Abnormal expressed long non-coding RNA IRAIN inhibits tumor progression in human renal cell carcinoma cells

Wang Zhiqiang; Liu Qian; Li Tieqiang; Li Xiaodong; Zhang Guangwei; Li Yang; Zi Hao; Zhu Chaoyang

doi:10.1515/biol-2016-0026

Artikel Open Access

Abnormal expressed long non-coding RNA IRAIN inhibits tumor progression in human renal cell carcinoma cells

Wang Zhiqiang , Liu Qian , Li Tieqiang , Li Xiaodong , Zhang Guangwei , Li Yang , Zi Hao und Zhu Chaoyang

Veröffentlicht/Copyright: 23. September 2016

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Abstract

Objectives

The long non-coding RNA (lncRNA) IRAIN has been verified to have key roles in tumor biology. The aim of this study was to explore its expression and biological functions in human renal cell carcinoma (RCC) cells.

Methods

Quantitative RT-PCR was applied to detect the RNA expression of IRAIN in RCC tissues and cell lines when compared with respective controls. MTT and flow cytometry methods were respectively used to monitor the cell proliferation and apoptosis of 786-O cells after IRAIN was overexpressed. Altered expression of cyclin D1 and Bax was determined by immunoblotting. Xenograft models were finally carried out to confirm the roles of IRAIN in RCC in vivo.

Results

IRAIN expression was found to be remarkably decreased in RCC tissues and cell lines. Its overexpression in 786-O cells significantly inhibited cell proliferation and promoted apoptosis. We further demonstrated that cyclin D1 was reduced while apoptosis promoting protein Bax was elevated in IRAIN-overexpressed 786-O cells. Importantly, we found that IRAIN overexpression could suppress in vivo tumorigenesis of RCC, reflected by tumor volume and tumor weight measurement.

Conclusion

IRAIN might serve as a novel tumor suppressing lncRNA and a potential therapeutic target in RCC treatment.

Keywords: IRAIN; RCC; proliferation; apoptosis; xenograft

1 Introduction

Renal cancer represents as the seventh most frequent cancer in men and the ninth most frequent cancer in women over the world, with an increasing morbidity by 2% per-decade [1]. Among these renal cancer patients, approximately 90% suffer from renal cell carcinoma (RCC). According to related statistics, over one hundred thousand cases die of RCC, and the incidence and mortality increase during the last 60 years [2]. Radical nephrectomy remains as the most effective choice for RCC treatment, especially for those patients at early and local stages. However, organ metastasis occurs in 20%-40% of RCC patients after surgery, which leads to worse outcomes [3]. The fact that RCC is commonly resistant to chemotherapy and radiotherapy also augments the difficulty of RCC therapy [4]. Therefore, investigating the molecular and pathogenic basis of RCC progression will certainly benefit the cure options of this disease.

Long non-coding RNAs (lncRNAs) are a class of non-protein coding RNAs longer than 200 nucleotides. Although they are initially thought of as “transcription noise” in early years after discovery, they emerged as popular RNA stars in the biological and medical studies in recent years, particularly in the cancer research area [5-8]. Also, numerous studies had pointed out that lncRNAs could serve as promising targets for cancer therapy, as well as diagnostic and/or prognostic biomarkers in selected cancers [9,10].

LncRNA IRAIN is an intragenic lncRNA found by Hu’s group within the insulin-like growth factor type I receptor (IGF1R) locus, and is transcribed in an antisense direction from an intronic promoter in the IGF1R gene [11]. They also found that IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk acute myeloid leukemia (AML) patients, suggesting the anti-tumor activity of IRAIN in AML [11]. However, another report on non-small cell lung cancer (NSCLC) demonstrated the tumor promoting functions of this lncRNA, by promoting cell proliferation in vitro, while having almost no impact on apoptosis and migration [12]. The roles of IRAIN in RCC have not been recognized till now. The objective of this study was to show the expression and functions of IRAIN in RCC cells.

