# Polysaccharides Extracted fromAngelica sinensis (Oliv.) Diels Relieve the Malignant Characteristics of Glioma Cells through Regulating the MiR-373-3p-Mediated TGF-β/Smad4 Signaling Pathway

Authors: Chunhong Qu; Weifeng Dong
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Objectives. Angelica sinensis polysaccharide (ASP) is a traditional herbal medicine accompanied by antitumor potential. This study aims to explore the therapeutic potential of ASP on glioma, as well as the underlying mechanisms involving microRNA-373-3p (miR-373-3p) and the TGF-β/Smad4 signaling pathway. Methods. U251 cells (a human glioma cell line) were treated with different concentrations of ASP. miR-373-3p was silenced in U251 cells by the transfection of the miR-373-3p inhibitor. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. Cell migration and invasion were detected by wound healing and transwell assays, respectively. The miR-373-3p expression was measured by RT-qPCR. The protein expressions of TGF-β and Smad4 were evaluated by both western blotting and immunofluorescence. Results. ASP inhibited the viability, migration, and invasion, and enhanced the apoptosis of U251 cells in a dose-dependent manner. ASP increased miR-373-3p expression and decreased TGF-β and Smad4 expressions in U251 cells. Silencing of miR-373-3p weakened the effects of ASP on inhibiting cell viability, migration, and invasion, as well as promoting cell apoptosis. In addition, deleting miR-373-3p weakened the inhibiting effects of ASP on the TGF-β/Smad4 pathway in U251 cells. Conclusions. ASP suppresses the malignant progression of glioma via regulating the miR-373-3p-mediated TGF-β/Smad4 pathway.


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## Body

## 1. Introduction

Glioma is the most widespread kind of glial cell-derived primary intracranial tumor, accounting for 30–40% of all primary tumors in the adult central nervous system [1, 2]. Gliomas are diagnosed in over 1,000,000 new patients every year worldwide, and patients with glioblastoma multiforme, which is the most aggressive type of glioma, have a median survival period of around 15 months, with a 5-year survival rate of about 5% [2–4]. Traditionally, surgical resection, radiotherapy, and chemotherapy are the leading strategies for the treatment of glioma, whereas the prognosis is still not satisfactory especially due to metastasis, recurrence, and resistance [5, 6]. With the development of novel therapeutic strategies, traditional herbal medicine brings a new dawn against glioma [7].Angelica sinensis (A. sinensis) is a traditional herbal medicine used to treat gynecologic, cardio-cerebrovascular, neurological, and nephrotic disorders [8]. Angelica sinensis polysaccharide (ASP) isolated from the roots of A. sinensis is a key bioactive component [9]. Evidence has determined that ASP possesses a variety of pharmacological activities, including immunomodulation, hematopoiesis, antioxidation, antiosteoarthritis, neuro-protection, and hepatoprotection [10]. Notably, ASP also exhibits antitumor potential against diverse cancers. According to Zhou et al., ASP promotes apoptosis in breast cancer cells both in vitro and in vivo [11]. Cao et al. have found that ASP inhibits cervical cancer cell proliferation whereas promotes apoptosis [12]. In addition, Yang et al. demonstrated that ASP inhibits neuroblastoma cell growth, migration, and invasion [13]. However, knowledge of the action mechanisms of ASP in glioma remains limited.MicroRNAs (miRNAs) are short noncoding RNAs that exert key regulatory functions in carcinogenesis by modulating post-transcriptional gene expression [14]. MiR-373-3p is an oncomiRNA that is upregulated in diverse cancers, for instance, lung adenocarcinoma [15], tongue squamous cell carcinoma [16], testicular germ cell tumor [17], and esophageal squamous cell carcinoma [18]. On the contrary, miR-373-3p can also act as an antioncomiRNA in pancreatic carcinoma [19], choriocarcinoma [20], hepatocellular carcinoma [21], cervical carcinoma [22], and glioma [23–25]. Jing et al. proved that the miR-373 level is reduced in glioma tissues [23]. miR-373 was reported to be a new therapeutic target for clinical glioblastoma patients [26]. Furthermore, several miRNAs, such as miR-10a [27], −22 [28], −223 [29], and −675 [13], have also been found to be implicated in the action mechanisms of ASP. However, whether the action mechanism of ASP in glioma is associated with miR-373-3p remains unclear.The TGF-β/Smad4 signaling system is an essential signal transduction pathway involved in tumorigenesis [30]. Previous studies have determined that some miRNAs exert important regulatory roles in cancers by regulating the TGF-β/Smad4 pathway, including miR-539 in prostate cancer [31], miR-183 in ovarian cancer [32], and miR-34a in colorectal cancer [33]. We suspected that there may be a certain relationship between miR-373-3p and the TGF-β/Smad4 pathway, contributing to the effects of ASP on glioma. In this study, the effects of ASP were investigated in glioma cells in the aspect of cell viability, apoptosis, migration, and invasion. The action mechanisms of ASP involving miR-373-3p and the TGF-β/Smad4 pathway were further evaluated. This study aimed to discover a potential glioma therapeutic drug and reveal related molecular mechanisms.


