LncRNA MBNL1-AS1 Suppresses Cell Proliferation and Metastasis of Pancreatic Adenocarcinoma through Targeting Carcinogenic miR-301b-3p

Pancreatic adenocarcinoma (PAAD) has been a huge challenge to public health due to its increasing incidence, frequent early metastasis, and poor outcome. The molecular basis of tumorigenesis and metastasis in PAAD is largely unclear. Here, we identified a novel tumor-suppressor long noncoding RNA (lncRNA) MBNL1-AS1, in PAAD and revealed its downstream mechanism. Quantitative real-time PCR (qRT-PCR) data showed that MBNL1-AS1 expression was significantly downregulated in PAAD tissues and cells, which was closely associated with metastasis and poor prognosis. Cell counting kit-8 (CCK-8) assay, transwell assay, and western blot verified that overexpression of MBNL1-AS1 suppressed cell proliferation, migration, and epithelial mesenchymal transformation (EMT) behavior in PAAD cells. By using a dual luciferase reporter gene system, we confirmed that miR-301b-3p was a direct target of MBNL1-AS1. Further mechanismic study revealed that upregulation of miR-301b-3p abolished the inhibitory effect of MBNL1-AS1 overexpression on cell proliferation, tumorigenesis, migration and EMT. Our results demonstrate that MBNL1-AS1 plays a tumor-suppressive role in PAAD mainly by downregulating miR-301b-3p, providing a novel therapeutic target for PAAD.


Introduction
Pancreatic adenocarcinoma (PAAD) is a malignant tumor of the digestive tract. Its incidence rate is increasing all over the world, and its prognosis is usually poor [1]. Although the treatment for other cancers has made great progress in recent years, the survival rate of PAAD has been stagnant, and the 5-year survival rate remains between 5% and 10% [2]. It is prone to distant metastasis in the early stage, and there are no specifc symptoms and signs in the early stage of PAAD, which poses a huge challenge for the improvement of PAAD prognosis. Understanding the molecular mechanism of pancreatic tumorigenesis and metastasis may help to develop new diagnostic and therapeutic methods.
Long noncoding RNAs (lncRNAs) are considered as tumor regulators and have attracted increasing attention in the feld of cancer research over the past decades [3]. LncRNA is defned as a class of noncoding RNAs (ncRNAs) with a length of more than 200 nucleotides [4]. LncRNAs account for a large proportion of the human genome and participate in a set of physiological or pathological process through interacting with DNAs, mRNAs, ncRNAs, and proteins [5]. Numerous studies have showed that lncRNAs are specifcally expressed in tumors and regulate multiple tumor biological processes, including sustaining proliferation signaling, evading growth suppressors, enabling replicative immortality, resisting cell death, etc. [6][7][8]. Among the aberrantly expressed lncRNAs, MBNL1-AS1 is downregulated and regarded as a novel tumor-suppressive lncRNA in several common cancers, like colon cancer, breast cancer, and non-small lung cancer [9][10][11]. MBNL1-AS1 has been proven to repress cell proliferation and migration in these cancers. In addition, MBNL1-AS1 is reported to be upregulated in acute myocardial infarction, and silencing MBNL1-AS1 reduces myocardial injury in animal models [12]. From the current research results, we know that MBNL1-AS1 reduces cell survival in both cancer and noncancer cells. However, it is still unclear whether and how MBNL1-AS1 plays tumor-suppressive roles in PAAD.
MicroRNAs (miRNAs) are a class of short ncRNAs consisting of about 22 nucleotides. Tey usually form incomplete base pairs with the 3′-untranslated region (3′-UTR) of the target gene and play a negative role in regulating gene expression [13,14]. Specifc miRNAs have been reported to regulate of a large amount of cellular biological processes, such as cell proliferation, diferentiation, migration, epithelial mesenchymal transformation (EMT), etc. [15]. Among them, a lot of functional miRNAs are identifed in the cancer progression. For example, miR-106b-5p contributes to the lung metastasis of breast cancer [16]. miR-19a facilitates metastasis and EMT in prostate cancer [17]. miR-331-3p promotes drug resistance in pancreatic cancer [18]. Recently, miR-301b-3p is reported to function as a tumor promoter in breast cancer, lung cancer, and colorectal cancers [19][20][21]. Interestingly, a few evidences have suggested that miR-301b links with gemcitabine resistance and cell invasion in pancreatic cancer, and miR-301b plays its role in pancreatic cancer through downregulating the expression of tumor-suppressor TP6 [22,23]. Tus, miR-301b might be an efective therapeutic target for PAAD treatment; however, little is known about its detailed regulatory mechanism in PAAD.
Herein, we detected the expressions of MBNL1-AS1 and miR-301b-3p in PAAD and analyzed the correlation between their expressions and metastasis and the overall survival rate of PAAD patients. Moreover, the role of MBNL1-AS1 and miR-301b-3p in PAAD and their interaction mechanisms were revealed in the present work. Te study aims to uncover the molecular mechanism of PAAD development and provide potential targets for the diagnosis and treatment of PAAD.

