Microarray expression profile of lncRNAs and the upregulated ASLNC04080 lncRNA in human endometrial carcinoma

  • Authors:
    • Wen Zhai
    • Xu Li
    • Shouzhen Wu
    • Yan Zhang
    • Huan Pang
    • Wei Chen
  • View Affiliations

  • Published online on: February 17, 2015     https://doi.org/10.3892/ijo.2015.2897
  • Pages: 2125-2137
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Abstract

Long non-coding RNAs (lncRNAs) have been recognized as a regulator of gene expression, and the deregulation of lncRNAs have been reported to be correlated with carcinogenesis and cancer progression. To explore the function of lncRNA in endometrial carcinoma, we analyzed the expression profiles of lncRNAs and coding genes in 3 paired endometrial carcinoma and adjacent non-tumor tissues, using a microarray. The results of microarray analysis indicated a significant difference in lncRNA and coding gene expression between endometrial carcinoma and their paired adjacent non-tumor tissues. A total of 53 lncRNAs (fold change >2.0, p-value <0.05) were found to be differently expressed in endometrial carcinoma compared to the normal controls. Among these ASLNC04080 was the most significantly upregulated lncRNA in microarray data, highly expressed in 22 out of 24 endometrial carcinoma tissues and HEC-1-B cell line. ASLNC04080 is 1867nt in length, consist of 6 exons, and locates at 1 p35.3(chr1: -28905061 - -28909492). In addition, 46 coding gene transcripts were differentially expressed (fold change >2.0, p-value <0.05) between endometrial carcinoma and adjacent non-tumor tissues. Pathway and gene ontology analysis demonstrated that these deregulated transcripts were involved in multiple signal pathways, biological processes, cellular components and molecular functions. Moreover, the ASLNC04080 lncRNA expression was correlated with 19 coding genes, and may contribute to endometrial carcinoma genesis and progression by co-regulating with coding gene. Expression inhibition of lncRNA ASLNC04080 in HEC-1-B cells caused repression of cell proliferation, increased cell apoptosis, and G1 phase arrest. These results suggested a potential function of ASLNC04080 in endometrial carcinoma genesis and progression.

Introduction

Endometrial carcinoma, comprising of several types of malignancies arise from the endometrium or lining of the uterus, is the most common gynecologic malignancy among women in the United States, with an estimated 52,630 new case and 8,590 deaths in 2014 (1). Endometrial carcinoma cases in Chinese women increased in the last decade. Based on clinical features and pathogenesis endometrial carcinomas have been classified into two types (2). Type I endometrial carcinomas occur commonly in perimenopausal women, with low grade, related to obesity and estrogen exposure. Type II endometrial carcinomas are more common in older women, unrelated to hormone excess, with a worse outcome than Type I. Previous studies have reported that various gene mutations, and expression deregulation are related to endometrial carcinoma genesis. PTEN and K-ras mutations occur in Type I endometrial carcinomas, often with Wnt, AKT and PI3KCA deregulation. TP53 and PPP2R1A mutations are frequent in Type II endometrial carcinomas, with HER2/neu overexpression and p16 inactivation (3,4).

LncRNAs are a spectrum of RNAs transcripted by RNA polymerase II, but not translated into proteins, with more than 200 nucleotides in length. LncRNAs which have previously been identified as transcription noise are proved to be involved in the process of multiple gene expression regulation (5,6). Increasing evidence suggests that the deregulation of lncRNAs is linked to disease, especially carcinoma genesis. UCA1 (710) upregulated in bladder carcinoma, is a functional lncRNA molecule involved in cell growth, cell cycle, cell invasion and tumorigenesis. LncRNA AFAP1-AS1 (11) is hypomethylated and upregulated in Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC) tissues and cell lines. Inhibition of its expression in EAC cells was able to diminish cell growth, migration, and invasion, as well as increase apoptosis. MALAT1 (12) is first discovered in non-small cell lung cancer and its over-expression associates with high metastatic potential and poor patient prognosis in variety of cancer. A recent report showns that the promoter hypermethylation silencing of tumor suppressor PCDH10 contributed to MALAT1 upregulation in endometrioid endometrial cancer (EEC) (13).

