The cells were incubated for 24 h. Fixing the cells in the lower chamber and staining with crystal violet for 30 min. Then count the number of invading cells.
H&E StainingTumor tissues were embedded in wax blocks and cut into 5 µm sections. Drying the sections and placing in an incubator of 65 °C for approximately 6 h, dewaxed and hydrated. After 15 min, the hematoxylin was washed off. After differentiation for a few seconds, staining with eosin for 2 min. Then the sections were sealed with neutral gum and photographed.
Immunohistochemistry StainingThe tumor tissue was fixed, sectioned and dried. The antibody dilution ratio of E-cadherin, N-cadherin, Vimentin, Snail, matrix metalloproteinase (MMP)-2, MMP-9 and Ki67 are 1 : 5000, 1 : 3000, 1 : 1000, 1 : 1000, 1 : 1000, 1 : 1000, and 1 : 50. Subsequently, the sections were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibody (diluted at 1 : 10000) for 2 h at room temperature. Afterwards, the sections were counterstained with hematoxylin and dried. Finally, the sections were sealed with neutral gum and images were observed by a microscope.
Western BlottingB16F10/MEG3-sh-B16F10 Cells were treated with GNA for 24 h. The cells were lysed by lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor. Subsequently, the protein concentration was determined by bicinchoninic acid assay kit, and 20 µg total protein was added to each well. The proteins were separated by 4–12% Bis-Tris polyacrylamide gel electrophoresis (PAGE) electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane for detection. The membrane was blocked by Tris Buffered Saline with Tween 20 (TBST) (containing 5% non-fat milk) at room temperature for 2 h and washed by TBST for 10 min for 3 times. Then the membrane was incubated with primary antibodies overnight at 4 °C and washed by TBST for 10 min for 3 times. The membrane was incubated with secondary antibody (1 : 10000) for 2 h at room temperature and washed by TBST for 10 min for 3 times. The proteins were visualized using the enhanced chemiluminescence kit and the Alpha View SA gel imager. The data was analyzed using the ImageJ software.
Flow Cytometry Detection of GFP Fluorescence Intensity in MetastasesFresh lung and liver tissues from metastatic melanoma mice were ground, filtered. Lysing red blood cells, washing and centrifuging to obtain the cells. GFP fluorescence intensity in lung and liver cells was detected by flow cytometry.
The femur and tibia of one leg of each group of mice were taken, then bone marrow cavity was rinsed with Dulbecco’s modified Eagle’s medium. Centrifuging, lysing red blood cells and washing to obtain bone marrow, and GFP fluorescence intensity in the bone marrow was detected by flow cytometry.
RT-qPCR AnalysisTotal RNA for RT-qPCR was isolated using the RNA Isolation Kit (Bimake, U.S.A.). After reverse transcription, the mixture containing 1 µg of cDNA and SYBR Green (Bimake) was subjected to qPCR (A&B, U.S.A.). Data processing and analysis were performed according to the 2−ΔΔCT method. qPCR was performed by using the following primers: MEG3: forward: 5′-AGCACAGTGGAGCCAGGAGTC-3′; reverse: 5′-AGCACAGTGGAGCCAGGAGTC-3′.
Statistical AnalysisAll data were presented as the means ± standard deviation (S.D.). The comparison of two samples was analyzed using the student’s t-test. Experiments were repeated three times. Statistical analysis was conducted by one-way ANOVA using statistical package SPSS 26.0 (Chicago, IL, U.S.A.). p < 0.05 was considered a statistically significant difference. A p-value <0.05 denoted statistical significance.
DTIC has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma for over 40 years and is still the standard of care in the medical treatment of advanced melanoma. Therefore, DTIC was used as a positive control in mouse metastatic melanoma model. We found that the size of pulmonary metastasis foci was significantly reduced after administration of GNA and DTIC (Fig. 1A). The volume of pulmonary metastases foci in each group was measured and statistically analyzed (Fig. 1B), we found GNA reduced the size of pulmonary metastasis foci in a dose-dependent manner. These results indicate that GNA can inhibit pulmonary metastasis of melanoma. In addition, the weight of mice (Fig. 1C) increased steadily in all groups in the early stage, but the weight of mice in control group decreased significantly in the late stage. In contrast, GNA and DTIC improved weight loss issues in mice significantly, indicating that GNA and DTIC can help maintain stable vital signs in metastatic melanoma mice. The results of pulmonary metastasis, hepatic metastasis and osseous metastasis (Fig. 1D) in each experimental group of mice by flow cytometry further indicated that GNA and DTIC inhibited the metastatic of melanoma in mice.
