| Objective: Breast cancer is the most common malignancy of women,which is accounting for23%of female cancer and accounting for14%of thecancer deaths in women[1]. The incidence, progression, metastasis and drugresistance of breast cancer is a multi-gene and multi-factor interaction result.Numbers of studies show that:breast cancer possesses a high heterogeneity.Different molecular phenotypes of breast cancer due to different responses tospecific treatment and the outcmes or prognosises are different, too. With therapid development of molecular biology and the emergence of various kindsof high-tech biotechnology, to explore molecular mechanism and to findrelevant biomarkers of breast cancer are becoming a hot spot.Metadherin (MTDH)/astrocyte elevated gene-1(AEG-1), may be closelyassociated with the incidence, progression and drug resistance of breast cancerif it is overexpression. The specific mechanism of resistance caused by MTDHoverexpression in breast cancer cells is not fully understood. Presently, thepossible mechanisms may be as follows:(1) MTDH leads cells from cellcycle G1to S phase, thereby reducing the relative proportion of cells inG,which controls the apoptosis of tumor;(2) high expression of MTDHmakes multidrug resistance gene (multidrug resistance gene1, MDR1)expression increased. As the increased expression of this protein, the drugresistance to tumors is increased, which reducing the concentration ofchemotherapeutic agents. Thereby, the exploration of MTDH role in thebiological behavior of breast cancer cells as well as regarding the MTDH as apotential target for treatment of breast cancer is significant.In our study,we inhibited the MTDH expression of MCF-7/ADR cells inbreast cancer through RNAi interference technology, then we observed the proliferation, metastasis and drug resistance in these special MCF-7/ADRcells. By this way, we explore the role of MTDH in MCF-7/ADR cellsbiological behavior, which can provide experimental and theoretical basis forpersonalized and targeted treatment of breast cancer by MTDH-siRNA.Method: This study successfully constructed four interference siRNA:MTDH-siRNA-1, MTDH-siRNA-2, MTDH-siRNA-3, empty-siRNA. UsingRNAi interference technology enables MTDH gene silencing. Realtime PCRand Western blot were used to determine the mRNA and protein expression ofMTDH in MCF-7/ADR before and after the silence. The growth curve ofMCF-7/ADR cell depicted in breast cancer by MTT.The drug sensitivity ofMCF-7/ADR cell lines before and after silence to1mg/L of adriamycin and8mg/L of taxol at24h was assessed by MTT. Statistical analysis wasperformed using SPSS13.0. P<0.05was considered statistically significant.Results:1The best silencing effect MTDH-siRNA screened by Realtime PCR andWestern BlotRealtime PCR results showed that: Compared with the non-transfectedgroup, the MTDHmRNA expressions in MCF-7/ADR breast cancer cells inMTDH-siRNA-1, MTDH-siRNA-2, MTDH-siRNA-3were decreased by46.88%,77.88%,30.12%, of which MTDH-siRNA-2group showed the mostobvious expression of the interference effect on MTDHmRNA (P<0.05),while the empty vector transfected group compared with the non-transfectedgroup, the MTDHmRNA expression was elevated by1.17%, and there was nostatistically significant (P>0.05).Western Blot results showed that: Compared with the non-transfectedgroup, the MTDH protein expressions in MCF-7/ADR breast cancer cells inMTDH-siRNA-1, MTDH-siRNA-2, MTDH-siRNA-3were decreased by84.81%,97.26%,75.26%, of which MTDH-siRNA-2group showed the bestsilencing effect on expression of protein (P<0.05), while the empty vectortransfected group compared with the non-transfected group, the MTDHprotein expression was decreased by1.08%，and the difference was no statistically significant(P>0.05).2MTDH mRNA expression in MCF-7/ADR cells before and after thesilenceRealtime PCR results showed that: Compared with the non-transfectedgroup, the mRNA expressions in MCF-7/ADR breast cancer cells inMTDH-siRNA-2（0.2ug）, MTDH-siRNA-2（0.4ug）, MTDH-siRNA-2（0.8ug）were decreased by63.22%,76.40%,87.06%, of which MTDH-siRNA-2（0.8ug）group showed the best expression of the interference effect onMTDH mRNA (P<0.05), while the empty vector transfected group