Methods and Materials
2.1. Clinical Samples. PAAD tissues and the adjacent normal tissues were collected from 52 cases of PAAD patients in the First Afliated Hospital of Xinjiang Medical University. All the patients received surgical treatment in our hospital from 2019 to 2021 and were diagnosed with PAAD by postoperative pathology. Te patients accepted no treatment before the operation, and their clinicopathological data and follow-up data were complete. Te surgical resection tissues were stored at −80°C. All the protocols involved with the human tissue samples were in accordance with the World Medical Association Declaration of Helsinki. Te study protocol was approved by the ethics committee afliated with the First Afliated Hospital of Xinjiang Medical University (approval number: 2021-032), and the study has been granted an exemption from requiring written informed consent.

Western Blot Analysis.
Total protein was obtained by RIPA lysis bufer (Solarbio, Beijing, China), and the concentration was measured by a BCA assay kit (Yeasen, Shanghai, China). Ten 50 μg protein samples was separated on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and transferred to polyvinylidene fuoride (PVDF) membranes (Millipore, MD., USA). Te membranes were blocked with 5% nonfat milk for 1.5 h and incubated with anti-E-cadherin, N-cadherin, and Vimentin at 4°C overnight. Te next day, the membranes were washed and incubated with horseradish peroxidase-labeled IgG antibody at room temperature for 1 h. Images of blots were captured by using the gel imaging system (Bio-Rad, CA., USA), and the original blots are presented in Supplementary fle 1.

Dual Luciferase Reporter Gene
System. According to the binding site between miR-301b-3p and MBNL1-AS1 analyzed by bioinformatic tools, the wild-type sequence of MBNL1-AS1 and the mutant sequence of MBNL1-AS1 were designed, synthesized, and inserted into the vector pRL-TK (Promega, Beijing, China). PANC-1 and SW1990 cells were seeded on a 96-well plate and transfected with the reconstructed vector containing MBNL1-AS1 sequences, miR-301b-3p mimic, or NC mimic, by using Lipofectamine 2000 regents (Invitrogen, CA., USA). 48 h later, the cells were lysed for measurement of luciferase activity.

Apoptosis.
Cell apoptosis was detected using the AnnexinV-FITC/PI Apoptosis Detection Kit (BD, NJ, USA). Briefy, cells were digested with 0.25% pancreatin, washed with precooled PBS, and centrifuged at 2000 rpm for 10 min. Cell precipitation was suspended with 300 μL binding bufer, mixed evenly with 5 μL Annexin V-FITC and incubated at room temperature for 15 min in the dark. 5 μL PI was added into cell mixture 5 min before measurement, and 200 μL binding bufer was added into them immediately before measurement. Flow cytometry was utilized for the measurement of the apoptotic rate.

Transwell
Assay. 100 μL PANC-1 or SW1990 cells (5 × 10 4 cells/well) were seeded in the upper chambers of a 24-well Transwell chamber (BD, NJ, USA), while 500 μL DMEM supplemented with 10% FBS was added to the lower chamber. After being cultured for 48 h, migrated cells in the lower chamber were fxed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Sigma, MO., USA) for 15 min at room temperature. After being washed and dried, cells were photographed under a microscope (Olympus, Tokyo, Japan).