(A) Representative metastatic lesions in the lungs of metastatic melanoma mice. n = 8/group. (B) Volume of metastatic lesions in the lungs of metastatic melanoma mice. n = 8/group. (C) Changes in body weight of mice. n = 8/group. (D) GFP fluorescence detection by flow cytometry. (E) H&E staining and melanin staining results of lung metastases in melanoma mice. Scale bar = 50 µm. Data represent mean ± S.D., n = 8/group. * p < 0.05, ** p < 0.01 compared to control group. (GNA, Gambogenic acid. DTIC, Dacarbazine).
The results of H&E staining (Fig. 1E) shows that the cells in GNA and DTICgroups were arranged loosely than those in control group, with obvious cell nucleus crinkling and rupture, and obvious areas of apoptosis and necrosis could be seen. In addition, the results of melanin staining (Fig. 1E) showed that GNA could reduce melanin deposition in a dose-dependent manner. The above results suggest that GNA inhibits the metastasis of melanoma in vivo.
GNA Inhibits the Invasion and Metastasis of B16F10 CellsNext, we explored the effect of GNA on the invasion and migration of melanoma cells (B16F10 and B16). The results of colony formation assay showed that GNA inhibited the proliferation of B16F10 and B16 cells dose-dependently (Fig. 2A). Transwell and wound healing assays were used to determine the migration and invasion ability of B16F10 and B16 cells. The results of wound healing assay showed that GNA inhibited the migration of B16F10 and B16 cells dose-dependently (Fig. 2B). In the transwell assay, the migration and invasion ability of B16F10 and B16 cells were reduced significantly in the GNA groups compared to the control group (Fig. 2C). These results suggest that GNA can inhibit melanoma metastasis in vitro.
(A) Images of B16 and B16F10 cells colony formation. B16 and B16F10 cells were cloned 15 d after treatment of GNA. The corresponding colony formation rate is counted. ** p < 0.01 vs. control in B16 and B16F10 cells. (B) After the B16 and B16F10 cells were scratched, and the wound healing was observed under the microscope at 0 and 24 h after treatment of GNA, scale bar = 100 µm. The wound healing rate of each group of cells was calculated relative to 0 h. ** p < 0.01 vs. control at 24 h. (C) Images of B16 and B16F10 cells after 24 h invasion and migration treated with GNA. The quantitative statistics of B16 and B16F10 cell invasion and migration. Scale bar = 100 µm. Data represent mean ± S.D., n = 3. * p < 0.05, ** p < 0.01 compared to control group. (GNA, Gambogenic acid).
To investigate whether GNA inhibits melanoma metastasis in vitro and in vivo was associated with epithelial–mesenchymal-transition (EMT), we further examined the expression of EMT-related proteins in mouse metastatic melanoma model and melanoma cells. MMPs promotes tumour metastasis by degrading the cytoplasmic matrix within and around tumour tissue, of which MMP-2 and MMP-9 have the most significant degradation effect, and are therefore considered to be one of the marker proteins of mesenchymal cells. The results of Immunocytochemistry (Fig. 3A) and its quantification analysis (Supplementary Fig. S1) suggested that GNA inhibited epithelial mesenchymal transformation in mouse transplanted melanoma. Furthermore, the results of Western blot (Fig. 3B) showed that the changes in epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin, Vimentin, MMP-2 and MMP-9) of B16F10 cells, further illustrating the effect of GNA on EMT. The results suggest that GNA can inhibit melanoma metastasis in vitro and in vivo, which is associated with improved EMT.
(A) Representative images of immunohistochemical staining of metastatic melanoma mice. Scale bar = 100 µm. (B) B16F10 cells were treated with GNA (0.25, 0.5, 1 µM) for 24 h, and the protein bands was tested by Western blot. Data represent mean ± S.D., n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control group. (GNA, Gambogenic acid. DTIC, Dacarbazine).