The mechanism of endometrial carcinoma genesis is complex and related with multiple gene mutation and deregulation. The exploration of gene expression profile, especially the potential functional lncRNA will help to gain better knowledge and to discover new therapeutic candidates for endometrial carcinoma. We performed a microarray analysis to study the expression of lncRNAs and coding genes in 3 endometrial carcinomas and their paired adjacent non-tumor tissues. Our result demonstrated that the expression profile of lncRNAs is significantly different between endometrial carcinomas and non-tumor endometrium. The novel ASLNC04080 lncRNA is upregulated in endometrial carcinomas and HEC-1-B cell line. Additionally the deregulation of coding genes was also detected in endometrial carcinomas, and these deregulated coding transcripts were involved in multiple biological processes, cellular components, molecular functions and pathways which were related to carcinogenesis and cancer progression. We constructed a co-expression network of lncRNAs and coding gene transcripts based on the expression level relation between lncRNAs and coding gene transcripts. According to the co-expression network ASLNC04080’s expression was correlated to 19 coding transcripts, showing a potential co-regulation function of lncRNA ASLNC04080. Taken together, these results suggest the altered expression levels of lncRNAs may contribute to endometrial carcinomas genesis and multiple molecular processes. In addition, ASLNC04080 could be a functional lncRNA molecule with potential use as biomarker or therapeutic target.

Materials and methods

Patient samples

All human endometrial carcinomas and their paired adjacent nontumor tissues were obtained from patients of the First Affiliated Hospital, School of Medicine of Xi’an Jiaotong University. Tissue samples were collected with informed consent from patients, as approved by the Hospital Ethics Committees. All tissue samples were pathologically confirmed. Three pairs of these patient samples were randomly selected for human lncRNA microarray analysis.

DNA microarray

RNA quality was assessed by Nanodrop ND-1000 and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. The RNA extraction was performed by KangChen Bio-tech, Shanghai, China.

The Human 12×135 k Long Non-coding RNA Array was manufactured by Roche NimbleGen. Each array represents all long transcripts, both protein coding mRNAs and lncRNAs (long non-coding RNAs) in the human genome. More than 23000 lncRNAs are collected from the authoritative data sources including NCBI RefSeq, UCSC, RNAdb, lncRNAs from literature and UCRs. The microarray analysis was performed by KangChen Bio-tech.

RNA labeling and array hybridization

Double-strand cDNA (ds-cDNA) was synthesized from 5 μg of total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the Nimblegen Gene Expression Analysis protocol (Nimblegen Systems, Inc., Madison, WI, USA). Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a nanodrop ND-1000. For Cy3 labeling of cDNA, the Nimblegen One-Color DNA labeling kit was used according to the manufacturer’s guideline detailed in the Gene Expression Analysis protocol (Nimblegen Systems, Inc.). ds-cDNA (1 μg) was incubated for 10 min at 98°C with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100 units of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added and the mix incubated at 37°C for 2 h. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation. Microarrays were hybridized at 42°C for 16–20 h with 4 μg of Cy3 labelled ds-cDNA in Nimblegen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - Nimblegen Systems, Inc.). Following hybridization, washing was performed using the Nimblegen Wash Buffer kit (Nimblegen Systems, Inc.). After being washed in an ozone-free environment, the slides were scanned using the Axon GenePix 4000B microarray scanner. The microarray analysis was performed by KangChen Bio-tech.

Data analysis

Slides were scanned at 5 μm/pixel resolution using an Axon GenePix 4000 B scanner (Molecular Devices Corp.) piloted by GenePix Pro 6.0 software (Axon). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and mRNA level (*_RMA.calls) files were generated after normalization. All mRNA level files were imported into Agilent GeneSpring Software (version 11.0) for further analysis. Differentially expressed lncRNAs and mRNAs were identified through Fold Change filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 11.0). GO analysis and Pathway analysis was performed using the standard enrichment computation method. The analysis was performed by KangChen Bio-tech.

GO analysis

The Gene Ontology project provides a controlled vocabulary to describe gene and gene product attributes in any organism (http://www.geneontology.org). The ontology covers three domains: Biological Process, Cellular Component and Molecular Function. Fisher’s exact test is used to find if there is more overlap between the DE list and the GO annotation list than would be expected by chance. The p-value denotes the significance of GO terms enrichment in the DE genes. The lower the p-value, the more significant the GO term (p≥0.05 is recommended).

Pathway analysis

Pathway analysis is a functional analysis mapping genes to KEGG pathways. The p-value (EASE-score, Fisher p-value or Hypergeometric-p-value) denotes the significance of the Pathway correlated to the conditions. The lower the p-value, the more significant the pathway is (the recommend p-value cut-off is 0.05).

Co-expression network

We constructed a coding-noncoding gene co-expression network to investigate the relation between lncRNAs and their coding genes, 6 lncRNAs up- or down-regulated in endometrial carcinoma tissues were selected to draw the network. i) The data were preprocessed by using the median gene expression value of all transcripts expressed from the same coding gene, without special treatment of the lncRNA expression value. ii) Then the data were screened for differentially expressed lncRNAs and mRNAs and removed from the dataset. iii) The R-value was used to calculate the correlation coefficient of the PCC between lncRNA and coding genes (only lncRNA-coding PCC, not including lncRNA-lncRNA or coding-coding PCC). iv) Based on Pearson’s correlation coefficient selecting PCC ≥0.95 as meaningful related pair v) The co-expression network was drawn using Cytoscape. In the network, a round node represents the coding gene, a box node represents the lncRNA, a red node represents an upregulated lncRNA/mRNA and a green node represents an under-regulated lncRNA/mRNA. A red solid line indicates a positive correlation, and a blue dashed line indicates a negative correlation.

