Xiaotan Sanjie Decoction Inhibits Gastric Cancer Cell Proliferation, Migration, and Invasion through lncRNA-ATB and miR-200A

This study is aimed at exploring whether Xiaotan Sanjie decoction (XTSJ) inhibits gastric cancer (GC) proliferation and metastasis by regulating lncRNA-ATB expression. qRT-PCR and Western blot were used to analyze lncRNA-ATB and downstream-regulated genes/proteins in human GC cells. CCK8, Edu, and flow cytometry assays were used to detect the inhibitory effect of XTSJ on cell proliferation and apoptosis. Moreover, transwell and wound healing assays were used to detect the inhibitory effect of XTSJ on migration and invasion. qRT-PCR and Western blot were used to detect regulated genes and proteins levels. The HGC-27 cell line was used for follow-up analysis due to the high level of lncRNA-ATB and cell characteristics. XTSJ inhibited the proliferation and metastasis of HGC-27 in a dose-dependent manner. Further research found that XTSJ downregulated lncRNA-ATB, Vimentin, and N-cadherin, while it upregulated miR-200a and E-cadherin in a dose-dependent manner. XTSJ also upregulated Caspase 3, Caspase 9, Bax, and downregulated Bcl-2. Furthermore, XTSJ inhibited tumor growth in vivo and downregulated EMT signaling pathways. These results indicate that XTSJ may affect EMT and Bcl-2 signaling pathways by regulating lncRNA-ATB and miR-200a, thus inhibiting proliferation, migration, and invasion of HGC-27 cells. Therefore, XTSJ may be an effective treatment for the high levels of lncRNA-ATB in GC.


Introduction
Gastric cancer (GC) has a poor prognosis and is the leading cause of cancer-related deaths [1]. Research has shown that traditional Chinese medicine can treat cancer [2,3]. Therefore, studying the molecular pathways of traditional Chinese medicine can effectively enhance GC treatment.
lncRNAs are long-chain noncoding RNAs regulating gene transcription and downstream biological signals in tumors [4,5]. Preliminarily studies have shown that lncRNAs acts as "sponge" to modulate downstream biological signals by competitively binding or chelating micro-RNAs [6]. TGF-β-activated lncRNA-ATB can enhance epithelial-mesenchymal transition(EMT)-related metastasis and proliferation by competitively binding to the miR-200 family in certain malignant tumors [7,8]. Recent research showed that lncRNA-ATB upregulation in GC enhances vascular infiltration and overall survival while lncRNA-ATB silencing inhibits cell proliferation [9]. Therefore, lncRNA-ATB and miR-200a are potential therapeutic targets for GC treatment.
Xiaotan Sanjie decoction (XTSJ) can effectively prolong the survival of GC patients, decrease TGF-β and IL-8 levels, and inhibit the expression of fibroblast activation protein (FAP) [10,11]. This research aimed to assess whether XTSJ prevents GC proliferation and metastasis by regulating lncRNA-ATB and miR-200a.

Preparation of Drugs.
Prof. Wei Pinkang provided the XTSJ. XTSJ components have been described in previous papers. Its active components have been analyzed via HPLC [12]. The 5-fluorouracil (5-Fu) was obtained from Lilly France (Suzhou, China).

CCK-8 Assay.
The GC cells (5 × 10 3 cells/well) were reinoculated and treated with XTSJ for 24, 48, and 72 hours. An appropriate amount of CCK-8 solution was added to the sample, then incubated for 2 hours. A SpectraMax i3 microplate reader (Molecular Devices, CA, USA) was used to measure absorbance.
2.6. Transwell Migration and Invasion Assays. The HGC-27 cells (4 × 10 4 cells/well) were reinoculated into the upper chamber coated with Matrigel for invasion assay. For the migration assay, HGC-27 cells (4 × 10 4 cells/well) were reinoculated into the upper chamber (4 × 10 4 cells/well) without Matrigel. The upper chamber was filled with serum-free medium, while the lower chamber was filled with medium containing 10% FBS. The transwell chambers were fixed with a 4.0% paraformaldehyde solution for 15 minutes, then stained with a 0.1% crystal violet solution for 10 minutes. A Leica DMi1 inverted microscope (Leica, Wetzlar, Germany) was used to obtain images.

2.7.
Wound-Healing Assay. The HGC-27 cells (4 × 10 5 cells/ well) were reinoculated until they reached 90% confluence. A 200 μl sterile pipette tip was then used to scratch the center of each well. A Leica DMi1 inverted microscope was then used to assess the wound after 0, 24, and 48 hours.
Quantitative analysis of each sample was conducted using the 2 -△△Ct method.
2.10. In Vivo Experiment. BALB/c nude mice (6-week-old, female) were reared in a sterile environment. The animal experiment was approved by the Ethics Committee of the Tongde Hospital of Zhejiang Province. The tumor model was established by subcutaneously inoculating the HGC-27 cells (1 × 10 6 cells/well) into the right side of mouse back. The mice were treated using intragastric of XTSJ (1 g/kg for low-dose and 2 g/kg for high-dose) or intraperitoneal injection of 5-FU (30 mg/kg) every day.
The mice were euthanized after seven days. The tumor tissues were dissected and collected. Pathological examination and SABER-FISH were used to assess the expression of EMT related antibodies and the location of lncRNA-ATB in tissues [13,14].

