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Overexpression of long non-coding RNA GHET1 promotes the development of multidrug resistance in gastric cancer cells
Biomedicine & Pharmacotherapy 92 (2017) 580–585
Available online at
ScienceDirect www.sciencedirect.com
Overexpression of long non-coding RNA GHET1 promotes the development of multidrug resistance in gastric cancer cells Zhang Xianwenb , Bo Pinga,* , Liu Lianga , Zhang Xizhib , Li Junb a b
Medical College, Yangzhou University, Yangzhou, 225009, China Department of Oncology, Clinical College, Yangzhou University, Yangzhou, 225009, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2017 Received in revised form 24 April 2017 Accepted 24 April 2017
Background and amid: Multidrug resistance (MDR) is the main obstacle to successful chemotherapy for patients with gastric cancer (GC). The GHET1 affects a variety of cancer development, and increased GHET1 promotes gastric carcinoma cell proliferation. This study amid to investigate the effect of GHET1 on the development of MDR in gastric cancer cells. Methods and results: In this study, we observed an increased expression levels of GHET1 in drug-resistant gastric cancer patients and cisplatin-resistant gastric cancer cell lines (BGC823/DPP and SGC7901/DDP cell line). Next, the two cell lines were used to in vitro validation of the effect of GHET1 expression on the MDR development. Silence of GHET1 in the two cisplatin-resistant gastric cancer cell lines inhibited the MDR with rising inhibition rate tested by MTT (methyl thiazolyl tetrazolium) assay. The apoptotic cell rate was analyzed by staining with Annexin V/PI. The results showed that the IC50 (half maximal inhibitory concentration) of the two cisplatin-resistant gastric cancer cell lines transfected with si-GHET1 were significantly reduced. Contrary to inhibition of GHET1 in the two cisplatin-resistant gastric cancer cell lines, we found that overexpression of GHET1 reduced sensitivity of BGC823 and SGC7901 cells to cisplatin with decreased inhibition rate and apoptotic cell rate and increased IC50. Furthermore, the qRT–PCR and western blot results showed that overexpression of GHET1 downregulated Bax expression and upregulated Bcl-2, MDR1 and MRP1 expression in BGC823 and SGC7901 cells. Conclusion: Highly expressing GHET1 promoted the development of MDR which was related to the Bax, Bcl-2, MDR1 and MRP1 genes expression in gastric cancer cells. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: CHET1 Gastric cancer Multidrug resistance
1. Introduction Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes, the fourth most common type of cancer and the second leading cause of cancer-related deaths in the world [1,2]. Conventional chemotherapy is the main treatment for cancer and benefits patients in the form of decreased relapse and metastasis and longer overall survival [3]. Cisplatin (DDP) is the most commonly used chemotherapeutic agent for the treatment of gastric cancer [4]. Unfortunately, acquired resistance to DDP is common which represents a major obstacle to the effective treatment of gastric cancer [5]. Multidrug resistance gene 1 (MDR1) [6] and multi-drug resistance protein (MRP1) [7], both
* Corresponding author at: Medical College, Yangzhou University, Yangzhou 225009, China. Tel.: +86 0514 87978872. E-mail address: [email protected] (P. Bo). http://dx.doi.org/10.1016/j.biopha.2017.04.111 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
are classical drug-resistant molecules and have been found to play important roles in mediating MDR in gastric cancers. LncRNA with lengths of more than 200 nt have been linked to every stage of cell life, including cell proliferation, differentiation, apoptosis, and motility [8], and have been identified as oncogene or tumor suppressors or a predictor of prognosis in gastric cancer [9,10]. Based on the function and functional mechanisms of lncRNAs, further investigation into the relationship between lncRNAs and MDR gastric cancer development is necessary. Yan et al. [11] found that lncRNAs HOTAIR promoted cisplatin resistance via targeting miR-126 to activate the PI3K/AKT/MRP1 pathway in gastric cancer. Zhang et al. [12] reported that overexpression of lncRNAs PVT1 promoted the development of multidrug resistance in gastric cancer cells. Fang [13] and Chao [14] have both found that lncRNAs UCA1 increased multidrug resistance of gastric cancer. Lan and colleagues reported that silencing of lncRNAs ANRIL inhibited the development of multidrug resistance in gastric cancer cells [15]. Zhang et al. [16] have found
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that silencing of HULC enhanced chemotherapy-induced apoptosis in human gastric cancer. A novel lncRNA GHET1 (gastric carcinoma high expressed transcript 1) was found to be aberrantly expressed in gastric carcinoma, and the higher expression of GHET1 was associated with bigger tumor size and worse survive. In addition, GHET1 was proven to promote gastric carcinoma cell proliferation via increasing c-Myc mRNA stability and expression [17]. In this study, we further analyzed the effect of GHET1expression on the development of multidrug resistance in gastric cancer. 2. Material and methods
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curves. Three independent experiments were performed in triplicate. 2.5. RNA extraction and quantitative real-time PCR TRIzol reagent (TaKaRa) was used to extract total RNA from the tissues and cells according to the manufacturer’s instructions. RTqPCR was performed using the Sybr green reaction mix and a Light 480 sequence detection system (Roche, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as an internal control and the 2 DDCt method was employed to determine the relative quantitation of GHET1, Bax, bcl-2, MDR1 and MRP1 expression levels.
