Expression profiling of lncRNAs and mRNAs in placental site trophoblastic tumor (PSTT) by microarray

Abstract

As a rare type of gestational trophoblastic disease, placental site trophoblastic tumor (PSTT) is originated from intermediate trophoblast cells. Long noncoding RNAs (lncRNAs) regulate numerous biological process. However, the role of lncRNAs in PSTT remains poorly understood. In the present study, expression levels of lncRNAs and mRNAs in four human PSTT tissues and four normal placental villi were investigated. The results of microarray were validated by the reverse transcription and quantitative real-time polymerase reaction (RT-qPCR) and immunohistochemistry analyses. Furthermore, GO and KEGG pathway analyses were performed to identify the underlying biological processes and signaling pathways of aberrantly expressed lncRNAs and mRNAs. We also conducted the coding-non-coding gene co-expression (CNC) network to explore the interaction of altered lncRNAs and mRNAs. In total, we identified 1247 up-regulated lncRNAs and 1013 down-regulated lncRNAs as well as 828 up-regulated mRNAs and 1393 down-regulated mRNAs in PSTT tissues compared to normal villi (fold change ≥ 2.0, p < 0.05). GO analysis showed that mitochondrion was the most significantly down-regulated GO term, and immune response was the most significantly up-regulated term. A CNC network profile based on six confirmed lncRNAs (NONHSAT114519, NR_103711, NONHSAT003875, NONHSAT136587, NONHSAT134431, NONHSAT102500) as well as 354 mRNAs was composed of 497 edges. GO and KEGG analyses indicated that interacted mRNAs were enriched in the signal-recognition particle (SRP)-dependent cotranslational protein targeting to membrane and Ribosome pathway. It contributes to expand the understanding of the aberrant lncRNAs and mRNAs profiles of PSTT, which may be helpful for the exploration of new diagnosis and treatment of PSTT.

Keywords: lncRNA; microarray analysis; placental site trophoblastic tumor (PSTT).

Figure 1

Differential mRNA and lncRNA expression of PSTT and villi by microarray. The volcano plot represents total identified mRNAs (A) and lncRNAs (B) expression between the PSTT and normal villi. The horizontal black lines show the default 2.0-fold change and the vertical black lines represent a P-value of 0.05. The red and blue plots represent up- and downregulated RNAs with FC ≥ 2.0 and P < 0.05. Heat map and hierarchical clustering analysis of the top 20 up- and downregulated mRNAs (C, E) and lncRNAs (D, F) between PSTT and normal villi.

Figure 2

Validation of the microarray results of mRNAs and lncRNAs by RT-qPCR and immunohistochemistry. RT-qPCR was performed to test the differentially expressed mRNAs (A) and lncRNAs (C) (n=4 for PSTT group and control group, respectively); the fold change of each mRNA (B) and lncRNA (D) between PSTT tissues and control group was determined microarray and RT-qPCR. The expression of PLAC8 and EGR1 was obviously higher than normal villi, while ADAMTS6 expression is much lower in PSTT compared to normal villi (F). ns, No significance; *, P < 0.05; **, P < 0.01, P < 0.001, Student t-test. Scale bar = 100μm.

Figure 3

GO and KEGG analysis of altered mRNAs. The GO analysis of down-regulated mRNAs (A) and up-regulated mRNAs (B). KEGG pathways showed TOP 10 significantly enriched pathways of down-regulated mRNAs(C) and up-regulated mRNAs(D).

Figure 4

CNC networks by validated lncRNAs. The CNC networks were performed by 6 validated lncRNAs and 354 interacted mRNAs. The CNC networks were composed of 497 edges and 360 nodes with 207 positive interactions (continuous lines) and 290 negative interactions (dotted lines). The green nodes represented down-regulated mRNAs and blue ones denoted up-regulated mRNAs.

Figure 5

GO and KEGG analyses of interacted mRNAs in CNC networks. GO (A) and KEGG (B) analyses of CNC interacted mRNAs