RNA extraction and cell culture

Total RNA from tissue samples, whole blood and cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and integrity were determined by spectrophotometry and standard RNA gel electrophoresis.

The human endometrial carcinoma (HEC-1-B); cervical carcinoma (Siha HeLa) and ovarian cancer (3AO SKOV3) cell lines were cultured in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% bovine calf serum. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.

Expression analysis of ASLNC04080 by RT-PCR and qRT-PCR

Total RNA isolated from tissue samples, whole blood and cells was reverse transcribed by using PrimeScript™ RT Reagent kit with gDNA Eraser (Takara, Dalian, China). Takara Taq™ (Takara) was used for 32 PCR cycles, annealing temperature was 65°C. SYBR® Premix Ex Taq™ was used for real-time PCR, annealing temperature was 61°C. Primer sequences were as follows: ASLNC04080: forward primer, 5′-CGCTATGTGTGGTGCCTGGGGTG-3′ and reverse primer, 5′-CAGCGCCTGAGTGGGTTTCGG-3′; 18S: forward primer, 5′-GCTCAGCGTGTGCCTACCCTAC-3′ and reverse primer, 5′-GTAGTAGCGACGGGCGGTGTGTA-3′.

Rapid amplification of cDNA ends (5′- and 3′-RACE)

One microgram of total RNA of whole blood was purified further by treating with RNase-Free DNaseI (Takara), then reverse transcribed with the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA) according to manufacturer’s instructions. Specific 5′- and 3′-RACE cDNA ends were amplified with the universal primer mix provided in the kit and gene specific primers (GSPs) with the advantage 2 PCR polymerase mix (Clontech). The PCR products were subcloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and several recombinant clones were isolated for sequencing. The GSP sequences: 5′RACE Gene-Specific Primers (Out Primer 5′-AGCGCCTGAGTGGGTTTCGG-3′; Inner Primer 5′-GGGGGTCATACTCCCCGAAAGG-3′). 3′RACE Gene-Specific Primers (Out Primer 5′-TTAATAGATTAGGCACAGGATGGGT-3′; Inner Primer 5′-CAGGATGGGTGTTTAATTCTCGGCAA-3′).

Amplification of truncated full length cDNA sequence

Total RNA isolated from whole blood and cells was reverse transcribed by using PrimeScript II 1st Strand cDNA Synthesis kit (Takara). The Takara LA Taq® (Takara) was used for 30 cycles PCR amplification, annealing temperature was 62°C. The truncated full length cDNA sequence primers: forward primer 5′-ACCGCACCCGGCAGTAGTAC-3′, reverse primer 5′-ATTGATCACCTCTGAAGTTCAGTAGCA-3′. The PCR products were subcloned into pGEM-T Easy vector (Promega) and several recombinant clones were isolated for sequencing.

Small interfering RNA transfection

Three different small interfering RNAs (siRNA) targeted ASLNC04080 (si-RNA616, si-RNA1315, si-RNA1535; (Gene Pharma, Shanghai, China) and nonsense siRNA control were transected to HEC-1-B cells by X-tremeGENE siRNA Transfection Reagent (Roche, Mannheim, Germany). The sequences of the 3 siRNAs were as follows: si-RAN616: 5′-CAGGGUUCAUUUCCAGACU-3′; si-RNA1315: 5′-GGCGUACUUAAGCAGAUGA-3′; si-RNA1535: 5′-GGAGUUGGGACAAUCUCUA-3′. The knockdown effect of siRNA was detected by qRT-PCR.

Cell proliferation assays

HEC-1-B cells were plated 3000 cells per well onto 96-well plates, after 24 h siRNA1535 and nonsense siRNA control were transfected. Every 24 h cell proliferation reagent CCK8 (Dojindo, Kumamoto, Japan) was added 10 μl per well and then incubated at 37°C for 2 h. Optical density was measured at 450 nm using a microplate reader (EnSpire, USA), and the proliferation activity curve was drawn.

Cell apoptosis assays

HEC-1-B cells were plated at 15×104 cells per well into 6-well plates. After 48 h of treatment with siRNA-1535 and nonsense siRNA control, HEC-1-B cells were harvested and stained with Annexin V and PI using Annexin V-FITC/PI apoptosis detection kits (Beyotime, Haimen, China) and then examined by flow cytometry (BD FACSCantoII, BD Biosciences, San Jose, CA, USA). Cellular proteins were extracted 48 h after siRNA transfection. Caspase-3 (Cell Signaling Technology, Danvers, MA) expression was detected by western blotting.

