| BACKGROUND&OBJECTIVEBreast cancer is a malignant tumor which occurred in the mammary gland epithelial.Mammary gland is not vital organs of the body.In situ breast cancer were not life-threatening.But if breast cancer cells loss the characteristics which normal cells have,links between the cells become loose that lead breast cancer cells fall off easily.Once cancer cell falls off,transfer form.That means breast cancer cells spread throughout the body by blood or lymph,which is life-threatening.Breast cancer becomes a common tumor which threat to women's physical and mental health currently Therefore,to clarify the molecular mechanism of breast cancer metastasis and to find the prevention and treatment of breast cancer is an important topic in the research of breast cancer currently.In recent years,the ErbB family act as a more and more attractive target for cancer among the researchers.The study found that ErbB family plays a very important role in a variety of tumors such as breast,lung small cell lung cancer,stomach cancer,colon cancer,has become one of the candidate targets for Many cancer therapies.ErbB family,known as EGFR family,belongs to I receptor tyrosine kinase family,is a kind of enzyme protein which across the membrane,is made up of EGFR/ErbBl HER1?Neu/ErbB2/HER2?ErbB3/HER3 and ErbB4/HER4.ErbB2,known as HER2/Neu/lien/P185,abnormal expansion in breast cancer,and is closely related to the occurrence and prognosis of breast cancer.Erbin as ErbB2 binding protein,is one of the important members of LAP(leucine-richi repeats and PDZ domains,LRRs and PDZ).Erbin not only plays an important role in ErbB signaling,but also play an important role in polarization of epithelial cells.Erbin's C-end contains a PDZ domain,which can be combined with the protein phosphorylation ErbB2,regulates ErbB2 and its position.Though there are a lot of research about the Erbin,but its role in development of tumor is still not clear.But the role of Erbin plays in the occurrence,development and metastasis of tumor is still not clear.Methods1?Correlation analysis of clinicopathological features and expression of Erbin in breast cancer.Detect Erbin protein expression in breast cancer tissue microarray which contains clinicopathological features such as gender,age,organ,pathology diagnosis,classification,TNM stage,Tumor Node Metastasis),and type With immunohistochemical.First put the chip into dimethyl to benzene dewaxing(7 to 10 min x 3 times)? hydration by gradient alcohol(anhydrous ethanol,anhydrous ethanol,95%ethanol,95%ethanol,95%,90%ethanol,80%ethanol,75%ethanol,75%ethanol 1 to 2 min each)?repair antigen by 0.01 M pH6.0 citrate buffer when in the environment of the high pressure for 3 min?wash with 0.02 M pH7.2 7.4 PBS(phosphate buffer solution,phosphate buffer)(3 min x 3 times)?use3%hydrogen peroxide to eliminate endogenous peroxidase activity for 10 to 15 min at room temperature wash with 0.02 M pH7.2 7.4 PBS(phosphate buffer solution,phosphate buffer)(3 min x 3 times)-rabbit polyclonal antibody against people Erbin(1:300 dilution)?4? for 16-20hours?place at room temperature for 30 minutes?wash with 0.02 M pH7.2 7.4 PBS(phosphate buffer solution,phosphate buffer)(3 min x 3 times)?Goat anti Rabbit IgG-HRP at room temperature 30 min?wash with 0.02 M pH7.2 7.4 PBS(phosphate buffer solution,phosphate buffer)(3 min x 3 times)?DAB?hematoxylin staining?differentiation by0.1%hydrochloric acid alcohol?back to the blue by tap water?conventional dehydration transparent?sealing piece with neutral resin.Analysis integrated optical density(IDO)of every point in tissue microarray by Image analysis software Image pro plus 6.0(IPP6.0).randomly selected five horizons Each point in tissue microarray,determine and the IDO,the mean IDO of five horizons can represent total protein expression.2?Effect of Erbin knockdown on biological characteristics of human breast cancer cell lines.2.1 