2.1. Human tissue specimens 2.6. Protein extraction and western blotting A total of 40 gastric carcinoma tissues that 20 samples from cisplatin sensitive patients and another 20 from drug-resistant patients were obtain when patients underwent radical resections at Subei people’s Hospital, Yangzhou, China. All patients were informed consent, and this study was performed with the approval of the Medical college of Yangzhou university Institutional Review Board. 2.2. Gastric cancer cell and drug-resistant cell line The human gastric cancer cell lines BGC823 and SGC7901 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). And the cisplatin resistant strains, BGC823/DPP and SGC7901/DDP were derivative from BGC823 and SGC7901 by cisplatin (DDP). All the cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Invitrogen Life Technologies) at 37 C in a humidified incubator with 5% CO2 atmosphere. To maintain the MDR phenotype, BGC823/DPP and SGC7901/DDP cells were cultured in the presence of 0.5 mg/mL DPP. 2.3. Cell transfection with plasmid and small interfering RNA The GHET1 overexpression plasmid was constructed by inserting the amplified GHET1 cDNA into pcDNA3.1 vector (Invitrogen, Shanghai, China), named pcDNA-GHET1. Small interference RNA (siRNA) targeting GHET1 was designed and synthesized by Santa Cruz Biotechnology, as well as negative control siRNA, which was used to knockdown experiments. All cells were grown in six-well plates to 70% confluency and transfections were performed using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s instructions. BGC823/ DPP and SGC7901/DDP cells were transfected with si-NC or siGHET1, while BGC823 and SGC7901 cells were transfected with pcDNA or pcDNA-GHET1. The expression of GHET1 was detected by real-time PCR, and the transfected cells were used for further analysis. 2.4. In vitro drug sensitivity assay The gastric cancer cell lines and the gastric cancer drug cell lines were transfected with si-CGET1 and pcDNA-GHET1, respectively, and seeded into 96-well plates (5*103 cells/well) to maintain overnight. The DDP solution were freshly prepared before experiment and the cells were treated with different concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mg/mL) for 48 h. The effect of GHET1 on sensitivity of the gastric cancer cells to anticancer drugs were evaluated using MTT assay. The absorbance at 490 nm was read on a spectrophotometer. The concentration of DDP at 50% growth inhibition (IC50) was estimated using relative survival
Cells were lysed using RIPA buffer (Pierce) containing protease inhibitors. Total cell lysates were prepared in a 1*sodium dodecyl sulfate (SDS) buffer. Identical quantities of proteins (30–50 mg) were separated by 8–12% SDS/PAGE and transferred onto PVDF membranes. After an incubation with antibodies specific for Bax, bcl-2, MDR1 and MRP1 (Santa Cruz Biotechnology) or GAPDH (Sigma) with gentle shaking at 4 C overnight, the blots were incubated with Horseradish peroxidase-conjugated goat antirabbit IgG antibody (Santa). Antibody-bound proteins were detected by Beyo-ECL Plus kit. The data were analyzed via densitometry using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) and normalized to the expression of the internal control (GAPDH). 2.7. Cell apoptosis by flow cytometry Cells (1*106/ml) were washed and re-suspended in binding buffer, followed by adding annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (Sigma). After incubation for 15 min at room temperature, annexin-binding buffer was added, and samples were kept on ice. Then flow cytometry was used to count the stained cells. 2.8. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 software and the data are presented as the mean SD. For comparisons, Student’s t-test was performed as indicated. All P values were obtained using the SPSS 18.0 software package (SPSS, Chicago, IL, USA). A value of p < 0.05 was considered to indicate a statistically significant difference 3. Result 3.1. GHET1 is associated with cisplatin resistance in gastric cancer To determine whether GHET1 is involved in MDR development in gastric cancer, its expression levels were determined by qRT-PCR in gastric cancer tissue of cisplatin sensitive and drug-resistant patients, as well as the cisplatin resistant strains and the parental cell line. As Fig. 1A showed that, the GHET1 level in gastric cancer tissue of cisplatin drug-resistant patients were higher than that in drug sensitive patients. And then, we have analyzed the GHET1 expression in the two cisplatin resistance lines and their parental line. As Fig. 1B showed that, the GHET1 expression in cisplatin resistant strains (BGC823/DPP and SGC7901/DDP) were also higher than that in their parental line (BGC823 and SGC7901 cells), respectively. These data indicated that GHET1 was a multidrug resistance-related lncRNA in gastric cancer.
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Fig. 1. GHET1 expression is closely related to the sensitivity of gastric cancer to cisplatin. A: the GHET1 level in gastric cancer tissue of cisplatin sensitive patients and drugresistant patients. B: the expression of GHET1 in BGC823/DPP and SGC7901/DDP cell lines were significantly higher than that in BGC823 and SGC7901, respectively. **p < 0.01.