Cell cycle analysis

HEC-1-B cells were plated at 15×104 cells per well into 6-well plates. After 48 h of treatment with siRNA1535 and nonsense siRNA control, HEC-1-B cells were harvested, washed with ice-cold phosphate-buffered saline, fixed with 70% ethanol overnight, and pretreated with 5 mg/ml ribonuclease for 30 min at 37°C and then stained with PI (100 μg/ml). Cell cycle profile was determined by flow cytometry (BD FACSCanto II, BD Biosciences) analysis of DNA content of cell nuclei.

Results

ASLNC04080 is upregulated in endometrial carcinoma and cell lines

We performed a microarray analysis of lncRNA in 3 paired endometrial carcinoma and adjacent non-tumor tissues. In the lncRNA expression profiling data, a total of 23,837 lncRNAs expressed in endometrial carcinoma were detected. A comparison of lncRNA expression level between endometrial carcinoma and adjacent non-tumor tissues identified 53 lncRNAs which were significantly differentially expressed (fold change ≥2.0, p≤0.05) (Fig. 1A and C and Table I). ASLNC04080 was the most upregulated lncRNA (fold change =3.38, p=0.038), and CD109474 was the most downregulated lncRNA (fold change =4.72, p=0.034) in the endometrial carcinoma group.

Table I

Differentially expressed lncRNAs with >2-fold change in 3 paired endometrial carcinoma tissues (C) vs. adjacent non-tumor tissues (N).

Table I

Differentially expressed lncRNAs with >2-fold change in 3 paired endometrial carcinoma tissues (C) vs. adjacent non-tumor tissues (N).

SEQ_IDLog2 fold change C/NRegulation
ASLNC002942.5359635Up
ASLNC022392.0272071Up
ASLNC040803.3796694Up
ASLNC062452.5081081Up
ASLNC065372.5098338Up
ASLNC088842.236056Up
ASLNC091302.2931793Up
ASLNC095072.4517279Up
ASLNC102472.355146Up
ASLNC124962.2451406Up
ASLNC144622.097873Up
ASLNC148322.020886Up
ASLNC155272.5785253Up
ASLNC170472.3866124Up
ASLNC174642.1540515Up
ASLNC230652.1494098Up
ASLNC233332.0054722Up
ASLNC234142.4249542Up
ASLNC236712.4036572Up
AV7320452.1128297Up
BC1465942.3366451Up
BF4481782.5298557Up
BF9587402.079252Up
BI0464822.8131595Up
BI5202652.009207Up
BX6429243.1237326Up
BX6489122.0242465Up
DA1956062.1546383Up
DB2694432.336296Up
DB3352542.1803222Up
DB3417242.82081Up
DB3497012.6501746Up
H442332.1850893Up
exon24392.012377Up
exon28172.0926008Up
exon34972.1918285Up
exon4182.4487195Up
exon46522.4781675Up
exon8442.45636Up
ASLNC000382.559887Down
ASLNC000872.1803436Down
ASLNC041172.09159Down
ASLNC051932.5719202Down
ASLNC054852.8765132Down
ASLNC095912.8102934Down
ASLNC102232.6993935Down
ASLNC109152.2386236Down
ASLNC141862.162685Down
ASLNC183942.2218604Down
ASLNC215132.0064554Down
BF5077082.3711028Down
CD1094744.7178984Down
HMlincRNA14542.032924Down

[i] P-values <0.05 were considered significant.

The RT-PCR result showed ASLNC04080 expression difference in 3 paired endometrial carcinoma and adjacent non-tumor tissues which we used for microarray analysis (Fig. 2B). We evaluated the expression level of lncRNA ASLNC04080 in 24 paired endometrial carcinoma and adjacent non-tumor tissues as well as gynecological cancer cell lines. In our result ASLNC04080 expression level in endometrial carcinoma tissues was upregulated in 22 out of 24 paired samples (Fig. 2A). The ASLNC04080 transcripts were also detected in endometrial carcinoma (HEC-1-B), cervical carcinoma (Siha HeLa) and ovarian cancer (3AO SKOV3) cell lines (Fig. 2C). Additionally, according to UCSC information, ASLNC04080 was expressed in a series of lymphocytes (Fig. 2D). We detected the expression of ASLNC04080 in whole blood from both endometrial carcinoma patients as well as healthy people (Fig. 2E).