Screening high level of Erbin protein expression of breast cancer cell lines.Detecte level of Erbin protein expression of different breast cancer cell line such as SK-3,MCF-7,435 s,MD231 and BT549 by Western Blot.Take 20 pg protein mix with 2 x SDSLoading Buffer buffer in a ratio of 1:1?100-degree water bath for 10 min?ice cooling for 2 min.?on sample after centrifugal?6%SDS-PAGE electrophoresis?adopting protein Marker as reference?stop electrophoresis when the distance of bromophenol blue and the bottom is0.5 cm?transfer the protein of PAGE glue to PVDF(polyvinylidene fluoride,polyvinylidene fluoride)membranes by Bio-RAD miniature electric transfer system at 4 ?,300 mA for 100 min?take out the PVDF membrane with tweezers carefully?incubation for 1 h at room temperature with 20 ml sealing fluid(PBST containing 5%skimmed milk powder)-pour out sealing fluid?rabbit polyclonal antibody against people(1:1000dilution)incubation at 4 ? overnight-?washing membrane by 25 ml PBST(Phosphate Buffered Saline with Tween-20)(4min×4 times)?Goat anti Rabbit IgG-HRP(1:5000 dilution)incubate at room temperature for 1 hour?washing membrane by 25 ml PBST(4min×2times,8min×2times)?dry film then add chemiluminescence reagent incubate for 1 min?exposure for 1 min in the chemiluminescence apparatus?get the picture?internal control with a-tubulin.2.2 Construction and identification Erbin stable konckdowen breast cancer cell lines.Design and filter out the most effective sequence RNA interference technique.Transfer the clone to lentivirus to constructe vector which Erbin expression stable knockdown.Transfect the vector into 293 T cells then obtain virus supernatant.After detect the concentration,add virus in human breast cancer cell lines BT549,MD231 training for 48 hours then join puromycin for drug screening.Identificate the level of Erbin expression of stable knockdown on human breast cancer cell lines MD231-shRNA-KD,BT549 shRNA-KD and negative control cells MD231-shRNA-NC,BT549-shRNA-NC by Western Blot.2.3 Plate colong formation efficiency was compared after Erbin expression was knocked down in BT549 and MD231cells.CCK-8 assays show that Erbin stable knocked-down can promote cell proliferation in MD231 cells.Migration ability was compared after Erbin expression was knocked down in BT549,MD231 cells by Transwell chamber and scratch experiment.2.4 Proliferation and migration ability in vivo were compared after Erbin expression was knocked down inMD231 cells by nude mice local transfer model.3?Effect of Erbin knockdown on biological characteristics in vivo of Rat source cell lines.3.1 Construction and identification Erbin stable knockdown CT26 colorectal cancer cells in mice CT26 and,melanoma cells in mice B16.Design of rat Erbin stable knockdown target sequence by RNA interference technique.Transfer the clone to lentivirus to constructe vector which Erbin expression stable knockdown.Transfect the vector into 293 T cells then obtain virus supernatant.After detect the concentration,add virus in human breast cancer cell lines CT26,B16 training for 48 hours then join puromycin for drug screening.Identificate the level of Erbin expression of stable knockdown cell lines CT26-shRNA-KD,B16 shRNA-KD and negative control cells CT26-shRNA-NC,B16-shRNA-NC by Western Blot.3.2 Construction liver metastases model of mice by colorectal cancer cells CT26.Select 20 Babl/c mice which 6 weeks old,divided into two groups at random by male and female half.Narcotize Babl/c mice With 0.2%pentobarbital sodium by intraperitoneal injection(5?l/g).When anesthesia completely,after alcohol cotton ball wiping the skin of mice,cut a incision about 3 cm along the rib arch then oppression hemostasis with gauze.Expose the spleen,find blood vessel and ligamentsof the head of the spleen,free spleen head after the ligation.Pull the spleen out the abdominal cavity by eye tweezers gently.Inject 2×106/100ul cells in