Xenograft
Tumors. PANC-1 cells in the logarithmic growth phase were digested with 0.25% trypsin and resuspended in PBS after washing. 200 μL cell suspension (1 × 10 6 cells) was subcutaneously injected into the right armpit of nude mice using a 1 mL syringe. Tumor growth was recorded within 28 d after injection. Te mice were sacrifced at 28 day and tumors were weighed.
2.11. Statistical Analysis. GraphPad Prism 8.0 (GraphPad Software, CA., USA) was used for data analysis and graphics drawing. Each experiment was independently repeated at least 3 times, and all data were presented as the mean ± standard deviation. Data in two groups was compared by using Student's t-test, and p < 0.05 was considered statistically signifcant.

MBNL1-AS1 Was Downregulated in PAAD and Closely
Related to PAAD Progression. We frst collected 52 cases of surgical resection tissues from PAAD patients and detected the expression of MBNL1-AS1 in PAAD tumor tissues and adjacent normal tissues. Te qRT-PCR data in Figure 1(a) indicated that the MBNL1-AS1 expression of tumor tissues was lower than that of normal tissues. Ten these tumor tissues were divided into two groups according to the average value of MBNL1-AS1 expression, 28 cases with high MBNL1-AS1 levels and 24 cases with low MBNL1-AS1 levels, and the relationship between MBNL1-AS1 and their clinicopathological features was shown in Table 1. Low expression of MBNL1-AS1 was signifcantly associated with poor diferentiation degree, high TNM stage, and lymph node metastasis, while there was no signifcant correlation between MBNL1-AS1 and other variables, including age, gender, tumor location, or alcohol history (Table 1). Additionally, Kaplan-Meier analysis data indicated that lower MBNL1-AS1 expression was associated with a poorer prognosis and shorter overall survival of PAAD (Figure 1(b)). Meanwhile, the MBNL1-AS1 expression in PAAD cell lines (Capan-2, AsPC-1, SW1990, and PANC-1) was lower than that in normal pancreatic ductal epithelial cells (Figure 1(c)). Taken together, the data suggested that MBNL1-AS1 was downregulated in PAAD and closely related to PAAD progression and metastasis.

Overexpression of MBNL1-AS1 Suppressed Cell Proliferation, Migration, and EMT in PAAD.
Due to the signifcance of abnormal expression in SW1990 and PANC-1, the two cell lines were chosen for the following experiments. To investigate the role of MBNL1-AS1 in PAAD, pcDNA-MBNL1-AS1 was transfected into SW1990 and PANC-1 for overexpressing MBNL1-AS1 (Figure 2(a)). Ten, CCK-8 assay was performed to evaluate cell proliferation, and the results in Figure 2(b) showed that overexpression of MBNL1-AS1 suppressed the cell viability of the PAAD cells. Next, transwell assay was used to evaluate cell migration, and we found that cell migration ability was also inhibited by overexpression of MBNL1-AS1 (Figure 2(c)). Finally, the expression of EMT markers was measured through western blot (Figure 2(d)). Te data indicated that MBNL1-AS1 overexpression elevated the expression of an epithelial marker (E-cadherin) but decreased the expression of mesenchymal marker (N-cadherin and Vimentin), suggesting that MBNL1-Genetics Research 3 AS1 hindered EMT behavior in PAAD cells. Collectively, these results demonstrated that upregulating MBNL1-AS1 suppressed proliferation, migration and EMT in PAAD cells.

MiR-301b-3p was Targeted by MBNL1-AS1 in PAAD.
Trough bioinformatics analysis, we found that miR-301b-3p could bind to MBNL1-AS1 theoretically, which was verifed by the next dual luciferase reporter gene system (Figure 3(a)). We found that miR-301b-3p mimic reduced the luciferase activity in PAAD cells transfected with the wild-type sequence of MBNL1-AS1, rather than that in cells transfected with the mutant sequence of MBNL1-AS1. And miR-301b-3p expression in PANC-1 and SW1990 could be reduced by overexpressing MBNL1-AS1 (Figure 3(b)). Subsequently, we detected miR-301b-3p expression in PAAD tissues and normal tissues. As expected, miR-301b-3p was upregulated in PAAD tissues (Figure 3(c)) and was negatively related to MBNL1-AS1 expression (Figure 3(d)). Likewise, we categorized these tumor tissues into a miR-301b-3p high-expression group and a miR-301b-3p low-expression group according to the average level of miR-301b-3p. Contrary to MBNL1-AS1, miR-301b-3p expression was closely related to diferentiated degree, TNM stage, and lymph node metastasis ( Table 2).   Moreover, Kaplan-Meier analysis illustrated that the patients with higher miR-301b-3p expression had a poorer prognosis (Figure 3(e)). In addition, the miR-301b-3p expression in PAAD cell lines was shown to be higher than that in normal pancreatic ductal epithelial cells (Figure 3(f )). Altogether, these data miR-301b-3p was a target of MBNL1-AS1 and involved in the PAAD progression.
To sum up, it is suggested that MBNL1-AS1 hindered PAAD development partly by targeting the tumorpromoting miR-301b-3p.

