Asiaticoside Suppresses Gastric Cancer Progression and Induces Endoplasmic Reticulum Stress through the miR-635/HMGA1 Axis

Objective Gastric cancer is a prevalent malignant tumor with high morbidity and poor prognosis. Asiaticoside (AC) has antitumor effects, while its role in gastric cancer is elusive. Thus, this study investigated the effect of AC on gastric cancer progression. Methods Cell viability and migration were determined using the CCK-8 and Transwell migration assay. Endoplasmic reticulum stress was detected through measuring the expressions of GRP78, Chop, and hnRNPA1 by Western blot. The luciferase assay confirmed the relationship between miR-635 and High Mobility Group AT-Hook 1 (HMGA1). The effect of AC on tumor growth was evaluated by establishing a xenograft tumor. The survival rate of mice was analyzed by Kaplan-Meier analysis. Results AC suppressed gastric cancer cell viability and restrained cell migration. AC inhibited the expressions of the cell proliferation marker PCNA and EMT-related marker N-cadherin and increased E-cadherin expression. AC elevated the levels of GRP78 and Chop and suppressed the level of hnRNPA1. In addition, AC restrained gastric cancer proliferation and migration ability and induced endoplasmic reticulum stress by upregulating miR-635 expression. Furthermore, HMGA1 was proven to be a target of miR-635. AC constrained gastric cancer cell proliferation and migration and promoted endoplasmic reticulum stress by regulating HMGA1. Moreover, AC suppressed in vivo tumor growth and improved the survival time of mice. Additionally, AC elevated the expressions of miR-635, E-cadherin, GRP78, and Chop and inhibited Ki-67, HMGA1, N-cadherin, and hnRNPA1 expressions in tumor tissues of mice. Conclusion AC suppressed gastric cancer progression and induced endoplasmic reticulum stress via the miR-635/HMGA1 axis, providing a valuable drug against gastric cancer.


Introduction
Gastric cancer is a prevalent malignant tumor globally [1]. The incidence of gastric cancer ranks fifth among all malignancies in recent years, and over 1,000,000 cases are diagnosed each year in the world [2,3]. At present, surgical resection is the common therapeutic strategy for patients at an early stage [1]. Nevertheless, the majority of patients are diagnosed at the advanced stage [4]. Surgical resection combined with system chemotherapy is the conventional strategy for advanced patients [1]. However, this therapeutic strategy is unsatisfactory, and the prognosis remains poor [5]. Hence, it is imperative to search promising therapeutic strategies against gastric cancer.
In recent years, active compounds from herbal plants are extensively used in the treatment of diseases [6]. Asiaticoside (AC) is the critical compound extracted from Centella asiatica [7]. Accumulating evidence reveals that AC exerts vital functions in many diseases [8][9][10][11]. For example, AC alleviates the RANKL-induced osteoclastogenesis through modulating NFATc1 and NF-κB signaling pathways [8]. AC also exerts anti-inflammation function to attenuate transient cerebral ischemia-reperfusion injury and allergic response [9,10]. Besides, AC alleviates bleomycin-induced pulmonary fibrosis through activation of the Cyclic Adenosine Monophosphate (cAMP) and Ras-proximate-1 (Rap1) signaling pathway [11]. Recently, the antitumor role of AC in some cancers has emerged [12][13][14][15]. Huang et al. proved that AC promoted cancer cell apoptosis and strengthened the anticancer activity of vincristine [12]. Besides, AC suppressed cell proliferation, migration, and invasion in multiple myeloma drug-resistant cancer [13]. Zhou et al. showed that AC inhibited cell proliferation via regulating the NF-κB pathway in colorectal cancer [14]. Ma et al. demonstrated that AC suppressed cell proliferation and chemotherapeutic drug resistance in hepatocellular carcinoma [15]. However, the effect of AC on gastric cancer progression remains elusive.
MicroRNAs (miRNAs) belong to the small noncoding RNAs family, which usually function as regulators in many diseases via restraining the target gene expression [16]. For instance, miR-142-3p suppresses stem cell properties and enhances sensitivity to radiotherapy via regulating β-catenin in breast cancer [17]. miR-378 restrains cell aggressive behaviors through inhibiting SDA1 Domain Containing 1 (SDAD1) in colon cancer [18]. Besides, the regulatory roles of miRNAs in gastric cancer have been reported. Lei et al. showed that miR-143 and miR-145 repressed gastric cancer metastasis via inhibiting Myosin VI (MYO6) expression [19]. miR-206 suppressed gastric cancer cell proliferation via inhibiting cyclinD2 expression [20]. As a member of miRNAs, miR-635 is involved in the regulation of cancers. Tian et al. reported that miR-635 suppressed osteosarcoma progression via inducing cell apoptosis [21]. Zhang et al. discovered that miR-635 repressed tumorigenesis in non-smallcell lung cancer [22]. Besides, miR-635 is decreased in gastric cancer and regulates gastric cancer progression [23]. However, it is unclear whether miR-635 mediates the modulation of AC on gastric cancer development.