spleen tail by 1 ml syringe(about 3 min).During injection,parallel to the spleen capsule as far as possible,If hurt spleen,hemorrhagic shock will occur.After ligation of splenic pedicle then removal of the spleen after 5 min.suture the abdominal wall,then clean the wound by alcohol cotton ball.Observe the liver metastasis after mercy killing the mice four weeks later,3.3 Construction the whole body metastasis model of mice by melanoma cells B16.Select 20 C57BL/6 mice which 6 weeks old,divided into two groups at random by male and female half.Inject 2×106 cells/100 u 1 into mice via tail vein.Observe the metastasis after mercy killing the mice three weeks later,4.The relationship between Erbin and Epithelial-Mesenchymal Transition.Detecte level of EMT related markers on human breast cancer cell lines MD231-shRNA-KD,BT549 shRNA-KD and negative for cell MD231-shRNA-NC,BT549 shRNA-NC cell by Western Blot,to explore whether Erbin involved in EMT process or not.5.Constructe breast cancer model of Erbin PDZ domain structure mutation of MMTV-PyVT mice.1.The mice bred and observationBecause the mice MMTV-PyVT with rat breast tumor viruses and milking capacity is lost,so(MMTV-PyVT)(?)hybrid cross with WT.In addition MMTV-PyVT can only carry a rat breast tumor virus fragments increased the difficulty of the mice to foster.Erbin ? C/? C cross out of Erbin ? C/+(?)and Erbin ? C/+(?).(MMTV-PyVT)(?)came with Erbin ? C/? C? cross out PyVT&Erbin ? C/+.Then will PyVT&Erbin? C/+(?)came to Erbin ? C/? C(?)hybrid out PyVT&Erbin ? C/?C.2.Identification of miceCut the mice toes?put it in a 200?l eppendorf tube?add 20?lA liquid?put on the PCR reaction(97 ? for 1 hour,4 ? for enter)? add 70 ?l B liquid to maintain?Then take out the eppendorf tube ?centrifugal 3 min,4000 rpm?store at 4 ?.The primers that MMTV-PyVT mice genotype identification using are:PYVT-F 5'-GGA AGC AAG TAC TTC ACAAGG G-3'PYVT-R 5'-GGA AAG TCA CTA GGA GCA GGG-3'PYVT-pos-F 5' CAA ATG TTG CTT GTC TGG TG-3'PYVT-pos-R 5' GTC AGT CGA GTG CAC AGT TT-3'The primers that Erbin C-end PDZ domain structure mutant mice genotype identification using are:Erbin-1 5 '-CACTC TGTAATCAGT TCTTA GCAG-3'Erbin-WT 5'-GGTAA GACAG AAACT GGCAC CAG-3'Erbin-MT 5'-CACTC CAACC TCCGC AAACT C-3'Using 15 ?1 PCR reaction system According to the following PCR reaction conditions Electrophoresis with 2%agarose gel containing up to 1%EB.3.Mercy kill MMTV-PyVT mice and PyVT&Erbin ? C/+ after six months ?dehydration of breast cancer and lung-embedding? hematoxylin-eosin stainingResults1?Correlation analysis of clinicopathological features and expression of Erbin in breast cancer.Detect Erbin expression level of breast cancer tissue microarray by immunohistochemical.Erbin located in the cytoplasm,when the expression of Erbin is high it can sometimes located in the nucleus.The expression of Erbin has no significative relation with age,grading(P>0.05),but has closely relation with pathological diagnosis(F = 7.214,P = 0.000)and TNM(F=5.9977,P=0.000)(Tumor Node Metastasis)stages.2.Effect of Erbin knockdown on biological characteristics of human breast cancer cell lines.2.1 Detect the level of Erbin protein expression of breast cancer cell line SK-3,MCF-7,435 s,MD231 and BT549 by Western Bolt.successfully screened breast cancer cell lines BT549,MD231 whose level of Erbin protein express were high.2.2 Successfully,construct Erbin stable knockdown breast cancer cell lines MD231 ?shRNA-KD,BT549 shRNA-KD and negative control cells MD231-shRNA-NC,BT549-shRNA-NC by RNA interference technique.2.3 Cell proliferation in vitro was compared after Erbin stable knockdown of human breast cancer cells line BT549 by CCK 8,after knockdown speed of growth rate is speed up significantly(F = 13.808,P=0.001).Cell proliferation in vitro was influented byDifferent time point significantly(F = 1477.308,P = 0.000).Two factors between groups of cells in each group does not exist