Discussion
In recent years, PAAD has attracted increasing attention for its growing incidence and high mortality [24,25]. Owing to the rapid growth of PAAD cells, abundant blood vessels and lymphatic vessels around the pancreas, and the incomplete envelope of the pancreas itself, PAAD is prone to metastasis at the early stage [26,27]. EMT, a transition that epithelial cells lose polarity and connections between cells, acquire the ability of infltration and migration, and become cells with interstitial morphology and characteristics, is the main Log-rank X 2 =4.971 P=0.026 High miR-301b-3p Low miR-301b-3p    driving force of migration [28]. In this study, we explored the driving molecular of cell proliferation, migration, and EMT behavior in PAAD, hoping to improve the understanding of the PAAD development. Accumulating evidence showed the dysregulation of specifc ncRNAs could be observed in almost all cancers, and some of these ncRNAs play critical roles in the cancer development and progression [29]. Over the past few decades, a large number of ncRNAs (including lncRNAs and miR-NAs) have been recognized as cancer-associated regulators [30][31][32][33]. Te present work frstly showed that MBNL1-AS1 expression was signifcantly downregulated in PAAD tissues and cells and demonstrated that overexpression of MBNL1-AS1 suppressed cell proliferation, migration, and EMT behavior in PANC-1 and SW1990 cells. MBNL1-AS1 is a novel lncRNA discovered in recent years. Previous studies have proved that MBNL1-AS1 plays a tumor-suppressive role in several types of cancers, such as breast cancer, lung cancer, and bladder cancer [34][35][36]. Te role of MBNL1-AS1 in PAAD has not been clarifed until our study.
According to the competitive endogenous RNA (ceRNA) hypothesis, lncRNAs exert their biology roles usually by sponging miRNAs to regulate genes expression [37]. To date, some miRNAs have been verifed to be the target of MBNL1-AS1, including miR-135a-5p in lung cancer and bladder cancer, miR-412-3p in colon cancer, and miR-338-5p in retinoblastoma [9,34,36,38]. In the present study, miR-301b-3p was demonstrated to be the target molecular of MBNL1-AS1, and its expression was elevated in PAAD tissues and cells. As previously reported, miR-301b-3p is essential for the initiation of diverse common cancers, including PAAD [22,[39][40][41]. MiR-301b-3p promotes cell proliferation and migration by inhibiting a lot of downstream efector genes, which function as tumor suppressors in cells [21,[42][43][44]. Finally, through a function recovery experiment, we verifed that upregulation of miR-301b-3p abolished the inhibitory efect of MBNL1-AS1 overexpression on cell proliferation, apoptosis, tumorgenesis, migration, and EMT. Tese results preliminarily illustrated the underlying regulatory mechanism of PAAD tumorgenesis and metastasis. However, our present work only illustrated the interaction of MBNL1-AS1 and miR-301b-3p in PAAD because of the limited time and conditions. Actually, many miRNAs could be targeted by MBNL1-AS1. Terefore, further studies are needed to screen other functional miRNAs and improve the molecular mechanisms in the future.

Conclusion
In conclusion, we frst identifed MBNL1-AS1 as a tumorsuppressive lncRNA in PAAD, and MBNL1-AS1 reduces cell proliferation, migration, and EMT behavior of PAAD cells via sponging and downregulating miR-301b-3p. Te fndings shed light on a signifcant role of ncRNAs in the PAAD progression and might provide a theoretical basis for the development of PAAD therapies.

Data Availability
Te data used in this study are all included in this paper. Te original data are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
CMS and WH made contributions to the conception and design of the research. CMS and A conducted investigation and data curation. QLC and TYH were responsible for supervision. CMS and A drafted and revised the manuscript. QLC and HL reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. CS and AA contributed equally to this work.