MicroRNA-708 inhibits the proliferation and chemoresistance of pancreatic cancer cells

: Pancreatic cancer is one of the most aggressive malignancies with poor prognosis and high mortality. Recent studies showed that microRNAs are dysregulated and involved in the initiation and progression of pancreatic cancer. In this study, we found that miR-708 was significantly downregulated in pancreatic cancer tissues and cell lines. Lentivirus-mediated overexpression of miR-708 could significantly inhibit the proliferation and invasion, while enhanced chemosensitivity to gemcitabine in both Panc-1 and SW1990 cells. Luciferase reporter assay showed that miR-708 bound the 3’-untranslated region of survivin and suppressed the expression of survivin in pancreatic cancer cells. In pancreatic cancer tissues, survivin protein was highly expressed and negatively correlated with miR-708 expression. Furthermore, the restoration of survivin expression could partially antagonize proliferation inhibition and apoptosis induction by miR-708 in pancreatic cancer cells. The Panc-1 cells with overexpression of miR-708 also showed decreased proliferation capability in nude mouse model compared with parental cells. In conclusion, our results suggest that miR-708 inhibits pancreatic cancer and could be a novel potential candidate to treat pancreatic cancer. the tumorigenesis and progression of pancreatic cancer.


Introduction
As one of the most common aggressive malignancies in the world, pancreatic cancer is the fourth major cause of cancerrelated deaths with high mortality (Jin et al., 2018). Despite the increased surgical resection rate in recent decades, the 5-year survival rate has not been substantially improved for pancreatic cancer (Siegel et al., 2017). The poor prognosis is mainly attributed to the advanced stage when pancreatic cancer is diagnosed, and inherent cancer cell resistance to chemotherapeutic drugs (Zhang et al., 2019). While medicinal plants have been recently explored to screen effective agents to overcome cancer chemoresistance, the progress is still limited (Orozco Montes et al., 2019;Villa-Hernández et al., 2018). F u r t h e r u n d e r s t a n d i n g o f o n c o g e n e s i s a n d chemotherapy resistance mechanisms is pivotal to promote therapeutic effects for pancreatic cancer. Until now, the identification of new sensitive biomarkers and novel pharmacological targets remains an urgent task.
MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules as long as approximately 22 nucleotides. MiRNAs can suppress the translation of target genes by binding to the 3' untranslated region (3'-UTR) and regulate cellular proliferation, apoptosis, and differentiation *Address correspondence to: Donglin Sun, czyysdl@163.com; Haijiao Yan, haijiao8237@163.com (Abreu et al., 2017). Multiple miRNAs have been involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC) by modulating diverse cell signaling pathways . Lin et al. (2018) reported that miR-1179 was downregulated in pancreatic cancer, and the restoration of miR-1179 expression could inhibit tumor proliferation both in vitro and in vivo by suppressing the expression of target gene E2F5. Recently, increasing evidence has suggested that aberrant miRNA expression was associated with the resistance to anticancer drugs (Cagle et al., 2019;Si et al., 2019). Our team previously reported that miR-17-5p inhibitor could enhance chemosensitivity to gemcitabine by the upregulation of Bim expression in pancreatic cancer cells (Yan et al., 2012). Lower miR-708 expression was reported recently in the late stage of ovarian cancer, and patients with higher miR-708 would have the potential of a better survival rate (Lin et al., 2015). miR-708 expression was also found to be decreased in gastric cancer tissues, and ectopic expression of miR-708 could inhibit proliferation and invasion of gastric cancer cells in vitro . However, the role of miR-708 in the chemoresistance of pancreatic cancer remains elusive. Therefore, this study aimed to investigate the function of miR-708 in pancreatic cancer. We found that miR-708 expression was significantly decreased in pancreatic cancer tissues and cell lines. Furthermore, lentivirus-mediated overexpression of microRNA-708 could inhibit the proliferation, invasion, and chemoresistance to gemcitabine in pancreatic cancer cells. Our results provide novel insights into the crucial role of miR-708 in the tumorigenesis and progression of pancreatic cancer.

Tissue samples and cell lines
The matched 52 pancreatic carcinoma tissues and adjacent normal pancreatic tissues were obtained from patients at the Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Soochow University, from 2014 to 2017. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University. (Approval No. 2013038, October 2013). The samples were confirmed histologically, and all patients provided informed consent. The immortalized human pancreatic ductal epithelial cell line H6C7 was kindly provided by Dr. M.S. Tsao (Ontario Cancer Institute, University of Toronto, Canada), and was maintained in serum-free medium containing keratinocyte (Invitrogen, Carlsbad, CA, USA). The Panc-1 and SW1990 cell lines of pancreatic carcinoma were provided by the Culture Center of Wuhan University (Wuhan, China) and cultured in DMEM medium containing 10% fetal bovine serum (FBS). All the cells were routinely grown in a humidified atmosphere with 5% CO 2 at 37 o C.