interaction effect(F ?1.607,P=0.182).Using one-way analysis of variance(One-Way ANOVA),proliferation capacity between experimental group and the control group at different time points was difference,in addition to the first day(F = 0.147,P =0.719),the fourth day(F = 0.788,P = 0.425),the fifth day(F = 0.021,P = 0.893).Cell proliferation in vitro was compared after Erbin stable knockdown of human breast cancer cells line MD231 by CCK 8,after knockdown speed of growth rate is speed up significantly(F=28.968,P=0.000).Cell proliferation in vitro was influented byDifferent time point significantly(F=2756.275,P=0.000)).Two factors between groups of cells in each group does not exist interaction effect(F=3.804,P=0.007).Using one-way analysis of variance(One-Way ANOVA),proliferation capacity between experimental group and the control group at different time points was difference,in addition to the second day(F=1.176,P=0.339),the fourth day(F=3.283,P=0.144),the fifth day(F=3.344,P=0.141),the seventh day(F=3.117,P=0.152).Plate colong formation efficiency results found that compare to Erbin stable knockdown of BT549 cells MD231 cells,clone formation rate of the control group is reduced significantly.(BT549 cells F = 1.597,P = 0.000;MD231 cells F = 3.428,P = 0.000).Migration ability of Erbin stable knockdown of BT549 cells,MD231 cells and the control cell was different significantly compare by transwell chamber(BT549 cells F = 2.579,P-0.000;MD231 cells F = 0.441,P =0.000).The results of Cell scratch were different significantly between Erbin stable knockdown of MD231 cells the control cell(MD231 cells F-94.280,P = 0.000).2.4 Proliferation and migration ability in vivo were compared after Erbin expression was knocked down inMD231 cells by nude mice local transfer model.The results show that local accumulated into tumor volume of Erbin stable knockdown of breast cancer cell lines MD231 is less than the control group obviously(F-8.431,P-0.010)Local number into tumor of Erbin stable knockdown of breast cancer cell lines MD231 is less than the control group obviously(F = 8.615,P =0.010);Local tumor score of Erbin stable knockdown of breast cancer cell lines MD231 is less than the control group obviously(F=9.707,P=0.007);Local transfer accumulated diameter of tumor which metastasis of Erbin stable knockdown of breast cancer cell lines MD231 is greater than the control group significantly(F = 29.64,P = 0.000);Number of tumor which is metastasis of Erbin stable knockdown of breast cancer cell lines MD231 is greater than the control group significantly(F = 32.10,P = 0.000);.The metastasis score of Erbin stable knockdown of breast cancer cell lines MD231 is less than the control group obviously(F=35.038,P=0.000).3.Effect of Erbin knockdown on biological characteristics in vivo of Rat source cell lines.3.1 Construction and identification Erbin stable knockdown CT26 colorectal cancer cells in mice CT26 and,melanoma cells in mice B16.Identificate the level of Erbin expression of stable knockdown cell lines CT26-shRNA-KD,B16 shRNA-KD and negative control cells CT26-shRNA-NC,B16-shRNA-NC by Western Blot.3.2 Construction liver metastases modelof mice by colorectal cancer cells CT26.Liver metastases cumulative volume Erbin stable knockdown mice colon cancer cells CT26 greater than the control group obviously(F = 5.108,P = 0.0038);liver metastases of Erbin stable knockdown.mice colon cancer cells CT26 greater than the control group obviously(F = 10.816,P = 0.005);The metastases score of Erbin stable knockdown mice colon cancer cells CT26 significantly greater than the control group(F = 11.11,P = 0.004).3.3 Construction the whole body metastasis model of mice by melanoma cells B16.The weight of lung of Erbin stable knockdown of mice melanoma B16 was greater than the control group(F = 5.974,P = 0.028);Number of tumor lung which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F = 