## 2. Methods

### 2.1. Cell Treatments

The human glioma cell line U251 (American Type Culture Collection, Manassas, VA, USA) was incubated at 37°C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS). Some U251 cells were treated with various dosages of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h. In addition, another part of U251 cells was stably transfected with a lentivirus-packed miR-373-3p inhibitor or inhibitor negative control (inhibitor NC) (RiboBio, Guangzhou, China) for 72 h using the HighGene transfection reagent (ABclonal, Wuhan, China). The transfected cells were further treated with 0.4 mg/mL ASP for 48 h. U251 cells without treatments were used as the control.


### 2.2. Cell Counting Kit-8 (CCK-8) Assay

A CCK-8 (Beyotime, Shanghai, China) was used to detect cell proliferation. Simply, 100μL·U251 cell suspension was plated into 96-well plates and treated with ASP for 12, 24, and 48 h, respectively. Then, 10 μL CCK-8 was injected into each well. After incubating for 2 h at 37°C, a microplate reader (DR-3518G, Hiwell-Diatek, Wuxi, China) was used to determine the optical density (OD) at 450 nm.


### 2.3. Flow Cytometry

An annexin V-FITC apoptosis detection kit (Beyotime) was used to measure cell apoptosis. After suspending in 300μL binding buffer, cells were incubated for 15 min with 5 μL annexin V-FITC and further treated for 10 min with 10 μL propidium iodide (PI) at 25°C in darkness. The apoptosis was assessed by using a flow cytometer (CytoFLEXS, Beckman, Miami, FL, USA), and the apoptotic rate was quantified by CellQuest software (BD Biosciences, San Jose, CA, USA).


### 2.4. Wound Healing Assay

U251 cells suspended in fresh DMEM were seeded into 6-well plates at a density of 5 × 104/mL. After 12 h of culturing at 37°C, a wound was scratched in each well using a pipette tip. Then, the cells were rinsed in phosphate buffer saline (PBS), and the remaining cells were continuously cultured for 24 h in a 5% CO2 incubator at 37°C. The wound distance was measured under a microscope (CKX53, Olympus, Japan) at 0 and 24 h post scratching.


### 2.5. Transwell Assay

Cell invasion was detected using Transwell chambers. Each well of the Transwell upper chamber with precoated Matrigel was inoculated with 5 × 104 U251 cells suspended in serum-free DMEM. The lower compartment was supplied with 500 μL DMEM containing 10% FBS. After 24 h, the cells in the lower compartment were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Cells were observed in five randomly selected regions under a microscope (CKX53, Olympus).


