Qigesan reduced the migration and invasion of esophageal cancer cells via inhibiting TGF-β1 pathway

Background:The present study aim to investigate the mechanism of Qigesan (QGS) on migration and invasion of esophageal cancer cells. Methods: First, we detected the cells in the normal group and the QGS group using microarray, and analyzed the ontology function and signaling pathway of target genes, and screened out TGF-β as one of the important targets of QGS. The subsequent verification experiments were divided into four groups: control group, model group (TGF-β1 stimulation group), QGS group (TGF-β1+QGS group) and positive control group (TGF-β1+TGF-β1 receptor inhibitor group). The migration and invasion abilities, as well as the expressions of related proteins and mRNA of each experimental group were detected. The migration and invasion ability of cells were detected by cell scratch test, and the Gene expression levels of E-cadherin, Vimentin, Smad2 and Smad7 mRNA in TGFβ1/Smad pathway were detected by RT-qPCR . The expression of E-cadherin, Vimentin, p-Smad2/3, Smad2/3 and Smad7 were detected by WB. Results:The results showed that TE-1 cells were reversed from fibroblast induced by TGF-β1 to epithelial cells after being treated QGS with the concentration of 20ug/mL for 36 h. Besides, QGS inhibited the migration and invasion of TE-1 cells and down-regulated the Gene and protein expression of Vimentin, Smad2 mRNA and Vimentin, p-Smad2/3 and Smad2/3. Meanwhile, The gene and protein overexpression E-cadherin and Smad7 was observed in QGS-treated cells. Conclusions:Together, these data indicated that QGS interfered with the epithelial-mesenchymal transition (EMT) process of TE-1 cells and reduced the migration and invasion of TE-1 cells via regulating TGF-β pathway, which maybe provided idea for the treatment of esophageal cancer.


Introduction
Esophageal cancer is a kind of digestive tract tumor with high morbidity and mortality, and it is the eighth most common malignant tumor in the world with mortality ranking sixth in the global cancer [1] . In 2018, there were about 572,000 new patients with esophageal cancer, and 508,600 people died of esophageal cancer [2] .
China is one of the areas with high incidence of esophageal cancer, among which Lin County is a high-risk area, and its incidence type is mostly squamous cell carcinoma [3] . When patients are diagnosed with Esophageal cancer, most of them are in the middle or advanced stage with metastasizing, resulting in a low 5-year survival rate [4][5] . It is reported that the migration and invasion of esophageal cancer is an important factor affecting the survival rate of patients with esophageal cancer, and epithelial mesenchymal transition is the premise of tumor migration and invasion [6] .
Epithelial-mesenchymal transition (EMT) plays an important role in the occurrence and development of tumors, and mediates tumor energy metabolism,heterogeneity, drug resistance, invasiveness, stem cells and other processes [7,8] . It was reported that EMT changed the polarity of tumor cells, rearranged the cytoskeleton, degraded extracellular matrix, lose intercellular connections, and promoted tumor cells to metastasize with the blood and lymphatic system [9] . Once they reached a new suitable location, they went through the reverse EMT process. Mesenchymal-epithelial transition (MET) allowed cells to regain epithelial polarity and invade the tissues of the colonization site [10][11] . EMT of esophageal squamous cell carcinoma avoided immune surveillance by inhibiting the activity of natural killer cells (NK) and reducing the release of effective molecules [12] . TGF-β is an important factor affecting EMT of tumor cells, and the process of inducing EMT of tumor cells mainly involving Smad-dependent and Smad-independent signaling pathway [13] .
Jiawei Qigesan improved the toxic side effects caused by radiotherapy and chemotherapy in patients undergoing radical esophagectomy, improved the quality of life of patients, reduced the recurrence rate and prolong the disease-free survival time [16] . Jiawei Qigesan inhibited lung metastasis of esophageal cancer in nude mice in animal experiment [17] . Our previous studies showed that the combination of Qigesan's drug-containing serum and cisplatin enhanced the inhibitory effect of chemotherapeutic drugs on esophageal cancer EC9706 cells under hypoxia environment, increased the expression of phosphatase, tensin analogue (PTEN) mRNA and programmed death protein 4 (PDCD4), and decreased the expression of miR-21 [18] . The QGS combined with cisplatin enhanced the sensitivity of EC9706 cells to cisplatin and achieve the effect of enhancing the curative effect [19] . Qigesan was proved to improve the quality of life of esophageal cancer model mice by protecting organs and tissues, and improving the suppression and imbalance of immune mechanism [20] . The above reports indicated that QGS played key roles in treating esophageal cancer by inhibiting the metastasis of esophageal cancer.
However, there were few reports on the intervention and mechanism of QGS on cancer cells migration and invasion. In this study, the intervention ways of QGS on migration and invasion of esophageal cancer were screened by gene expression microarray technology, and verified by experiments as follows.