2 Materials and Methods

2.1 Patients & Tissues

30 pairs of RCC tissues and their adjacent non-tumor tissues were collected from patients in Huaihe Hospital of He’nan University from 2014-2015. The research was approved by the Ethics Committee of Huaihe Hospital of He’nan University, and written informed consent was obtained from all patients. All tissue samples were immediately frozen by liquid nitrogen and stored at -80°C until used.

2.2 Cell lines and Reagents

Human embryonic kidney 293T cells and renal cell carcinoma cell lines UOK117, 786-O and SN12 were cultured in proper medium with 10% FBS. The cyclin D1, Bax and tubulin antibodies were purchased from Cell Signaling Technology.

2.3 Lenti-Virus

The lenti virus containing the lncRNA IRAIN overexpression vector were purchased from the Genepharma Company, as well as the control virus. 786-O cells were infected by lentivirus with a MOI of 5 for 24 h.

2.4 Reverse Transcription and qRT-PCR

Total RNA from human tissues or cultured cells was extracted using TRiZol reagent and the cDNA was synthesized by reverse transcription. Quantitative realtime PCRs (qRT-PCRs) were carried out by SYBR green reagent on a ABI7500 system. Primers of IRAIN were from Sangon Biotech, and the GAPDH was used for normalization. Data was collected in triplicate to assess the statistical significance.

2.5 Western Blotting

Cells were lysed using cell lysis RIPA buffer containing protease and phosphatase inhibitors. 30 μg of whole cell extracts were subjected to 11% SDS-PAGE gel and transferred to NC membranes. The membranes were blocked with 5% nonfat milk for 1 h at RT and then incubated with the cyclin D1 (1:1000), Bax (1:1000) and tubulin (1:5000) primary antibodies at 4°C overnight, followed by the incubation of the appropriate HRPconjugated secondary antibodies for 1h at RT. Finally, all blots were visualized with ECL and analyzed by ImageJ software.

2.6 MTT Assay

To assess cell growth, 24 h after infection with IRAINoverexpression lenti virus or control virus, cells were trypsinized and seeded in 96-well plates. At 24 h, 48 h and 72 h, 100 μl MTT (5l) were added to each well and the cells were incubated for additional 4 h at 37°C. After discarding the medium, 100 μl DMSO were added and the absorbance at 570 nm was measured by using a spectrophotometer.

2.7 Cell Apoptosis Assay

To assess late cell apoptosis, cells were seeded in a 60 mm dish. 24 h later, cells were infected with IRAINoverexpression either the lenti virus or the control virus. 48 h later, cells were harvested and fixed with cold 70% ethanol overnight. The next day, cells were washed twice with cold PBS, and incubated with 100 μg/ml PI and 0.5 μg/ml RNase A for 1 h at 37°C before subjecting to FACS analysis.

2.8 In vivo xenograft tumor growth

Four-week-old male nude mice [BALB/cA-nu (nu/nu)] were obtained from Nanjing Experimental Animal Center and maintained in SPF conditions. The mice were separated randomly into two groups and were subcutaneously injected with lenti virus-infected 786-O cells at each flank. Tumor volume was measured every week with a caliper and calculated using equation: V = a × b2 × 0.5326. On the 45th day, the mice were killed and xenografts were weighed.

2.9 Statistical analysis

All values were calculated by the SAS software v8 and Microsoft Excel 2003. Data were shown as mean ± SD. Differences between the groups were calculated using paired- or grouped- student’s t-test. P values < 0.05 were considered statistically significant. *: P < 0.05, **: P < 0.01.

