Original Article MEG3 Regulates Cell Cycle Progression Via Control of P53 Expression in Gastric Cancer

Int J Clin Exp Pathol 2016;9(7):6887-6895 www.ijcep.com /ISSN:1936-2625/IJCEP0026301

Original Article MEG3 regulates cell cycle progression via control of P53 expression in gastric cancer

Xiaomin Yang1, Hao Ren1, Ran Wei1,2, Xin Zhang1, Xiaoli Zhang1, Chuanxin Wang1, Yi Zhang1

1Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, China; 2Wakayama Medical University, Wakayama, Wakayama, Japan Received February 20, 2016; Accepted May 21, 2016; Epub July 1, 2016; Published July 15, 2016

Abstract: Gastric cancer is the second leading cause of cancer deaths worldwide. Recently lncRNA has been reported playing important role in many diseases especially in the progress of tumor formation. However the roles of many lncRNAs in gastric cancer remain largely unclear. LncRNA-MEG3 has been invested playing key role in many diseases especially in central nervous system diseases. In our study, we examined the expression of MEG3 and P53 mRNA in 59 gastric cancer tissues and their adjacent tissues. In addition we tested the expression level of lncRNA- MEG3 in gastric epithelial cells and gastric cancer cell lines such as AGS, BGC823, SGC7901, N87 using qRT-PCR and found that the expression level of MEG3 in gastric cancer tissues and cell lines were much lower than that of adjacent tissues and GES1 cell line. Transfection of pCDNA-MEG3 could significantly inhibit the cell cycle in BGC823 cell line. What’s more, we also found the expression of protein P53 was upregulated in a great extent in the group of pCDNA-MEG3 compared with the. In conclusion our study suggested that lncRNA-MEG3 is down expression in gastric cancer and regulate the process of carcinogenesis and may serve as a therapeutic target of gastric cancer in future.

Keywords: Gastric cancer, lncRNA-MEG3, P53, cell cycle, therapeutic target

Introduction scripts can, among other effects, change the chromosome structure [9], and modulate gene Gastric cancer is the fourth most prevalent activity [10] as well as enhance gene transc- cancer worldwide and the second leading ription [11] These aforementioned molecular cause of cancer-related death [1]. In China, mechanisms have been cited as suggesting gastric cancer is the second leading cause of that lncRNAs participate in diverse biological death from cancer [2, 3]. Although great impr- processes, especially tumorigenesis. The bio- ovements have been made in the early detec- logical features of cancer influenced by lncRNAs tion and treatment of gastric cancer, the molec- include cell proliferation and cell cycle [12], ular mechanisms of the development of gastric migration [13], differentiation, moving, apopto- cancer remain unclear [4, 5]. sis [14] In gastric cancer, many lncRNAs can Therefore, in this study, we explored the mech- function as either tumor suppressors or onto- anisms of gastric cancer. The Human Genome genesis with complicated mechanisms. For Project (HGP) found that more than 90% of the example, GAS5, an lncRNA, can influence gas- genome is noncoding RNA. Long noncoding tric cancer cell proliferation, partly by regulating RNA (lncRNA) is a class of endogenous, poorly E2F1 and P21 expression [14]. conserved RNA that lacks protein-coding potential because of the absence of ORF. In contrast MEG3 was first identified on mouse distal chro- to the length of small ncRNAs such as miRNAs, mosome 12 while locating on human’s chromo- siRNAs, and piRNAs, the length of long noncod- some 14q32 [15, 16]. It expressed in many nor- ing RNA varies from 200 hundred nucleotides mal tissues such as human pituitary but lost in to tens of thousands of bases [6-8]. lncRNA kinds of human cancers and tumor cell lines studies have reported that noncoding tran- including urothelia carcinoma [17], tongue MEG3 and P53 in gastric cancer squamous cell carcinoma [18], liver fibrogene- Transfection sis [19] and lung cancer [20]. Studies have suggested that hypermethylation of differentially Cells (BGC823 cell line) were seeded into six- methylated regions (DMRs) contribute to the well plates and the plasmid with pCDNA-MEG3 silence of MEG3 gene in human tumors [21, (Gene Chem, Shanghai) and empty vectors 22]. In addition it has been proved that MEG3 were transfected into the cells by Lipofectamine can induce the accumulation of P53 and stimu- 2000 reagent (Invitrogen, Shanghai, China) late the expression of the growth differentia- according to the manufacturer’s protocols. tion factor 15 (GDF15) [16]. After 24 h and 72 h, transfected cells were harvested for protein extraction and cell cycle Here, paired gastric cancer tissue samples analysis. were collected to detect the relative expression Total RNA extraction and quantitative reverse- of MEG3 by RT-PCR and the relationship bet- transcription polymerase chain reaction analy- ween clinicopathological features and expres- ses sion of MEG3 was analyzed. We demonstrated that upregulation of lncRNA-MEG3 can inhibit Total RNA was isolated from frozen tissues cell proliferation while cells accumulated in G1 and cultured cells using TRIzol reagent (Invitr- phase. To make further, western blot analysis ogen, USA) according to the manufacturer’s pr- was used to detect whether the expression of otocols. The RNA concentration was measur- P53 changed or not afterthe upregulation of ed by ultraviolet-visible spectrophotometer at MEG3. Hence we come to a conclusion that A260/280. Then cDNA was generated using lncRNA-MEG3lncRNA-MEG3 can affect the cell PrimeScriptTM RT reagent Kit (Takara, Dalian, cycle via regulating the key player P53. China) with 500 ng total RNA, 2 ul 5×PrimeScript Buffer, 0.5 ul PrimeScript RT Enzyme Mix I, 0.5 Materials and methods ul Oligo dT Primer (50 μM), 0.5 ul Random 6

