Acidic Tumor Microenvironment Promotes Pancreatic Cancer through miR-451a/MEF2D Axis

Pancreatic cancer (PC), as a highly malignant and aggressive solid tumor, is common in the digestive system. The acidic microenvironment is one of the critical markers of cancer. Nonetheless, there are few studies on how the acidic microenvironment affects the development of PC. This study focused on investigating the specific molecular mechanisms of the acidic microenvironment in PC. In our study, qRT-PCR was conducted for examining microRNA (miR)-451a and myocyte enhancer factor 2D (MEF2D) expressions in PANC-1 cells. Then, detailed functional effects of an acidic environment on miR-451a and MEF2D in PANC-1 cells were detected by CCK-8, colony formation, flow cytometry, wound healing, transwell, mitochondrial functionality measurement, JC-1 staining, DCFH-DA staining, and sphere formation assays. The relationship between miR-451a and MEF2D was confirmed by luciferase reporter analysis. Under acidic conditions, the increase of proliferation, migration, and invasion of PANC-1 cells was observed. Moreover, the mitochondrial oxidative respiration-related gene miR-451a was reduced in acidic conditions. In addition, we found that, in PANC-1 cells under an acidic environment, miR-451a overexpression enhanced oxygen consumption, mitochondrial membrane potential (MMP) loss, and ROS generation and inhibited proliferation, migration, invasion, and stemness via sponging MEF2D. In a word, our results revealed that the acidic microenvironment regulated PC progression by affecting the miR-451a/MEF2D axis, indicating a novel avenue for the future treatment of PC.


Introduction
Pancreatic cancer (PC), as the most common malignant solid tumor of the digestive system, is characterized by high malignancy and invasiveness, and poor prognosis, with less than 8% 5-year survival rate, which is one of the greatest challenges in modern oncology research [1][2][3]. Based on the Global Cancer Statistics report in 2018, the incidence of PC was 2.5%, while the mortality rate was as high as 4.5% [4]. Moreover, PC may become the second most fatal malignancy by 2030 [5]. e high mortality rate of PC is mainly due to its strong ability to invasion and metastasis. Studies show that PC has begun to invade and metastasize in the early stages [6]. us, it is essential to explore the molecular mechanisms of PC invasion and metastasis.
Emerging evidence has indicated that the acidic microenvironment is one of the critical markers of the tumor [7,8]. Studies revealed that most tumor cells exhibit the metabolic adaptability of the "Warburg" effect, that is, regardless of whether there is sufficient oxygen, tumor cells tend to produce energy through glycolysis, which significantly increases glucose metabolism and then produces a large amount of lactic acid [9,10]. In addition, insufficient perfusion and chaotic vascular structure further increase the accumulation of lactic acid and hydrogen ions in the tumor microenvironment [11]. erefore, compared with normal tissue, the extracellular environment of tumor tissue is acidic, with a pH range of 5.5 to 7.0 [12]. In the early stage of tumor cell growth, the acidic environment makes the cells more aggressive, allowing cancer cells to invade normal tissues [13]. In addition, the generation of an acidic microenvironment induced with tumors including PC can inhibit tumor cell apoptosis and promote cell proliferation, invasion, and immune escape [7,14].
us, whether the acidic microenvironment contributes to the progression of PC aroused our concern. MicroRNAs (miRNAs) are single-stranded noncoding RNAs with a length of about 21-23 nt, involved in regulating post-transcriptional gene expression. rough targeting the 3′-UTR region of target genes, miRNAs regulate growth and survival through gene inhibition, thereby inhibiting translation or accelerating mRNA degradation [15]. Currently, miRNAs have been found to possess substantial impacts on cell activities, such as proliferation, differentiation, and apoptosis and are involved in the progression of most malignant tumors, including PC [16,17].
Interestingly, to get with the acidic hypoxic microenvironment, many miRNAs are abnormally expressed in PC cells [18,19]. Clarence et al. reported that miR-451a as a tumor suppressor was downregulated in the hypopharyngeal carcinogenesis exposure to weakly acidic bile [20]. Among those dysregulating miRNAs in the acidic microenvironment, miR-451a, as an attractive candidate, was low expressed under acidic conditions. Importantly, myocyte enhancer factor 2D (MEF2D), a member of the MEF2 family exhibiting functional effects on the progression of tumors including PC [21], might be a potential target of miR-451a according to bioinformatics prediction. erefore, whether miR-451a regulates the proliferation, migration, invasion, and apoptosis of PC cells through MEF2D under an acidic microenvironment was investigated in this study.