3 Results

3.1 Reduced expression of IRAIN in RCC

To explore the role of IRAIN in RCC cells, we first examined its expression change in RCC tissues and cell lines. A total of 30 cases of RCC tissues (Tumor, T) matched with their adjacent normal counterparts (Normal, N) were collected. The expression of IRAIN was determined by qRT-PCR. GAPDH was used as the internal control. As shown in Figure 1A, we found that IRAIN was remarkably reduced in RCC tissues compared with the matched normal renal tissues. Moreover, we detected the expression of IRAIN in several RCC cell lines, and human 293T cells were considered as the normal control cells. We also found that in RCC cell lines, which included UOK117, 786-O, SN12, IRAIN had significantly decreased expression when compared to that in 293T cells (Figure 1B). Among these cell lines, IRAIN levels were lowest in 786-O cells, hence we mainly carried out functional assays in 786-O cells.

Figure 1

Reduced expression of IRAIN in RCC tissues and cell lines. (A) The expression levels of lncRNA IRAIN in 30 RCC tissues and adjacent non-tumor tissues were assessed by quantitative real-time PCR. The relative mRNA expression levels are presented as fold change = 2DCtNDCtT of tumors versus matched non-tumor tissues. (B) The expression levels of lncRNA IRAIN in human embryonic kidney 293T cells and three renal cell carcinoma cell lines were assessed by quantitative real-time PCR.

3.1.1 IRAIN impaired RCC cell proliferation and promoted apoptosis

From the above findings, we observed a decreased expression of IRAIN in RCC cells. We next performed gain-of-function studies in 786-O cells in which IRAIN was least expressed. IRAIN was forced expressed in 786-O cells by infecting the cells with lenti virus containing IRAIN full-length transcripts. The result from Figure 2A reflected that IRAIN was indeed overexpressed in 786-O cells, as detected by qRT-PCR. Cell proliferation monitored by the MTT method shown in Figure 2B demonstrated that IRAIN overexpression caused reduced proliferation capacity of 786-O cells. Additionally, we measured cell apoptosis rate by the flow cytometry technique. The result shown in Figure 2C illustrated that IRAIN overexpression significantly induced cell apoptosis in 786-O cells. Molecularly, we measured the protein expression of proliferation-related cyclin D1 and apoptosis-promoting protein Bax, and found that cyclin D1 was decreased in IRAIN-overexpressed cells, whereas Bax was elevated after IRAIN was overexpressed (Figure 2D). These results suggested that IRAIN was a tumor suppressor in RCC cells by inhibiting cell proliferation and promoting apoptosis.

Figure 2

Overexpression of IRAIN inhibited 786-O cell proliferation and promoted cell apoptosis. (A) Expression of IRAIN was significantly upregulated by ~6-fold in 786-O cells infected with lenti-virus. (B) Cell proliferation was analyzed using MTT assay. Each value represents the mean of three replicates. The proliferation of 786-O cells infected with LV-IRAIN was significantly suppressed at 72h with LV-Con cells. (C) Overexpression of IRAIN induced apoptosis. Flow cytometric analysis showed that enhanced IRAIN expression had a significant effect on apoptosis induction in 786-O cells at 72h compared with control cells. (D) Protein expression levels of cell proliferation marker cyclin D₁ and cell apoptosis marker bax were measured by western blot analysis. 786-O cells were infected with LV-Con or LV-IRAIN, and the expression levels of markers were detected by western blotting at 72h after infection. Tubulin was used as an internal control.

3.2 The tumor suppressing effect of IRAIN in RCC cells in mice

Afterwards, we confirmed the above in vitro functions of IRAIN in xenograft models in mice. 786-O cells stably expressing IRAIN or blank control were injected subcutaneous at two sides of the back. The tumorigenesis process in vivo persisted for 45 days. Every one week, tumor volume was calculated and growth curve was plotted accordingly (Figure 3A). Finally, tumors were stripped from the mice and tumor were weighed for the two groups (Figure 3B). The tumor growth curve showed that IRAIN overexpression led to reduced growth rate, and the final tumor weight showed that IRAIN overexpressed caused decreased mean tumor weight in this group. We further extracted total RNA from these tumors and demonstrated that IRAIN was truly overexpressed in the experimental group (Figure 3C). Therefore, the in vivo data strongly confirmed the tumor suppressing effects of IRAIN in RCC

Figure 3

IRAIN suppressed xenograft tumorigenesis of 786-O cells. (A) Weekly measurements of average tumor volumes in both LV-Con and LV-IRAIN groups. (B) The range of individual terminal tumor weights was plotted between two groups at the 45th day when the mice were killed. (C) Expression of IRAIN was significantly upregulated by ~4-fold in tumors of the LV-IRAIN group.

