A Study On XBP1mRNA Involved In The Progress Of Breast Cancer

Objectives:Alleviating the endoplasmic reticulum (ER) stress-associated unfolded protein response is indispensable for survival of eukaryotic cells. As a key downstream transcriptional factor involved in unfolded protein response, XBP1triggers cascades to attenuate mammal ER stress, and the regulation of its expression involves an unconventional mRNA splicing that removes26nucleotides intron and is conducted by the endonuclease IRE la and an unknown RNA ligase. In contrast to the conventional splicing that exclusively takes place in the nucleus, the location of unconventional splicing still remains controversial. Moreover, it is also unclear whether the unconventional splicing of XBP1mRNA could be activated by a pathway independent of unfolded protein response.Using breast cancer cell line as the model, we investigate the location of unconventional splicing of XBP1mRNA splicing and reveal that the unconventional splicing of XBP1mRNA canbe activated in the nucleus by a pathway independent of ER stress. Dissecting the mechanism underlying XBP1mRNA unconventional splicing may be helpful to find out a new therapeupitcal target for breast cancer.Methods:1. We estimated the amount of XBP1s mRNA in breast cancer cell lines using RT-PCR.2. We constructed an ER stress indicator system (ER-stress activated indicator, ERAI), then expressed it stably in MCF-7cells to study mechanism underlying the unconventional splicing of XBP1mRNA.3. We treated MCF-7/ERAIm454-557cells with1mM DTT for30minutes, and then assayed the spliced mRNAs of ERAIm454-557and XBP1in the absence or continued presence of DTT at intervals.4. We knockdowned IRE la to test whether ER stress-independent splicing of ERAIm454-557mRNA also required IRE1α, then detected the location of IRE1α by immunofluorescent experiments. 5. We separated nuclear and cytoplasmic fractions of MCF-7/ERAIm454-557and extracted their total RNA to compare the difference between the splcing of XBP1and ERAIm454-557mRNA, and identified the subcellular localization of the unconventional splicing of mRNA.6. We used actinomycin D (Act D) to block transcription in MCF-7/ERAIm454-557cells, and then the splicing in thenuclear and cytoplasmic fractions using RT-PCR analyses.We treated MCF-7/wt cells with a high concentration of DTT (20mM) for the time course analysis of XBP1mRNA splicing in the nucleus and cytoplasm.7. We generated different constructs containing XBP1deletion fragments fused with mCherry at N-terminus, stably expressed these mCherry-tagged XBP1deletions in MCF-7cells, and then analyzed their unconventional splicing.8. We generated several constructs containing distinct sequences including26nt intron, stably expressed these genes, respectively, in MCF-7cells and measured their splicing in the absence of ER stress.9. We loaded the total cytoplasmic RNA of MCF-7cells after DTT treated, then detected the degradation of XBP1s mRNA that increased with time.10. We stably expressed two shRNA constructs against XBP1in MCF-7/ERAIm454-557using lentivirus to test their effects on the spliced ERAIm454-557mRNA in the absence or presence of ER stress induction.11. Soft Agar assays were performed to detect the effect of XBP1s in cell proliferation and clone growth.Results:1. Both transformed and non-transformed breast cells had the basic spliced XBP1mRNA in the absence of ER stress induction.2. Screened an ideal tool to study the basic splicing of XBP1mRNA without ER stress induction.3. The splicing of endogenous XBP1mRNA was much more sensitive to ER stress induction than ERAIm454-557mRNA.4. The knockdown of IRE1α significantly reduced the basic spliced mRNA of ERAIm454-557, suggesting the dependence of ER stress-independent splicing of ERAIm454-557mRNA on IRE1α.The immunofluorescent imaging results showed that DTT treatment significantly induced the nuclear translocation of IRE1α.5. The spliced mRNA of ERAIm454-557was found in the nuclear fraction in the absence of ER stress induction. In the ER stress condition, the spliced mRNAs of both ERAIm454-557and XBP1significantly increased in the cytoplasm but not in the nucleus.6. In the normal condition, Act D did not induced significant splicing of XBP1mRNA, but it increased the ratio of spliced ERAIm454-557mRNA in the nucleus. Act D largely enhanced the ratio of spliced mRNAs of both XBP1and ERAIm454-557in the presence of0.5mM of DTT.Upon the induction of ER stress by DTT, the spliced XBP1mRNA dramatically increased to the saturation level in the cytoplasm while gradually enhanced over time in the nucleus.This suggested that ER stress promoted ER stress-independent splicing of XBP1mRNA in the nucleus.7. The deletion of5’-nucleotides significantly increased the splicing of XBP1mRNA in the absence of ER stress induction.8. ERAIm485-530that only contained stem-loop pair sequence still had a weak splicing in the absence of ER stress. This was confirmed by the fluorescent image, suggesting the presence of spliced ERAIm485-530, and the ER stress-independent splicing of ERAIm485-530mRNA occurred in the nucleus.9. The spliced XBP1mRNA in cytoplasm induced by DTT was susceptible to degradation that increased with time.10. RT-PCR results showed that the two shXBP1constructs did not apparently reduced the level of XBP1mRNA in the absence of ER stress. Interestingly, shXBP1constructs significantly inhibited the splicing of XBP1mRNA upon ER stress induced by DTT, and they decreased spliced XBP1mRNA and enhanced unspliced XBP1mRNA. In the meantime, the level of spliced ERAIm454-557mRNA was obviously reduced by shXBPl constructs in the absence of ER stress. ER stress-induced splicing of ERAIm454-557mRNA was also greatly suppressed by shXBP1constructs.11. Slight overexpression of XBP1s promotted MCF-7cells proliferation and clony formation ability.Conelusions:1. The unconventional splicing of XBP1mRNA can occur independent of ER stress, and the sequences flanking stem-loop pair were critical to the unconventional splicing.2. ER stress-independent splicing of XBP1mRNA occurred in the nucleus and required IRE1α.3. The existed spliced XBP1mRNA in MCF-7cells resulted from the ER stress-independent unconventional splicing in the nucleus.4. Low levels of XBPls promoted the growth of MCF-7cells.