LncRNA AB209630 inhibits gemcitabine resistance cell proliferation by regulating PI3K/AKT signaling in pancreatic ductal adenocarcinoma

Abstract

BACKGROUND:

Recent study revealed that abnormal long noncoding RNAs (lncRNAs) expression are association with chemotherapy resistance of pancreatic ductal adenocarcinoma (PDAC).

OBJECTIVE:

The present study was aimed to investigate the effects of lncRNA AB209630 expression for gemcitabine resistance in PDAC cells.

METHODS:

In the study, increased expression of lncRNA AB209630 could suppress cell proliferation and cell colony formation ability in gemcitabine resistance cells of PDAC. Furthermore, western blot results demonstrated that upregulation of lncRNA AB209630 suppressed the PI3K/AKT signaling pathway in gemcitabine resistance cells. Besides, we found that lncRNA AB209630 expression was dramatically downregulated in PDAC tissues compared to adjacent normal tissues. Lower PDAC expression predicted a poor prognosis in PDAC patients.

CONCLUSIONS:

Thus, these results indicated that lncRNA AB209630 may be a potential target of PDAC.

1. Introduction

Pancreatic cancer (PC) is one of the most lethal malignancies worldwide. Approximately 90% of pancreatic cancers are identified as pancreatic ductal adenocarcinoma (PDAC). Every year, more than 40,000 patients die from this disease [1]. Patients who were diagnosed at advanced stage have the median survival of 3–6 months and the 5-year survival rate is less than 7 [2]. PDAC has shown highly resistant to chemotherapy in clinical practice [3]. Thus, to investigate novel strategies to reduce drug resistance of PDAC is urgent.

Recent studies have indicated that the contributions of long non-coding RNAs (lncRNAs) to pancreatic carcinogenesis and progression. Abnormal expression of lncRNAs was involved in resistant or sensitivity to chemotherapy of some tumors. For instance, long non-coding RNA UCA1 promotes cisplatin/gemcitabine resistance through CREB modulating miR-196a-5p in bladder cancer cells [4]. The long non-coding RNA HOTTIP promotes progression and gemcitabine resistance by regulating HOXA13 in pancreatic cancer [5]. Curcumin sensitizes pancreatic cancer cells to gemcitabine by attenuating PRC2 subunit EZH2, and the lncRNA PVT1 expression [6]. AB209630, a long non-coding RNA, has been recently identified as tumor suppressor in tumor progression. lncRNA AB209630 was decreased expression in hypopharyngeal squamous cell carcinoma, influences proliferation, invasion, metastasis, and survival [7]. The reduced expression of lncRNA AB209630 was observed in hepatocellular carcinoma tissues suppresses cell proliferation and metastasis in human hepatocellular carcinoma [8]. However, the role of lncRNA AB209630 in PDAC progression and gemcitabine resistance remains unknown.

In the study, we found that lncRNA AB209630 expression was significantly downregulated in PDAC tissues. Higher expression of lncRNA AB209630 suppressed cell proliferation ability. Furthermore, we demonstrated that upregulation of lncRNA AB209630 suppressed the PI3K/AKT signaling pathway. Thus, these results indicated that lncRNA AB209630 may be a potential target of pancreatic cancer.

2. Materials and methods

2.1 Clinical tissue samples

This study was approved by the Ethical Committee of Department of Oncology, Shanghai Medical College, Fudan University. A total of 53 pairs of PDAC tissues and adjacent normal tissue samples were obtained from January 2011 to February 2014 at Department of Oncology, Shanghai Medical College, Fudan University. All of PDAC patients underwent surgical resection and patients had no any therapy before surgery. Fresh tissues were store at $-$ 80 ${}^{\circ }$ C for RNA analyses.

2.2 Cell line culture

Human PDAC cell lines Panc-1 and BXPC-3 were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 ${}^{\circ }$ C in a humidified atmosphere of 5% CO ${}_2$ .

2.3 Development of gemcitabine-resistant PANC-1 and BXPC-3 cells

PANC-1 and BXPC-3 cells were selected by continuous treatment of PANC-1 and BXPC-3 cells with 600 nM gemcitabine (Selleck, Houston, TX, USA), when the confluence of cells reached 50% resulting in subclones resistant to gemcitabine. Two independent GR clones of PANC-1 (PANC-1/GEM) and BXPC-3 (BXPC-3/GEM) cells were established, respectively.

2.4 Cell proliferation assay

Cell proliferation capacity was detected using CCK-8 Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Transfected cells were detected at different time points (0, 24, 48, 72, and 96 h), the CCK8 solution (10 $\mu$ m) was added to each well of the plates. Then, cells were incubated at 37 ${}^{\circ }$ C for 2 h. Cell proliferation was detected with a microplate reader (Biotek ELX-800, USA) and the absorbance at 450 nm was measured.