21.043,P = 0.001);The cumulative diameter tumor lung which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F = 25.06,P = 0.000);The The score of lung tumors which were metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F = 18.193,P = 0.001).Number of tumor liver which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F= 17.21,P=0.001);The cumulative diameter tumor liver which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=14.716,P=0.002);The score of liver tumors which were metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=16.533,P=0.001).Number of tumor kidney which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=11.949,P=0.004);The cumulative volume tumor kidney which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=11.286,P=0.005);The score of kidney tumors which were metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=20.346,P=0.000):The number of organ which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=7.248,P=0.018);Number of tumorOther organs which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=17.759,P=0.001)The cumulative volume tumor kidney which is metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=16.333,P=0.001);The score of Other organs tumors which were metastasis of Erbin stable knockdown of mice melanoma B16 was greater than the control group significantly(F=17.55,P=0.001).4?The relationship between Erbin and Epithelial-Mesenchymal Transition.The expression of ZO-1,beta-catenin,Claudin 1,the EMT related markers of Erbin stable knockdown breast cancer cell lines BT549,MD231 are higher than the control group significantly.5.Constructe breast cancer model of Erbin PDZ domain structure mutation of MMTV-PyVTmice.1.The mice bredPyVT&Erbin?C/+;PyVT&Erbin?C/?C(?)2.Identification of miceMMTV genotype identification:MMTV-PyVT positive mice can appear a 556 bp banding.Erbin genotype identification:mice homozygous Erbin(Erbin A C/C)A mice can appear in a 300 bp and 400 bp band;Erbin wild-type mice(Erbin +/+)can have a stripe between 400 bp and 500 bp;Erbin heterozygote(Erbin ? C/?)in mice in 300 bp to 400 bp and 400 bp to 500 bp two all.3.Erbin PDZ domain structure mutation of MMTV-PyVT pulmonary metastasis of breast cancer in miceBy using Independent Sample t Test,analysis results showed that the weight of lung of PyVT&Erbin ? C/+ is no significantly different compared with control group(F=0.279,P=0.612).The number of lung cancer which is metastasis PyVT&Erbin ? C/+ is greater than the control group(F=20.463,P=0.002).The cumulative volume of lung cancer which were metastasis PyVT&Erbin ? C/+ have no difference compared with control group(F=2.78,P=0.133).The score of lung cancer which are metastasis of PyVT&Erbin ? C/+ is greater than the control group(F=14.032,P=0.006).Conclusion1?Erbin located in the cytoplasm,when the expression of Erbin is high it can sometimes located in the nucleus.The expression of Erbin has no significative relation with age,grading,but has closely relation with pathological diagnosis and TNM(Tumor Node Metastasis)stages.2.Erbin is closely related to appreciation ability in vitro,clone formation ability migration abilityof breast cancer cells in vitro.That is to say Erbin knockdown maybe play an important role in tumor cell metastasis.3?Erbin knockdown of colon cancer cells of mice melanoma cells of mice,make tumor cell metastasis more obvious in vivo,that is to say,Erbin knockdown plays an important role in Rat source cell metastasis.4.The expression of ZO-1,beta-catenin,Claudin 1,the EMT related markers of Erbin stable knockdown breast cancer cell lines BT549,MD231 are higher than the control group significantly.5.The number of lung metastasis and score Py VT&Erbin A C/+ mice on significantly greater than the control group,while the weight of lung and lung metastasis accumulated diameter there are no differences. |
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