Quantitative Real-Time Polymerase Chain Reaction
(qRT-PCR). Total RNAs of AGC and HGC27 cells were obtained utilizing TRIzol, which were used to produce cDNA following the manufacturer's instruction of the Taq-Man MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR assay was conducted using SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were as follows: 95°C for 2 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min. The primer sequences of miR-635 were as follows: F, 5 ′ -TATAGCATATGCAGGGTG-3 ′ , and R, 5 ′ -CGCATTCGGAGTGCGAGTT-3 ′ [24]. U6 served as the control gene. The miR-635 expression was determined utilizing the 2 −ΔΔCt method.   Journal of Immunology Research the luciferase activity was tested utilizing the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Xenograft Tumor
Model. Ten BALB/c nude mice aged 4-6 weeks were bought from Beijing Laboratory Animal Research Center (Beijing, China). The mice were allocated into the control and AC groups (n = 5). AGS cells (1 × 10 5 cells) were injected subcutaneously into the left flank of mice. After 1 week of injection, 10 mg/kg AC and each volume saline were administered to mice in the AC group and control group by oral gavage every 2 days, respectively [14]. Tumor diameters were monitored every week until 5 weeks. Mice were euthanized using CO 2 inhalation (30% flow rate), and the tumor tissues were collected to conduct qRT-PCR and immunohistochemistry. All mouse experiments complied with the national and international regulations and policies. The study was permitted by the Experimental Animal Ethics of the Second Affiliated Hospital of Wenzhou Medical University. The staining images were examined using a microscope.
2.11. Statistical Analysis. All data were displayed as mean ± standard deviation (SD). Data analysis was completed utilizing SPSS Statistics 22.0 (SPSS, Chicago, IL, USA). The differences between two groups were assessed using the independent t-test. One-way ANOVA determined the differences among multiple groups. The survival rate was analyzed using Kaplan-Meier analysis. P value below 0.05 was identified as statistically significant.

Asiaticoside Restrains Cell Aggressive Behaviors and Promoted Endoplasmic Reticulum Stress in Gastric Cancer.
To investigate the effect of AC on gastric cancer, AGS and HGC27 cells were incubated with AC (0.5 μM, 1 μM, 2 μM, 4 μM, or 8 μM). Cell viability of AGS and HGC27 cells was evaluated after treatment with AC for 24 h. AC decreased cell viability of AGS and HGC27 cells dose-dependently (P < 0:05, Figure 1(a)). Besides, the inhibition rate of 2 μM AC on cell viability was 49.03% for AGS cells and 46.82% for HGC27 cells (Figure 1(a)). Therefore, 2 μM AC was used for the subsequent experiments. The Transwell migration assay found that migration ability of AGS and HGC27 cells was suppressed after treatment with 2 μM AC (P < 0:01, Figure 1(b)). Additionally, AC inhibited the expressions of PCNA and N-cadherin in both AGS and HGC27 cells (P < 0:01, Figure 1(c)). E-cadherin was enhanced after treatment with 2 μM AC (P < 0:01, Figure 1(c)). Furthermore, AC remarkably elevated the expressions of GRP78 and Chop and suppressed the level of hnRNPA1 (P < 0:01, Figure 1(d)). Therefore, AC suppressed the proliferation and migration ability and induced endoplasmic reticulum stress in gastric cancer.