3.2. Knockdown of GHET1 reverse the MDR gastric cell resistance To investigate whether GHET1 could affect sensitivity of gastric cancer cell, we transfected BGC823/DPP and SGC7901/DDP cells with si-GHET1 or si-NC. After transfection with si-GHET1, the two cell lines showed remarkably decreased GHET1 expression level (Fig. 2A). And then, after the BGC823/DPP and SGC7901/DDP cell lines transfecting with si-GHET1 or si-NC for 24 h, these cells were treated with different concentrations of cisplatin for another 48 h, and the inhibition rates were analyzed by MTT assay. With increasing concentration of cisplatin, the BGC823/DDP cell transfected with si-GHET1 always have a higher inhibition rate than that
transfected with si-NC (Fig. 2B). The SGC7901/DDP cell status was in keeping with the BGC823/DDP cell (Fig. 2C), which implied that knockdown of GHET1 reduced drug resistance of BGC823/DDP and SGC7901/DDP cells. Next, we examined 50% inhibitive concentration (IC50) of cisplatin in BGC823/DDP and SGC7901/DDP cells, and the results showed that BGC823/DDP and SGC7901/DDP cells transfected with si-GHET1 have a low IC50 (Fig. 2D), which suggested that knockdown of GHET1 reduced drug resistance of cisplatin resistant strains in gastric cancer. In addition, the cell apoptosis rate of cisplatin resistant strains transfected with siGHET1 were detected by staining with Annexin V and PI using flow cytometry. As showed in Fig. 2E, a higher cell apoptosis rate
Fig. 2. Knockdown of GHET1 reversed the MDR gastric cell resistance. A: detection of interference efficiency of GHET1 interference sequence. B and C: the BGC823/DPP and SGC7901/DDP cell lines that transfected with si-GHET1/NC were treated with different concentrations of cisplatin for 48 h, then the inhibitive rate of cell lines were tested with the MTT assay. D: the IC50 of BGC823/DPP and SGC7901/DDP cell line that transfected with si-GHET1/NC were analyzed by MTT. E: the BGC823/DPP and SGC7901/DDP cell line that transfected with si-GHET1/NC were treated with 1 ug/ml cisplatin for 48 h, then apoptotic cells were detected by staining with Annexin V/PI using flow cytometry. **p < 0.01.
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occurred in BGC823/DDP and SGC7901/DDP cells transfected with si-GHET1, further suggesting that knockdown of GHET1 reduced drug resistance of cisplatin resistant strains in gastric cancer. 3.3. GHET1 reduces sensitivity of gastric cancer cells to anticancer drugs To determine the role of GHET1 in drug resistance of gastric cancer, we transfected BGC823 and SGC7901 cells with pcDNAGHET1 or pcDNA. As Fig. 3A showed, BGC823 and SGC7901 cells transfected with pcDNA-GHET1 have dramatically increased GHET1 expression level. Afterwards, after BGC823 and SGC7901 cells transfected with pcDNA-GHET1 or pcDNA, these cells were treated with different concentrations of cisplatin for 48 h, and MTT assay was used to analyze the inhibition rates. With increasing concentration of cisplatin, the BGC823 cell transfected with pcDNA-GHET1 always have a lower inhibition rate than that transfected with pcDNA (Fig. 3B), and the same situation also happened in SGC7901 cells (Fig. 3C), suggesting that the GHET1 decreased the sensitivity of gastric cancer cells to cisplatin. As Fig. 3D showed that the BGC823 and SGC7901 cells transfected with pcDNA-GHET1 have a high IC50, which further suggested that the GHET1 could decrease the sensitivity of gastric cancer cells to cisplatin. Moreover, the cell apoptosis of BGC823 and SGC7901 cells transfected with pcDNA-GHET1 were detected by staining with Annexin V and PI using flow cytometry. As showed in Fig. 3E, a lower cell apoptosis rate occurred in BGC823 and SGC7901 cells transfected with pcDNA-GHET1, further suggesting that the GHET1 reduced the sensitivity of gastric cancer cells to cisplatin. 3.4. GHET1 promotes resistance of gastric cancer cells that was associated with Bax, Bcl-2, MDR1 and MRP1 B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) are a pair of apoptosis genes, which can inhibit or promote apoptosis [18]. Multi-Drug Resistance Protein 1 (MDR1) and
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Multidrug Resistance Related Protein 1 (MRP1) are expressed on the cell surface, and the high capacity transporters are responsible for modulating chemo-sensitivity of cancer [19]. To explore whether GHET1 promote the development of MDR in gastric cancer cells was associated with Bax, Bcl-2, MDR1 and MRP1 expression levels, the BGC823 and SGC7901 cells were transfected with pcDNA-GHET1 or pcDNA, and RT-qPCR and western blot were used to detect Bax, Bcl-2, MDR1 and MRP1 expression levels. The Bax expression was significantly decreased in BGC823 and SGC7901 cells transfected with pcDNA-GHET1, while bcl-2 expression was significantly increased (Fig. 4A, B and E), indicating that overexpression of GHET1 inhibited cell apoptosis. The qRTPCR and western blotting results revealed that overexpression of GHET1 increased the expression of MDR1 and MRP1, indicating that overexpression of GHET1 increased sensitivity gastric cancer cells (Fig. 4C, 4D and 4E). As Fig. 4F showed a pathway illustration identified in this study. In the BGC823 and SGC7901 cell, overexpression of GHET1 downregulated Bax expression and upregulated Bcl-2, MDRA and MRP1 expression, which resulted in increasing cisplatin resistance and inhibiting cell apoptosis. In the BGC823/DPP and SGC7901/DDP cell, knockdown of GHET1 leaded to increase cisplatin sensitivity and promote cell apoptosis. 4. Discussion MDR plays a critical role in tumor initiation and progression by promoting cell proliferation and inhibiting apoptosis [20], and the development of cancer cell against cancer chemotherapy is a major obstacle to the effective treatment of gastric cancer [5]. In the present study, we examined the GHET1 expression level in gastric carcinoma tissues of cisplatin sensitive patients and drug-resistant patients, as well as the MDR gastric cancer cell lines and their chemo-sensitive parental line. We also identified the function of GHET1 in cisplatin resistant strains (BGC823/DPP and SGC7901/ DDP cells) and their parental line (BGC823 and SGC7901 cells) using gain-of-function and loss-of-function approaches in vitro.