### 2.6. Real Time-Quantitative PCR (RT-qPCR)

Total RNAs were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). After reverse transcription using a cDNA synthesis mix (Tiangen, Beijing, China), the cDNAs were used as the amplification template. The following program was used to perform RT-qPCR on an RT-qPCR system (Mx3000P, StrataGene, Santa Clara, CA, USA): 95°C for 3 min, and 40 cycles of 95°C for 15 s and 62°C for 40 s. The primers included miR-373-3p forward, 5′-GAA GUG CUU CGA UUU UGG GGU GU-3′; miR-373-3p reverse, 5′-ACC CCA AAA UCG AAG CAC UUC UU-3′; U6 forward, 5′-CTC GCT TCG GCA CA-3′; U6 reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′. The relative expression level was determined by the 2−∆∆Ct method, with U6 serving as the internal control.


### 2.7. Western Blotting

To isolate the total proteins, U251 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime). The protein samples were segregated by 10% SDS-PAGE, which were then deposited onto polyvinylidene difluoride membranes. After that, the membranes were blocked with 5% nonfat milk for 1 h before being incubated with primary antibodies anti-TGF-β (1 : 2000, AF1027, Affinity, Cincinnati, OH, USA) or anti-Smad4 (1 : 2000, AF5247, Affinity) for 12 h at 4°C. GAPDH was regarded as an internal control (1 : 3000, AF7021, Affinity). After being washed with Tris-buffered saline with Tween 20 (TBST), the membranes were further treated with a secondary antibody (HRP-IgG, 1 : 2000, ab205718, Abcam, Cambridge, England) for 1 h at 25°C in the dark. An ECL kit (Pierce, Rockford, IL, USA) was used to visualize the protein bands, which were then photographed using a Gel imaging system (Tanon 3500, Shanghai, China).


### 2.8. Immunofluorescence

The protein expressions of TGF-β and Smad4 were further detected by immunofluorescence. The cells were treated with 4% paraformaldehyde for 15 min and then penetrated with 1% Triton X-100 for 10 min before being blocked with 3% bovine serum albumin for 30 min. Subsequently, the cells were incubated with the corresponding primary antibodies: anti-TGF-β (1 : 500, AF1027, Affinity) or anti-Smad4 (1 : 500, AF5247 Affinity) for 12 h at 4°C. Following washing with PBS, cells were further cultured with Cy3-labeled IgG (red fluorescence for TGF-β, 1 : 200, A0516, Beyotime) or Alexa Fluor 488-labeled IgG (green fluorescence for Smad4, 1 : 500, ab150077, Abcam) for 1 h at 25°C in the dark. After 5 min of counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL), the cells were captured under a microscope (CKX53, Olympus).


### 2.9. Statistical Analysis

GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA) was applied for statistical analyses. Data were presented as mean ± standard deviation (SD), and the differences across multigroups were determined using one/two-way analysis of variance (ANOVA) followed by Tukey’s test. AP value < 0.05 presented as statistically significant.


## 2.1. Cell Treatments

The human glioma cell line U251 (American Type Culture Collection, Manassas, VA, USA) was incubated at 37°C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS). Some U251 cells were treated with various dosages of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h. In addition, another part of U251 cells was stably transfected with a lentivirus-packed miR-373-3p inhibitor or inhibitor negative control (inhibitor NC) (RiboBio, Guangzhou, China) for 72 h using the HighGene transfection reagent (ABclonal, Wuhan, China). The transfected cells were further treated with 0.4 mg/mL ASP for 48 h. U251 cells without treatments were used as the control.


## 2.2. Cell Counting Kit-8 (CCK-8) Assay

A CCK-8 (Beyotime, Shanghai, China) was used to detect cell proliferation. Simply, 100μL·U251 cell suspension was plated into 96-well plates and treated with ASP for 12, 24, and 48 h, respectively. Then, 10 μL CCK-8 was injected into each well. After incubating for 2 h at 37°C, a microplate reader (DR-3518G, Hiwell-Diatek, Wuxi, China) was used to determine the optical density (OD) at 450 nm.