Asiaticoside Restrains Gastric Cancer Cell Aggressive Behaviors and Induced Endoplasmic Reticulum Stress by
Upregulating the miR-635 Expression. miR-635 presented lowly expression in gastric cancer and suppressed gastric cancer development [23]. Thus, we explored whether miR-635 mediated the regulation process of AC on gastric cancer aggressive behaviors and endoplasmic reticulum stress. Firstly, miR-635 expression in gastric cancer cells was detected. It was observed that miR-635 was decreased in AGS and HGC27 cells (P < 0:01, Figure 2(a)). Then, the influence of AC on miR-635 expression was detected in AGS and HGC27 cells. Results showed that AC considerably elevated the miR-635 expression (P < 0:01, Figure 2(b)).
After the miR-635 inhibitor was introduced into AGS and HGC27 cells, the promotion effect of AC on miR-635 expression was abolished (P < 0:01, Figure 2(c)). Besides, the inhibitory action of AC on cell viability and migration ability was reversed by downregulated miR-635 (P < 0:01, Figures 2(d) and 2(e)). Furthermore, the inhibitory effect of AC on the expressions of PCNA and N-cadherin was abrogated upon miR-635 silencing (P < 0:01, Figure 2(f)). Instead, the expression of E-cadherin induced by AC was suppressed by downregulated miR-635 (P < 0:01, Figure 2(f)). Moreover, the promotion role of AC in GRP78 and Chop expression levels and the inhibitory effect of AC on hnRNPA1 expression were abolished by decreased miR-635 (P < 0:01, Figure 2(g)). Thus, AC inhibited gastric cancer cell proliferation and migration and promoted endoplasmic reticulum stress by upregulating miR-635.
3.3. HMGA1 Is a Target of miR-635. Given that miRNAs usually exerted regulation functions through modulating the expressions of target genes, we next screened the target genes of miR-635 in the TargetScan database and found that HMGA1 was targeted by miR-635. The putative targeting sequences between miR-635 and HMGA1 are listed in Figure 3(a). To deeply testify the relationship between miR-635 and HMGA1, the luciferase assay was performed. Overexpressed miR-635 suppressed the luciferase activity of the psi-CHECK-2-HMGA1-3 ′ -UTR reporter but had no effect on the psiCHECK-2-MUT-HMGA1-3′-UTR reporter (P < 0:05, Figure 3(b)). HMGA1 protein level was suppressed by overexpressed miR-635 and increased by downregulated miR-635 (P < 0:01, Figures 3(c) and 3(d)). Furthermore, AC treatment significantly suppressed the HMGA1 protein level which was reversed by downregulated miR-635 (P < 0:01, Figure 3(e)). Hence, HMGA1 was a target of miR-635.

Asiaticoside Suppresses Gastric Cancer Cell Aggressive
Behaviors and Induced Endoplasmic Reticulum Stress by Regulating HMGA1. To determine whether HMGA1 3 Journal of Immunology Research participated in the modulation of AC on gastric cancer cell proliferation, migration, and endoplasmic reticulum stress, the HMGA1 overexpression plasmid was transfected into AC-treated AGS and HGC27 cells. Results showed that AC suppressed the HMGA1 expression, which was abrogated by HMGA1 overexpression plasmid (P < 0:01, Figure 4(a)).