Fig. 3. GHET1 reduces sensitivity of gastric cancer cells to anticancer drugs. A: analysis of overexpression of efficiency of pcDNA-GHET1 in BGC823 and SGC7901 cell. B and C: the BGC823 and SGC7901 cells that transfected with pcDNA-GHET1 were treated with different concentrations of cisplatin for 48 h, then the inhibition rate of cell lines were tested with the MTT assay. D: the IC50 of BGC823 and SGC7901 cell that transfected with pcDNA-GHET1 were analyzed by MTT. E: the BGC823 and SGC7901 cell that transfected with pcDNA-GHET1 were treated with 1 ug/ml cisplatin for 48 h, then apoptotic cells were detected by staining with Annexin V/PI using flow cytometry. **p < 0.01.
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Fig. 4. The GHET1 promoted resistance of gastric cancer cells that was associated with Bax, Bcl-2, MDR1 and MRP1 mRNA. A-D: the RT-Qpcr was used to detect the expression of apoptosis related gene (Bax and Bcl-2) and drug resistance related gene (MDR1 and MRP1) in BGC823 and SGC7901 cell that transfected with pcDNA-GHET1. E: the western blot was used to detect the expression of apoptosis related gene (Bax and Bcl-2) and drug resistance related gene (MDR1 and MRP1) in BGC823 and SGC7901 cell that transfected with pcDNA-GHET1. (F) A pathway illustration identified in this study. **p < 0.01.
Our results demonstrated that GHET1 was up-regulated in gastric carcinoma tissues of the drug-resistant patients, and high expression of GHET1 was correlated with the sensitivity of gastric cancer cell to cisplatin. Enhanced GHET1 expression reduced the sensitivity of BGC823 and SGC7901 cells to cisplatin, while inhibition of GHET1 expression inhibited the MDR of BGC823/ DDP and SGC7901/DDP cells. Therefore, our results indicated that GHET1 functioned as a drug resistant genes in gastric carcinoma. It is well known that protein-coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs [21]. Although researches of lncRNAs have dominated the field of RNA regulation in gastric cancer, such as MALAT1 [10], NEAT1 [22], HOTAIR [23] and so on, accumulating evidence has indicated that these lncRNAs may also play an important role in the development of multidrug resistance. MALAT1 could regulate multidrug resistance of hepatocellular carcinoma cells via modulating autophagy [24]. NEAT1 could mitigate multidrug resistance by inhibiting ABCG2 in leukemia [25]. HOTAIR could promote cisplatin resistance in gastric cancer [11] and confer tamoxifen resistance in breast cancer [26]. GHET1 had been proved to be an oncogene in colorectal cancer [27] and bladder cancer [28]. In this study, we found that higher expression of GHET1 promoted the development of multidrug resistance in gastric cancer cell. The Bcl-2 family of genes includes some important regulators of apoptosis. Among these genes, Bcl-2 and Bax control cell death, thus contributing to both tumor growth and drug sensitivity. MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and drug resistance in human cancer is associated with overexpression of MDR1 gene. The multidrug resistance protein 1 (MRP1) was originally discovered as a cause of multidrug
resistance in tumor cells. In this study, we further determined whether the expression level of GHET1 correlated with the apoptosis-related or drug resistance-related genes. Western blot was conducted to evaluate the protein expression level of Bax, Bcl2, MRP1 and MDR1 in gastric cancer cells transfected with pcDNAGHET1. As the results showed that, BGC823 and SGC7901 cells transfected with pcDNA-GHET1 both have a decreased Bax expression and increased Bcl-2, MDR1 and MRP1 expression, further suggesting GHET1 promoted the development of multidrug resistance in gastric carcinoma cell. Whether GHET1 also physically associates with other genes except c-Myc, and the direct or indirect physical interaction between them requires further investigation. In general, we demonstrated that expression of GHET1 were significantly upregulated in gastric cancer tissues of drug resistance patients. Moreover, GHET1 expression was also obviously increased in the BGC823/DDP and SGC7901/DDP cell lines. We also provided evidence for GHET1 function on MDR development in gastric cancer and revealed enhancer-like roles of GHET1 in MDR1 and MRP1 expression. This result indicated that GHET1 depletion may be a promising therapeutic strategy for gastric cancer MDR reversal. Conflict of interest The authors declare no conflict of interest. Acknowledgement This project was supported by the National Nature Science Foundation of China (Grant No. 81673736)
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Available online at
ScienceDirect www.sciencedirect.com
Overexpression of long non-coding RNA GHET1 promotes the development of multidrug resistance in gastric cancer cells Zhang Xianwenb , Bo Pinga,* , Liu Lianga , Zhang Xizhib , Li Junb a b
Medical College, Yangzhou University, Yangzhou, 225009, China Department of Oncology, Clinical College, Yangzhou University, Yangzhou, 225009, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2017 Received in revised form 24 April 2017 Accepted 24 April 2017
