| As the most common noninvasive breast cancer,DCIS contributes to almost 20% of newly diagnosed breast cancer cases in China.The prognosis of DCIS is extremely favourable since tumor cells did not break through the basement membrane,but there is still a small proportion of DCIS patients still relapse after treatment.At present,little is known about researches on DCIS related prognosis and the molecular mechanism for DCIS metastasis because of the lack of following-up data.MiRNAs are small noncoding RNAs with conservative sequence,stable structure,sequential expression.Proliferation,apoptosis,differentiation,migration and other important life events in cells,even tumorigenesis or inhibiting tumorigenesis,have already been proved that can be regulated by miRNAs via inhibiting the expression of target genes.Therefore,miRNAs may play regulatory role in the tumorigenesis,growth and development of DCIS,and affect the prognosis of DCIS.Objective:The aim of this study is to screen key miRNAs that contribute to DCIS metastasis and demonstrate potential mechanism of mi RNA-654-5p.Methods:1.Clinical data and follow-up information of DCIS and DCIS-Mi cases were collected,including age,tumor size,nuclear grade,lymph node status,ER(Estrogen receptor,ER),PR(Progesterone receptor,PR),HER2(Human epidermal growth factor receptor-2,HER2),Ki-67 positive rate,operation mode and postoperative adjuvant treatment.The clinicopathological features of DCIS and DCIS-Mi and survival analysis were performed to find out the risk factors associated with poor prognosis(finding recurrence / metastasis during follow-up period).2.The samples used for miRNA microarray were formalin-fixed andparaffin-embedded(FFPE)tissue specimens after microdissection of good and poor prognosis cases(poor prognosis was defined as either recurrence or metastasis during follow-up period,good prognosis was defined as no recurrence nor metastasis during the follow-up period).High grade nuclei of DCIS and DCIS-Mi FFPE samples with more than 75% tumor cells were selected respectively.The qPCR method was used to screen the critical miRNA related to prognosis.3.Wound healing assay and transwell assay were used to detect the mobility of MDA-MB-231 cells in which the expression mi RNA was up-regulated.4.The proliferation of MDA-MB-231 transfered with miRNA mimics was detected by CCK-8.The slides of cells using IHC staining for Ki-67 was to detecte the cell proliferative potential.5.Annexin V PE/7-AAD Apoptosis Detection Kit was applied to detecte the apoptosis level of MDA-MB-231 cells after up regulation of miRNA expression.6.The expression of genes about proliferation,apoptosis,migration were examined by real-time quantitative PCR after using statistical analysis.7.The protein levels of EMT related protein were examined by western blot.8.Immunofluorescence staining(phalloidin)for F-actin was to observe the cytoskeleton form after high expression of mi RNA in MDA-MB-231 cells.Results:1.Compared with DICS-Mi,the clinicopathological features of DCIS were had less cases of high nuclear grade and lymph node metastases,more ER positive,PR positive cases and less high-expressed HER2 cases.There was no difference in clinicopathological features between DCIS and DCIS-Mi patients in poor prognosis.2.The tumor size,lymph node metastasis and ER expression were related to the prognosis of DCIS(P=0.000,P=0.000,P=0.024),and tumor size and lymph node metastasis were the independent prognostic factors,HR(95%CI)was 0.068(0.13-0.362)and 0.018(0.003-0.099)respectively.All of statistical results were statistically significant.3.To screen high risk miRNAs that lead to bad prognosis of DCIS patients,miRCURY LNA? microRNA Array was used on FFPE samples with different prognosis.According to the microRNA array analysis,77 miRNAs were selected from all 2085 mi RNAs which were expressed greater than2-fold consistently(P<0.05).4.Total miRNAs were isolated from FFPE samples of 16 pure DCIS cases(4 of favourable prognosis,12 of poor prognosis).5 miRNAs differentially expressed in good/poor prognosis group were selected by qPCR verification :miRNA-654-5p(P=0.048),miRNA-141-5p(P=0.037),miRNA-767-5p(P=0.027),mi RNA-4507(P=0.024),mi RNA-4255(P=0.048).The expression of miRNA-654-5p showed the best consistentcy.5.As the same verification process,5 differentially expressed miRNAs in good/poor prognosis group were screened out from DCIS-Mi samples(5 of favourable prognosis,17 of poor prognosis)by qPCR quantification,involving miRNA-152-5p(P=0.042),miRNA-196a-5p(P=0.048),miRNA-4271(P=0.048),miRNA-5094(P=0.015),miRNA-5696(P=0.005).6.The wound area in the treatment group(transfected with miRNA-654-5p mimics)was significantly less than that in the negative control group(NC group)after 24 hours(P<0.05).7.After 48 hours of culture in the wells,more cells migrated to the lower wells in the treatment group(397.750±107.166)than in the NC group(126.500±26.834),which showed significant defference(P<0.05).8.Cytoskeleton showed that compared with the NC group,cells with high miRNA-654-5p expression lost their polarity and tended to be more fusiform,and the stress fibers arranged in experimental group were clear and the movement ability was enhanced.9.CCK-8 results showed that the capability of proliferation in two groups were not statistically significant(P>0.05);there was no statistical differencebetween two groups about the amount of Ki-67 positive cells(P >0.05).Results of the two experiences mentioned above showed that miRNA-654-5p with no relationship with cell proliferation.10.There is no difference in the number of apoptotic cells in treatment group and NC group at the(P>0.05).11.QPCR results showed that the expressions of apoptosis and proliferation related genes in treatment group and NC group had no relationship with the expression of miRNA-654-5p.The expression of snail,metastasis-associated gene,was upregulated in experimental group because of miRNA-654-5p(P =0.021).12.Western blot results showed that compared with the NC group,the expression of EMT-related protein including snail and vinmentin were increased,while the expression of E-cadherin decreased in the treatment group,and the results were all statistically different(P<0.05).Conclusions:1.The clinicopathological features of DCIS and DCIS-Mi are different,the expression of invasive clinicopathological features of DCIS was weak.DCIS in high nuclear grade means a greater likelihood of malignant behavior.HER2 is less expressed in poor prognosis DCIS cases.Tumor size,lymph node metastasis and ER expression can be used as independent prognostic factors of DCIS.2.The distinct-expressed mi RNAs in poor prognosis of DCIS and DCIS-Mi indicated that miRNA would express differently with different stages of breast cancer progression,called time specificity.3.MiRNA-654-5p was screened that highly expressed in poor prognosis DCIS patients consistently.The high expression of miRNA-654-5p may be associated with poor prognosis according to the clinicopathological features and the statistical analysis of q PCR and other molecular experiments.4.MiRNA-654-5p promoted motility in breast epithelial cells.The up-regulation of mi RNA-654-5p altered the expression of EMT related proteins which might indicate that may enhance cell migration through EMTand promote metastasis. |
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