## 2.3. Flow Cytometry

An annexin V-FITC apoptosis detection kit (Beyotime) was used to measure cell apoptosis. After suspending in 300μL binding buffer, cells were incubated for 15 min with 5 μL annexin V-FITC and further treated for 10 min with 10 μL propidium iodide (PI) at 25°C in darkness. The apoptosis was assessed by using a flow cytometer (CytoFLEXS, Beckman, Miami, FL, USA), and the apoptotic rate was quantified by CellQuest software (BD Biosciences, San Jose, CA, USA).


## 2.4. Wound Healing Assay

U251 cells suspended in fresh DMEM were seeded into 6-well plates at a density of 5 × 104/mL. After 12 h of culturing at 37°C, a wound was scratched in each well using a pipette tip. Then, the cells were rinsed in phosphate buffer saline (PBS), and the remaining cells were continuously cultured for 24 h in a 5% CO2 incubator at 37°C. The wound distance was measured under a microscope (CKX53, Olympus, Japan) at 0 and 24 h post scratching.


## 2.5. Transwell Assay

Cell invasion was detected using Transwell chambers. Each well of the Transwell upper chamber with precoated Matrigel was inoculated with 5 × 104 U251 cells suspended in serum-free DMEM. The lower compartment was supplied with 500 μL DMEM containing 10% FBS. After 24 h, the cells in the lower compartment were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Cells were observed in five randomly selected regions under a microscope (CKX53, Olympus).


## 2.6. Real Time-Quantitative PCR (RT-qPCR)

Total RNAs were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). After reverse transcription using a cDNA synthesis mix (Tiangen, Beijing, China), the cDNAs were used as the amplification template. The following program was used to perform RT-qPCR on an RT-qPCR system (Mx3000P, StrataGene, Santa Clara, CA, USA): 95°C for 3 min, and 40 cycles of 95°C for 15 s and 62°C for 40 s. The primers included miR-373-3p forward, 5′-GAA GUG CUU CGA UUU UGG GGU GU-3′; miR-373-3p reverse, 5′-ACC CCA AAA UCG AAG CAC UUC UU-3′; U6 forward, 5′-CTC GCT TCG GCA CA-3′; U6 reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′. The relative expression level was determined by the 2−∆∆Ct method, with U6 serving as the internal control.


## 2.7. Western Blotting

To isolate the total proteins, U251 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime). The protein samples were segregated by 10% SDS-PAGE, which were then deposited onto polyvinylidene difluoride membranes. After that, the membranes were blocked with 5% nonfat milk for 1 h before being incubated with primary antibodies anti-TGF-β (1 : 2000, AF1027, Affinity, Cincinnati, OH, USA) or anti-Smad4 (1 : 2000, AF5247, Affinity) for 12 h at 4°C. GAPDH was regarded as an internal control (1 : 3000, AF7021, Affinity). After being washed with Tris-buffered saline with Tween 20 (TBST), the membranes were further treated with a secondary antibody (HRP-IgG, 1 : 2000, ab205718, Abcam, Cambridge, England) for 1 h at 25°C in the dark. An ECL kit (Pierce, Rockford, IL, USA) was used to visualize the protein bands, which were then photographed using a Gel imaging system (Tanon 3500, Shanghai, China).


## 2.8. Immunofluorescence

The protein expressions of TGF-β and Smad4 were further detected by immunofluorescence. The cells were treated with 4% paraformaldehyde for 15 min and then penetrated with 1% Triton X-100 for 10 min before being blocked with 3% bovine serum albumin for 30 min. Subsequently, the cells were incubated with the corresponding primary antibodies: anti-TGF-β (1 : 500, AF1027, Affinity) or anti-Smad4 (1 : 500, AF5247 Affinity) for 12 h at 4°C. Following washing with PBS, cells were further cultured with Cy3-labeled IgG (red fluorescence for TGF-β, 1 : 200, A0516, Beyotime) or Alexa Fluor 488-labeled IgG (green fluorescence for Smad4, 1 : 500, ab150077, Abcam) for 1 h at 25°C in the dark. After 5 min of counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL), the cells were captured under a microscope (CKX53, Olympus).