mers (100 μM) and RNase Free dH2O up to 10 Patient and tissue samples ul incubated at 37°C for 15 min and 85°C for 5 sec. For quantitative real-time polymerase Matched gastric cancer and adjacent non- chain reaction (qRT-PCR), we used the SYBR® tumor tissues were obtained from 59 patients Premix Ex TaqTM II (Takara, Dalian, China) in who underwent primary surgical resection at the CFX-96 real-time PCR System (BIO-RAD, Qilu Hospital of Shandong University between USA). The PCR mixture solution included 12.5 February 2015 and September 2015. All tis- ul SYBR Premix Ex Taq II, 1 ul forward primer sues were immediately snap-frozen in liquid (10 uM), 1ul reverse primer (10 uM), 2 ul cDNA nitrogen and stored at -80°C until total RNA product and 8.5 ul nuclease-free water and was isolated. The tumor was staged by Tumor incubated at 95°C for 15 s, 40 cycles of 95°C Node Metastasis (TNM) Staging. This project for 5 s and 60°C for 30 s followed by the melt was approved by the Clinical Research Ethics curve to test the specificity of the PCR product. Committee of Qilu Hospital of Shandong GAPDH was used to normalize the relative University. expression of RNA in our experiments. The PCR primers for MEG3, P53 mRNA and GAPDH were Cell lines and cultures as follows: MEG3 sense 5’GCCTGCTGCCCATC- Human gastric cancer cell lines (BGC823, TACAC3’ and reverse 5’CCTCTTCATCCTTTGC- SGC7901, AGS and N87) and human gastric CATC3’; P53 sense 5’GTTCCGAGAGCTGAATGA- epithelium-immortalized cell line (GES1) were GG3’ and reverse 5’TCTGAGTCAGGCCCTTCT- gifted from Shongdong Province Key Laboratory GT3’; GAPDH sense 5’AGCCACATCGCTCAGAC- for Tumor Target Molecules, Medical Research AC3’ and reverse 5’GCCCAATACGACCAAATCC3’. and Laboratory Diagnostic Center. The BGC823, The 2-ΔΔCT method was used to calculate the AGC-7901, N87 and GES1 were maintained in relative expression of P53 mRNA and lncRNA- MEG3 in all samples. an atmosphere of 5% CO2 in RPMI-1640 medium (Hyclone, Beijing, China) supplemented wi- Western blotting analysis th 10% fetal bovine serum (Hyclone, Beijing, China) at 37°C while AGS maintained in F12 For western blotting, BGC823 cells were trans- medium with the same condition. fected with pCDNA-MEG3 plasmid and the

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Figure 1. LncRNA-MEG3 and P53 mRNA expression was tested by qRT-PCR. A. The relative expression of MEG3 in gastric cancer tissues (n=59) were significantly lower than those in paired adjacent tissues (P=0.003); B. The P53 mRNA expression levels in carcinoma lesions was significantly lower compared to those of noncancerous lesions (P=0.004). *P<0.05, **P<0.01.

