Introduction
Pancreatic cancer (PANC) is one of the most common fatal gastrointestinal malignancies. It is still a major global public health problem for its 5 year survival rate is less than 5% [
1]. Late diagnosis, aggressive tumor growth, high metastasis and recurrence, and chemotherapy resistance lead to worse therapeutic effects and poor prognosis of PANC [
2‐
4]. It is common in pancreatic cancer to have genetic and epigenetic abnormalities, as well as aberrant activation of tumor-driver genes [
5,
6]. Therefore, it is of great significance to identify new oncogenes involved in pancreatic carcinogenesis and improve the early screening and diagnosis rate for improving the overall survival of PANC patients.
The miRNAs are a group of small non-coding RNAs that modify mRNA translation or stability to regulate their target genes [
7]. Since their discovery in 1993, miRNAs have been extensively studied for their role in tumor progression. There has been evidence that long non-coding RNAs (lncRNAs) are involved in cancer regulation via sponge miRNAs, interacting with downstream mRNA to create a regulatory network [
8‐
10]. Previous studies have found that miR-532 plays a regulatory role in a variety of nausea tumors, but its direction of action on cancer is divergent. In breast cancer and gastric cancer, miR-532 promotes cancer cell proliferation and metastasis [
11,
12], but it inhibits cancer cell proliferation and metastasis in lung cancer and prostate cancer [
13‐
15]. In colorectal cancer (CRC), miR-532 has been reported as a proto-oncogene and tumor suppressor [
16,
17].
The role of miR-532 in PANC has not been reported. Alizadeh Savareh B et al. [
18] constructed a machine-learning method to determine the diagnostic model of pancreatic cancer using circulating microRNA signatures. According to the statistical analysis of the survival rate, miR-532 was considered to have a meaningful correlation with the prognosis of pancreatic cancer patients. Our previous study found that miR-532 may play a tumor suppressor role in PANC. In this study, we will explore its upstream and downstream mechanism, and explore its potential value in the diagnosis and treatment of PANC.
Materials and methods
PANC tissues and cell lines
Seventeen pairs of pancreatic cancer tissues and adjacent normal pancreatic tissues that underwent surgical treatment in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2020 to February 2022 were collected. All tumor tissues were confirmed as adenocarcinoma by histological identification. All patients understood that the collected tissue would be used for scientific research and provided written informed consent.
Human pancreatic cancer cell lines PANC03.27, Capan-1, Capan-2, SW1990, hPAF-II, Panc 10.05, BXPC-3, CFPAC-1 and immortalized human pancreatic ductal epithelial cell line (HPNE) were purchased from the Cell Bank of Chinese Academy of Sciences (China, Shanghai). Cells were cultured in DMEM supplemented with 10% inactivated FBS (Gibco, USA), 1 × 105 U/L penicillin, and 100 mg/L streptomycin (Gibco). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. HPNE cells were grown in RPMI-1640 medium (Gibco) supplemented with 10% inactivated fetal bovine serum (FBS).
Cell transfection
Obio Technology Corp (China) provided the lentivirus constructing TWIST1 overexpress. SiRNA for knockdown LZTS1-AS1 and all the miR-532 mimics, inhibitors, and negative control used for transfection were purchased from GenePharma (Shanghai, China). Transfections were performed according to the manufacturer’s instructions with Lipofectamine 2000 (Invitrogen, USA) (Additional file
1).
Quantitative RT-PCR
According to the manufacturer's protocol, total RNA was extracted from crushed tissue or cultured cells using a Trizol reagent (Invitrogen). For RT-PCR, 1 g cDNA and SYBR Green RT-PCR kit (Takara, Japan) were used, and an RNA reverse transcription kit (Takara) was used for reverse transcription. For the reaction, 95 °C was pre-denaturated for 3 min, followed by 40 cycles (30 s at 95 °C, 45 s at 58 °C), followed by a 6 min extension at 72 °C. The primer sequences of the primers used were provided in Additional file
2: Table S1. They were synthesized by Shanghai Sangon Biotech Co., Ltd. (China). The 2
−ΔΔCt method was used to calculate the relative expression of targets. Repeat the calculation for each sample at least 3 times.
CCK-8 assay
In accordance with the instructions of the supplier, the CCK-8 kit was purchased from Boster Biological Technology Co., Ltd. (China). Cells in the logarithmic growth phase were seeded into 96-well plates at a concentration of 5 × 103 per well. The optical concentration (OD) values at 450 nm were measured before and after 72 h of culture with a microplate analyzer. Repeat at least 3 times for each sample.
Cell apoptosis and cell cycle assays
A dye combination of Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) was used to stain transfected cells (BD Pharmingen, USA) after transfection. A flow cytometry analysis (BD Biosciences, CA, USA) was performed on annexin V + cells. Cells were harvested and fixed with 70% cold ethanol at − 20 °C for cell cycle analysis. Following that, cells were stained with the Cell Cycle Kit (BD Pharmingen) following the manufacturer's instructions.