Background and amid: Multidrug resistance (MDR) is the main obstacle to successful chemotherapy for patients with gastric cancer (GC). The GHET1 affects a variety of cancer development, and increased GHET1 promotes gastric carcinoma cell proliferation. This study amid to investigate the effect of GHET1 on the development of MDR in gastric cancer cells. Methods and results: In this study, we observed an increased expression levels of GHET1 in drug-resistant gastric cancer patients and cisplatin-resistant gastric cancer cell lines (BGC823/DPP and SGC7901/DDP cell line). Next, the two cell lines were used to in vitro validation of the effect of GHET1 expression on the MDR development. Silence of GHET1 in the two cisplatin-resistant gastric cancer cell lines inhibited the MDR with rising inhibition rate tested by MTT (methyl thiazolyl tetrazolium) assay. The apoptotic cell rate was analyzed by staining with Annexin V/PI. The results showed that the IC50 (half maximal inhibitory concentration) of the two cisplatin-resistant gastric cancer cell lines transfected with si-GHET1 were significantly reduced. Contrary to inhibition of GHET1 in the two cisplatin-resistant gastric cancer cell lines, we found that overexpression of GHET1 reduced sensitivity of BGC823 and SGC7901 cells to cisplatin with decreased inhibition rate and apoptotic cell rate and increased IC50. Furthermore, the qRT–PCR and western blot results showed that overexpression of GHET1 downregulated Bax expression and upregulated Bcl-2, MDR1 and MRP1 expression in BGC823 and SGC7901 cells. Conclusion: Highly expressing GHET1 promoted the development of MDR which was related to the Bax, Bcl-2, MDR1 and MRP1 genes expression in gastric cancer cells. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: CHET1 Gastric cancer Multidrug resistance
1. Introduction Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes, the fourth most common type of cancer and the second leading cause of cancer-related deaths in the world [1,2]. Conventional chemotherapy is the main treatment for cancer and benefits patients in the form of decreased relapse and metastasis and longer overall survival [3]. Cisplatin (DDP) is the most commonly used chemotherapeutic agent for the treatment of gastric cancer [4]. Unfortunately, acquired resistance to DDP is common which represents a major obstacle to the effective treatment of gastric cancer [5]. Multidrug resistance gene 1 (MDR1) [6] and multi-drug resistance protein (MRP1) [7], both
* Corresponding author at: Medical College, Yangzhou University, Yangzhou 225009, China. Tel.: +86 0514 87978872. E-mail address: [email protected] (P. Bo). http://dx.doi.org/10.1016/j.biopha.2017.04.111 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
are classical drug-resistant molecules and have been found to play important roles in mediating MDR in gastric cancers. LncRNA with lengths of more than 200 nt have been linked to every stage of cell life, including cell proliferation, differentiation, apoptosis, and motility [8], and have been identified as oncogene or tumor suppressors or a predictor of prognosis in gastric cancer [9,10]. Based on the function and functional mechanisms of lncRNAs, further investigation into the relationship between lncRNAs and MDR gastric cancer development is necessary. Yan et al. [11] found that lncRNAs HOTAIR promoted cisplatin resistance via targeting miR-126 to activate the PI3K/AKT/MRP1 pathway in gastric cancer. Zhang et al. [12] reported that overexpression of lncRNAs PVT1 promoted the development of multidrug resistance in gastric cancer cells. Fang [13] and Chao [14] have both found that lncRNAs UCA1 increased multidrug resistance of gastric cancer. Lan and colleagues reported that silencing of lncRNAs ANRIL inhibited the development of multidrug resistance in gastric cancer cells [15]. Zhang et al. [16] have found
X. Zhang et al. / Biomedicine & Pharmacotherapy 92 (2017) 580–585
that silencing of HULC enhanced chemotherapy-induced apoptosis in human gastric cancer. A novel lncRNA GHET1 (gastric carcinoma high expressed transcript 1) was found to be aberrantly expressed in gastric carcinoma, and the higher expression of GHET1 was associated with bigger tumor size and worse survive. In addition, GHET1 was proven to promote gastric carcinoma cell proliferation via increasing c-Myc mRNA stability and expression [17]. In this study, we further analyzed the effect of GHET1expression on the development of multidrug resistance in gastric cancer. 2. Material and methods
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curves. Three independent experiments were performed in triplicate. 2.5. RNA extraction and quantitative real-time PCR TRIzol reagent (TaKaRa) was used to extract total RNA from the tissues and cells according to the manufacturer’s instructions. RTqPCR was performed using the Sybr green reaction mix and a Light 480 sequence detection system (Roche, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as an internal control and the 2 DDCt method was employed to determine the relative quantitation of GHET1, Bax, bcl-2, MDR1 and MRP1 expression levels.