Western Blot Assay.
Protein was isolated from PANC-1 cells and measured through the BCA kit (Beyotime Biotechnology, China). First, the protein was extracted using 12% SDS-PAGE and then shifted into PVDF membranes (Millipore, USA), which were next incubated using 5% skimmed milk, followed by overnight incubation with primary antibodies under 4°C. After rinsing the membranes, they were incubated for 1 h using HRP-labeled secondary antibody (1:4,000, SA00004-10; Proteintech, China) under ambient temperature. Finally, the enhanced chemiluminescence kit (ECL; Millipore, Bedford, USA) was utilized to observe protein blots, whereas ImageJ software (NIH, version 4.3) was adopted for quantification. All primary antibodies used

Colony Formation Assay.
Colony formation assay was performed as previously described [23]. PANC-1 cells were cultured within 6-well plates, and the medium was replaced every 2-3 days for a total of two weeks. Subsequently, cells were stained with 0.5% crystal violet for 15 min and imaged via a light microscope (Nikon, Japan).
2.6. Flow Cytometry. Apoptosis was detected using flow cytometry. PANC-1 cells were trypsin and rinsed by PBS, and then re-suspension was done within a 500 μL binding buffer. After that, Annexin V-FITC (5 μL) and PI (10 μL) were utilized to treat PANC-1 cells in the ark for 15 min. At last, a flow cytometer (BD Biosciences, USA) was utilized to determine apoptotic cells [24].

Wound
Healing Assay. Cells (2 × 10 5 cells/well) were seeded into a 12-well culture plate and cultured in DMEM supplemented with 10% FBS. PANC-1 cells from different groups reached 100% confluence, and 200 μL pipette tip was used to scratch a wound. An inverted microscope (Olympus, Japan) was adopted to observe cell images at 0 and 48 h (200×) [24].

Transwell
Assay. PANC-1 cells were cultured into the upper compartment with the basal medium in the migration assay. Lower chambers were supplied with 600 μL RPMI-1640 medium containing 10% FBS. After 48 h, cells were immobilized with methanol and stained with 0.1% crystal violet and observed under a microscope (Leica, Germany) and counted. In invasion assay, the used membrane was precoated with Matrigel (Franklin Lakes, NJ, USA). e other methods were like cell migration [24].

qRT-PCR.
After the total RNA extraction, cDNA was prepared with the extracted total RNA by the RNeasy plus micro kit through reverse transcription with specific instructions. e starting material of qRT-PCR was carried out using the StepOne system (Life Technologies Corp). Sequences of all primers were designed by Primer Premier software 4.0 (Premier, Canada), which are shown in Table 1. U6 or β-actin was normalized by the 2 −ΔΔCT approach [25].

Luciferase Reporter Assays.
is study subcloned MEF2D wild-type (WT) or mutant (MT) to the pmirGLO dual-luciferase vectors (Promega, USA) for generating pmirGLO-MEF2D WT/MUT to co-transfect into PANC-1 cells with NC mimics or miR-451a mimic. Luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) at 48 h. Renilla luciferase activity was used as the endogenous control [24].

Oxygen Consumption.
In brief, PANC-1 cells from different groups were washed by PBS and added with 150 μL fresh medium. Next, 10 μL R01 oxygen fluorescent probe was added and thoroughly mixed, and 100 μl oxygen sealing solution was added per well. Finally, the Seahorse XFe174 96 flux analyzer (Agilent, Santa Clara, CA) detected O 2 consumption.