Preparation of QGS
The Chinese materia medica of QGS come from Tong Ren Tang Group (Beijing, China). See Table 1 for QGS composition. Beat the medicine into coarse powder, reflux that medicine with 70% ethanol in a ratio of m (medicine weight, unit: g): v (volume of 70% ethanol, unit: ml) = 1: 8, filtering, concentrate, uniformly mixing the concentrated solution and ethyl acetate in a volume ratio of 2:1 in a separatory funnel for extraction.The ethyl acetate extract was dried and weighed in vacuum at low temperature to obtain the extract quality, and the final paste yield (mass ratio of the finally obtained paste medicine to the original medicinal materials) was 0.82%. 1.00 g drugs was dissolved in 50mL dimethyl sulfoxide (DMSO), mixed well to fully dissolved , and then stored at -20℃ for later use.

Regents
The

MTT assay was used to detect the effect of QGS on TE-1 cell activity
The TE-1 cells were cultured in 96-well plate at the density of 8×10 3 cells/well.

Experimental grouping
The cells were divided into control group, model group (adding TGF-β1 with the concentration of 15ng/mL), QGS group (adding QGS on the basis of model group) and SB431542 group (adding TGF-β1 receptor inhibitor SB431542 on the basis of model group). The drug concentrations of TGF-β1 and SB431542 were determined by previous experiments. When testing the cell invasion ability, it was necessary to add the step of spreading glue after scribing, the volume ratio of medium to Matrigel matrix glue was found in the pre-experiment.

The effect of QGS on migration and invasion of TE-1 cells was detected by cell scratch test
Detection of gene expression with RT-qPCR method.
The cells with the density of 1.5×105 cells/well were cultured in 24-well plate

Western blot assay
Cell lysate was prepared from blank control group, model group, Qigesan group and SB431542 group. The lysate was centrifuged in a centrifuge for 4℃, 12000× g, 10 min.

Statistical analysis
Differences between groups were determined by one-way analysis of variance with the LSD and Dunnet's T3 post-hoc tests using the SPSS 25.0 software. All results were presented as a mean±standard error. Differences were considered to be significant when P < 0.05.

The effects of Qigesan on TE-1 cells viability and the morphological changes of cells in each experimental group
After treating cells for 12h, 24h, 36h, 48h and 60h, there were significant differences between different concentrations of QGS group and control group (P < 0.05). After

Effects of QGS on migration and invasion of TE-1 cells
On the whole, the change of cell area in each group increased with the increase of time.
Compared with the control group, the migration and invasion abilities of the model group were enhanced (P < 0.05). Compared with the model, the ability of cell migration and invasion in QGS group and SB431542 group was inhibited and weakened (P < 0.05). However, there was no significant difference in migration and invasion ability between QGS group and the SB431542 group (P > 0.05) (Figure 3,4).

Effect of QGS on mRNA expression of TGF-β1 pathway related genes
Compared with the control group, the expression levels of Vimentin and Smad2 mRNA of the model group were significantly increased (P < 0.05) (Figure 5a,5b), while the mRNA expression levels of E-cadherin and smad7 were significantly decreased (P < 0.05) (Figure 5c,5d). Compared with the model group, the expression levels of Vimentin and Smad2 mRNA in QGS group and SB431542 group were significantly decreased (P < 0.05) (Figure 5a,5b), while the expression levels of E-cadherin and Smad7 mRNA were significantly increased (P < 0.05) (Figure 5c,5d). There was no significant difference in mRNA expression between QGS group and SB431542 group (P > 0.05).

Effect of QGS on expression of TGF-β1 pathway related proteins
Compared with the control group, the protein expression levels of Vimentin, p-Smad2/3 and Smad2/3 in the model group were significantly increased (P < 0.05) ( Figure   6a,6b,6c), while the protein expression levels of E-cadherin and Smad7 were significantly decreased (P < 0.05) (Figure 6d,6e). Compared with the model group, the protein expression levels of Vimentin, p-Smad2/3 and Smad2/3 in the QGS group were significantly decreased (P < 0.05) (Figure 6a,6b,6c), while the protein expression levels of E-cadherin and Smad7 were significantly increased (P < 0.05) (Figure 6d,6e). There was no significant difference in mRNA expression level between QGS group and SB431542 group (P > 0.05).