Sequence structure of ASLNC04080

The 5′RACE and 3′RACE were performed to acquire 5′-end and 3′-end cDNA sequence of ASLNC04080 (Fig. 3A). According to the overlapping region, we spliced 5′-end (744 bp) and 3′-end (1488 bp) sequence forming the full length cDNA sequence (1867 bp) (Fig. 3B). The sequence message was submitted to NCBI, GeneBank, Accession no. KJ782215. The ASLNC04080 cDNA sequence identity is 99.5% of the part human chromosome 1 cosmid (chr1: -28905061 - -28909492) sequence, by mapping with Blat search program from UCSC. The full length ASLNC04080 cDNA contained 6 exons mapped with the 1 p35.3 (Fig. 3C), and the identity is 99% (Query cover 39%) of the SNHG12.

The truncated full length cDNA sequence (184-1838, 1655 bp) was acquired from human whole blood and endometrial carcinoma cell line HEC-1-B (Fig. 3D), and identity of 99% (Query cover 88%) of the full length ASLNC04080 cDNA (KJ782215).

Coding gene expression profile in endometrial carcinoma

The microarray analysis also included information on the coding gene. A total of 18,738 coding transcripts (mRNA) could be detected in the 3 paired tissue samples. Compared the mRNA expression level between endometrial carcinoma and adjacent non-tumor tissues, there were 46 mRNA differentially expressed; of those 26 were upregulated and 20 were downregulated in the endometrial carcinoma group (fold change ≥2.0, p≤0.05) (Fig. 1B and D; and Table II).

Table II

Differentially expressed mRNAs with >2-fold change in 3 paired endometrial carcinoma tissues (C) vs. adjacent non-tumor tissues (N).

Table II

Differentially expressed mRNAs with >2-fold change in 3 paired endometrial carcinoma tissues (C) vs. adjacent non-tumor tissues (N).

Gene symbolLog2 fold change C/NRegulation
COL1A22.1063402Up
PLA2G1B2.4415922Up
OR1S12.391917Up
GEMIN72.4388766Up
C10orf1142.1364949Up
FIGNL23.4194834Up
IL312.0702891Up
FAM23A2.0272226Up
ACOX32.8613539Up
NKPD12.0944095Up
FAM148C2.1054876Up
AKR1C12.4084988Up
TLE22.0259166Up
CDH72.0604987Up
HOXC62.1476102Up
C9orf612.0307186Up
KIR2DL12.1328676Up
TINAG2.2404068Up
MGRN12.1519182Up
CCR103.6094117Up
EMID22.6075573Up
BAX2.5370033Up
DAND52.2088263Up
HOXC62.1748328Up
ZBTB122.3850377Up
SCN1B2.1365902Up
GCH12.2663758Down
H2BFWT2.1060047Down
DDAH12.0587702Down
LOC1203762.1094232Down
CRYM3.8512769Down
RDH52.0616512Down
SLC4A42.5983744Down
EGR32.0082827Down
TSPAN82.9661324Down
APBA32.10935Down
XBP12.010461Down
SERPINB32.4469104Down
KRT232.4278355Down
SERTAD42.100912Down
KIAA13243.5217378Down
SLC38A52.3809822Down
PARP152.4587605Down
CACNB23.3419204Down
ZNF3202.033858Down
PRSS122.3177733Down

[i] P-values <0.05 were considered significant.

With Pathway and Gene Ontology analysis we found that these deregulated coding genes were involved in multiple pathways and gene ontology. Pathway analysis indicated that 12 pathways corresponded to upregulated transcripts (Table III), and 12 pathways corresponded to downregulated transcripts (Table IV). Ras signaling pathway corresponded to 15 transcripts from both up- and down-regulated data (Tables III and IV).

Table III

Upregulated coding gene transcripts corresponding to 12 pathways.

Table III

Upregulated coding gene transcripts corresponding to 12 pathways.

Pathway IDDefinitionFisher - P-valueGenes
hsa05032Morphine addiction - Homo sapiens (human)0.004254111 ADORA1//GABRB1//GABRQ//PDE3A//PRKCG
hsa00512Mucin type O-Glycan biosynthesis - Homo sapiens (human)0.00528077 GALNT13//GALNTL6//ST3GAL2
hsa04972Pancreatic secretion - Homo sapiens (human)0.005316287 ATP2A1//ATP2B3//PLA2G1B//PRKCG//PRSS2
hsa04974Protein digestion and absorption - Homo sapiens (human)0.01848372 COL12A1//COL1A2//COL4A6//PRSS2
hsa04672Intestinal immune network for IgA production - Homo sapiens (human)0.01969566 CCR10//CCR9//CD86
hsa00140Steroid hormone biosynthesis - Homo sapiens (human)0.02776873 AKR1C1//CYP19A1//UGT2B28
hsa04510Focal adhesion - Homo sapiens (human)0.03121468 COL1A2//COL4A6//MYLPF//PAK4//PRKCG//VEGFC
hsa00592α-Linolenic acid metabolism - Homo sapiens (human)0.03328378ACOX3//PLA2G1B
hsa04950Maturity onset diabetes of the young - Homo sapiens (human)0.03328378HNF4A//NKX2-2
hsa05211Renal cell carcinoma - Homo sapiens (human)0.04036226 GAB1//PAK4//TCEB2
hsa00514Other types of O-glycan biosynthesis - Homo sapiens (human)0.04651509GLT25D2//RFNG
hsa04014Ras signaling pathway - Homo sapiens (human)0.0466148 ANGPT4//GAB1//PAK4//PLA2G1B//PRKCG//VEGFC

Table IV

Downregulated coding gene transcripts corresponding to 12 pathways.