Real-time PCR
Total RNA was extracted from clinical tissues or cultured cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions. cDNA was synthesized using TaqMan MicroRNA Reverse Transcription kit, and realtime PCR was performed using SYBR Green qPCR Master Mix Kit (Takara, Dalian, China) on a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences were as follows: miR-708, forward: 5'-CGC GGA TCC GAC TTC ATT CCC CTA ACC C-3' and reverse: 5'-CCG GAA TTC TGG CAC GCA GGA GAC AGT-3'; and U6, forward: 5'-CGC TTC GGC AGC ACA TAT AC-3' and reverse: 5'-AAA TAT GGA ACG CTT CAC GA-3' . The expression level of miR-708 was normalized to U6 and calculated according to the 2 -ΔΔCt method. The primers of survivin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were designed and synthesized by GenePharma (Shanghai, China). GAPDH was served as the internal reference for survivin mRNA.

Construction of vectors
To overexpress miR-708, a fragment of 88 bp containing the miR-708 precursor and the flanking sequence was synthesized and cloned into BamHI and EcoRI sites of lentiviral vector pHBLV-U6-ZsGreen-Puro (Promega, Madison, WI, USA), and recombinant lentivirus was constructed by co-transfection of miR-708 lentiviral vector and packaging plasmids to 293T cells using Lipofectamine 2000 (Invitrogen). Panc-1 and SW1990 cells were infected with lentivirus for 8 h in the presence of 8 mg/mL polybrene. Forty-eight hours after infection, 2 μg/mL puromycin was added to the culture medium to select the infected cells for one week. The two cell lines stably expressing miR-708 were established successfully.
To overexpress sur vivin, a 426 bp fragment of survivin cDNA was cloned into plasmid pcDNA3.1, and the recombinant plasmid was subsequently generated and transfected to cancer cells for 24 h using Lipofectamine 2000.

Cell invasion assay
Invasion assay was performed using Matrigel-coated Transwell chamber (BD Biosciences, San Jose, CA, USA). Cancer cells were starved in serum-free medium overnight and were harvest and resuspended in FBS-free DMEM. A total of 5 × 10 4 cells in 100 μL DMEM were plated into the upper chambers. Then the lower chamber was covered with DMEM supplemented with 20% FBS. After incubation for 24 h at 37°C, non-invasive cells on the upper surface of the membrane were gently removed by cotton swabs, and the invaded cells were fixed with 4% formaldehyde and stained by 0.5% crystal violet. The stained cells were counted under an Olympus inverted microscope in five random visual fields.

Western blot analysis
Total protein was extracted after the cells were lysed in lysis buffer, then the concentration of total protein was measured by BCA assay. Equal proteins (40 μg/lane) were separated by 15% sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Sigma). After blocking by 5% skimmed milk, the membranes were incubated with primary antibodies for survivin, p-AKT, NF-kB P65, E-cadherin, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After extensive washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The protein bands were finally visualized using the SuperSignal enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). GAPDH expression was detected as the internal control.
Apoptosis assay by flow cytometry Cells were harvested and washed in cold PBS and stained with annexin V and PI using the annexin V-FITC Apoptosis Detection Kit (BD Biosciences). After incubation in the dark at room temperature for 20 min, the fluorescence intensity of each group of cells was immediately detected by flow cytometry (Coulter Biosciences, Brea, CA, USA).

Nude mouse model
Female BABL/c nude mice (4 weeks old) were purchased from the animal center of the Chinese Academy of Science (Shanghai, China). The animal experiments were approved by the Institutional Animal Care and Treatment Committee of Third Affiliated Hospital of Soochow University. 2 × 10 6 of tumor cells in a volume of 100 mL medium were injected subcutaneously into the right flank of each nude mouse (6 mice per group). After implantation for 7 days, tumor volume was measured every 7 days, using the following formula: volume = length (mm) × width 2 (mm)/2. All the mice were euthanized after 28 days.

