Inhibitory Effect Of MicroRNA-100 On Malignant Transformation Of Breast Cells And Its Mechanism

Research background:Breast cancer is the highest incidence and the most harmful malignant tumor among women in the world,with more than 1.6 million new cases and about 520000 deaths every year.It is very important to study the mechanism of breast cancer.At present,more and more studies have shown that the abnormal expression of miRNA is closely related to the occurrence and development of breast cancer.MicroRNA(miRNA)is a kind of non-coding small RNA(miRNA),which is composed of 19 nucleotides and 25 nucleotides.It is widely found in plants,animals and viruses.MiRNA plays an important role in tumorigenesis and development by regulating the expression of a variety of genes and participating in many physiological processes such as cell proliferation,differentiation and apoptosis.Among them,the role of abnormal expression of miR-100 in tumorigenesis and development has been widely concerned.Among them,the abnormal expression of miR-100 plays an important role in the development of tumor.The miR-100 is located on chromosome 11 and consists of 22 nucleotides in length.Studies have shown that miR-100 can regulate apoptosis,autophagy,proliferation,migration,stem cell self-renewal and drugs by targeting different molecules such as mammalian target of rapamycin(mTOR),PLK1 and SMARCA5 Sensitivity.Our previous study found that the expression of miR-100 was significantly down-regulated in breast cancer tissue samples,but higher in normal tissues in patients with breast cancer.Therefore,we want to explore whether the mir-100 is related to the occurrence and development of breast cancer.Research objective:To investigate the effect of miR-100 on the malignant transformation of normal breast epithelial cells and the malignant phenotype of breast cancer cells,and to further explore whether it’s molecular mechanism was related to "stem" expression of tumor cells and epithelial-mesenchymal transition.Research content:1.To detect the expression level of miR-100 in breast cancer tissues,normal breast epithelial cells MCF-10 A,and breast cancer cells MCF-7 in clinical.2.Normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7 were transfected with the lentiviral vector of miR-100 minics and miR-100 inhibitor,respectively,and puromycin was used to screen and establish stable cell lines expressing green fluorescent protein.To detecthe expression of miR-100 in lentivirus transfected stable cell lines MCF-10 A and MCF-7.3.To detect the effects of miR-100 on the malignant biological behavior of normal breast epithelial cells and breast cancer cells by stable down-regulation of miR-100 expression of MCF-10 A and stable up-regulation of miR-100 expression of MCF-7cell lines.These include:(1)Plate colony assay,MTT assay and Transwell assay were used to detect the effect of miR-100 on colony formation,viability and invasion of MCF-10 A and MCF-7.(2)Western Blot,mammosphere formation assay and soft agar colony formation assay were used to detect the effect of miR-100 expression on breast cancer stem cell marker CD44 and CD24 protein expression and mammosphere formation ability.4.To explore whether the mechanism of regulation of miR-100 on the behavior of breast cells is related to epithelial stromal transformation: morphological observation and comparison the changes of epithelial phenotype and interstitial phenotype,and todetect the effect of miR-100 to epithelial-like marker E-cadherin by Western blot.and to interstitial-like marker N-cadherin protein expression.Research result:1.It was found that the expression level of MiR-100 in normal breast tissues and cell lines was significantly higher than that in breast cancer tissues and breast cancer cells,suggesting that it might be a tumor suppressor gene in the carcinogenesis and development of breast cancer.2.Successfully constructed normal breast epithelial MCF-10 A cells with stable down-regulation of miR-100 expression and breast cancer MCF-7 cells with up-regulated miR-100 expression.3.MiR-100 has a negative regulatory effect on the malignant behavior of breast cells:inhibition of the expression of breast cells proliferation,up-regulation of invasion of malignant indicators;On the contrary,up-regulation of its expression can inhibit the malignant behavior of breast cancer cells,such as the weakening of proliferation,invasion and other malignant indicators.The details are as follows:(1)The results of plate cloning showed that down-regulation of the expression of miR-100 significantly enhanced the ability of clone formation of MCF-10 A cells,while the up-regulation of miR-100 significantly decreased the ability of clone formation of MCF-7 cells.(2)The results of MTT showed that down-regulation of the expression of miR-100 significantly enhanced the activity of MCF-10 A cells,while the activity of MCF-7cells decreased obviously after up-regulation of miR-100.(3)The results of the invasion experiment showed that: the invasiveness of MCF-10 A cells was enhanced by down-regulating the expression of miR-100,while the invasiveness of MCF-7 cells decreased significantly after up-regulation of miR-100.4.MiR-100 has a negative regulatory effect on breast cancer stem cells:Afterup-regulating the expression of miR-100 in breast epithelial cells,it has inhibitory effect on tumor stem cells.(1)Down-regulate the expression of miR-100,the expression of CD44 is increased and the expression of CD24 is decreased.The expression of miR-100 was up-regulated,and the result was opposite.(2)After down-regulating the expression of miR-100,the ability of microsphere formation and non-anchored growth of MCF-10 A cells was increased,while the ability of microsphere formation and non-anchored growth of MCF-7 cells decreased after up-regulation of miR-100.5.The regulatory effect of miR-100 on malignant phenotype of breast cells may be mediated by EMT:After down-regulation of miR-100 expression,the morphology of MCF-10 A cells changed from epithelial-like to stromal-like,and the expression of stromal-like protein increased,while the expression of epithelial-like protein decreased;On the other hand,the up-regulation of the morphology of miR-100,interstitial epithelioid changes were observed in MCF-7 cells.The expression of stromal protein decreased,while the expression of epithelial-like protein increased.Conclusion:The decrease of miR-100 expression in mammary epithelial cells(MCF-10A)can lead to the increase of malignant phenotypes such as proliferation,invasion,cloning ability and breast cancer stem cell level of breast epithelial cells,and thus play the role of malignant transformation of benign cells.The mechanism may be related to the stem cell transformation caused by the occurrence of EMT after its deletion.The upregulated expression of miR-100 in breast cancer cells can inhibit the malignant behavior of breast cancer cells,such as proliferation,invasion,clone forming ability,invasion ability,tumor stem cell activity,and so on.Its mechanism is also closely related to the inhibition of EMT.These results suggest that: on the one hand,as a tumor suppressor gene,the deletion or functional inhibition of miR-100 may be the potential molecularmechanism of breast cancer.On the other hand,increasing its expression in breast cancer cells can inhibit the malignant behavior of breast cancer,suggesting that it may be a potential effective target for the treatment of breast cancer.