2.1. Human tissue specimens 2.6. Protein extraction and western blotting A total of 40 gastric carcinoma tissues that 20 samples from cisplatin sensitive patients and another 20 from drug-resistant patients were obtain when patients underwent radical resections at Subei people’s Hospital, Yangzhou, China. All patients were informed consent, and this study was performed with the approval of the Medical college of Yangzhou university Institutional Review Board. 2.2. Gastric cancer cell and drug-resistant cell line The human gastric cancer cell lines BGC823 and SGC7901 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). And the cisplatin resistant strains, BGC823/DPP and SGC7901/DDP were derivative from BGC823 and SGC7901 by cisplatin (DDP). All the cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Invitrogen Life Technologies) at 37 C in a humidified incubator with 5% CO2 atmosphere. To maintain the MDR phenotype, BGC823/DPP and SGC7901/DDP cells were cultured in the presence of 0.5 mg/mL DPP. 2.3. Cell transfection with plasmid and small interfering RNA The GHET1 overexpression plasmid was constructed by inserting the amplified GHET1 cDNA into pcDNA3.1 vector (Invitrogen, Shanghai, China), named pcDNA-GHET1. Small interference RNA (siRNA) targeting GHET1 was designed and synthesized by Santa Cruz Biotechnology, as well as negative control siRNA, which was used to knockdown experiments. All cells were grown in six-well plates to 70% confluency and transfections were performed using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s instructions. BGC823/ DPP and SGC7901/DDP cells were transfected with si-NC or siGHET1, while BGC823 and SGC7901 cells were transfected with pcDNA or pcDNA-GHET1. The expression of GHET1 was detected by real-time PCR, and the transfected cells were used for further analysis. 2.4. In vitro drug sensitivity assay The gastric cancer cell lines and the gastric cancer drug cell lines were transfected with si-CGET1 and pcDNA-GHET1, respectively, and seeded into 96-well plates (5*103 cells/well) to maintain overnight. The DDP solution were freshly prepared before experiment and the cells were treated with different concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mg/mL) for 48 h. The effect of GHET1 on sensitivity of the gastric cancer cells to anticancer drugs were evaluated using MTT assay. The absorbance at 490 nm was read on a spectrophotometer. The concentration of DDP at 50% growth inhibition (IC50) was estimated using relative survival
Cells were lysed using RIPA buffer (Pierce) containing protease inhibitors. Total cell lysates were prepared in a 1*sodium dodecyl sulfate (SDS) buffer. Identical quantities of proteins (30–50 mg) were separated by 8–12% SDS/PAGE and transferred onto PVDF membranes. After an incubation with antibodies specific for Bax, bcl-2, MDR1 and MRP1 (Santa Cruz Biotechnology) or GAPDH (Sigma) with gentle shaking at 4 C overnight, the blots were incubated with Horseradish peroxidase-conjugated goat antirabbit IgG antibody (Santa). Antibody-bound proteins were detected by Beyo-ECL Plus kit. The data were analyzed via densitometry using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) and normalized to the expression of the internal control (GAPDH). 2.7. Cell apoptosis by flow cytometry Cells (1*106/ml) were washed and re-suspended in binding buffer, followed by adding annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (Sigma). After incubation for 15 min at room temperature, annexin-binding buffer was added, and samples were kept on ice. Then flow cytometry was used to count the stained cells. 2.8. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 software and the data are presented as the mean SD. For comparisons, Student’s t-test was performed as indicated. All P values were obtained using the SPSS 18.0 software package (SPSS, Chicago, IL, USA). A value of p < 0.05 was considered to indicate a statistically significant difference 3. Result 3.1. GHET1 is associated with cisplatin resistance in gastric cancer To determine whether GHET1 is involved in MDR development in gastric cancer, its expression levels were determined by qRT-PCR in gastric cancer tissue of cisplatin sensitive and drug-resistant patients, as well as the cisplatin resistant strains and the parental cell line. As Fig. 1A showed that, the GHET1 level in gastric cancer tissue of cisplatin drug-resistant patients were higher than that in drug sensitive patients. And then, we have analyzed the GHET1 expression in the two cisplatin resistance lines and their parental line. As Fig. 1B showed that, the GHET1 expression in cisplatin resistant strains (BGC823/DPP and SGC7901/DDP) were also higher than that in their parental line (BGC823 and SGC7901 cells), respectively. These data indicated that GHET1 was a multidrug resistance-related lncRNA in gastric cancer.
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Fig. 1. GHET1 expression is closely related to the sensitivity of gastric cancer to cisplatin. A: the GHET1 level in gastric cancer tissue of cisplatin sensitive patients and drugresistant patients. B: the expression of GHET1 in BGC823/DPP and SGC7901/DDP cell lines were significantly higher than that in BGC823 and SGC7901, respectively. **p < 0.01.