2.5 Colony formation assay

Transfected PANC-1/GEM and BXPC-3/GEM cells were seeded and cultured in six-well plates at a density of 300 cells/per well. Cells were cultured for 14 days at 37 ${}^{\circ }$ C in a humidified atmosphere of 5% CO ${}_2$ . Then, colonies were fixed with 4% paraformaldehyde for 20 min and stained with a 0.1% crystal violet solution for 20 min. Finally, the numbers of cell colonies were counted under a microscope.

2.6 qPCR analysis

Total RNAs were extracted using TRIzol reagent (Invitrogen). The cDNA was transcribed by RNA using Reverse Transcriptase M-MLV kit (TAKALA, Dalian, China) according to the manufacturer’s instructions. SYBR Premix Ex Taq was used for qRT-PCR reaction on Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The mRNA expression was calculated using 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{CT}}$ methods. LncRNA AB209630 expression was normalized to GAPDH expression. Primers for lncRNA AB209630 were as follow: lncRNA AB209630-F, 5’-GGGCTATTGTCCCTAAGTTGAT-3’, and lncRNA AB209630-R5’-TGTCTTGTAGAGCATAAGGAAACC-3’; GAPDH-F, 5’-GGGAAACTGTGGCGTGAT-3’, and GAPDH-R, 5’-GAGTGGGTGTCGCTGTTGA-3’.

Figure 1.

Development of gemcitabine-resistant PANC-1 and BXPC-3 cells. (A)–(B) Acquired gemcitabine-resistant PANC-1/GEM and BXPC-3/GEM cells were generated by exposing the corresponding wild-type (WT) cells to gemcitabine. (C)–(D) lncRNA AB209630 expression was increased by transfecting pcDNA3.1-AB209630 plasmid into PANC-1/GEM and BXPC-3/GEM cells compared to pcDNA3.1-vector. * $P<0.05$ .

2.7 Western blot analysis

Protein were lysed using RIPA buffer and added with a protease inhibitor cocktail (Sigma). The proteins were separated on sodium dodecyl sulfate-polyacryla-mide gel electrophoresis (SDS/PAGE), and then were transferred to polyvinylidene difluoride membranes (PVDF, Millipore). The membranes were incubated with primary antibodies with PI3K (1:1000, Santa Cruz Biotechnology, CA, USA), p-PI3K (1:1000, Santa Cruz Biotechnology, CA, USA), AKT (1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (1:1000, Santa Cruz Biotechnology, CA, USA) overnight at 4 ${}^{\circ }$ C. Following the membranes were probe with horse-radish peroxidase (HRP)-conjugated secondary antibody at room temperature, proteins blots were visualized by an enhanced chemiluminescence system.

2.8 Statistical analysis

Statistical analysis was analyzed using SPSS 18.0 software (IBM, Chicago, IL, USA). Differences between groups were compared using Student’s t test or one-way ANOVA. Values of $P<0.05$ were considered statistically significant.

3. Results

3.1 Upregulation of lncRNA AB209630 expression inhibits cells proliferation in PANC-1/GEM and BXPC-3/GEM cells

In the study, we constructed the gemcitabine-resis-tant PDAC cells (PANC-1/GEM and BXPC-3/GEM) by continuous treatment of PANC-1 and BXPC-3 cells with 600 nM gemcitabine. The resistance status was determined by calculating the half maximal inhibitory concentration (IC50) of gemcitabine using MTT cytotoxicity assays. Results showed that PANC-1/GEM or BXPC-3/GEM cells had an increased IC50 value than wild PANC-1 or BXPC-3 cells (Fig. 1A–B).

Figure 2.

Upregulation of lncRNA AB209630 expression inhibits cells colony formation ability in PANC-1/GEM and BXPC-3/GEM cells. (A)–(B) CCK8 cell assays were performed to assess cell proliferation by transfecting pcDNA3.1-AB209630 plasmid into PANC-1/GEM and BXPC-3/GEM cells compared to pcDNA3.1-vector. (C)–(D) Cell colony assays were performed to assess cell proliferation by transfecting pcDNA3.1-AB209630 plasmid into PANC-1/GEM and BXPC-3/GEM cells compared to pcDNA3.1-vector. * $P<0.05$ .

Furthermore, cell proliferation was detected by using CCK8 assays. The Results showed that cell proliferation ability was inhibited by transfecting with pcDNA3.1-AB209630 vector into PANC-1/GEM or BXPC-3/GEM cells, compared to the control groups (Fig. 2A–B). Subsequently, we performed the cell colony formation assays. Consistent with above results, PANC1/GEM or BXPC-3/GEM cells tranfected with pcDNA3.1-AB209630 vector showed reduced colonies number, compared to pcDNA3.1-vector group (Fig. 2C–D). Thus, these results indicated that upregulation of lncRNA AB209630 expression inhibited cells proliferation in gemcitabine-resistant PANC-1 and BXPC-3 cells.