4 Discussion

RCC is currently the most common type of renal cancer and accounts for 3% of all adult cancers [13]. The most prevalent histological form of all RCC cases is the clearcell type, called clear cell RCC, which accounts for 75% of cases [13]. Featured by inactivation of a critical tumor suppressor, von Hippel Lindau (VHL) [14], clear cell RCC has been treated by targeting the VHL-hypoxia-inducible factor (HIF)-VEGF axis. LncRNAs have been found to be dysregulated in multiple cancers and to be important in the regulation of carcinogenesis, thus suggesting that this class of RNA transcripts may be used as biomarkers in cancer. LncRNAs have been found to participate in many different biologic processes, including basic processes like DNA epigenetic regulation, alternative splicing, RNA decay, transcription, translation, and functional processes like cell cycle control, apoptosis and cell differentiation [15,16].

Insulin-like growth factor 1 (IGF-1) signaling mediated by IGF1R is a crucial growth regulatory pathway, and its enhanced activation is thought to play a vital role in cancer cell proliferation, migration, and apoptosis. IRAIN is a 5.4 kb non-coding RNA within the IGF1R locus, transcribed from an intronic promoter in antisense orientation. However, the functions of IRAIN remain largely unknown in malignancies. There is only one report characterizing its role in non-small cell lung cancer [12]. In their study, the expression level of IRAIN was remarkably increased in NSCLC tissues and connected with tumor size and smoking status. In addition, knockdown of IRAIN suppressed NSCLC cell proliferation in vitro [12].

We focused on investigating the expression and function of IRAIN in RCC cells. Our data suggested that IRAIN expression was downregulated in RCC, including clinical specimens and cultured cell lines. Functional assays demonstrated that IRAIN could significantly inhibit cell proliferation and induced apoptosis. The molecular changes of cyclin D1 and Bax protein expression further supported the tumor suppressing roles of IRAIN in RCC cells. Importantly, our in vivo results finally confirmed the anti-tumor activity of IRAIN in xenograft models. Nevertheless, there are still some questions need to be further answered. For instance, the contribution of IRAIN for metastasis behaviors of RCC cells remains unknown. Time permitting, we next would pay more attention to this regulatory aspect and discuss the impact of IRAIN overexpression or inhibition on RCC cell migration and invasion. Another concern is about the exact mechanisms of IRAIN on regulating cell proliferation and apoptosis in RCC. Although we showed that cyclin D1 and Bax proteins were both altered following IRAIN overexpression, the detailed mechanisms were unclear.

In summary, our results presented here illustrated that lncRNA IRAIN was decreased in RCC and functioned as a potential tumor suppressor by limiting cell proliferation and inducing apoptosis.

Conflict of interest: Authors declare nothing to disclose.

