| As a single-pass transmembrane receptor belonging to member of type I cytokine receptor superfamily,prolactin receptor(PRLR) plays a critical role in cellular signal transduction initiated by its ligand prolactin(PRL). ?S2 PRLR is a variant of PRLR, firstly cloned and reported by Tan et al, in which, the S2 subdomain(100 aa) of the extracellular domain is missing(the other subdomain is S1). In the presence of its ligand, PRL, PRLR on the plasma membrane will be dimerized because each PRL has two binding sites for PRLR, and the cellular signal transduction will be followed immediately. A sizable body of preliminary experiments showed that ?S2 PRLRs were able to dimerize in the absence of PRL(constitutive dimerization) and to induce proliferation of human breast cancer cells(MCF-7, T47D) implying its pathogenetic significance in human breast cancer.A series of biochemical event will be followed after PRLR dimerization, such as recruitment of JAK2, phosphorylation of PRLR and STAT5, dimerization of phosphrylated STAT5, nuceus translocation of STAT5 dimer and binding to the specific promoter region and initiating gene expression(JAK2-STAT pathway). We hypothesis that the function-alteration of ?S2 PRLR due to the missing of S2 subdomain will change the transcripome of breast cancer cells(its target gene spectrum and the expressions).In the present study, cultured human breast cancer cells(MCF-7) were transfected with adenovirus carrying LF-PRLR(LF-PRLR cells) or ?S2 PRLR(?S2 PRLR cells) cDNA or not carrying any external DNA(Con). After a futher 48h- incubation of the cells, total RNAs were extracted by using TRIzol reagent and followed by RNA-sequencing(RNA-Seq).The RNA-Seq data were up-loaded onto genomebrowser setup by University of California Santa Cruz(genome.ucsc.edu) and a custome specific channel was used. The alignment and analysis shows:(1) the genes significantly affected by ?S2 PRLR over-expression are those located at chromesome 11,12,14,16,17,19?20,but not at 4?13?18,21.(2) the genes regulated by ?S2 PRLR includes not only encoding genes, but also non-coding ones including microRNAs and small nucleolar RNAs, etc. 19648(including 17594 encoding genes and 2054 non-coding genes), 20247(including 18093 encoding genes 2154 non-coding genes) and 19500 genes(including 17472 encoding genes and non-coding 2028 genes) were detected in Con, LF-PRLR and ?S2 PRLR cells respectively in the present RNA-Seq.(3) Of the whole transcriptome, 192 genes were only detected in ?S2 PRLR cells,but not in LF-PRLR and Con cells; however, other 483 genes were expressed in LF-PRLR Con cells, but not in ?S2 PRLR cells; and other 18924 genes were detected in all the three groups of cells.(4) several tumour-associated genes(27 genes) were observed to be expressed in ?S2 PRLR cells,but not in LF-PRLR and Con cells.(5) nine apoptosis-associated genes were detected only in ?S2 PRLR cells, but not in LF-PRLR and Con cells.(6) MicroRNA were also the regulated target of ?S2 PRLR. Expression of eight microRNA genes were detected exclusively in ?S2 PRLR cells, but not in other two groups of cells.(7) ?S2 PRLR plays roles in the regulation of small nucleolar RNA(snoRNA) expression. Ten snoRNAs were detected only in ?S2 PRLR cells, but not in the other two groups of cells.Taking together, we concluded that(1) genes affected by ?S2 PRLR includs not only the encoding but also the noncoding ones;(2) the effect of ?S2 PRLR on transcriptome of breast cancer cells are different from that of its intact counterpart, LF-PRLR;(3) of the genes affected by ?S2 PRLR, tumour-associated genes, apoptosis-associated genes, microRNA and snoRNA are all included.In the present study, we firstly investigated the effect of the newly reported variant of prolactin receptor(?S2 PRLR) on the transcriptome of human breast cancer cells(MCF-7) by using RNA-sequencing. The result will greatly facilitate the understabding of the molecular mechanisms underlaying the disease. |
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