3.2. Knockdown of GHET1 reverse the MDR gastric cell resistance To investigate whether GHET1 could affect sensitivity of gastric cancer cell, we transfected BGC823/DPP and SGC7901/DDP cells with si-GHET1 or si-NC. After transfection with si-GHET1, the two cell lines showed remarkably decreased GHET1 expression level (Fig. 2A). And then, after the BGC823/DPP and SGC7901/DDP cell lines transfecting with si-GHET1 or si-NC for 24 h, these cells were treated with different concentrations of cisplatin for another 48 h, and the inhibition rates were analyzed by MTT assay. With increasing concentration of cisplatin, the BGC823/DDP cell transfected with si-GHET1 always have a higher inhibition rate than that
transfected with si-NC (Fig. 2B). The SGC7901/DDP cell status was in keeping with the BGC823/DDP cell (Fig. 2C), which implied that knockdown of GHET1 reduced drug resistance of BGC823/DDP and SGC7901/DDP cells. Next, we examined 50% inhibitive concentration (IC50) of cisplatin in BGC823/DDP and SGC7901/DDP cells, and the results showed that BGC823/DDP and SGC7901/DDP cells transfected with si-GHET1 have a low IC50 (Fig. 2D), which suggested that knockdown of GHET1 reduced drug resistance of cisplatin resistant strains in gastric cancer. In addition, the cell apoptosis rate of cisplatin resistant strains transfected with siGHET1 were detected by staining with Annexin V and PI using flow cytometry. As showed in Fig. 2E, a higher cell apoptosis rate
Fig. 2. Knockdown of GHET1 reversed the MDR gastric cell resistance. A: detection of interference efficiency of GHET1 interference sequence. B and C: the BGC823/DPP and SGC7901/DDP cell lines that transfected with si-GHET1/NC were treated with different concentrations of cisplatin for 48 h, then the inhibitive rate of cell lines were tested with the MTT assay. D: the IC50 of BGC823/DPP and SGC7901/DDP cell line that transfected with si-GHET1/NC were analyzed by MTT. E: the BGC823/DPP and SGC7901/DDP cell line that transfected with si-GHET1/NC were treated with 1 ug/ml cisplatin for 48 h, then apoptotic cells were detected by staining with Annexin V/PI using flow cytometry. **p < 0.01.
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occurred in BGC823/DDP and SGC7901/DDP cells transfected with si-GHET1, further suggesting that knockdown of GHET1 reduced drug resistance of cisplatin resistant strains in gastric cancer. 3.3. GHET1 reduces sensitivity of gastric cancer cells to anticancer drugs To determine the role of GHET1 in drug resistance of gastric cancer, we transfected BGC823 and SGC7901 cells with pcDNAGHET1 or pcDNA. As Fig. 3A showed, BGC823 and SGC7901 cells transfected with pcDNA-GHET1 have dramatically increased GHET1 expression level. Afterwards, after BGC823 and SGC7901 cells transfected with pcDNA-GHET1 or pcDNA, these cells were treated with different concentrations of cisplatin for 48 h, and MTT assay was used to analyze the inhibition rates. With increasing concentration of cisplatin, the BGC823 cell transfected with pcDNA-GHET1 always have a lower inhibition rate than that transfected with pcDNA (Fig. 3B), and the same situation also happened in SGC7901 cells (Fig. 3C), suggesting that the GHET1 decreased the sensitivity of gastric cancer cells to cisplatin. As Fig. 3D showed that the BGC823 and SGC7901 cells transfected with pcDNA-GHET1 have a high IC50, which further suggested that the GHET1 could decrease the sensitivity of gastric cancer cells to cisplatin. Moreover, the cell apoptosis of BGC823 and SGC7901 cells transfected with pcDNA-GHET1 were detected by staining with Annexin V and PI using flow cytometry. As showed in Fig. 3E, a lower cell apoptosis rate occurred in BGC823 and SGC7901 cells transfected with pcDNA-GHET1, further suggesting that the GHET1 reduced the sensitivity of gastric cancer cells to cisplatin. 3.4. GHET1 promotes resistance of gastric cancer cells that was associated with Bax, Bcl-2, MDR1 and MRP1 B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) are a pair of apoptosis genes, which can inhibit or promote apoptosis [18]. Multi-Drug Resistance Protein 1 (MDR1) and
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Multidrug Resistance Related Protein 1 (MRP1) are expressed on the cell surface, and the high capacity transporters are responsible for modulating chemo-sensitivity of cancer [19]. To explore whether GHET1 promote the development of MDR in gastric cancer cells was associated with Bax, Bcl-2, MDR1 and MRP1 expression levels, the BGC823 and SGC7901 cells were transfected with pcDNA-GHET1 or pcDNA, and RT-qPCR and western blot were used to detect Bax, Bcl-2, MDR1 and MRP1 expression levels. The Bax expression was significantly decreased in BGC823 and SGC7901 cells transfected with pcDNA-GHET1, while bcl-2 expression was significantly increased (Fig. 4A, B and E), indicating that overexpression of GHET1 inhibited cell apoptosis. The qRTPCR and western blotting results revealed that overexpression of GHET1 increased the expression of MDR1 and MRP1, indicating that overexpression of GHET1 increased sensitivity gastric cancer cells (Fig. 4C, 4D and 4E). As Fig. 4F showed a pathway illustration identified in this study. In the BGC823 and SGC7901 cell, overexpression of GHET1 downregulated Bax expression and upregulated Bcl-2, MDRA and MRP1 expression, which resulted in increasing cisplatin resistance and inhibiting cell apoptosis. In the BGC823/DPP and SGC7901/DDP cell, knockdown of GHET1 leaded to increase cisplatin sensitivity and promote cell apoptosis. 4. Discussion MDR plays a critical role in tumor initiation and progression by promoting cell proliferation and inhibiting apoptosis [20], and the development of cancer cell against cancer chemotherapy is a major obstacle to the effective treatment of gastric cancer [5]. In the present study, we examined the GHET1 expression level in gastric carcinoma tissues of cisplatin sensitive patients and drug-resistant patients, as well as the MDR gastric cancer cell lines and their chemo-sensitive parental line. We also identified the function of GHET1 in cisplatin resistant strains (BGC823/DPP and SGC7901/ DDP cells) and their parental line (BGC823 and SGC7901 cells) using gain-of-function and loss-of-function approaches in vitro.