comparedwith the non-transfected group, the MTDH mRNA expression was increasedby2.16%.The difference was no statistically significant(P>0.05).Comparing with the non-transfected group, the mRNA expressions inMCF-7/ADR breast cancer cells in MTDH-siRNA-2（24h）, MTDH-siRNA-2（48h）, MTDH-siRNA-2（72h）were decreased by69.62%,81.44%,93.87%,of which MTDH-siRNA-2（72h）group revealed the best expression of theinterference effect on MTDH mRNA (P<0.05), while the empty vectortransfected group compared with the non-transfected group, the MTDHprotein expression was increased by10.32%，and the difference was nostatistically significant(P>0.05).3MTDH protein expressions in MCF-7/ADR cells before and after thesilenceWestern blot results showed that: Compared with the non-transfectedgroup, the MTDH protein expressions in MCF-7/ADR breast cancer cells inMTDH-siRNA-2（0.2ug）, MTDH-siRNA-2（0.4ug）, MTDH-siRNA-2（0.8ug）were decreased by43.54%,86.37%,82.75%, of which MTDH-siRNA-2（0.4ug）group showed the best interference effect on expression of protein (P<0.05), while the empty vector transfected group compared with thenon-transfected group, the MTDH protein expression was increase by4.90%，and the difference was no statistically significant(P>0.05).Comparing with the non-transfected group, the MTDH proteinexpressions in MCF-7/ADR breast cancer cells in MTDH-siRNA-2（24h）, MTDH-siRNA-2（48h）, MTDH-siRNA-2（72h）were decreased by77.19%、94.21%、91.97%, of which MTDH-siRNA-2（48h）group showed the bestinterference effect on expression of protein (P <0.05), while the empty vectortransfected group compared with the non-transfected group, the MTDHprotein expression was increase by7.33%， and the difference was nostatistically significant(P>0.05).4The change of the MCF-7/ADR cell growth curves before and after thesilenceDetermined by the growth curves reaveal that: if the tumor growth for24h, the OD values in non-transfected group and MTDH-siRNA-2group were0.561±0.060vs0.318±0.038; when at48h, they became0.918±0.066vs0.454±0.037; as the tumor growth for72h, the OD values were1.302±0.142vs0.640±0.084. The results showed that the transfected group grawsignificantly slowly than than non-transfected group (P<0.05).From the above we can come to the conclusion that MTDH in MCF-7/ADRcell proliferation in human breast cancer is important factor.5The sensitivity of MCF-7/ADR cells to doxorubicin before and after thesilenceBy determined by MTT to detect the drug sensitivity of which MCF-7/ADR cells before and after the silence to1mg/L adriamycin at24h. Resultsshowed that the inhibition rate of1mg/L doxorubicin in non-transfected groupand MTDH-siRNA-2group at24h were32.86%±5.49%vs55.40%±3.49%. With statistical analysis, it is considered that the inhibition rate ofMCF-7/ADR cell to doxorubicin both before and after the silence wasstatistically significant (P <0.05).6The sensitivity of MCF-7/ADR cells to paclitaxel before and after thesilenceThe detection of the drug sensitivity to paclitaxel of8mg/L inMCF-7/ADR cells before and after the silenced at24h by MTT: the inhibitionrates of the MTDH-siRNA-2group and the non-transfected group wererespectively32.86%±5.49%vs55.40%±3.49%. The statistical analysis showed that the inhibition rate of the MCF-7/ADR cell to paclitaxel beforeand after the silence was statistically significant (P <0.05).Conclusions:1When the human breast cancer MCF-7/ADR cells are stransientlytransfected by siRNA,the expression of MTDHmRNA and protein in thesebreast cancer cells would be inhibitted obviously.2If the MTDH genes are suppressed by siRNA, the proliferation ofMCF-7/ADR cells in breast cancer are significantly inhibited. It suggests thatMTDH plays an important role in human breast cancer cell proliferation.3As the MTDH gene is inhibited by siRNA, the drug sensitivity ofMCF-7/ADR cells to doxorubicin is increased, and this reveals that thehigher expression of MTDH gene may be associated with breast cancer cellsresistant to doxorubicin.4When the MTDH gene is suppressed by siRNA, the drug sensitivity ofMCF-7/ADR cells to paclitaxel is increased. So we can say that thehigher expression of MTDH gene may be connected with breast cancer cellsresistant to paclitaxel. |
PDF Full Text Request