Discussion
Εsophageal cancer (ΕC) is one of the most aggressive diseases worldwide and deadliest types of cancer. The early symptoms are not obvious, which makes it difficult to diagnose them in time. Esophagectomy is still the main treatment, and some methods are being tried,such as endoscopic treatment for early EC, prophylactic chemoradiation, Perioperative treatment, targeted therapy and immunotherapy, etc. However, the choice of these treatments is affected by the type and stage of the disease, tumor location, individual differences of patients, and there is no global consensus on the optimal regimen [21] .
Metastasis and invasion of esophageal cancer is an important reason affecting the therapeutic effect, and EMT is the main factor causing metastasis and invasion.The EMT program is often activated reversibly, It will lead to the appearance of intermediate cells, which may promote the proliferation and spread of cancer cells at different stages of tumor development [22] .It was reported that EMT program in melanoma cells potentiates migration rate and development of lung colonies into immunodeficient host of cells grown in standard pH [23] . Another study showed that EMT also upregulated the expression of FHOD1, may contribute to tumour progression [24] .Yan [25] found that overexpression of integrin-linked kinase (ILK) promotes migration and invasion of colorectal cancer cells by inducing epithelial-mesenchymal transition via NF-κB signaling.These studies have proved that EMT is directly related to tumor migration and invasion. TGF-β signaling pathway is an important way to participate in the regulation of EMT,and TGF-β1 is considered as an effective inducer of epithelialmesenchymal transition in tumor microenvironment. Research has found that TGF-β1 induces epigenetic silence of TIP30 to promote tumor metastasis in esophageal carcinoma [26] . A clinical study on esophageal cancer patients in Xinjiang, China showed that:TGF-b1/Smad signaling pathway regulates Epithelial-to-Mesenchymal Transition in esophageal squamous cell carcinoma [27] .
Traditional Chinese medicine also plays a certain role in the treatment of esophageal cancer, it was reported that Anti-metastatic effects of Aidi on human esophageal squamous cell carcinoma by inhibiting epithelial-mesenchymal transition and angiogenesis [28] . A study on esophageal cancer found that Chinese herb medicine matrine induce apoptosis in human esophageal squamous cancer KYSE-150 cells through increasing reactive oxygen species and inhibiting mitochondrial function [29] .
Berberine inhibits the metastatic ability of prostate cancer cells by suppressing Epithelial-to-Mesenchymal Transition (EMT)-associated genes with predictive and prognostic relevance [30] . Other Chinese herbal medicine components or extracts have also been proved to be related to the drug resistance [31] and cycle [32] of esophageal cancer.
At the beginning of this study, we analyzed TE-1 cells in the control group and QGS group by gene chip technology, and found that the main biological processes involved in changed genes are cytoskeletal protein binding, ATP binding, adenyl nucleotide binding,adenyl ribonucleotide binding and so on;the KEGG PATHWAY involved by changed genes are TGF-β signaling pathway, cell cycle, pathways in cancer, etc.
Cytoskeletal protein binding and TGF-β signaling pathway are both related to cell migration and invasion, therefore, we conducted a follow-up experiment to verify the effect of QGS on the migration and invasion of TE-1 cells. showed the effect similar to SB431542, which reduced the migration and invasion ability of TE-1 cells. There was a report that Qigesan inhibits migration and invasion of esophageal cancer cells via inducing connexin expression and enhancing gap junction function [33] ,Our experimental results support this view. In the following experiments, we detected related molecules Vimentin, p-Smad2/3, Smad2/3, Ecadherin, Smad7. Smad is a TGF-β/Smad pathway protein, Vimentin is one of the interstitial molecular markers, and E-cadherin is the epithelial molecular marker [34][35][36] .
In conclusion, this study suggests that TGF-β signaling pathway is involved in EMT, and Qigesan inhibits the migration and invasion of TE-1 cells by regulating TGF-β signaling pathway.
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by National Natural Science Foundation of China (Nos. 81873285 and 82074313). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Authors' contributions YLC and LHY designed the study and modified the manuscript. YWS and RZ performed the main experiments. YWS wrote the manuscript. DHM and YSW revised the manuscript. XC and ZXZ performed the extracts preparation. YSW analyzed the data. SSR drafted all figures and tables. All authors read and approved the final version of the manuscript.