Western blot assay
In this study, cells were collected, disrupted with RIPA cleavage buffer (Thermo Fisher Scientific, USA), centrifuged, and the protein concentration was measured with BCA Kit (Thermo Fisher Scientific). SDS-PAGE was performed on 50 mg of protein followed by transfer to PVDF membranes (Millipore, USA). The membranes were blocked in 5% non-fat milk at room temperature for 1 h and then treated overnight at 4 °C according to the antibody protocol described above. The following antibodies purchased from Protientech (USA) were used: anti-ATG5 (66744-1-Ig), anti-ATG12 (67341-1-Ig), anti-LC3 (14600-1-AP), anti-Beclin1 (11306-1-AP), anti-p62 (18420-1-AP); anti-E-cadherin (20874-1-AP), anti-N-cadherin (66219-1-Ig), anti-Vimentin (10366-1-AP), anti-α-SMA (67735-1-Ig) and anti-GAPDH (60004-1-Ig). Moreover, the respective Goat anti-rabbit IgG secondary antibody (Abcam, ab205718) was used to incubate these membranes according to protocol. In order to quantify the protein blots, the Thermo Fisher Scientific ECL system was used in conjunction with Image J software to analyze the data.
Transfected cells were seeded into 6-well plates at 700 cells per well and maintained in DMEM containing 10% FBS for two weeks. A 30 min staining with 0.1% crystal violet followed by imaging and counting was performed after the cells had been fixed with methanol.
Immunohistochemistry
A 0.3% V/V solution of hydrogen peroxide was used to block the endogenous peroxide activity within cells. Then the cells were incubated with anti-Cyclin D1 (Proteintech, 26939-1-AP) or anti-MMP-9 (Proteintech, 10375-2-AP) overnight at 4 °C, and then incubated with the corresponding biotinylated secondary antibodies. As a counterstain, hematoxylin was used to counterstain the cells after the DAB reactions were developed using the DAB Kit (BD Bioscience, USA). In order to score staining, relative quantitative methods were used based on the intensity and the rate of positive staining.
Xenografts in nude mice
The CFPAC-1 cells were stably transfected. BALB/C mice (4 weeks old, 18–22 g) were injected subcutaneously with around 1 × 107 cells. Measurements of the tumor size (W) and length (L) were taken every week, and the tumor volume (V) was calculated using the equation V = (W2 × L)/2. The entire experimental procedure lasted for 4 weeks, and mice that died during this period were not included in the study. The mice were sacrificed after 4 weeks, and tumors were removed and weighed. The animal studies were performed in accordance with the institutional ethics guidelines for animal experiments, which were approved by the animal management committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (2021 IEC No.228).
Transmission electron microscopy (TEM)
CFPAC-1 cells were fixed in 2.5% glutaraldehyde without washing at 37 °C and further fixed with 2% osmium tetroxide buffer. The fixed cells were then dehydrated with graded ethanol series and embedded in Epon. Electron microscopy was performed using a Talos F200X S/TEM transmission electron microscope (Thermo Fisher Scientific).
Transwell assay
600 μl culture medium containing 10% FBS was added to the lower chamber. A single cell suspension containing 8 × 104 cells was seeded on a 200 μl serum-free medium for cell migration and 200 μl matrix gel for cell invasion in the upper chamber. After 24 h of culture, cells in the upper chamber were fixed with 4% paraformaldehyde for 5 min, stained with 0.1% crystal violet for 5 min, and transmembrane cells were counted under a light microscope (Olympus, Japan).
Dual-luciferase reporter assays
The wild-type (wt) and mutant (mut) 3′-UTR of LZTS1-AS1 were constructed by GenePharma. The luciferase construct and miR-532 mimic were co-transfected into CFPAC-1 cells following the manufacturer's protocol. 48 h after transfection, the luciferase activity was detected by a dual luciferase reporter kit (Promega). Each experiment was repeated 3 times.
RNA immunoprecipitation (RIP) assay
After being washed using PBS twice, CFPAC-1 cells were lysed with RIPA lysis buffer (ThermoFisher Scientific, USA) containing a protease inhibitor cocktail. The lysate was then incubated with magnetic beads of human anti-Ago2 antibody (Proteintech, 67934-1-Ig) and normal rabbit IgG as control. The beads were washed twice with 700 mM NaCl wash buffer. RNA in immunoprecipitates was isolated using TRIzol reagent (Invitrogen) and subjected to qRT-PCR analysis.
Statistical analysis
All data are presented as means ± standard deviation (SD). Statistical significance analysis and statistical graphing of the data were performed using Prism GraphPad 8.0 software. One-way ANOVA and Student’s t-test were used for data analysis. Kaplan–Meier curve and log-rank test were used to analyze the survival of patients. Expression correlation analysis was performed using Pearson’s correlation coefficient. P < 0.05 was regarded as a statistically significant difference.