XTSJ Inhibits Migration and Invasion in HGC-27 Cells.
Transwell assays demonstrated that XTSJ significantly reduced cell number in the migration assays at high concentrations (0.15 mg/ml). XTSJ also significantly reduced   (Figures 3(a) and 3(b)). Wound healing assays found that XTSJ effectively maintained the wound healing range and inhibited cell invasion (Figures 3(c) and 3(d)). These results suggest that XTSJ can effectively inhibit HGC-27 metastasis.

XTSJ Inhibits the Expression of lncRNA and Related
EMT and Bcl-2 Signaling Pathways. lncRNA-ATB and downstream-regulated genes and proteins were analyzed using qRT-PCR and Western blotting to reveal how XTSJ inhibits the proliferation and metastasis of HGC-27. qRT-PCR showed that XTSJ reduced the levels of lncRNA-ATB, Vimentin, and N-cadherin while increasing miR-200a and E-cadherin levels in a dose-dependent effect (Figure 4(a)). Similarly, Western blot assays showed that XTSJ reduced ZEB-1, Vimentin, and Ncadherin levels while increasing E-cadherin levels (Figures 4(b) and 4(c)). Western blot also showed that XTSJ reduced Bcl-2 level while it increased Caspase 3, Caspase 9, and Bax levels (Figures 4(d) and 4(e)). These results suggest that XTSJ affects EMT and Bcl-2 signal pathways by downregulating lncRNA-ATB and upregulating miR-200a.
3.5. The Effect of XTSJ on GC In Vivo. Pharmacodynamic analysis was conducted using HGC-27 transplanted tumor nude mice model. XTSJ inhibited the transplanted tumors in mice, similar to chemotherapeutic drugs (5-FU) (Figures 5(a) and 5(b)). SABER-FISH results showed that lncRNA-ATB was upregulated in the model group while it was downregulated in XTSJ group (Figure 5(c)). Immunohistochemical (IHC) analysis showed that XTSJ reduced the levels of antibodies, such as Ki67, Vimentin, and N-cadherin, while it increased E-cadherin levels ( Figure 5(d)). These results indicate that XTSJ can affect EMT signaling pathway in vivo ( Figure 6).

Discussion and Conclusion
Gastric cancer (GC) has a poor prognosis, especially GC cells at low to medium differentiation stages that easily undergo proliferation, migration, invasion, and tumorigenesis [15]. Therefore, studies should assess the biological mechanism of proliferation and metastasis in GC.
Similarly, lncRNA-ATB was remarkably expressed in GC cells than in adjacent tissues. Increased lncRNA-ATB increases the infiltration depth, distant metastasis, and late tumor lymph node metastasis, affecting the overall survival of GC patients [30]. lncRNAs may act as sponges by competitively binding to microRNAs (miRNAs), thereby inhibiting the active functions [31]. Moreover, lncRNA-ATB silencing can upregulate miR-200 family and induce apoptosis through Bcl-2/caspase 3 pathway in lung cancer and prostate cancer [7,21]. lncRNA-ATB can also upregulate ZEB1 and ZEB2 by competitively binding to miR-200 family in various tumors [32,33], resulting in proliferation, migration, and invasion. However, lncRNA-ATB silencing can significantly inhibit ZEB1 and ZEB2 expression, thus inhibiting cell migration and invasion [17,34]. These results indicate that lncRNA-ATB can affect EMT by competitively binding with miR-200a, thus promoting tumor metastasis in GC [35,36]. Type I cadherin (epithelial cadherin and E-cadherin) is transformed into N-cadherin and Vimentin (mainly expressed in mesenchymal cells) during EMT [37]. In this study, HGC-27 was selected for follow-up experiments because it had lower E-cadherin and miR-200a levels and higher lncRNA-ATB, Vimentin, N-cadherin, and ZEB-1 levels. XTSJ has been proven in previous research to suppress proliferation, angiogenesis, invasion, and migration [10,12,38,39]. This might be due to the fact that XTSJ lowers lncRNA-ATB and promotes competitive binding to miR-200a, lowering ZEB-1-mediated EMT and Bcl-2-mediated apoptosis. Reduced levels of Vimentin and N-cadherin associated with a mesenchymal phenotype and increasing levels of E-cadherin associated with an epithelial phenotype defined EMT reversal. However, more research should be conducted to identify the major target and effector chemicals of XTSJ that inhibit lncRNA-ATB expression. Furthermore, no research has demonstrated XTSJ's therapeutic effectiveness or associated biomolecular signals in human GC.
In conclusion, XTSJ may affect EMT and Bcl-2 signaling pathways by regulating lncRNA-ATB and miR-200a, thus inhibiting proliferation, migration, and invasion of HGC-27 cells. Therefore, XTSJ may be an effective treatment for the high level of lncRNA-ATB in GC.

Data Availability
The data used to support the findings of this study are included within the article. LncRNA-ATB Mesenchymal phenotype Apoptosis Figure 6: Molecular mechanism of XTSJ. The corresponding mechanism may be related to XTSJ reducing LncRNA-ATB and increasing miR-200a competing with it to reduce the expression of downstream ZEB-1 and reverse EMT. The process of reversing EMT showed decreased expression of Vimentin and N-cadherin associated with mesenchymal phenotype and increased E-cadherin expression associated with an epithelial phenotype. In addition, XTSJ could reduce Bcl-2, increase Bax, Caspase 3, and Caspase 9 to induce apoptosis.