## 2.9. Statistical Analysis

GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA) was applied for statistical analyses. Data were presented as mean ± standard deviation (SD), and the differences across multigroups were determined using one/two-way analysis of variance (ANOVA) followed by Tukey’s test. AP value < 0.05 presented as statistically significant.


## 3. Results

### 3.1. ASP Inhibits the Malignant Properties of Glioma Cells

The role of ASP in glioma was evaluated in the malignant characteristics of U251 cells. ASP greatly declined the cell viability in a dose-dependent manner (Figure1(a), P<0.001). In contrast, U251 cell apoptosis was dose-dependently increased by ASP treatment (Figure 1(b), P<0.05). The intervention of ASP also retarded the migration and invasion of U251 cells with increasing doses (Figures 1(c) and 1(d), P<0.01).Figure 1
ASP inhibited the malignant characteristics of U251 cells. (a) Cell viability was measured by CCK-8 assay; (b) cell apoptosis was measured by flow cytometry; (c) cell migration was measured by wound healing assay; (d) cell invasion was measured by Transwell assay. U251 cells were treated with different concentrations of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ###P<0.001 vs. 0.1 mg/mL ASP; &&P<0.01, &&&P<0.001vs. 0.2 mg/mL ASP. ASP upregulates miR-373-3p and blocks the TGF-β/Smad4 signaling pathway in glioma cells.
(a)(b)(c)(d)Since miR-373-3p is an antioncogene in glioma, the regulatory function of ASP on miR-373-3p was analyzed. The expression of miR-373-3p was considerably upregulated in a dose-dependent manner after ASP treatment (Figure2(a), P<0.05). The effect of ASP on the TGF-β/Smad4 pathway was further evaluated. TGF-β and Smad4 protein expressions were both dose-dependently decreased by ASP treatment, as shown by western blotting (Figure 2(b), P<0.05).Figure 2
ASP upregulated miR-373-3p and inhibited the TGF-β/Smad4 pathway. (a) The expression of miR-373-3p was detected by RT-qPCR; (b) the protein expressions of TGF-β and Smad4 were detected by western blotting. U251 cells were treated with different concentrations of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h; ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ###P<0.001 vs. 0.1 mg/mL ASP; &&&P<0.001 vs. 0.2 mg/mL ASP. miR-373-3p knockdown weakens the antitumor activities of ASP in glioma cells.
(a)(b)The role of miR-373-3p in the action of ASP involvement has been identified. Transfection of the miR-373-3p inhibitor significantly reduced miR-373-3p expression in ASP-treated U251 cells (Figure3(a), P<0.001). The following functional experiments revealed that miR-373-3p silencing weakened the impacts of ASP on decreasing U251 cell viability (Figure 3(b), P<0.001) and promoting U251 cell apoptosis (Figure 3(c), P<0.05). Moreover, miR-373-3p silencing partially eliminated the inhibitory effects of ASP on U251 cell migration and invasion (Figures 3(d) and 3(e), P<0.001).Figure 3
Silencing of miR-373-3p weakened the antitumor effects of ASP in U251 cells. (a) miR-373-3p expression was detected by RT-qPCR; (b) cell viability was measured by CCK-8 assay; (c) cell apoptosis was measured by flow cytometry; (d) cell migration was measured by wound healing assay; (e) cell invasion was measured by transwell assay. U251 cells were transfected with the miR-373-3p inhibitor/inhibitor NC and then treated with 0.4 mg/mL ASP.∗P<0.05, ∗∗∗P<0.001 vs. Control; ##P<0.01, ###P<0.001 vs. ASP; &P<0.05, &&&P<0.001 vs. ASP + inhibitor NC.
(a)(b)(c)(d)(e)Silencing of miR-373-3p activates the TGF-β/Smad4 pathway in ASP-treated glioma cells. The downstream pathway of miR-373-3p involving the TGF-β/Smad4 pathway was further analyzed in ASP-treated U251 cells. Western blotting revealed that miR-373-3p silencing significantly weakened the inhibiting effects of ASP on the protein level of TGF-β and Smad4 (Figure 4(a), P<0.05). In addition, immunofluorescence indicated that the decreased fluorescence of TGF-β and Smad4 in ASP-treated U251 cells were also recovered by miR-373-3p silencing (Figure 4(b) and 4(c), P<0.01).Figure 4
Silencing of miR-373-3p weakened the inhibiting effects of ASP on the TGF-β/Smad4 pathway in U251 cells. (a) The protein expressions of TGF-β and Smad4 were detected by western blotting. (b) Relative fluorescence of TGF-β and Smad4 were detected by immunofluorescence. (c) Positive cells for TGF-β and Smad4 following immunofluorescence. U251 cells were transfected with the miR-373-3p inhibitor/inhibitor NC and treated with 0.4 mg/mL ASP. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. ASP; &&P<0.01, &&&P<0.001 vs. ASP + inhibitor NC.
(a)(b)(c)