Table IV

Downregulated coding gene transcripts corresponding to 12 pathways.

Pathway IDDefinitionFisher - P-valueGenes
hsa04015Rap1 signaling pathway - Homo sapiens (human)0.002010251 CSF1//FGF13//GNAQ//HGF//ITGAM//ITGB1//KITLG//MLLT4//PIK3CB
hsa05152Tuberculosis - Homo sapiens (human)0.00262324 ARHGEF12//ATP6V0A2//CYP27B1//HLA-DOA//IL10//ITGAM//NFYC//PIK3C3
hsa04014Ras signaling pathway - Homo sapiens (human)0.003095239 CSF1//ETS1//FGF13//HGF//KITLG//MLLT4//PIK3CB//PLA2G4A//SHC1
hsa05146Amoebiasis - Homo sapiens (human)0.003198103 COL5A2//GNAQ//IL10//ITGAM//PIK3CB//SERPINB3
hsa00380Tryptophan metabolism - Homo sapiens (human)0.0158507 ACMSD//KYNU//OGDHL
hsa05140Leishmaniasis - Homo sapiens (human)0.01747982 HLA-DOA//IL10//ITGAM//ITGB1
hsa04270Vascular smooth muscle contraction - Homo sapiens (human)0.03014367 ARHGEF12//GNAQ//GUCY1A3//KCNU1//PLA2G4A
hsa04970Salivary secretion - Homo sapiens (human)0.03164653 ATP1A2//GNAQ//GUCY1A3//STATH
hsa04912GnRH signaling pathway - Homo sapiens (human)0.03392592 GNAQ//GNRH1//MAP3K3//PLA2G4A
hsa04964Proximal tubule bicarbonate reclamation - Homo sapiens (human)0.03706998ATP1A2//SLC4A4
hsa05150Staphylococcus aureus infection - Homo sapiens (human)0.04175273 HLA-DOA//IL10//ITGAM
hsa04730Long-term depression - Homo sapiens (human)0.04544242 GNAQ//GUCY1A3//PLA2G4A

Gene ontology (GO) analysis was preformed to show that the differently expressed mRNA transcripts were associated with biological processes (BP), cellular components (CC) and molecular function (MF) (Fig. 4). Progesterone metabolic process is one of the most frequent fold enrichment biological processes. There were 13 upregulated and 18 downregulated transcripts involved in biological process of wound healing. Deregulated transcripts were involved in the molecular function voltage-gated channel activity, including voltage-gated ion channel activity, voltage-gated channel activity, voltage-gated cation channel activity and voltage-gated calcium channel activity

Expression of ASLNC04080 correlates with the coding genes

Based on the correlation analysis between differently expressed lncRNAs and mRNAs, we constructed a coding-non-coding gene co-expression network. Four upregulated and 2 downregulated lncRNAs in endometrial carcinoma tissues were selected to draw the network. The expression of 289 mRNAs was related (Pearson’s correlation coefficients: PCC ≥0.95) to these 6 lncRNAs (Fig. 5).

ASLNC04080 expression level was correlated with 19 coding gene transcripts (Fig. 5). Besides, these coding genes were involved in multiple pathways and gene ontology. For example CCR10 was upregulated in endometrial carcinoma and positively correlated with ASLNC04080 expression (PCC >0.95), and participated in intestinal immune network for IgA production. In addition, SYCP2 negatively correlated with ASLNC04080 expression (PCC >0.95), involved in the pathway of the cell cycle. These results implicated that ASLNC04080 has an inter-regulation relation with the coding gene, and could be a potential functional molecule in endometrial carcinoma genesis and progression.

Inhibition of ASLNC04080 expression influences HEC-1-B cell proliferation, apoptosis and cell cycle

We used 3 different siRNAs to inhibit ASLNC04080 expression in human endometrial carcinoma cell line HEC-1-B. All 3 siRNAs were tested to have >50% reduction efficiency of ASLNC04080 expression in HEC-1-B, and siRNA-1535 could reduce by >80% the ASLNC04080 expression (Fig. 6A). Therefore, we performed several in vitro assays to determine the functional consequences of ASLNC04080 by inhibiting ASLNC04080 expression via siRNA-1535.