Fig. 3. GHET1 reduces sensitivity of gastric cancer cells to anticancer drugs. A: analysis of overexpression of efficiency of pcDNA-GHET1 in BGC823 and SGC7901 cell. B and C: the BGC823 and SGC7901 cells that transfected with pcDNA-GHET1 were treated with different concentrations of cisplatin for 48 h, then the inhibition rate of cell lines were tested with the MTT assay. D: the IC50 of BGC823 and SGC7901 cell that transfected with pcDNA-GHET1 were analyzed by MTT. E: the BGC823 and SGC7901 cell that transfected with pcDNA-GHET1 were treated with 1 ug/ml cisplatin for 48 h, then apoptotic cells were detected by staining with Annexin V/PI using flow cytometry. **p < 0.01.
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Fig. 4. The GHET1 promoted resistance of gastric cancer cells that was associated with Bax, Bcl-2, MDR1 and MRP1 mRNA. A-D: the RT-Qpcr was used to detect the expression of apoptosis related gene (Bax and Bcl-2) and drug resistance related gene (MDR1 and MRP1) in BGC823 and SGC7901 cell that transfected with pcDNA-GHET1. E: the western blot was used to detect the expression of apoptosis related gene (Bax and Bcl-2) and drug resistance related gene (MDR1 and MRP1) in BGC823 and SGC7901 cell that transfected with pcDNA-GHET1. (F) A pathway illustration identified in this study. **p < 0.01.
Our results demonstrated that GHET1 was up-regulated in gastric carcinoma tissues of the drug-resistant patients, and high expression of GHET1 was correlated with the sensitivity of gastric cancer cell to cisplatin. Enhanced GHET1 expression reduced the sensitivity of BGC823 and SGC7901 cells to cisplatin, while inhibition of GHET1 expression inhibited the MDR of BGC823/ DDP and SGC7901/DDP cells. Therefore, our results indicated that GHET1 functioned as a drug resistant genes in gastric carcinoma. It is well known that protein-coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs [21]. Although researches of lncRNAs have dominated the field of RNA regulation in gastric cancer, such as MALAT1 [10], NEAT1 [22], HOTAIR [23] and so on, accumulating evidence has indicated that these lncRNAs may also play an important role in the development of multidrug resistance. MALAT1 could regulate multidrug resistance of hepatocellular carcinoma cells via modulating autophagy [24]. NEAT1 could mitigate multidrug resistance by inhibiting ABCG2 in leukemia [25]. HOTAIR could promote cisplatin resistance in gastric cancer [11] and confer tamoxifen resistance in breast cancer [26]. GHET1 had been proved to be an oncogene in colorectal cancer [27] and bladder cancer [28]. In this study, we found that higher expression of GHET1 promoted the development of multidrug resistance in gastric cancer cell. The Bcl-2 family of genes includes some important regulators of apoptosis. Among these genes, Bcl-2 and Bax control cell death, thus contributing to both tumor growth and drug sensitivity. MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and drug resistance in human cancer is associated with overexpression of MDR1 gene. The multidrug resistance protein 1 (MRP1) was originally discovered as a cause of multidrug
resistance in tumor cells. In this study, we further determined whether the expression level of GHET1 correlated with the apoptosis-related or drug resistance-related genes. Western blot was conducted to evaluate the protein expression level of Bax, Bcl2, MRP1 and MDR1 in gastric cancer cells transfected with pcDNAGHET1. As the results showed that, BGC823 and SGC7901 cells transfected with pcDNA-GHET1 both have a decreased Bax expression and increased Bcl-2, MDR1 and MRP1 expression, further suggesting GHET1 promoted the development of multidrug resistance in gastric carcinoma cell. Whether GHET1 also physically associates with other genes except c-Myc, and the direct or indirect physical interaction between them requires further investigation. In general, we demonstrated that expression of GHET1 were significantly upregulated in gastric cancer tissues of drug resistance patients. Moreover, GHET1 expression was also obviously increased in the BGC823/DDP and SGC7901/DDP cell lines. We also provided evidence for GHET1 function on MDR development in gastric cancer and revealed enhancer-like roles of GHET1 in MDR1 and MRP1 expression. This result indicated that GHET1 depletion may be a promising therapeutic strategy for gastric cancer MDR reversal. Conflict of interest The authors declare no conflict of interest. Acknowledgement This project was supported by the National Nature Science Foundation of China (Grant No. 81673736)
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