## 3.1. ASP Inhibits the Malignant Properties of Glioma Cells

The role of ASP in glioma was evaluated in the malignant characteristics of U251 cells. ASP greatly declined the cell viability in a dose-dependent manner (Figure1(a), P<0.001). In contrast, U251 cell apoptosis was dose-dependently increased by ASP treatment (Figure 1(b), P<0.05). The intervention of ASP also retarded the migration and invasion of U251 cells with increasing doses (Figures 1(c) and 1(d), P<0.01).Figure 1
ASP inhibited the malignant characteristics of U251 cells. (a) Cell viability was measured by CCK-8 assay; (b) cell apoptosis was measured by flow cytometry; (c) cell migration was measured by wound healing assay; (d) cell invasion was measured by Transwell assay. U251 cells were treated with different concentrations of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ###P<0.001 vs. 0.1 mg/mL ASP; &&P<0.01, &&&P<0.001vs. 0.2 mg/mL ASP. ASP upregulates miR-373-3p and blocks the TGF-β/Smad4 signaling pathway in glioma cells.
(a)(b)(c)(d)Since miR-373-3p is an antioncogene in glioma, the regulatory function of ASP on miR-373-3p was analyzed. The expression of miR-373-3p was considerably upregulated in a dose-dependent manner after ASP treatment (Figure2(a), P<0.05). The effect of ASP on the TGF-β/Smad4 pathway was further evaluated. TGF-β and Smad4 protein expressions were both dose-dependently decreased by ASP treatment, as shown by western blotting (Figure 2(b), P<0.05).Figure 2
ASP upregulated miR-373-3p and inhibited the TGF-β/Smad4 pathway. (a) The expression of miR-373-3p was detected by RT-qPCR; (b) the protein expressions of TGF-β and Smad4 were detected by western blotting. U251 cells were treated with different concentrations of ASP (0, 0.1, 0.2, and 0.4 mg/mL) for 48 h; ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ###P<0.001 vs. 0.1 mg/mL ASP; &&&P<0.001 vs. 0.2 mg/mL ASP. miR-373-3p knockdown weakens the antitumor activities of ASP in glioma cells.
(a)(b)The role of miR-373-3p in the action of ASP involvement has been identified. Transfection of the miR-373-3p inhibitor significantly reduced miR-373-3p expression in ASP-treated U251 cells (Figure3(a), P<0.001). The following functional experiments revealed that miR-373-3p silencing weakened the impacts of ASP on decreasing U251 cell viability (Figure 3(b), P<0.001) and promoting U251 cell apoptosis (Figure 3(c), P<0.05). Moreover, miR-373-3p silencing partially eliminated the inhibitory effects of ASP on U251 cell migration and invasion (Figures 3(d) and 3(e), P<0.001).Figure 3
Silencing of miR-373-3p weakened the antitumor effects of ASP in U251 cells. (a) miR-373-3p expression was detected by RT-qPCR; (b) cell viability was measured by CCK-8 assay; (c) cell apoptosis was measured by flow cytometry; (d) cell migration was measured by wound healing assay; (e) cell invasion was measured by transwell assay. U251 cells were transfected with the miR-373-3p inhibitor/inhibitor NC and then treated with 0.4 mg/mL ASP.∗P<0.05, ∗∗∗P<0.001 vs. Control; ##P<0.01, ###P<0.001 vs. ASP; &P<0.05, &&&P<0.001 vs. ASP + inhibitor NC.
(a)(b)(c)(d)(e)Silencing of miR-373-3p activates the TGF-β/Smad4 pathway in ASP-treated glioma cells. The downstream pathway of miR-373-3p involving the TGF-β/Smad4 pathway was further analyzed in ASP-treated U251 cells. Western blotting revealed that miR-373-3p silencing significantly weakened the inhibiting effects of ASP on the protein level of TGF-β and Smad4 (Figure 4(a), P<0.05). In addition, immunofluorescence indicated that the decreased fluorescence of TGF-β and Smad4 in ASP-treated U251 cells were also recovered by miR-373-3p silencing (Figure 4(b) and 4(c), P<0.01).Figure 4
Silencing of miR-373-3p weakened the inhibiting effects of ASP on the TGF-β/Smad4 pathway in U251 cells. (a) The protein expressions of TGF-β and Smad4 were detected by western blotting. (b) Relative fluorescence of TGF-β and Smad4 were detected by immunofluorescence. (c) Positive cells for TGF-β and Smad4 following immunofluorescence. U251 cells were transfected with the miR-373-3p inhibitor/inhibitor NC and treated with 0.4 mg/mL ASP. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. ASP; &&P<0.01, &&&P<0.001 vs. ASP + inhibitor NC.
(a)(b)(c)