Compared with cells transfected with negative control siRNA, HEC-1-B cell proliferation was significantly suppressed within 48 h (Fig. 6B) by inhibiting ASLNC04080 expression. The cell apoptosis assays were examined by flow cytometry after HEC-1-B transfection with ASLNC04080 and negative control siRNA. Knockdown of ASLNC04080 expression increased apoptosis in HEC-1-B cells (NC vs. siRNA-1535: 15.9±0.66% vs. 26.9±1.27%, T-test P<0.05) (Fig. 6C). Moreover, caspase-3 protein cleavage was detected only in siRNA-1535 transfected cells (Fig. 6D). We performed cell cycle assays after siRNA transfection 48 h. Knockdown of ASLNC04080 expression induced G1 phase arrest (NC vs. siRNA-1535: 41.14±1.10% vs. 49.85±0.77%, t-test P<0.05) (Fig. 6E).

These findings suggest that the ASLNC04080 lncRNA could regulate endometrial carcinoma cell HEC-1-B proliferation, apoptosis and cell cycle.

Discussion

Molecular alterations in endometrial carcinoma have been studied for many years (14,15). Microsatellite instability (MI), and gene mutations (PTEN, K-RAS and PIK3CA) have been shown involved in type I endometrial carcinoma. Type II endometrial carcinoma exhibits mutations of p53, loss of heterozygosity (LOH), and molecular alterations (STK1 5, p16, and c-erb-B2). Recently a comprehensive, multiplatform analysis of 373 endometrial carcinomas identified new hotspot mutations in POLE, and based on the genomic characterization they classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high (16). The efforts on the integrated analysis of molecules not only help us to construct new tumor classifications, but also affect treatment recommendations for patients, provides opportunities for genome-guided clinical trials and drug development. Besides the known coding genes, noncoding RNAs act as a new hallmark for endometrial carcinoma diagnosis and therapy, have attracted wide attention. A large quantity of miRNAs target important genes in tumor development and progression has been identified in endometrial carcinoma (17). Hiroki et al (18), identified 120 miRNAs deregulated in endometrial serous carcinoma. Moreover, their results showed that microRNA expression is associated with clinical pathology and prognosis of patients with endometrial serous adenocarcinoma.

LncRNA has been confirmed to regulate gene expression in histone modification (19), regulation of transcription (20) and splicing, and plays an essential role in cellular proliferation, development and metabolism (21). The deregulation of lncRNA is associated with physiological disorders and disease development (22). Increased lncRNAs correlated with carcinogenesis and tumor progression have been discovered accompanied with sequencing and microarray technological development. A series of differentially expressed lncRNAs (lncRNA-HEIH, lncRNA-MVIH, lncRNA-LALR1, lncRNA-LET and lncRNA-Dreh) have been identified between HBV-related HCC and paired peritumoral tissues by microarray (2325). With further studies, these lncRNAs were verified to be functional molecules contributing to HCC tumor growth, angiogenesis, metastasis and serving as a predictor for HCC patients’ poor recurrence-free survival after hepatectomy. Although a systematic study of lncRNA in endometrial carcinoma is lacking, a few functional lncRNAs have been shown deregulated in endometrial carcinoma. LncRNA NCT25 mutations were observed in endometrial tumor specimens (in 23 of 48 of the samples), and could be a mutational target specifically in endometrial cancer. MALAT1 upregulation is the result of tumor suppressor PCDH10 silencing in endometrioid cancer (13). Therefore, more functional lncRNA could be related to endometrial carcinoma genesis and progression, and act as potential hallmark for endometrial carcinoma diagnosis and therapy.

To investigate the lncRNA expression profile of endometrial carcinoma, we performed a microarray analysis containing lncRNA and coding gene information in 3 paired endometrial carcinoma and adjacent non-tumor tissues. The microarray data showed a significant difference of lncRNA expression pattern between endometrial carcinoma and adjacent non-tumor tissues. A total of 53 lncRNAs (C vs. N: 39 up-, 14 down-regulation, fold change >2.0, p<0.05) were deregulated in endometrial carcinoma. ASLNC04080 is the most upregulated lncRNA in endometrial carcinoma (fold change: 3.379, p=0.03778). The expression level of ASLNC04080 is higher in endometrial carcinoma tissues (22/24) compared with non-tumor tissues. ASLNC04080 expression could also be detected in endometrial carcinoma cell line HEC-1-B as well as other gynecological cancer cell lines (Siha, HeLa, 3AO and SKOV3). ASLNC04080 is transcribed from the chr1: -28905061 - -28909492 loci (1 p35.3), consisting 6 exons 1,867nt in length.