Previously, we sequenced the lncRNA transcriptome and found that the expression of MEG3 in melanoma was up-regulated after giving different doses of GNA, which is consistent with the sequencing results. Besides, the bioinformatics database (TCGA and GTEx) showed that the MEG3 gene was reduced significantly in melanoma patients (Fig. 4A). To determine this, we used the Kaplan–Meier Plotter to stratify melanoma based on MEG3 into high and low (Fig. 4B). Results show little association between MEG3 expression and survival of patients (p > 0.05). It is possible that the limited sample size within the TCGA database may not be sufficient to show significant difference. Furthermore, MEG3 gene expression in mouse metastatic melanoma model established by using melanoma cells with knockdown of MEG3 (Fig. 4C) showed that GNA up-regulated the expression of MEG3 gene in a dose-dependent manner. To further investigate whether GNA inhibit melanoma metastasis in vitro and in vivo was associated with MEG3, we knocked down the MEG3 gene in B16F10 and B16 cells and established metastatic melanoma model again (Figs. 4D, E). We found that sh-MEG3 group had the darkest metastatic tumor color compared with other groups (Fig. 4D). Besides, pulmonary metastasis, hepatic metastasis and osseous metastasis were evidently in the sh-MEG3 group of melanoma mice, while GNA reverses MEG3-induced metastasis in melanoma (Fig. 4E). The results suggest that GNA may inhibit pulmonary metastasis, hepatic metastasis and osseous metastasis in metastatic melanoma mice through upregulation of MEG3. The results of H&E staining (Fig. 4F) of the pulmonary metastasis showed that the tumor cells in the Control group had a normal round or oval shape, with a few showing an irregular shape. In sh-MEG3 group, the growth of tumor cells was exuberant, the morphology was normal, and cell nucleus volume was larger than the Control group. After administration of GNA, the cell volume was obviously shrunk, some mitosis and a few nucleated blood cells were observed, indicating that apoptosis and necrosis occurred. GNA may inhibit pulmonary metastasis in melanoma mice by up-regulating MEG3, which was further explained by melanin staining (Fig. 4F). The above results suggest that GNA may inhibit melanoma metastasis by up-regulating MEG3 expression in vivo.
(A) Expression of MEG3 in melanoma patients in the TCGA and GTEx database. (B) Overall survival of the melanoma patients with high (group G1) or low (group G2) levels of MEG3 using TCGA database. (C)The expression of MEG3 genes in metastatic melanoma mice was detected by RT-qPCR. (C: Control group, L: GNA 2 mg/kg, M: GNA 4 mg/kg, H: GNA 8 mg/kg, D: DTIC group). (D) Representative metastatic lesions in the lungs of melanoma mice. n = 8/group. (E) Flow cytometric detection of GFP fluorescence intensity in pulmonary metastasis, hepatic metastasis and osseous metastasis of metastatic melanoma mice. n = 8/group. (F) H&E staining and melanin staining results of lung metastases in metastatic melanoma mice. n = 8/group. Scale bar = 50 µm. (GNA, Gambogenic acid. DTIC, Dacarbazine).
Then, we explored the effect of GNA on the invasion and migration of B16F10 and B16 cells with knockdown of MEG3 in vitro assay. Melanoma cells in the sh-MEG3 group were more proliferative, migratory and invasive compared to the Control group (Figs. 5A–C). These results demonstrate that knockdown of MEG3 promotes the migration and invasion of B16F10 and B16 cells, suggesting that MEG3 may be a potential target gene. While GNA inhibits migration and invasion of sh-MEG3 B16F10 and sh-MEG3 B16 cells (Figs. 5A–C), which is consistent with those of in vivo experiments. Besides, MEG3 gene expression in B16F10 cells (Fig. 5D) showed that GNA up-regulated the expression of MEG3 gene, MEG3 may be one of the targets of GNA. We speculate that the inhibition of melanoma metastasis in vitro and in vivo by GNA may be related to the upregulation of MEG3 expression.
(A) Images of B16 and B16F10 cells colony formation. B16 and B16F10 cells were administering GNA or down-regulating of MEG3 expression. After 15 d of incubation and the corresponding colony formation rate is counted. * p < 0.05, ** p < 0.01 vs. Control in B16 and B16F10 cells. (B) After the B16 and B16F10 cells were scratched, and the wound healing was observed under the microscope at 0 h and 24 h, scale bar = 100 µm. The wound healing rate of each group of cells was calculated relative to 0 h. * p < 0.05, ** p < 0.01 vs. Control at 24 h. (C) The Images of invasion and migration of B16 and B16F10 cells. The number of migration and invasion cells of B16 and B16F10 were counted. (D) The expression of MEG3 genes in B16F10 cells was detected by RT-qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control. Scale bar = 100 µm. (GNA, Gambogenic acid).
To further investigate whether the inhibition of melanoma metastasis by GNA through upregulation of MEG3 expression in vitro and in vivo was yet associated with EMT, we examined the expression of EMT-related proteins in mouse metastatic melanoma model and melanoma cells with knockdown of MEG3. The results of Immunocytochemistry (Fig. 6A) and its quantification analysis (Supplementary Fig. S2) showed that dark brown mesenchymal markers (N-cadherin and Vimentin) and matrix metalloproteinases (MMP-2 and MMP-9) could be seen in sh-MEG3 group compared with Control group, while GNA could reverse the epithelial-mesenchymal transformation that sh-MEG3induced. Comparing with the sh-MEG3 group, the expression of Ki-67 in Control group was also reduced significantly. These results indicated that GNA inhibited epithelial mesenchymal transformation in mouse transplanted melanoma by upregulating the expression level of MEG3. The results of Western blot (Fig. 6B) in vitro were consistent with those in vivo. These results suggest that lncRNA MEG3 regulates EMT and is involved in GNA inhibition of melanoma invasion and metastasis in vivo and in vitro.