3.2 Upregulation of lncRNA AB209630 expression inhibits PI3K/AKT signaling pathway

PI3K/AKT signaling pathway was reported to be associated with gemcitabine resistance in some tumors [9, 10]. Western blot analysis showed that p-PI3K and p-AKT expression levels were significantly downregulated when PANC-1/GEM or BXPC-3/GEM cells were tranfected with pcDNA3.1-AB209630 vector, compared to pcDNA3.1-vector (Fig. 3A–B). However, PI3K and AKT expression levels were not significant changed, when PANC-1/GEM or BXPC-3/GEM cells were tranfected with pcDNA3.1-AB209630 vector, compared to pcDNA3.1-vecto (Fig. 3C–D). Thus, these results indicated that upregulation of lncRNA AB209630 expression inhibited PI3K/AKT signaling pathway.

Figure 3.

Upregulation of lncRNA AB209630 expression inhibits PI3K/AKT signaling in PANC-1/GEM and BXPC-3/GEM cells. (A) The relative expression levels of PI3K, p-PI3K, AKT, p-AKT was detected by western blot after transfecting pcDNA3.1-AB209630 plasmid into PANC-1/GEM cells compared to pcDNA3.1-vector. (B) The relative expression levels of PI3K, p-PI3K, AKT, p-AKT was detected by western blot after transfecting pcDNA3.1-AB209630 plasmid into BXPC-3/GEM cells compared to pcDNA3.1-vector. * $P<0.05$ .

Figure 4.

LncRNA AB209630 expression was lower in PDAC tissues and associated with prognosis. (A) The lncRNA AB209630 expression was detected using qRT-PCR in 53 pairs of PDAC tissues and adjacent normal tissue samples. GAPDH was identified as an internal control. (B) Patients with lower lncRNA AB209630 expression had a poor prognosis than those with higher lncRNA AB209630 expression by using Kaplan-Meier analysis and log rank test. * $P<0.05$ .

3.3 LncRNA AB209630 expression is downregulated in PDAC tissues and associates with prognosis

In the study, we first analyzed the expression of lncRNA AB209630 in PDAC tissues and adjacent normal tissues using qRT-PCR. The results found that lncRNA AB209630 expression was dramatically reduced in PDAC tissues compared to adjacent normal tissues (Fig. 4A). Furthermore, we analyzed the association of lncRNA AB209630 expression with prognosis of patients. Survival plots by Kaplan-Meier analysis showed that lncRNA AB209630 expression associated with poor overall survival time (Fig. 4B, log rank test, $P<0.05$ ). Thus, our results indicated that lncRNA AB209630 expression was lower in PDAC and associated with tumor prognosis.

4. Discussion

Inherent chemoresistance all contribute to the poor prognosis of PC patients. lncRNAs have been identified as crucial regulator in tumor progression. Some of lncRNAs were reported to be involved in tumor chemoresistance. LncRNA H19 is a major mediator of doxorubicin chemoresistance in breast cancer cells through a cullin4A-MDR1 pathway [11]. LncRNA NEAT1 contributes to paclitaxel resistance of ovarian cancer cells by regulating ZEB1 expression via miR-194.chemoresistance all contribute to the poor prognosis of PC patients [12]. LncRNA FENDRR sensitizes doxorubicin-resistance of osteosarcoma cells through down-regulating ABCB1 and ABCC1 [13]. TGF $\beta$ 1 promotes gemcitabine resistance through regulating the LncRNA-LET/NF90/miR-145 signaling axis in bladder cancer [14]. In the study, we demonstrated that upregulation of lncRNA AB209630 expression inhibited cells proliferation in gemcitabine-resistant PANC-1 and BXPC-3 cells. Furthermore, our results indicated that lncRNA AB209630 expression was lower in PDAC and associated with tumor prognosis.

PI3K/AKT signaling pathway participated in tumor chemoresistance. Phenoxodiol enhances the antitumor activity of gemcitabine in gallbladder cancer through suppressing Akt/mTOR pathway [15]. HS-104, a PI3K inhibitor, enhances the anticancer efficacy of gemcitabine in pancreatic cancer [16]. Evodiamine enhanced antitumor efficacy of gemcitabine on pancreatic cancer by regulating PI3K/Akt pathway [17]. Our results showed that p-PI3K and p-AKT expression levels were significantly downregulated when PANC-1/GEM or BXPC-3/GEM cells were tranfected with pcDNA3.1-AB209630 vector, compared to pcDNA3.1-vector, which indicated that upregulation of lncRNA AB209630 expression inhibited PI3K/AKT signaling pathway.

In conclusion, in the study, we found that lncRNA AB209630 expression was lower in PDAC tissues and lower PDAC expression associated with poor prognosis of patients. Furthermore, we demonstrated that lncRNA AB209630 overexpression suppressed gemcitabine resistance by inhibiting PI3K/AKT signaling pathway. Thus, our findings indicated that lncRNA AB209630 may serve as target of PDAC treatment.