Statistical analysis
Statistical analyses were carried out by SPSS 17.0 software (SPSS Inc., Chicago, IL, USA), and the comparison of numerical data among multiple groups was performed using the one-way ANOVA test. p < 0.05 was considered to be statistically significant.

miR-708 expression was downregulated in pancreatic cancer tissues and cell lines
To explore the role of miR-708 in pancreatic cancer, the miR-708 expression level in human pancreatic cancer tissues and matched adjacent normal regions were detected by real-time PCR. We found that the expression of miR-708 was significantly downregulated in pancreatic cancer tissues compared with the normal adjacent tissues ( Fig.  1(A), p < 0.01). We further confirmed that the miR-708 level was notably decreased in four pancreatic cancer cell lines, including Bxpc-3, Panc-1, Capan-1, and SW1990 compared to immortalized ductal epithelial cell line H6C7 (Fig. 1(B), p < 0.05). Among these cancer cell lines, Panc-1 and SW1990 showed relatively lower miR-708 level and were selected for subsequent experiments.

miR-708 overexpression suppressed proliferation, invasion and chemoresistance of pancreatic cancer cells in vitro
To reveal the function of miR-708 on the development of pancreatic cancer, the Panc-1 and SW1990 cell lines were infected with LV-miR-708 or negative control miRNA. RT-qPCR showed enhanced expression of miR-708 about 20-fold higher than control after infection of LV-miR-708 in both cell lines (Fig. 2(A), p < 0.01). We found a significant decrease in proliferation capability in cancer cells overexpressing miR-708 compared with control cells in a time-dependent manner, and the maximum inhibitory rate was 56.02 ± 3.98% and 53.24 ± 3.85% for Panc-1 and SW1990 on day 8, respectively ( Fig. 2(B), p < 0.05).
We also investigated the effect of miR-708 on gemcitabine chemosensitivity in pancreatic cancer cells in vitro. MTT assay showed that ectopic expression of miR-708 led to 18.13 ± 3.34 and 19.33 ± 3.55 loss of viability in Panc-1 and SW190 cells, respectively. Moreover, ectopic expression of miR-708 combined with gemcitabine could even cause 60.50 ± 6.14% and 61.33 ± 6.49% loss of viability ( Fig. 2(D), p < 0.01).

MicroRNA-708 targeted survivin
To illustrate the molecular mechanisms of miR-708 in regulating malignant phenotypes of pancreatic cancer cells, bioinformatics analysis by TargetScan was carried out to predict the potential targets of miR-708. Survivin was predicted as a target of miR-708 (Fig. 3(A)). To further clarify whether the 3'-UTR of survivin is the functional target of miR-708, luciferase reporters harboring the miR-708 binding sites in survivin 3'-UTR and the mutant were reconstructed (Fig. 3(B)). After the reporters were co-transfected with miR-708 mimics into Panc-1 cells, miR-708 mimics significantly decreased luciferase activity of wild-type reporter but not the mutant reporter (p < 0.05; Fig. 3(C)). In addition, the survivin protein level was reduced in a dose-dependent manner when Panc-1 cells were transfected with 50-200 nM of miR-708 mimics (Fig. 3(D)). Taken together, these results indicate that miR-708 negatively regulated survivin expression by binding to its 3'-UTR, and survivin is a direct target of miR-708.  Furthermore, both NF-κB and p-Akt levels decreased while E-cadherin, cleaved caspase-3, and cleaved caspase-7 levels increased when survivin expression was suppressed by miR-708 (Fig. 3(E)). These results suggest a proapoptotic role of miR-708.

Survivin was overexpressed in pancreatic cancer tissues and negatively correlated with miR-708 expression
To further explore the association between miR-708 and survivin, we detected the abundance of survivin in pancreatic cancer tissues and paired adjacent normal tissue. Compared with normal tissues, survivin mRNA levels significantly increased in pancreatic cancer tissues (Fig. 4(A)), The correlation between miR-708 expression and survivin level was analyzed by the Spearman's test, and miR-708 had significant inverse association with survivin mRNA expression in pancreatic cancer tissues (Fig. 4(B)).

Survivin antagonized miR-708 on pancreatic cancer cell growth and apoptosis
To explore whether survivin mediates the biological function of miR-708 in regulating the proliferation and apoptosis of pancreatic cancer cells, both Panc-1 and SW1990 cells were transfected with survivin construct to overexpress survivin (Fig. 5(A)). Flow cytometry assay showed that survivin overexpression partially inhibited the apoptosis induced by miR-708 in each cell line (Fig. 5(B)). The change of proliferation capacity of Panc-1 and SW1990 cells expressing both miR-708 and survivin indicated that survivin overexpression significantly rescued the inhibitory effect of miR-708 on the growth of pancreatic cancer cells (Fig. 5(C)).