Mitochondrial Membrane Potential Analysis.
PANC-1 cells from different groups were treated with JC-1 (0.5 mL) (Yeasen, Shanghai, China) for 15 min at 37°C. en, the cells were centrifuged at 400 rpm/min for 5 min followed by resuspension with 2 mL buffer solution. After centrifuging at 400 rpm/min for 5 min, the cells were resuspended with 0.3 mL buffer solution. Finally, images were collected using a fluorescent microscope (Olympus, Tokyo, Japan).

Detection of Reactive Oxygen Species (ROS) Generation.
Dichlorodihydrofluorescein diacetate (DCFH-DA) was adopted to evaluate ROS generation. Briefly, PANC-1 cells from different groups were incubated with 10 mM DCF-DA at 37°C for 15 min. After being washed three times with PBS, fluorescent microscopic images were observed under a fluorescent microscope (Olympus, Tokyo, Japan).

Statistical
Analysis. Data were analyzed using GraphPad Prism 5.0 and presented in mean ± SD. All experiments were repeated at least 3 times. Unpaired Student's t-test was used to compare the differences between two independent groups. e differences between multiple groups were compared by ANOVA and Tukey's post hoc analysis. P < 0.05 stood for statistical significance.

Tumor Acidic Microenvironment
Promotes the Proliferation, Migration, and Invasion of PC Cells. In order to detect whether the acidic microenvironment potentiates PC progression, PANC-1 cells were treated under normal (pH 7.4) and acidic conditions (pH 6.8) up to 48 h at 5% CO 2 . PANC-1 cells exposed to acidic conditions showed higher cell viability (Figure 1(a)). Meanwhile, the acid-treated PANC-1 cells presented increased proliferation-related proteins (PCNA and Ki-67) (Figure 1(b)), as well as significantly enhanced colony formation ability (Figure 1(c)) and reduced apoptotic cells (Figure 1(d)). In addition, wound healing and transwell assays illustrated that cell motility and invasion ability were markedly enhanced in acid-treated PANC-1 cells (Figures 2(a) and 2(b)). Also, MMP-2 and MMP-9 protein levels related to migration and invasion were significantly increased in acid-treated PANC-1 cells (Figure 2(c)). ese data illustrated that an acidic microenvironment could promote the development of PC.

Tumor Acidic Microenvironment Downregulated the Expression of Mitochondrial Oxidative Respiration-Related
Gene miR-451a. To explore whether the acidic environment promotes PC development by regulating ROS-related genes, we searched ROS-related genes through GeneCards software. We performed a functional enrichment analysis of ROS-related genes through g:Profiler software (Figure 3(a)). In the meantime, qRT-PCR analysis was conducted for assessing miR-451a expressions in PANC-1 exposed to an acidic environment. Figure 3(b) demonstrates that, among the top five genes, only the expression of miR-451a (mitochondrial oxidative respiration-related gene) was significantly downregulated under acidic conditions.

Tumor Acidic Microenvironment Regulates the Function of Mitochondria via the miR-451a/MEF2D Axis.
e results above further investigated whether the acidic environment affected mitochondrial function by modulating the miR-451a/MEF2D axis in PANC-1 cells. As shown in Figure 6(a), miR-451a overexpression inhibited the decrease of oxygen consumption caused by acidic conditions, while MEF2D upregulation partially restored the effects of miR-451a mimics on the oxygen consumption. Moreover, miR-451a mimics promoted the MMP loss and ROS generation in PANC-1 cells under acidic conditions, partially rescued by MEF2D upregulation (Figures 6(b), 6(c)).

Tumor Acidic Microenvironment Modulates the Proliferation, Migration, and Stem Cell Stemness of PC through the miR-451a/MEF2D
Axis. Finally, we further verified our hypothesis that an acidic environment modulated PC's proliferation, migration, and stem cell stemness through the miR-451a/MEF2D axis. CCK-8, wound, healing, and transwell analyses revealed that MEF2D overexpression partly reversed the reduction of functional cell capabilities caused by miR-451a mimics in an acidic environment (Figures 7(a)-7(c)). Furthermore, miR-451a upregulation significantly inhibited the increase of sphere formation and the increase of CD24, CD44, and ESA expression caused by the acidic conditions, while MEF2D overexpression reversed the functions of miR-451a mimics (Figures 7(d), 7(e)).