References

[1] Reeves D.J., Liu C.Y., Treatment of metastatic renal cell carcinoma, Cancer Chemother. Pharmacol., 2009, 64, 11-25.10.1007/s00280-009-0983-zSuche in Google Scholar PubMed

[2] Bex A., Jonasch E., Kirkali Z., Mejean A., Mulders P., et al., Integrating surgery with targeted therapies for renal cell carcinoma: current evidence and ongoing trials, Eur. Urol., 2010, 58, 819-28.10.1016/j.eururo.2010.08.029Suche in Google Scholar PubMed

[3] Larkin P.J., Miller J.A., Allen R.S., Chitty J.A., Gerlach WL, et al., Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum, Plant Biotechnol. J., 2007, 5, 26-37.10.1111/j.1467-7652.2006.00212.xSuche in Google Scholar PubMed

[4] Cho I.C., Chung J., Current status of targeted therapy for advanced renal cell carcinoma, Korean J. Urol., 2012, 53, 217-28.10.4111/kju.2012.53.4.217Suche in Google Scholar PubMed PubMed Central

[5] Quinn J.J., Chang H.Y., Unique features of long non-coding RNA biogenesis and function, Nat. Rev. Genet., 2016, 17, 47-62.10.1038/nrg.2015.10Suche in Google Scholar PubMed

[6] Archer K., Broskova Z., Bayoumi A.S., Teoh J.P., Davila A., et al., Long Non-Coding RNAs as Master Regulators in Cardiovascular Diseases, Int. J. Mol. Sci., 2015, 5, 16, 23651-67.10.3390/ijms161023651Suche in Google Scholar PubMed PubMed Central

[7] Li Y., Wang X., Role of long noncoding RNAs in malignant disease (Review), Mol. Med. Rep., 2016, 13, 1463-9.10.3892/mmr.2015.4711Suche in Google Scholar PubMed

[8] Zhang F., Zhang L., Zhang C., Long noncoding RNAs and tumorigenesis: genetic associations, molecular mechanisms, and therapeutic strategies, Tumour Biol., 2016, 37, 163-75.10.1007/s13277-015-4445-4Suche in Google Scholar PubMed

[9] Yuan C.L., Li H., Zhu L., Liu Z., Zhou J., et al., Aberrant expression of long noncoding RNA PVT1 and its diagnostic and prognostic significance in patients with gastric cancer, Neoplasma, 2016, 63, 442-9.10.4149/314_150825N45Suche in Google Scholar PubMed

[10] Lv M., Xu P., Wu Y., Huang L., Li W., et al., LncRNAs as new biomarkers to differentiate triple negative breast cancer from non-triple negative breast cancer, Oncotarget, 2016, 7, 13047-59.10.18632/oncotarget.7509Suche in Google Scholar PubMed PubMed Central

[11] Sun J., Li W., Sun Y., Yu D., Wen X., et al., A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies, Nucleic Acids Res., 2014, 42, 9588-601.10.1093/nar/gku549Suche in Google Scholar

[12] Feng J., Sun Y., Zhang E.B., Lu X.Y., Jin SD., et al., A novel long noncoding RNA IRAIN regulates cell proliferation in non small cell lung cancer, Int. J. Clin. Exp. Pathol., 2015, 8, 12268-75.Suche in Google Scholar

[13] Chow W.H., Devesa S.S., Warren J.L., Fraumeni JF Jr. Rising incidence of renal cell cancer in the United States, JAMA, 1999, 281, 1628-31.10.1001/jama.281.17.1628Suche in Google Scholar

[14] Clifford S.C., Prowse A.H., Affara N.A., Buys C.H., Maher E.R., Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis, Genes Chromosomes Cancer, 1998, 22, 200-9.10.1002/(SICI)1098-2264(199807)22:3<200::AID-GCC5>3.0.CO;2-#Suche in Google Scholar

[15] Wapinski O., Chang H.Y., Long noncoding RNAs and human disease, Trends Cell Biol., 2011, 21, 354-61.10.1016/j.tcb.2011.04.001Suche in Google Scholar

[16] Shi X., Sun M., Liu H., Yao Y., Song Y., Long non-coding RNAs: a new frontier in the study of human diseases, Cancer Lett., 2013, 339, 159-66.10.1016/j.canlet.2013.06.013Suche in Google Scholar

Received: 2016-6-25

Accepted: 2016-8-4

Published Online: 2016-9-23

Published in Print: 2016-1-1

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