| Objective: As one of the miRNA members, miR-10 b may plays an important role, and also has the same function and broad application prospects in recurrence and distant metastasis aspect of early breast invasive ductal carcinoma. High stability of miR-10 b is an important characteristic as a good biomarker. It still can be effective extracted and quantitated from the specimens which be fixed by the formalin and paraffin embedding for a few years. In situ hybridization technique(ISH) with low cost, simple operation and clinical propagable, can detect miRNA expression of a single cell, also can campare with the expression of miR-10 b of different cell lines without classification and separation, therefore,this study used in situ hybridzation to detect the expression of miR-10 b. Using the ISH to detect the different expression of miR-10 b in early breast invasive ductal carcinoma and the relationship of the expression of miR-l0 b with their clinicopathologic parameters, to analyze the median survival time and the expression of mi R-10 is relevant to the prognosis of early invasive ductal carcinoma. The expression of estrogen receptor,progesterone receptor(PR), human epidermal growth factor receptor 2 were also detected in the early breast cancer tissue specimens by immunohistochemical method, according to the 12 th St Gallen discovered international consensus meeting we divided them into different breast cancer molecular typing, analyse the relationship between miR-10 b and other different genotype with their median survival time and prognosis in early infiltrating ductal carcinoma.Finally,in order to lay the fundation for the further sdudy of the role of miR-10 b gene in the early invasive ductal carcinoma, we select target gene such as Notch1, NCOR2, HOXD10, NR4A3, CREB1, PPARA, PPARGCB by target genetic tests and lectures, analyse the relations of mi R-10 b with the recurrence and distant metastasis in early breast invasive ductal carcinoma. Methods: The expression of miR-10 b were detected in 193 cases of early breast invasive ductal carcinoma by ISH technology, dividing the 193 paraffin specimen into positive lymph node 124 cases and negative lymph nodes 69 cases by the clinical data; Using immunohistochemical testing the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, according to the 12 th St Gallen discovered international consensus meeting we divided them into different breast cancer molecular typing. Final target genes is applied to forecast that whether or not miR-10 b is related to transfer progress and can affect the prognosis in early breast invasive ductal carcinoma.The difference between miR-10 b and clinical index were analyzed by Pearson chi-square test; the Kaplan-Meier test to calculate the accumulative disease-free survival time; the Log-rank test median disease-free survival analysis; using the Cox proportional hazard model for multivariate survival analysis. Using a paired T-test method compared with earlly breast invasive ductal carcinoma of breast carcinoma tissue adjacent to carcinoma of target genes expression. Inspection level ?=0.05. Results: The expression of miR-10 b in early breast invasive ductal carcinoma with the lymph node metastasis and tumor recurrence and metastasis has statistical difference(P ?0.05); The median tumor-free survival time of the miR-10 b negative expression was higher than miR-10 b positive expression, the difference is statistically significant(P ?0.05); When the lymph node negative, the median tumor-free survival time of the mi R-10 b negative expression was higher than miR-10 b positive expression, the difference was statistically significant(P ?0.05); MiR-10 b and endocrine therapy acted as independent prognostic factor in early breast invasive ductal carcinoma(P?0.05), the positive expression of miR-10 b was risk factors(OR>1), endocrine therapy was protective factor(OR<1). When the The lymph node positive, the expression of miR-10 b in median tumor-free survival time had no statistical difference(P>0.05); The expression of miR-l0 b had no correlation with menstrual status, age, ER-?, PR and HER-2(P>0.05). Part two: In the early stage of invasive breast ductal, miR-10 b positive expression rate has the difference between the different expression of ER-?(P?0.05); The miR-10 b positive expression rate of HER-2(-) was significantly higher than that of HER-2(+), the difference is statistically significant(P?0.05). According to the results of immunohistochemical and FISH detection of the ER-??PR?HER-2, we divided into four different molecular subtypes and there were 139 cases can be accepted.The result showed that miR-10 b positive expression in luminal B was significantly lower than other types, the difference is statistically significant(P?0.05). When ER-?(+),PR(+),HER-2(-), early breast invasive ductal carcinoma with the median survival time is long, the prognosis is good, but the median disease-free survival analysis showed that the disease-free survival time of miR-10 b positive expression which is ER-?(+),PR(+),HER-2(-) in early invasive ductal breast cancer had no significant effect, the difference is statistically significant(P?0.05); Molecular subtypes in median survival time showed that median survival time of liumianl type A was longest, luminal B and Basal-like was center, over expression of HER-2 was shortest, the difference was statistically significant(P ? 0.05); Multivariate analysis of molecular subtypes was independent prognostic factors of breast infiltrative ductal carcinoma(P?0.05), miR-10 b positive expression was a risk factors(OR>1), but the results of multivariate analysis had no statistical significant(P>0.05). Part three: we selected target gene such as Notch1, NCOR2, HOXD10, NR4A3, CREB1, PPARA, PPARGCB by the target gene prediction software and lectures retrieval,By real-time fluorescent quantitative PCR experiment, miR-10 b real-time fluorescent amplification curve inflection point clear, the sample curve good parallelism, similar amplification efficiency, baseline straight; Amplification product melting curve show unimodal, primer is proved to be reasonable, specific amplification. The solubility curve of amplication was single peak,it showed that their was no nonspecific amplification. The expression of Notch1,NCOR2,HOXD10 was lower at cancer tissues than pericarcinous tissue(P?0.05), the difference is statistically significant. The expression of NR4A3, CREB1, PPARA rised at cancer tissues when it compared with pericarcinous tissue(P>0.05), there was no statistical significant. However,PPARGCB rate went up in cancer tissues(P?0.05), with statistical significance. Conclusion: The expression level of miR-l0 b has correlation with recurrence metastasis in early breast invasive ductal carcinoma, and plays an important role in recurrence transfer.MiR-l0 b positive expression could promote recurrence metastasis, especially in lymph node negative in early breast invasive ductal carcinoma, the median disease-free survival time is reduced. MiR-l0 b positively expression can be used as an early breast invasive ductal carcinoma recurrence of prognostic factors and the prognosis indicator for the early breast infiltrating ductal carcinoma. MiR-l0 b positively expression was negative associated with ER-?, HER-2, the expression of mi R-l0 b had significant differences among molecular subtypes, it was obvious lower in Luminal B type. The positive expression of miR-10 b, ER-?, PR and the negative expression of HER-2 had longer tumor-free survival time, prompting a good prognosis; the Luminal A and Luminal B types had longer tumor-free survival time, while HER-2 overexpression type and Basal like type had shorter tumor-free survival time, prompting a bad prognosis. The Cox multivariate regression analysis showed that molecular subtypes were independent prognostic factors for early breast invasive ductal carcinoma. The positive expression of miR-10 b negative regulate Notch1 and NCOR2 HOXD10 target genes, eventually leading to early recurrence of breast invasive ductal carcinoma metastasis.The mi R-10 b could be used as a prognostic reference indexes. |
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