(A) Representative images of immunohistochemical staining of transplanted melanoma mice. Scale bar = 100 µm. (B) B16F10 or sh-MEG3 B16F10 cells were treated with GNA (0.25 µM) for 24 h, and the protein bands was tested by Western blot. Data represent mean ± S.D., n = 3. * p < 0.05, compared to NC group. (GNA, Gambogenic acid).
Melanoma is a malignancy with a high risk of metastasis.29) Targeted research into the treatment of melanoma is therefore urgently needed. The development of EMT is closely related to the migration and invasion of tumor cells.30) EMT allows quiescent adherent cells to gain the ability to migrate through this process.31) Epithelial cells lose apical-basal polarity, cell-cell adhesion and transform into aggressive mesenchymal cells in this process.32) Blocking EMT is therefore key.
In our previous studies, GNA inhibited the proliferation28) and migration of melanoma cells,26) but the mechanisms involved were not clear. Therefore, we investigated the mechanism by which GNA inhibits the invasion and migration of melanoma. In this study, we firstly established metastatic melanoma model of B16F10-GFP-LUC cells and found medium and high concentrations of GNA reduced the number of pulmonary nodules and inhibited the pulmonary metastasis significantly in mice. H&E staining and melanin staining proved this point again. Besides, we found GNA inhibited pulmonary metastasis, hepatic metastasis and osseous metastasis of metastatic melanoma mice by flow cytometry. Then we performed colony formation assays, wound healing assays and transwell assays on B16 and B16F10 cells with different concentrations of GNA. The results of colony formation assays showed that GNA inhibited the proliferation of B16 and B16F10 cells. Meanwhile, wound healing assay and transwell assay showed that GNA inhibited the invasion and migration ability of B16 and B16F10 cells, further demonstrating that GNA could inhibit melanoma metastasis in vitro and in vivo. To investigate whether the inhibition of metastasis of melanoma cells in vitro and in vivo by GNA was related to EMT, we further investigated the expression of EMT-related proteins in mouse transplanted melanoma and melanoma cells and found that GNA inhibited the epithelial mesenchymal transition of tissue and cells of melanoma. GNA inhibited the metastasis of B16F10 cells in vitro and in vivo, which was associated with epithelial mesenchymal transition.
It has been shown that MEG3 expression is suppressed in a variety of tumours and that MEG3 can show its tumour suppressive activity by activating P53 and its target genes in different cancer cell lines.33) Based on the bioinformatics database (TCGA and GTEx) and our previous results of RNA sequencing, MEG3 is under-expressed in melanoma patients, while GNA up-regulates MEG3 expression in melanoma. In order to clarify whether the inhibition of melanoma metastasis by GNA is related to MEG3, under-expression of MEG3 was conducted in B16F10-GFP-LUC cells by lentivirus. Then we established a metastatic melanoma model again, and found that under-expression of MEG3 may promote the metastasis of melanoma. GNA may inhibit the metastasis of melanoma by upregulating MEG3 expression. We conducted MEG3 under-expressed in B16 and B16F10 cells by lentivirus, then performed colony formation assays, wound healing assays and transwell assays. The results confirmed that under-expression of MEG3 promoted invasion and migration of B16 and B16F10 cells compared with NC cells. The vitro studies further demonstrate that GNA can inhibit melanoma metastasis through up-regulating MEG3 expression. We then examined the expression of EMT-related proteins in mouse transplanted melanoma and melanoma cells and found that the lncRNA MEG3 could improve EMT and be involved in GNA inhibit invasion and metastasis of B16F10. Our innovation lies in the preliminary conclusion of the role of MEG3 in the inhibition of melanoma metastasis by GNA, the shortcoming of this study is that we did not combine the drugs for controlled comparison, and we can also thoroughly investigate the role of MEG3 in melanoma metastasis by over-expressing the MEG3 gene.
In conclusion, we have established GNA could improve epithelial mesenchymal transition by up-regulating the expression of the lncRNA MEG3 gene, thereby inhibiting melanoma metastasis in vitro and in vivo.
This work was financially supported by the National Natural Science Foundation of China [Grant Numbers: 81673650 and 81173600 to Q.L., 81903859 to H.C.], the Key Natural Science Project of Anhui University of Chinese Medicine [Grant Number: 2021zrzd08 to M.W.] and the first batch of talent support plan projects of Anhui University of Chinese medicine in 2022[Grant Number: 2022rcyb016 to M.W.].
MW performed experiments and analyzed data. CL and HC completed the statistical analysis. YT wrote the manuscript. MZ reviewed the entire article for English grammar. QL designed the experiments and revision of the manuscript.
The authors declare no conflict of interest.
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