Discussion
Leucine zipper tumor suppressor 1 (LZTS1) antisense RNA 1 (LZTS1-AS1) is a lncRNA located at Chr8p21.3, which has been reported to be associated with rheumatoid arthritis [
19]. Regarding the role of LZTS1-AS1 in pancreatic cancer, the study is the first report. Our study found that LZTS1-AS1 was highly expressed in pancreatic cancer tissues and associated with poor prognosis, and knockdown of LZTS1-AS1 in pancreatic cancer cells significantly inhibited cell proliferation, tumorigenicity, metastasis, and induced autophagy, suggesting its inhibitory effect on pancreatic cancer. These results add LZTS1-AS1 to the range of biomarkers for pancreatic cancer screening.
It has been reported that lncRNA plays a regulatory role in pancreatic cancer [
20‐
22]. Mechanistic studies have found that lncRNA can contribute to the progression of pancreatic cancer by modulating the expression of miRNA to form a lncRNA-miRNA-mRNA regulatory network [
23]. In this study, the starBase database was used to predict the binding site of LZTS1-AS1 with miR-532, and the dual luciferase gene reporter assay and RIP assay were used to verify the interaction between LZTS1-AS1 and miR-532 in pancreatic cancer cells. There was a negative correlation between LZTS1-AS1 and miR-532 expression levels in pancreatic cancer tissues. The effect of LZTS1-AS1 knockdown on the biological function of pancreatic cancer cells can be offset by inhibiting miR-532. These results indicate that LZTS1-AS1 promotes pancreatic cancer progression through sponge miR-532.
The regulatory effect of miR-532 on the proliferation and metastasis of cancer cells has been reported in many previous studies [
13‐
15], but its effect on autophagy is rarely explored. It has been reported that miRNA-532 can reverse the inhibitory effect of Rab3IP on the autophagy signaling pathway in gastric cancer [
24]. It has also been reported that autophagy-related circular RNA (ACR) is downregulated in chronic heart failure and may suppress hypoxia-induced cardiomyocytes by downregulating miR-532 [
25]. In this study, miR-532 was found to inhibit cell proliferation and induce autophagy in pancreatic cancer. Coincidentally, the role of miR-532 in pancreatic cancer is opposite to what has been reported for its role in gastric cancer [
24], both in cell metastasis and autophagy.
As early as 2013, it was found that Twist family BHLH transcription factor 1 (TWIST1) was hypermethylated in pancreatic cancer [
26]. Recent studies have found that TWIST1 promotes the Warburg metabolism of pancreatic cancer by transcriptionally regulating glycolytic genes [
27]. It can be said that TWIST1 plays multiple important roles in the occurrence and development of pancreatic cancer. Not surprisingly, TWIST1 plays a regulatory role in the proliferation, migration, invasion, and EMT of pancreatic cancer cells [
28‐
30], and TWIST1 is involved in the regulation as a miRNA target in these reports. In epithelial ovarian cancer, miR-532 has been identified as a prognostic marker and inhibits cell proliferation and invasion by targeting TWIST1 [
31]. Our study found that miR-532 also inhibited cell proliferation and metastasis by targeting TWIST1 in pancreatic cancer.
P62 is a substrate protein of autophagy and a key agonist of NRF2 [
32]. Degradation of p62 was reduced in autophagy-deficient cells. When p62 competitively binds KEAP1, NRF2 is released and enters the nucleus to activate downstream target gene transcription and promote tumor cell survival [
33]. Our study confirmed that miR-532 induces autophagy in pancreatic cancer cells through TWIST1, and the KEAP1/NRF2 pathway may be its downstream target gene. It has been reported that autophagy deficiency stabilizes TWIST1 protein through SQSTM1/p62 accumulation [
34]. SQSTM1 binds to TWIST1 to inhibit the degradation of TWIST1 in autophagosomes and proteasomes. SQSTM1-mediated stable expression of TWIST1 promotes EMT in vitro and promotes tumor growth and metastasis in mice. This may explain the transitional role of TWIST1 between autophagy and EMT. Moreover, TWIST1 has been reported to play an intermediate role between EMT and autophagy in cancers and diabetic kidney disease [
35‐
37]. In this study, TWIST1 was also involved in the regulation of EMT and autophagy in pancreatic cancer. However, whether the relationship among autophagy, EMT, and stable TWIST1 is the key to tumor proliferation and metastasis in pancreatic cancer remains to be further explored.
In conclusion, our study found that lncRNA LZTS1-AS1 is highly expressed in pancreatic cancer tissues and is associated with poor prognosis. It can promote proliferation, metastasis, and oncogenicity and inhibit autophagy of pancreatic cancer cells. Further investigation revealed that LZTS1-AS1 may exert a positive effect on pancreatic cancer by regulating TWIST1 through sponge miR-532. These findings provide a basis for LZTS1-AS1 as a novel biomarker for pancreatic cancer.
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