Table 1. Association between the expression of MEG3 with RIPA lysis buffer (Beyotime, Beijing, and the clinicopathological factors in human gastric- China) according to the manufacturer’s cancers instructions. Protein (20 ug) for each sam- Relative expression ple was resolved by 10% SDS-PAGE and Clinical parameters n P-value II of MEG3 I then transferred to PVDF membranes. The Gender 0.581 membranes were blocked using TBST buffer (TBS plus 0.1% Tween-20) with 5% fat- Male 39 0.618 (0.114-1.459) free milk and then incubated with rabbit Female 20 0.318 (0.090-1.201) anti P53 monoclonal antibody (1:1000, Age (years) 0.872 Cell Signaling Technology, Danvers, MA, ≥60 25 0.277 (0.102-1.392) USA). The rabbit anti β-actin monoclonal <60 34 0.502 (0.095-1.220) antibody (1:1000, Cell Signaling Techno- Diameter (cm) 0.953 logy, Danvers, MA, USA) as the internal ≥10 7 0.275 (0.149-0.755) control at 4°C overnight. The specific HRP- 10>X≥5 24 0.265 (0.101-1.278) conjugated secondary antibody (1:5000, <5 28 0.558 (0.091-1.467) Zhanshan Goldenbridge, Beijing, China) Differentiation 0.677 was used as secondary antibodies and ECL chromogenic substrate was used to Well 5 0.362 (0.108-1.087) quantify the expression level of specific Moderate 16 0.562 (0.065-1.426) protein and signals were quantified by Moderate-poor 31 0.498 (0.149-1.586) FluorChem E (Protein Simple, USA). Poor 7 0.176 (0.081-0.249) Lymphatic metastasis 0.425 Cell cycle analysis N0-N1 32 0.633 (0.100-1.320) N2-N3 27 0.274 (0.091-1.470) Cells were harvested by typsinization and Distant metastasis 0.017 washed three times using the cold phos- M0 54 0.401 (0.095-1.179) phate-buffered saline (PBS). 70% ethanol was used to fix the cells for cell cycle analy- M1 5 1.772 (1.108-2.824) sis at 4°C about 16 hours and then cells I: Median of relative expression, with 25th-75th percentile in paren- theses. II: P<0.05 was considered to indicate a statistical analysis. were washed by cold PBS. 200 ul cold PBS was added to weight the cells and mixed the presidium iodide (PI, BD) 500 ul with empty vector and cells were harvested 72 h the cells for 30 min in the dark. The BD post-transfection. Transfected cells were lysed FACSCato II was used to analyze the cell cycle

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Figure 2. MEG3 and P53 expression in gastric cancer cell lines 1×106 cells were collected and lysed with TRIzol reagent to extract total RNA and further more the relative expression of lncRNA-MEG3 (A) and P53 mRNA (B) was examined by qRT-PCR. The results displayed that not only lncRNA-MEG3 expression in gastric cancer cells was lower compared with GES1 cells but also P53 mRNA was lower significantly; (C) RIPA lysis buffer was used to extract the protein from cells and western blot to determine the protein expression with β-actin as the internal control.

Figure 3. The lncRNA-MEG3 up regulates P53 expression at the posttranscriptional level BGC823 cells were transfected with pCDNA-MEG3 plasmid and empty vector by Lip 2000. The expression of MEG3 expression was determined by RT-PCR (A) while P53 was detected using RT-PCR (B) and Western Blotting (C). The data suggested that the lncRNA-MEG3 can up-regulate the P53 expression at the posttranscriptional level.