## 4. Discussion

Glioma is the most prevalent tumor in the brain associated with high morbidity and mortality [34]. The prognosis of glioma patients treated with traditional therapeutic techniques such as surgical resection followed by adjuvant radiation and/or chemotherapy is restricted [5, 6]. Nowadays, emerging evidence has determined the positive antitumor potential of a variety of traditional herbal medicines, such as Glycyrrhiza uralensis, Panax ginseng, A. sinensis, Scutellaria baicalensis, Lycium barbarum, Coptis Chinensis, and Sophora flavescens [7]. Among these medicines, A. sinensis, commonly used for the treatment of gynecological diseases, exhibits a promising therapeutic potential against glioma [8]. For example, A. sinensis chloroform extract prevents glioblastoma cell growth and promotes apoptosis [35]; the methanol extract of A. sinensis promotes cell cycle progression in the G0-G1 phase and apoptosis of glioblastoma cells [36]. Z-ligustilide and n-butylidenephthalide extracted from A. sinensis also exhibit antitumor effects against glioblastoma [37, 38]. In this study, the antitumor capability of ASP, a bioactive component from A. sinensis, was analyzed. Similarly, with previous studies mentioned, ASP dose-dependently reduced the malignant features of glioma cells, evidenced by decreased viability, migration, and invasion, and increased cell apoptosis. Consistently, ASP has also been reported to inhibit the malignant progression of breast cancer [11], cervical cancer [12], and neuroblastoma [13]. Therefore, ASP is considered a promising therapeutic drug for glioma.MiRNAs acting as oncogenes or antioncogenes play important regulatory roles in the tumorigenesis and progression of glioma, such as the upregulating 15b, −17, −21, −92b, −130a, −221, −222, −320, and −376, as well as downregulating miR-29, −93, −122, −125b, −128, −203, −205, and −451a [39]. Evidence has determined that the action mechanisms of ASP in different diseases are associated with specific miRNAs, including miR-10a in epilepsy [27], miR-22 in myocardial infarction [28], miR-223 in spinal cord injury [29], and miR-675 in neuroblastoma [13]. In this study, the action mechanisms of ASP in glioma involving miR-373-3p were analyzed. The findings showed that ASP dose-dependently boosted the miR-373-3p level in glioma cells. MiR-373-3p is a specific miRNA that plays a dual role in different types of cancers [15–22]. miR-373 expression has been shown to be reduced in glioma tissues, which is associated with WHO grade, KPS score, poor overall survival, and progression-free survival [23]. Li et al. have reported that silencing of MSC-AS1 inhibits the development and chemoresistance of glioma cells to temozolomide by miR-373-3p overexpression [25]. Zhou et al. have found that SNHG16 increases glioma cell