Genome-wide Analysis Of The Effect Of ΔS2 SF1b PRLR Overexpression On Micro Rna Expression In Human Breast Cancer Cells

As a variant of short form 1b prolactin receptor(SF1b PRLR),ΔS2 SF1 b PRLR,in which the extra-cellular subdomain S2 was naturally lost,was reported to be expressed mainly in human productive cancer cells such as human breast cancer and prostate cancer cells,etc.We hypothesized that the loss of this structure,may change the molecular characteristics of SF1 b PRLR,which may cause changes in downstream cell transduction pathways and related micro RNA expression profiles in signal pathways,thereby affecting the biological characteristics of breast cancer cells.The present investigation was designed to verify the effect of ΔS2 SF1 b PRLR on the proliferation and micro RNA expression of human breast cancer cells(MCF7),in order to compare it with its intact countpart,SF1 b PRLR,and to explore its potential role in the occurrence and development of breast cancer and its molecular regulatory mechanism.SF1b PRLR or Δ S2 SF1 b PRLR c DNAs were recombined separately into lentivirus by using homologous recombination strategy,and then the prepared active virus particles carrying different genes including SF1 b PRLR or Δ S2 SF1 b PRLR c DNAs,or not carrying any target gene(Control)were transfected into cultured breast cancer cells(MCF7)separately.Stable cell lines including MCF7-ΔS2 SF1 b PRLR(ΔS2 SF1b),MCF7-SF1 b PRLR(SF1b)and MCF7-Con(Con)were achieved by multipal screening using puromycin.After a 48 h incubation in the presence of prolactin(PRL),the natural ligand of the receptor,total RNA for the later high-throughput sequencing was extracted by using TRIzol reagent,and the bio-informative or “-omic” analysis of the micro RNA expression spectrum alteration induced by this newly cloned variant prolactin receptor,as well as its intact counterpart,SF1 b,was performed in a genome-wide way.A separate set of cells were cultured and subjected to proliferation assay by using MTS reagent accordingly in order to clarify the change of the biological function because of the loss of the S2 subdomain.As results,the proliferation assay showed that:(1)overexpression of ΔS2 SF1 b significantly promoted proliferation of the cells(by 66.35%),the OD(492 nm)values of the control and ΔS2 SF1 b cells were 1.04 ± 0.11 and 1.73 ± 0.11 respectively(P<0.05).(2)The OD(492nm)value of ΔS2 SF1 b cells was obviously higher than that of its intact counterpart,and in SF1 b overexpressing cells,the OD(492 nm)value was 1.44 ± 0.14(P<0.05),implying the cell pro-propagation effect of ΔS2 SF1 b was significanly improvedby the loss of the extracellular subdomain S2.The sequencing result showed that ΔS2 SF1 b overexpression significantly altered the expression spectrum of micro RNA in cultured human breast cancer cells(MCF7).(1)There were 96 types of mic RNAs expressed only in the presence of Δ S2 SF1 b overexpression,whereas absolutely not expressed in control and SF1 b expressing cells.50 types of mic RNAs expressed only in the SF1 b cells,not in other two groups of cells.(2)In comparison with that of the control cells,245 and 206 types of micro RNA were increased(p≤0.05),whereas 304,277 decreased(p≤0.05)in SF1 b and ΔS2 SF1 b overexpressing cells respectively.(3)ΔS2 SF1 b played a very different,role from that of SF1 b in the regulation of expression of several types of micro RNA such as hsa-mi R-125a-5p＿R-1,hsa-mi R-99b-5p,hsa-mi R-27b-5p,hsa-let-7c-5p,hsa-mi R-24-3p＿R-2,hsa-mi R-27a-3p＿R-1,hsa-mi R-139-5p,and more importantly,ΔS2 SF1 b even played an opposite role of SF1 b in the control of expression of several types of micro RNA,for example,ΔS2 SF1 b promoted,whereas SF1 b,inhibited the expression of hsa-mi R-24-3p＿R-2,hsa-mi R-27a-3p＿R-1.GO function and KEGG enrichment analysis showed that the differentially expressing micro RNAs mainly targeted to the cancer-related genes,such as gene of MAPK,PI3K-Akt,and AMPK,m Tor,insulin,Fox O,Hippo,Erb B,TNF and other signaling pathways.(4)The prediction analysis of mic RNA revealed that:(1)the expression of PC-3p-2776＿684,PC-5p-3033＿620,etc.were significantly increased in case of ΔS2 SF1 b,but not of,SF1 b overexpression;(2)PC-5p-52538＿16,PC-5p-51117＿17 PC-5p-75774＿8 were expressed at a low level in the ΔS2 SF1 b overexpressing cells,and was not expressed at all in the SF1 b and control cells;(3)PC-5p-33907＿35As were always expressed with mi R-139,an anticancer related element,in case of overexpression of SF1 b in a partnership style,however,interestingly,both of them were disappeared(no expression)in the ΔS2 SF1 b overexpressing cells,and this phenomenon accounted,at least partly,for the different role of ΔS2 SF1 b and SF1 b in the control of cell growth.As a summary,we concluded that:(1)The lack of the extracellular domain S2 led to the weakening of the pro-proliferation(MCF7)of SF1 b,indicating that S2 subdoain is an critical structure of the prolactin receptor molecule.(2)Overexpression of ΔS2SF1b or SF1 b induced significant alteration of the expression of micro RNA in human breast cancer cells(MCF7),in comparason with the control cells,more importantly,ΔS2 SF1 b and SF1 b demonstrated very different natures and roles in the regulation of micro RNA expression in human breast cancer cells,indicating the importance and significance of the extracellular subdomain S2 for the bio-function,as well as the pathphysiological role in human breast cancer,of the receptor molecule.