Effects Of Silencing Moesin Expression By Lentiviral-mediated RNA Interference On The Biological Behavior Of Breast Cancer Cell Lines

PartⅠ: Expression of moesin in breast cancer tissue arraysand clinic pathologic significanceObjective: To investigate the expression of moesin, onc of thc ERM(ezrin/radixin/moesin) protein family, in breast cancer tissue arrays and explore itscorrelation with the clinical pathological features; to provide experimental basis forthe following research by detecting the expression of mocsin on the level of mRNAand protein in three breast cancer lines.Methods: Imunohistochemistry SP technique method was used to detect theexpression of moesin protein in 110 breast cancer tissues, 30 tissues adjacent to the cancerand 10 normal tissucs. The mRNA and protein expression of moesin in three breastcancer cell lines was detected by RT-PCR, real-time quantitivc PCR and Western Blot.Results: Moesin is expressed in tumor tissues and tissues adjacent to tumor, isnot expressed in normal tissues. Comparison in 30 tumor tissues, 30 tissues adjacentto tumor, 10 normal tissues shows that the abnormal positive expression ratio ofmoesin is 60.00%, 16.67%, 0.00% respectively(P＜0.05). In 80 breast cancer, thereis a close correlation between the abnormal positive expression of moesin with severalclinic pathological factors including ER negative, PR negative and lymph nodemetastasis (P＜0.05). Differences are seen between the expression on mRNA andprotein of moesin in three breast cancer cell lines ,expression level of moesin inMCF-7/ADR and MDA-MB-231is highcr than MCF-7/S (P＜0.05).Conclusion: Expression of moesin in breast cancer tissues may be associatcd withER statue and may play an important role in the carcinogenesis, progression andmetastasis. PartⅡ: Construction and identification ofmoesin-targeting RNAi Lentivirus expressive vectors and itseffect of gene silence on breast cancer cell linesObjective: To construct RNAi Lentivirus expressive vectors specific to moesingene and detect the effect of gene silence on breast cancer cell lines.Methods: The shRNA oligonucleotides targeting to moesin gene weresynthesized and cloned into pGCSIL-GFP to generate shRNA Lentivirus expressivevectors. The recombinants were named pGCSIL-GFP_moesin-TARGET 1/2/3/. RNAiLentivirus expressive vectors were identificated by sequencing. Moesin RNAiLentivirus expressive vectors were packaged by co-transfecting 293T with 3 plasmidssystem and the titre of Lentivirus particle was detected by titration dilution. The genesilencing effect was detected by real-time quantitive PCR and Western Blot on the levelof mRNA and protein after the RNAi Lentivirus particle was infected intoMDA-MB-231 and MCF-7/ADR.Results: Three RNAi Lentivirus expression vectors targeting moesin mRNAsequence were constructed successfully by sequencing. The Lentivirus particles werepackaged successfully to provide enough titre for the following experiments. Theinfection efficacy of both two breast cancer cell lines can reach 80%.The result ofreal-time quantitive PCR and Western Blot showed the specific silencing effect ofTARGET 1 was the best with the 85% depress of mRNA and 75% depress of protein inMDA-MB-231 and the 90% depress of mRNA and 85% depress of protein inMCF-7/ADR.Conclusion: RNAi Lentivirus expressive vectors specific to moesin weresuccessfully constructed. The effects of gene silencing of moesin were effectively andstable in MDA-MB-231 and MCF-7/ADR. PartⅢ. Effects of silencing moesin expression byLentiviral-mediated RNAi on biological behavior of breastcancer cell lines in vitroObjective: To investigate the effects of silencing moesin expression byLentiviral-mediated RNAi on cell proliferation, cell cycle, cell apoptosis, cellinvasion and metastasis and the change of drug resistance.Methods: After the MDA-MB-231 and MCF-7/ADR were infected with Lentivirusparticles, the proliferation were detected by MTT, cell cycle and apoptosis were detectedby Flow Cytometry, the mRNA expression of cell cycle related gene cyclin D1wasdetected by real-time quantitive PCR, the change of cell cycle and apoptosis ofMCF-7/ADR after treatment with 300ng/ml ADM were again detected by FlowCytometry, the invasive ability of MDA-MB-231was detected by Transwell assay, theprotein expression of CD44v6,MMP-7,E-cadherin were detected by Western Blot,the drug resistant ability of MCF-7/ADR was detected by MTT, p-gp proteinexpression was detected by Western Blot, the mRNA expression of Rho A,Rac1,Akt1were detected by real-time quantitive PCR and the Akt1,pAkt1 proteinexpression were detected by Western Blot.Results: The depression effect on cell proliferation was shown after silencingmoesin expression by Lentiviral-mediated RNAi (P＜0.05). Cell cycle detected byFCM shown that G0/G1 phase were increased and S phase were decreased (P＜0.05),and the mRNA expression of cyclin D1 can be degraded. Cell apoptosis inMDA-MB-231 shown no significant difference, but cell apoptosis in MCF-7/ADRcan be increased after treatment with 0.3μg/ml ADM (P＜0.05). The invasive ability ofMDA-MB-231 decreased significantly, CD44v6 and MMP-7 were depressed, butE-cadherin was increased. MTT showed that the IC50 of ADM in MCF-7/ADRchanged from 51.158±0.923μg/ml to 24.105±1.930μg/ml after RNAi, and the relativereversal rate is 53% (P＜0.05), at the same time the p-gp protein can bereduced(P＜0.05). The mRNA expression of Rho A,Rac1 were down-regulated after RNAi(P＜0.05), though the mRNA expression of Akt1 had no significant change, theprotein expression of pAkt1 were depressed(P＜0.05).Conclusion: Silencing moesin expressions by Lentiviral-mediated candown-regulate the cyclin D1 through reducing the expression level of Rho, Rac1,resulting in depressing the transition from G1 phase to S phase. The ability of drugresistance of MCF-7/ADR can be affected by down-regulating the expression of p-gp,this made the cell no longer adapt to the culture circumstance with 0.3μg/ml ADMtreatment, as the consequence the cell proliferation was depressed and the apoptosisrate increased. The invasive ability can be affected by CD44v6,MMP-7down-regulating and E-cadherin up-regulating. All these effects mentioned above maybe implemented by reducing the expression of Rho A, Rac1 and pAkt1.