MiR-7-5psuppress Proliferation And Induce Apoptosis By Targeting REGΓ In Breast Cancers

PART ⅠTHE EXPRESSION OF REGΓ IN BREAST CANCER CELLLINES AND PRIMARY BREAST CANCERSObjective: To investigate the expression of Proteasome activator subunit3(REGγ/PSME3/PA28γ/Ki antigen) in human breast cancer cell linesand human breast cancer tissue.Methods: the expression of REGγ was detected by Semi-quantitativeRT-PCR and immunoblot in a panel human breast cancer cell lines; theexpression of REGγ was detected by immunoblot in breast cancer tissues,and paired non-cancerous tissues as control; the REGγ expression levels inhuman breast cancer and normal breast tissues was analyzed by usingOncomine microarray online database (https://www.oncomine.org/).Results: REGγ was expressed in human breast cancer cell lines widely byRT-PCR, and data shown that the expression of REGγ in non-tumorigeniccell lines (HBL100and MCF10A) is relatively low, immunoblot shown that MDA-MB-231has the highest level of REGγ protein levels;immunoblot data shown that the protein level of REGγ was more higher inbreast cancer tissues compared with paired non-cancerous tissues;Compared to normal breast tissue, the expression of REGγ in DCIS andinvasive ductal carcinomas were increased at different degrees fromOncomine microarray database.Conclusion: mRNA and protein levels in human breast cancer REGγ celllines and tissues showed increased to varying degrees, may play animportant role in breast cancer process. PART ⅡREGΓ AS THE DOWNSTREAM TARGET OF MIR-7-5PObjective: Exploring the methods how targeted REGγ may provide novelideas for breast cancer treatment strategies development and screening ofmicroRNAcan target REGγ.Methods: MiRbase, Targetscan, PicTar and other database were used foranalyzing the downstream target gene of miR-7-5p; furthermore, theexpression of miR-7-5p was detected by real-time PCR in a panel humanbreast cancer cell lines; make sure that REGγ as the downstream target ofmiR-7-5p by luciferase reporter gene assay, verify the effect of miR-7-5p on REGγ mRNA and protein levels; the expression of miR-7-5p and REGγwere detected in human breast cancer tissues by real-time PCR andanalysed the correlation between the expression of miR-7-5p and REGγResults: MiRbase, Targetscan, PicTar and other database shows thatmiR-7-5p can specific binding REGγ3′UTR; the miR-7-5p expressionwas the lowest in MDA-MB-231cells; miR-7-5p significant inhibition ofluciferase activity, and miR-7-5p can bind the3′UTR of REGγ; miR-7-5ppromote REGγ mRNA degradation and inhibit mRNA translation.compared to adjacent tissue, miR-7-5p expression in human breast cancertissues was significantly reduced, whereas the expression REGγ increasedsignificantly, there has a negative correlation between miR-7-5p and REGγin breast cancers.Conclusion: miR-7-5p suppress the protein level of REGγ by bingding its3`UTR. PART ⅢTHE EXPRESSION OF REGΓ IN BREAST CANCER CELLLINES AND PRIMARY BREAST CANCERSObjective: in order to clarify miR-7-5p function in human breast cancerand associated mechanisms Methods: we reforced the expression of miR-7-5p in MDA-MB-231andMCF7(the relative low expression of miR-7-5p in our panel breast cancercell lines) cells for our study, and evaluate the effect of miR-7-5p on cellproliferation in MDA-MB-231and MCF7cells, and cell cycle, apoptosis,and the ability of colony formation in MDA-MB-231cells.Results: Quantitative PCR showed that overexpressing miR-7-5p successin MDA-MB-231and MCF7cells; CCK-8showed that miR-7-5p cansignificantly inhibit the proliferation of MDA-MB-231and MCF7cells;EdU cell staining show that miR-7-5p reduce the percentage of stainingcells in MDA-MB-231and MCF7cell lines; colony formation experimentsshowed that relative to the control group NC, miR-7-5p significantlyinhibited the capacity of colony formation in MDA-MB-231cells; flowcytometry, which used for detecting cell cycle and apoptosis, showed thatrelative to the control group NC, miR-7-5p cells can blocked G0/G1phaseand induced apoptosis of MDA-MB-231; immunoblot showed thatmiR-7-5p significantly inhibited the protein level REGγ, and promote theaccumμlation of p21and p27, which can induce cell cycle arrest in thecells，and induce apoptosis by cutting caspase3.Conclusion: miR-7-5p as a tumor suppressor in breast cancers byinhibiting proliferation and inducing apoptosis. PART ⅣTHE EXPRESSION OF REGΓ IN BREAST CANCER CELLLINES AND PRIMARY BREAST CANCERSObjective: To validate that miR-7-5p coμld targeted REGγ directly,thereby reducing REGγ protein levels, as a resμlt that inhibit proliferationof breast cancer cells.Methods: knocked down the expression of REGγ by shREGγ inMDA-MB-231cells, which has the highest expression in our panel breastcancer cell lines. REGγ expression was measured by quantitative PCR,knocked down REGγ and then evaluate the effect of REGγ on humanbreast cancer cell proliferation, cell cycle regμlation and apoptosis. CCK8and colony formation assay were used for evaluating the ability ofproliferation, and cell cycle, apoptosis were detected by flowcytometry.The molecular change was measured by immunoblot.Results: Quantitative PCR showed shREGγ can significantly reduce theexpression of REGγ in MDA-MB-231cells; compared with the emptyvector, shREGγ can reduce the clonogenic capacity of MDA-MB-231cells,induce G0/G1phase arrest and promote apoptosis, mainly caused p21accumμlation and caspase3cutoff in cells. These resμlts suggest that:miR-7-5p binding the3’UTR of REGγ directly, and suppresses REGγprotein level, inhibits proliferation of human breast cancer cells. Conclusion: knockdown REGγ can suppress cell proliferation and promoteapoptosis, and miR-7-5p act as a tumor suppressor by targeting REGγ.