The Expression Of LIN28A And Its Regulation Mechanisms In Invasive Breast Cancer

Objection:Breast cancer is the most common female malignant tumors, with three percent annual rate, breast cancer become a threat to the female in China. Epithelial-to-mesenchymal transition(EMT) is the biological processes of malignant tumor epithelial cells migration and invasion. As highly conserved RNA-binding proteins LIN28, there is widespread ectopic expression in a variety of tumors, and studies have found that, LIN28 overexpression is a strong predictor of poor prognosis in cancer patients. In this research, through detecting the expression of LIN28A in invasive breast cancer and the effect of LIN28A expression on breast cancer cell invasion, metastasis,proliferation and apoptosis, we want to know the regulation mechanisms of LIN28A in cell proliferation, invasion and apoptosis on breast cancer cell.Method:1 First, we use the quantitative real-time PCR to detect the mRNA expression of LIN28A,OCT4, E-cadherin and Vimentin in invasive breast cancer tissue and the corresponding adjacent tissues and clarify their relationships. Second,followed by the immunohistochemistry, we analysis the relationship between the expression of LIN28A and clinicopathological parameters, in order to clarify the relationship between LIN28A and EMT or Oct4 in invasive breast cancer, and finally by the TUNNEL assay, we explored the impact of LIN28A in apoptosis.2 Through building a stable LIN28A expression of MCF-7 cells, we discussed the influence of LIN28A in breast cell, including flow cytometry was used to viral transfection efficiency, transwell assay was used to cell invasion migration, clonogenic assay detected cell proliferation, flow cytometry was used to cell apoptosis and cell cycle situation. Finally, Real-time and Western-blot assay were used to the expression of the relevant gene and protein, specifically the regulatory mechanisms of LIN28A in breast cancer cells.Result: 1 The results of real-time PCR showed that, in 40 cases of invasive breast cancer tissues, the mRNA expression of LIN28A negatively correlated with the expression of E-cadherin(R=-0.34,P<0.05), positively correlated with the expression of OCT4(R=0.332,P<0.05), the same with vimentin(R=0.38,P<0.05). And the expression of E-cadherin negtively correlated with the mRNA expression of vimentin(R=-0.353,P<0.05); 2 Immunohistochemistry showed that, there was a relationship between the expression of LIN28A with tumor size, pathological stage, grade, lymph node metastasis and distant metastasis in invasive breast cancer(P<0.05). LIN28A positively correlated with Vimeatin(R=0.364,P <0.05), positively correlated with OCT4(R=0.423, P<0.05),negatively correlated with E-cadherin expression(R =-0.533,P<0.05), E-cadherin and Vimeatin expression was also a negative correlation(R =-0.453,P<0.05). Kaplan-Meier survival curve analysis showed that, patients with low expression levels of LIN28A survived significantly longer than those with high expression levels(P=0.021), indicated that invasive breast cancer, LIN28A was related with poor clinical prognosis; 3 TUNNEL assay showed that, high expression of LIN28A could decrease apoptosis in invasive breast cancer; 4 Puromycin stably expressing LIN28A of MCF-7 cell lines The results of flow cytometry assay indicated that, the transfection efficiency of control-pSBbi-GP and LIN28A-pSBbi-GP were 13.1 % and 12.6 % respectively when 400?l virus solution infected 293 T cell, when 800?l virus solution infected 293 T cell, the transfection efficiency were 19.5 % and 20.1 % respectively, Since the transfection efficiency is too low, it is infected with a virus stock MCF-7 cells, infection efficiency and control-pSBbi-GP LIN28A-pSBbi-GP was 29.6 %, 24.8 %.; 5 Real-time and Western-blot were used to detect stably transfected expression after LIN28A.Compared with the control-pSBbi-GP and parental, the expression of LIN28A mRNA in LIN28A-pSBbi-GP was increased, while the mRNA expression of let-7a reduced(P<0.05). Followed by Western-blot assay, the protein levels of LIN28A in LIN28A-pSBbi-GP was increased expression(P<0.05); 6 Clonogenic assayed Cell proliferation changes, results of this experiment showed that the number of parental clone group, control-pSBbi-GP group and LIN28A-pSBbi-GP group were 38.20±2.02?44.55±2.67 and 102.65±9.87, Compared with parental group and control-pSBbi-GP group, LIN28A-pSBbi-GP colonies significantly increased(P<0.05), however, there was no significant between the control-pSBbi-GP group and the parental group(P>0.05), prompting LIN28A gene overexpression could promote cell proliferation; 7 Transwell assay results showed that, in MCF-7 cells, compared with the parental group and control-pSBbi-GP group, LIN28A-pSBbi-GP group migration and invasion into the lower chamber cells increased significantly(P<0.05), prompting overexpression LIN28A gene could promote cell migration and invasion; 8 FCM was used to detect cell apoptosis and cell cycle changes, the results showed that compared with the parental group and the control-pSBbi-GP group, early apoptosis rate of LIN28A-pSBbi-GP group decreased significantly(P<0.05), and, after LIN28A overexpression, accelerated the G1/S phase transition; 9 Real-time and Western-blot results showed that, compared with the parental group and the control-pSBbi-GP group, LIN28A-pSBbi-GP group up-regulated the expression of Snail and Slug, CyclinD1 and eIF4 E expression increased, Bcl-XL expression up-regulated, suggesting that LIN28A overexpression may inhibit the let-7, and then induced the proliferation, inhibited the apoptosis in breast cancer cell; 10 Further study of the relationship between LIN28A and OCT4 by Real-time, and Western-blot experiments found that after overexpression LIN28A, OCT4 expression increased, decreased E-cadherin expression, increase Vimtern expression, suggesting that, LIN28A can also induce OCT4 expression, and promote breast EMT process.Conclusion:1 In invasive breast cancer, the expression of LIN28A negatively correlated with the expression of E-cadherin, positively correlated with the expression of vimentin, suggesting LIN28A may contribute to breast cancer development and metastasis by EMT, and LIN28A overexpression is an independent poor prognosis factor.2 By lentivirus transfection overexpression, LIN28A can significantly increase the proliferation, inhibit apoptosis. At the same time, let-7-related target genes upregulated, suggesting that by inhibiting let-7 expression, LIN28A can promote the EMT process in breast cancer cell. Through the LIN28/Let-7/HMGA2/Slug, Snail/E-cadherin and LIN28/let-7/BCL-XL signaling passway, LIN28A could promote the proliferation, inhibition apoptosis in breast cancer cells, In addition, combined with a previous study, we further study the relationship between LIN28A and OCT4 by Real-time, and Western-blot experiments found that after overexpression LIN28A, OCT4 expression increased, decreased E-cadherin expression, increase Vimtern expression, suggesting that, LIN28A also by inducing the expression of OCT4, promoting breast cancer EMT process. This would provide a new target for breast cancer treatment.