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Figure 4. The transfection efficiency of the plasmid. As shown in the picture, the transfection efficiency can up to 90% in both pCDNA-MEG3 and empty vector group. according to the protocols and ModFit software mRNA in cancer tissues from patients with gas- was used to quantify the cells. tric cancer was lower than that in matched non- tumor tissues (P=0.004<0.01, Figure 1B). Statistical analysis The association of MEG3 expression levels Differences from at least 3 separate experi- and clinicopathological factors ments among groups and evaluated by two- tailed Student’s t test using SPSS Statistics The relationship between lncRNA-MEG3 expr- v19.0 software (IBM). GraphPad Prism 5 was ession level and clinicopathological factors of used to drawing figures and P<0.05 was con- human gastric cancer was also been explored sidered to indicate a statistical analysis. in our study. The expression of MEG3 in patients with distant metastasis was significantly high- Results er than that in other tissues and data was shown in the Table 1. However, MEG3 expres- Expression of lncRNA-MEG3 and P53 mRNA in sion level didn’t show significantly association human gastric cancer and non-tumor tissues with other clinicopathological factors such as gender (P=0.581), age (P=0.872), diameters The lncRNA-MEG3 and P53 mRNA expression (P=0.953). levels of tumor and adjacent noncancerous tissues from the gastric cancer patients were MEG3 and P53 expression in gastric cancer tested by qRT-PCR. Significantly decreased of cell lines the expression level of MEG3 was observed in tumor tissues compared with the adjacent tis- The expression of lncRNA-MEG3 and P53 sues (P=0.003<0.01, Figure 1A). In addition, mRNA was quantified by qRT-PCR in gastric we found that the expression level of P53 cancer cell lines and normalized by GAPDH. As

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Figure 5. Low expression of MEG3 delayed cell cycle progression. The cells were harvested with PBS washing three times and fixed in 70% ethanol overnight. PI dyes were used to label all the cells and BD FACSCato II was used to analyze the cell cycle. As the result displayed in the piacture B, the lncRNA-MEG3 can induce G1 arrest significantly in the cell cycle process and suggested that low expression of MEG3 can promote the tumor growth via its influence on cell cycle. P<0.05 was considered to be significantly different.

Table 2. Cell cycle distribution of BGC823 cell lines with As shown in Figure 3, the protein different treatment expression of P53 in BGC823 cells G0/G1 (%) S (%) G2/M (%) P with the pCDNA-MEG3 was much high- Cells untreated 71.42±6.10 20.91±5.36 7.68±0.81 er than the group. Empty vector 72.78±3.96 19.15±2.19 8.07±2.11 Low expression of MEG3 delayed cell pCDNA-MEG3 76.06±2.16 17.92±1.53 6.02±1.47 <0.001 cycle progression the data shown in Figure 2, the expression of To explore the role of MEG3 in gastric tumor cell MEG3 in gastric cancer lines was significantly cycle distribution in vivo, flow cytometric analy- lower than that in the GES1 cells (P<0.01) and sis was conducted. The plasmid with pCDNA- what’s more, P53 mRNA expression displayed MEG3 and empty vector were transfected into similar to lncRNA-MEG3. In addition, the result gastric cell line BGC823. The results displayed of protein P53 determined by western blot that the group with pCDNA-MEG3 showed sig- showed expression deletion in SGC7901, nificantly G1 arrest (76.06±2.16%) compared BGC823 and N87 cell lines while less expres- with the empty plasmid group (72.78±3.96%), sion in AGS cells. while there are no significant difference between non-transfected BGC823 cells and The lncRNA-MEG3 regulates P53 expression the empty control (Figures 4, 5 and Table 2). The results suggested that lncRNA-MEG3 can P53 has been investigated to be a tumor sup- delay the cell cycle via G1 arrest and as a result pressor in many cancers. In our study, we the lower expression of lncRNA-MEG3 cause devoted to clarify the relationship between the tumor growing faster. lncRNA-MEG3 and P53 in gastric cancer. The plasmid with pCDNA-MEG3 and empty vector Discussion were transfected into gastric cell line BGC823. We found that compared with the group of A commonly definition for lncRNA is a class of empty vector, the expression of MEG3 in the RNA that longer than 200 nucleotides and with- group with pCDNA-MEG3 plasmid was up- out the function of translation into protein [23]. expression about 5000 fold or even more Many studies have investigated that the lncRNA (P<0.01). On the other hand, the expression of play key roles in many diseases including cen- P53 mRNA in BGC823 cell with pCDNA-MEG3 tral nervous system diseases [24-26] and was 1.21 (24 h), 1.16 (72 h) fold compared to especially cancers [23] such as bladder cancer the normal control (P<0.05). At the same time, [27], breast cancer [28], lung cancer [29]. In western blot was also used to detect the gastric cancer, many lncRNAs have been dem- changement of the protein expression of P53. onstrated to participate in the tumorgenecity