Journal of Immunology Research
Besides, AC suppressed cell viability and migration of AGS and HGC27 cells, but they were increased by overexpressed HMGA1 (P < 0:01, Figures 4(b) and 4(c)). Furthermore, AC inhibited the levels of PCNA and N-cadherin and increased E-cadherin level, which was reversed by overexpressed HMGA1 (P < 0:01, Figure 4(d)). Moreover, AC increased the expressions of GRP78 and Chop and inhibited hnRNPA1 expression, which was abolished by overexpressed HMGA1 (P < 0:01, Figure 4(e)). Taken together, AC restrained gastric cancer cell aggressive behaviors and promoted endoplasmic reticulum stress by regulating HMGA1.

Asiaticoside Abates Tumor Growth In Vivo.
To evaluate the antitumor effect of AC on gastric cancer, the xenograft tumor model was established by subcutaneously injecting AGS cells (1 × 10 5 cells) to nude mice. After 1 week, mice in the AC group and control group orally received 10 mg/ kg AC and each volume saline every 2 days, respectively. Results showed that the tumor volume in the AC group was significantly smaller than that in control group from the 14th day (P < 0:01, Figure 5(a)). Tumor weight presented obvious suppression after treatment with AC (P < 0:01, Figure 5(b)). Kaplan-Meier analysis results indicated that    Journal of Immunology Research the mice in the AC group exhibited longer survival times than those in the control group ( Figure 5(c)). The median survival time in the AC group and control group was 32 and 21 days, respectively ( Figure 5(c)). Besides, miR-635 expression was elevated in tumor tissues of mice in the AC group (P < 0:01, Figure 5(d)). Furthermore, AC administration suppressed the expressions of Ki-67, HMGA1, N-cadherin, and hnRNPA1 and increased the expressions of Ecadherin, GRP78, and Chop in tumor tissues (P < 0:01, Figure 5(e)). Collectively, AC abated tumor growth of gastric cancer.