According to the coding gene microarray data 46 mRNAs (C vs. N: 26 up-, 20 down-regulation, fold change >2.0, p<0.05) exhibited a different expression between paired endometrial carcinoma and adjacent non-tumor tissues. Previous studies demonstrated that PTEN mutations occur early in endometrial carcinogenesis and co-exist frequently with other deregulated molecules targeting AKT-PI3K-mTOR pathway. Through pathway analysis, we have found 6 up- and 9 down-regulated coding gene transcripts in endometrial carcinoma targeting Ras signaling pathway. Estrogen and progesterone accession and metabolism are related to endometrial carcinoma genesis and progression (26). In our data progesterone metabolic process is one of the most frequent fold enrichment biological processes. Two endometrial carcinoma upregulated coding gene transcripts CYP19A1 and AKR1C1 participated in this processes, and correlated with endometrial carcinoma genesis (27,28). Besides, 31 deregulated coding transcripts target the wound healing process. Growth evidence shows that voltage-gated channel activity alteration of carcinoma cell membrane is a characteristic of carcinoma cells (2931). In our results, the molecules target voltage-gated ion channel activity, voltage-gated channel activity, voltage-gated cation channel activity and voltage-gated calcium channel activity, displaying deregulation in endometrial carcinoma.

We constructed a coding-non-coding gene co-expression network (CNC), by analyzing the correlation of lncRNAs and the expression level of the coding gene transcripts. The expression of six selected lncRNAs were showed to be related to 289 coding gene transcripts. The BX642924 expression was correlated with 177 coding gene transcripts (Fig. 5), especially positively correlated to PRKCG (upregulated in endometrial cacionoma, PCC >0.98) and negatively correlated to KITLG (downregulated in endometrial cacionoma, PCC >0.0.95) expression. Both PRKCG and KITLG were involved in Ras signaling pathway, and have been showed related to tumor genesis and progression (32,33). According to these analyses we speculate that BX642924 may correlate with the Ras signaling pathway. The expression of three coding gene transcripts (ZNF275, PCDHGA8, PRSS21) was correlated with ASLNC02239 (upregulated in endometrial carcinoma) and ASLNC10223 (downregulated in endometrial carcinoma), showing a possible relation among these genes. More work is needed to confirm the relations and the underlying regulation mechanism of these coding and non-coding genes.

ASLNC04080 expression is correlated with 19 coding gene transcripts. KLK3 is the most positively related transcript with ASLNC04080 (PCC >0.97). Increasing evidence indicates that KLK3 (kallikrein-related peptidase 3) is implicated in carcinogenesis and acts as a biomarker or a diagnosis target in multiple types of cancer (34). Moreover, SYCP2 negatively correlates with ASLNC04080 expression (PCC >0.95), and have been shown to participate in the cell cycle pathway (35). Inhibition of ASLNC04080 expression in HEC-1-B cells, resulted in decreased cell proliferation, increased cell apoptosis and G1 phase arrest. Taking together, these findings suggest that ASLNC04080 is a functional lncRNA in human endometrial carcinoma and may contribute to carcinogenesis via interaction with other coding genes. More exploration of the function mechanism of ASLNC04080 is required.

To our knowledge, this is the first systematic research project of lncRNA expression profile in endometrial carcinoma. We found several lncRNAs expressed in endometrial carcinoma and correlated with multiple Gene Ontology and pathways involved in carcinogenesis. These findings could help us enrich the knowledge on the mechanism of endometrial carcinogenesis and find new diagnostic or therapeutic targets for endometrial carcinoma.

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May-2015
Volume 46 Issue 5

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Online ISSN:1791-2423

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Spandidos Publications style
Zhai W, Li X, Wu S, Zhang Y, Pang H and Chen W: Microarray expression profile of lncRNAs and the upregulated ASLNC04080 lncRNA in human endometrial carcinoma. Int J Oncol 46: 2125-2137, 2015.
APA
Zhai, W., Li, X., Wu, S., Zhang, Y., Pang, H., & Chen, W. (2015). Microarray expression profile of lncRNAs and the upregulated ASLNC04080 lncRNA in human endometrial carcinoma. International Journal of Oncology, 46, 2125-2137. https://doi.org/10.3892/ijo.2015.2897
MLA
Zhai, W., Li, X., Wu, S., Zhang, Y., Pang, H., Chen, W."Microarray expression profile of lncRNAs and the upregulated ASLNC04080 lncRNA in human endometrial carcinoma". International Journal of Oncology 46.5 (2015): 2125-2137.
Chicago
Zhai, W., Li, X., Wu, S., Zhang, Y., Pang, H., Chen, W."Microarray expression profile of lncRNAs and the upregulated ASLNC04080 lncRNA in human endometrial carcinoma". International Journal of Oncology 46, no. 5 (2015): 2125-2137. https://doi.org/10.3892/ijo.2015.2897