proliferation, migration, and invasion by downregulating miR-373-3p [24]. To combine with the abovementioned findings, we hypothesize that the miR-373-3p overexpression may contribute to the ASP antitumor role in glioma. Our subsequent assays further determined that silencing of miR-373-3p weakened the antitumor effects of ASP on glioma. Therefore, we concluded that ASP inhibits the malignant characteristics of glioma cells through upregulating miR-373-3p.The TGF-β/Smad4 signaling pathway is an important signal transduction pathway responsible for various physiological processes, such as cell proliferation, differentiation, apoptosis, migration, fibrosis, angiogenesis, immune suppression, and tumorigenesis [30, 40]. Previous studies have proved that the TGF-β/Smad4 pathway is an action target of many potential antitumor drugs, such as acetylsalicylic acid in breast cancer [41], baicalein in gastric cancer [42], and Hedyotis diffusa willd in colorectal cancer [43]. Jin et al. have reported that bleomycin inhibits the proliferation and promotes the apoptosis of glioma cells by repressing the TGF-β/Smad4 pathway [44]. In this study, the action mechanisms of ASP in glioma involving the TGF-β/Smad4 pathway were further analyzed. The results showed that ASP dose-dependently downregulated TGF-β and Smad4 in glioma cells. Since TGF-β participates in the pathogenesis of glioma via supporting tumor growth and self-renewal of initiating stem cells [45], inhibiting the TGF-β/Smad4 pathway may contribute to the antitumor effects of ASP in glioma. In addition, the TGF-β/Smad4 pathway has also been determined as the downstream pathway of specific miRNAs in diverse cancers, such as miR-539 in prostate cancer [31], miR-183 in ovarian cancer [32], and miR-34a in colorectal cancer [33]. Our following assays revealed that miR-373-3p silencing weakened the inhibiting activities of ASP on the TGF-β/Smad4 pathway in glioma cells, indicating a negative regulatory relationship between miR-373-3p and the TGF-β/Smad4 pathway. To combine with the action of ASP on miR-373-3p, we concluded that ASP blocks the TGF-β/Smad4 pathway through upregulating miR-373-3p.


## 5. Conclusion

ASP suppresses glioma cell proliferation, migration, and invasion while promoting apoptosis, confirming its therapeutic potential against glioma. miR-373-3p-mediated blocking of the TGF-β/Smad4 pathway is one of the antitumor mechanisms of ASP on glioma. However, this study only explored the ASP effects on glioma at the cellular level. In-depth action mechanisms of ASP on glioma need to be further studied in vivo. Additionally, more active constituents of A. sinensis responsible for the antiglioma activity need to be identified in further research.



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*Source: 7469774-2022-07-09.xml*