Discussion
Gastric cancer is a common malignance accompanied by high morbidity and poor prognosis [1-3, 5, 25]. Therefore, it is pressing to discover curative therapeutic strategies against gastric cancer. AC participates in the regulation of some cancers such as multiple myeloma, colorectal cancer, and colorectal cancer [13][14][15]. Thus, we conjectured that AC may modulate gastric cancer progression.
To investigate the influence of AC on gastric cancer progression, gastric cancer cells were treated with AC. The gastric cancer cell viability and migration were reduced after treatment with AC. Besides, AC suppressed the expressions of the cell proliferation marker PCNA and EMT-related marker N-cadherin and increased the expression of the EMT-related marker E-cadherin. In other words, AC restrained gastric cancer cell proliferation, migration, and EMT. These findings were in keeping with the reported studies [13][14][15]. Zhou et al. proved that AC inhibited the cell proliferation ability of colorectal cancer cells [14]. Ma et al.   Journal of Immunology Research found that AC restrained the proliferation capacity of hepatocellular carcinoma [15]. AC also inhibited multiple myeloma cell aggressive behaviors [13]. Besides, He et al. confirmed that AC repressed the EMT process of pancreatic cancer [26]. Furthermore, many studies demonstrate that endoplasmic reticulum stress exists in gastric cancer, and blocking endoplasmic reticulum stress suppresses gastric cancer cell aggressive behaviors [27][28][29]. For example, Liu et al. found that YAP accelerated gastric cancer development through regulating endoplasmic reticulum stress [27]. Therefore, we studied the action of AC on the endoplasmic reticulum stress in gastric cancer and proved that AC strengthened the levels of GRP78 and Chop and suppressed the hnRNPA1 level. That is to say, AC induced endoplasmic reticulum stress in gastric cancer. Similarly, Gurfinkel et al. discovered that AC induced endoplasmic reticulum disorder and calcium homeostasis dysregulation during inducing cell apoptosis in prostate cancer cells [30]. Thus, these findings revealed that AC suppressed the cell proliferation and migration ability and EMT and induced endoplasmic reticulum stress in vitro in gastric cancer. To better explore the action of AC on gastric cancer progression, we determined the influence of AC on tumor growth in vivo. Results indicated that AC treatment suppressed tumor growth and improved mouse survival. AC also suppressed the expression of Ki-67, HMGA1, N-cadherin, and hnRNPA1 and enhanced the expression of E-cadherin, GRP78, and Chop in tumor tissues. In other words, AC inhibited cancer proliferation, migration, and EMT and induced endoplasmic reticulum stress in vivo. These results were in harmony with the study performed by Zhou et al. [14]. They found that AC inhibited colorectal tumor growth dose-dependently [14].
Hence, AC suppressed tumor growth of gastric cancer in vivo. Together, AC suppresses gastric cancer progression and induces endoplasmic reticulum stress in vitro and in vivo. miR-635 is an important cancer regulator in cancers including osteosarcoma, non-small-cell lung cancer, and nasopharyngeal carcinoma [21,22,31]. However, there are few articles on the role of miR-635 in gastric cancer, with only Cao et al. finding that miR-635 is decreased in gastric cancer and modulates gastric cancer progression via regulating KIFC1 expression [23]. However, its function on the modulation of AC on gastric cancer development remained elusive. Therefore, we determined whether miR-635 participated in this process. Results indicated that miR-635 was decreased in gastric cancer cells, but AC treatment increased its expression. The inhibitory influences of AC on cell proliferation, migration, and EMT were abrogated by downregulated miR-635. Besides, the promotion effect of AC on endoplasmic reticulum stress was suppressed by downregulated miR-635. The effect of miR-635 on gastric cancer progression was consistent with the previous study [23]. Up to now, this study first reported that AC suppressed gastric cancer cell aggressive behaviors and induced endoplasmic reticulum stress through upregulating miR-635 expression. miRNAs usually play regulation roles via controlling gene expression [16]. Therefore, the target genes of miR-635 were searched. Among the target genes of miR-635, HMGA1, a small nuclear protein belonging to the HMGA family, is an oncofoetal gene and regulates genesis and development of various neoplasms [32]. Interestingly, HMGA1 displays a critical role in the detection and progression of gastrointestinal tumors [33]. HMGA family members are  Journal of Immunology Research considered reliable biomarkers able to efficiently track a gastrointestinal tumor in the future [33]. Therefore, HMGA1 was chosen as the target of miR-635 for follow-up study. Results indicated that HMGA1 was targeted by miR-635, and HMGA1 expression was inversely modulated by miR-635. Besides, AC suppressed the HMGA1 protein level, which was reversed by downregulated miR-635. Previous studies showed that HMGA1 functioned as an oncogene and promoted gastric cancer progression [34,35]. Therefore, we inferred that HMGA1 might exert function in the modulation of AC on gastric cancer progression. To verify the hypothesis, the gastric cancer cells were treated with AC and transfected with the HMGA1 plasmid to change the expression of HMAG1. Afterward, results revealed that the inhibitory influences of AC on cell proliferation, migration, and EMT were reversed by overexpressed HMAG1.   Furthermore, the promotion effect of AC on endoplasmic reticulum stress was suppressed by overexpressed HMAG1. The effects of HMAG1 on gastric cancer were in harmony with the published studies. Akaboshi et al. reported that overexpressed HMAG1 maintained the cell proliferation ability of gastric cancer cells [36]. Cao et al. found that overexpressed HMAG1 increased gastric cancer cell proliferation, migration, and invasion ability [34]. Jin et al. revealed that HMGA1 potentiated the aggressive development of gastric cancer via inducing EMT [35]. Therefore, these results indicated that AC suppressed gastric cancer cell aggressive behaviors and induced endoplasmic reticulum stress by regulating HMGA1 expression.

Conclusion
Asiaticoside suppressed gastric cancer cell aggressive behaviors and stimulated endoplasmic reticulum stress via regulating the expressions of miR-635 and HMGA1. Besides, asiaticoside restrained tumor growth in vivo. Therefore, asiaticoside may be a valuable drug against gastric cancer.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no competing interest.