6892 Int J Clin Exp Pathol 2016;9(7):6887-6895 MEG3 and P53 in gastric cancer process via complicated pathways [14, 30]. As tion. The research also has the condition to we all know, P53 functioned as a tumor sup- limit, the size of tissue samples was not enough pressor by activating ordownregulating gene to do more analysis and the survival time also expression. It has been confirmed that P53 needs to keep observation. In addition, making can regulate the cell cycle by the p53-p21- the genetically engineered mouse models DREAM-CDE/CHR pathway [31]. In our study, would be of great method to clarify the physio- the lncRNA-MEG3 was significantly downex- logical functions and specific molecular mecha- pressed in both gastric cancer tissues and cell nisms. Maybe lncRNA-MEG3 can be a thera- lines. To explore the relationship between peutic target of gastric cancer in the future and MEG3 and P53, the pCDNA-MEG3 plasmid was helpful in individualized medication. constructed and transfected into gastric cancer line BGC823. The results showed that up- Acknowledgements expression of MEG3 can positively regulate the This work was supported in partly by grants expression of P53 protein but the P53 mRNA from the Shandong Province Science Found- showed negative changement. It can be sus- ation for Key Programs (2008GG30002017 pect that lncRNA-MEG3 regulate the P53 and 2010GSF10274), the University Innovation expression mainly at posttranscriptional level. Program from Jinan, Shandong Province (201- Maybe lncRNA-MEG3 activates the P53 expres- 004050) and National Key Clinical Medical Sp- sion by interacting with P53 DNA binding ecialties foundation and partly supported by domains or activating the P53 promoter or Science Foundation of Qilu Hospital of Shan- other P53 target genes. Further more studies dong University. need to be conducted to clarify the specific molecular mechanism of MEG3 and P53 Disclosure of conflict of interest interactions. None. We further demonstrated that lncRNA-MEG3 plays role in the cell cycle distribution by over- Address correspondence to: Yi Zhang, Department expression the P53. Therefore the downexpres- of Clinical Laboratory, Qilu Hospital, Shandong sion of lncRNA-MEG3 contribute to the develop- University, 107 Wenhua West Road, Jinan 250012, ment of gastric cancer. In addition, the relation- Shandong, P. R. China. Tel: +86 531 82166802; ship between different expression level of Fax: +86 531 82166802; E-mail: yangxiaomin91@ MEG3 and the gastric tumor pathological stage yahoo.com.hk distant metastasis (P<0.05). The results sug- References gested that the gastric cancer with higher expression of lncRNA-MEG3 was limited the [1] Resende C, Thiel A, Machado JC, Ristimaki A. cells proliferation compared with the lower Gastric Cancer: Basic Aspects. Helicobacter group and then the group of higher expression 2011; 16: 38-44. choose the distant metastasis. [2] Du W, Ji H, Cao S, Wang L, Bai F, Liu J, Fan D. EpCAM: a potential antimetastatic target for Our findings indicated that the lncRNA-MEG3 gastric cancer. Dig Dis Sci 2010; 55: 2165- can regulate the protein P53 expression at 2171. posttranscriptional level in the development of [3] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, gastric cancer. However the specific molecular Bray F, Jemal A, Yu XQ, He J. Cancer statistics mechanisms remain unclear. What’s more, it in China, 2015. CA Cancer J Clin 2016. [4] Chen W, Zheng R, Zeng H, Zhang S. The up- has been demonstrated the lncRNA-MEG3 can dated incidences and mortalities of major can- enhance the ability of p53 by binding to the cers in China, 2011. Chin J Cancer 2015; 34: growth differentiation factor 15 (GDF15) to 53. stimulate the expression of the GDF15 using [5] Lan H, Zhu N, Lan Y, Jin K, Teng L. Laparoscopic HCT116 and U2OS cell lines [16]. gastrectomy for gastric cancer in China: an overview. Hepatogastroenterology 2015; 62: Conclusion 234-239. [6] Feingold EA, Good PJ, Guyer MS, Kamholz S, In brief, the expression of lncRNA-MEG3 was Liefer L, et al. The ENCODE (ENCyclopedia of positively correlated with the distant metasta- DNA elements) Project. Science 2004; 306: sis of gastric tumor and the cell cycle distribu- 636-640.

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