Overexpression of miR-708 inhibited pancreatic cancer growth in xenograft model
Panc-1 cells infected with LV-miR-708 and control cells were injected into mice to evaluate the growth capability of xenograft tumors in vivo. The volume of xenograft tumors resulting from the injection of Panc-1 cells overexpressing miR-708 was significantly reduced compared with the control group (Fig. 6, p < 0.05).

Discussion
As the most aggressive malignancy of the digestive system, pancreatic cancer still leads to high mortality of the patients. Illustrating the molecular mechanisms that contribute to the tumorigenesis and progression of pancreatic cancer is crucial to the development of novel therapeutic strategies. In the present study, we found that miR-708 expression was significantly downregulated in pancreatic cancer tissues and cells.
Lentivirus-mediated overexpression of miR-708 could suppress the proliferation, invasion, and chemoresistance to gemcitabine in pancreatic cancer cells. MiR-708 negatively regulated survivin protein expression at a post-transcriptional level. Restoration of survivin expression could partially rescue proliferation inhibition and apoptosis induction by miR-708. Moreover, Panc-1 cells stably overexpressing miR-708 showed decreased proliferation capability in a nude mouse model in vivo. These results reveal the important role of the miR-708/survivin axis in modulating the development and progression of pancreatic cancer.
Recent evidence showed that aberrant miRNA expression was involved in the development and progression of malignant tumors, including pancreatic cancer (Zhu et al., 2018;. miR-708 was reported to be highly expressed in the brain as well as the eyes and play a role in endoplasmic reticulum stress (Jang et al., 2012). Previous studies have reported the different expression levels of miR-708 in several cancer cells, which indicated that miR-708 may play different roles in a cancer type-dependent manner. For example, Li et al. (2015) found that miR-708 was downregulated in hepatocellular carcinoma (HCC) tissues and enforced expression of miR-708 suppressed the invasion and migration of HCC cell lines HepG2 and SMMC-7721. Qin et al. (2017) reported that restoration of miR-708 could sensitize ovarian cancer cells to cisplatin. However, Lei et al. (2014) reported that miR-708 was highly expressed in colorectal cancer (CRC) tissues, and inhibition of miR-708 suppressed cell proliferation and invasion in vitro. In this study, we found that miR-708 expression was significantly downregulated in pancreatic cancer cells. Our results confirmed that the restoration of miR-708 expression could significantly suppress the proliferation, induce apoptosis, and enhance chemosensitivity to gemcitabine in both Panc-1 and SW1990 cells.
As the smallest member of inhibitors of apoptosis protein (IAP) family, survivin participates in multiple biological processes, including restraining apoptosis and regulating cell division Bi et al., 2018). Aberrantly high expression of survivin was associated with cancer progression and poor prognosis (Han et al., 2018). Survivin blocks apoptosis downstream components of both mitochondrial pathway and death receptor pathways by directly suppressing the activity of caspase-3, caspase-7, and caspase-9 . These findings indicate that survivin may be a promising therapeutic target for cancer.
In this study, miR-708 was found to bind the 3′-UTR of survivin and negatively regulate the expression of survivin protein in pancreatic cancer cells. Moreover, miR-708 expression was negatively correlated with the survivin mRNA level in pancreatic cancer tissues. Restoration of survivin could partially reverse apoptosis and proliferation inhibition induced by miR-708 in both pancreatic cell lines of Panc-1 and SW1990. These data confirmed miR-708 as a novel upstream regulator of survivin.
Survivin was reported to activate NF-κB p65 via upregulating the transcriptional activity of IKKβ promoter in esophageal squamous cell carcinoma (Zeng et al., 2016). EMT is a critical process for epithelial-derived malignant tumor cells to acquire the ability of migration and invasion. Loss of E-cadherin as the tumor-suppressor protein is the central event for the development of malignant melanoma (Spangler et al., 2011). Activation of Akt, a serine/threonine kinase, has been found in multiple cancers. Akt activation promoted the progression of melanoma via upregulating NF-κB and inhibiting apoptosis (Zhao et al., 2010). AKT activation has been reported to be associated with the overexpression of survivin in melanoma cells (Simonetti et al., 2015). Our data showed that both NF-κB and p-Akt levels decreased when survivin was suppressed by miR-708, while E-cadherin was increased. We also confirmed that miR-708 overexpression led to increased levels of cleaved caspase-3 and caspase-7, further supporting the proapoptotic function of miR-708.
In summary, our results confirmed that miR-708 was downregulated in pancreatic cancer tissues and cells. Restoration of miR-708 significantly inhibited the proliferation, invasion, and chemoresistance of pancreatic cancer cells via directly targeting survivin. Therefore, the upregulation of miR-708 could be utilized as a novel approach for the treatment of pancreatic cancer.