Discussion
is study discovered that the acidic microenvironment promoted cell proliferation, migration, and invasion and reduced apoptosis in PC. Importantly, our results found that the acidic microenvironment decreased miR-451a expression, related to mitochondrial oxidative respiration, which enhanced the oxygen consumption, MMP loss, and ROS generation and inhibited the proliferation, migration, invasion, and stem cell stemness of PC cells under acidic conditions. In addition, we predicted and confirmed a novel target of miR-451a, MEF2D, representing an oncogenic protein that could regulate tumor suppression in PC. Like miR-451a mimics, knockdown of MEF2D also increased the oxygen consumption, MMP loss, and ROS generation with a consequent increase of apoptosis. Furthermore, MEF2D overexpression reversed the effects of miR-451a mimics on the functional effects of PC. To the best of our knowledge, our study firstly demonstrated that the acidic microenvironment accelerated PC progression by modulating the miR-451a/MEF2D pathway. e acidic tumor microenvironment is caused by the accumulation of acidic metabolites in the tumor microenvironment due to enhanced glycolysis, hypoxia, and tissue insufficiency [26]. Increasing studies have shown that the acidic microenvironment exerted essential effects on tumor    Journal of Oncology proliferation, invasion, and migration and immune escape and affects the effective treatment of tumors, making tumor cells insensitive to radiotherapy and chemotherapy, which makes tumor treatment more difficult [27,28]. For example, under an acidic environment, BRAF v600E melanoma cells manifested active proliferation, enhanced antiapoptotic ability, and exhibited more resistance to the BRAF inhibitor vemurafenib [29]. Noteworthy, it has been found that the acidic microenvironment induces epithelial-mesenchymal transition in PC cells [18] and thus affects the migration and invasion of cancer cells. Analogously, in our study, acidic conditions increased PANC-1 cell proliferation, migration, and invasion. MiRNAs have been confirmed to participate in posttranscriptional gene expression regulation, acting as oncogenes or tumor suppressors [30,31]. In addition, the evidence demonstrated that tumor cells exhibit increased intrinsic ROS stress due to increased metabolic activity and mitochondrial malfunction [32]. In this study, we focused on the effects of miRNAs on the functional activities of PC.
rough bioinformatics analysis, miR-451a (related to ROS) expressions were downregulated in PANC-1 cells under acidic conditions. miR-451a has been reported to have antineoplastic effects on multiple cancers, including lung cancer [33], papillary thyroid cancer [34], colorectal cancer [35], and breast cancer. Our study found that miR-451a upregulation reversed the effects of acid conditions on oxygen consumption, MMP loss, and ROS generation in PC cells, as well as the proliferation, migration, invasion, and stem cell stemness.
e above results suggested that the acidic tumor microenvironment played a crucial role in PC progression, inhibiting miR-451a.
MEF2D, a member of the MEF2 family, is a crucial developmental factor, showing an essential role in the    tumorigenesis of various malignant tumor cells. Firstly, it was reported in the experimental study of leukemia that MEF2D promoted the malignant transformation of normal cells [36]. Subsequent evidence showed that MEF2D exerted significant roles in the progression of hepatocellular carcinoma, colorectal cancer, lung cancer, and osteosarcoma [37][38][39][40]. Song et al. found that MEF2D controlled cell proliferation, migration, and invasion abilities in pancreatic cancer via the Akt/GSK-3β signaling pathway [41]. Our data illustrated that MEF2D, a target gene of miR-451a, was highly expressed under acidic conditions. e silence of MEF2D increased the oxygen consumption, MMP loss, and ROS generation with a consequent increase in apoptosis. In addition, MEF2D overexpression reversed the effects of miR-451a mimics on the functional activities of PC, which was consistent with previous reports [41].
In summary, we reported a novel molecular signaling pathway that the acidic tumor microenvironment promotes PC progression through the miR-451a/MEF2D axis, which provides a novel therapeutic target for PC.

Data Availability
All data generated or analyzed during this study are included within this article.