| Breast cancer is one of the most common malignant tumors in the world,causing serious threat to women's health.Breast cancer has the highest incidence of cancer in women both in the world and in China.According to the '2018 China tumor status and trends' report released by the National Cancer Center,compared with western developed countries,breast cancer in China has higher mortality rates and earlier onset age.Therefore,further studies on identification effective therapeutic targets have great significance on the prognosis and diagnosis of breast cancer patients.The ubiquitin-proteasome proteolysis system,responsible for degrading proteins,is involved in nearly all cellular processes.Dysregulation of the ubiquitin system leads to a variety of diseases such as cancers.Cullin protein family containing conserved cullin homology domain is a major type of E3 ligase.CUL4A(Cullin 4A),a member of the cullin protein family,forms CRL4A(cullin 4A-RING ubiquitin ligase)complex with a ring finger protein ROC1,an adapter protein DDB1,and DDB1-cullin4 associated factors(DCAFs).DTL,also known as CDT2,DCAF2 or RAMP(retinoic acid-regulated nuclear matrix associated protein),contains seven WD40 domains,is a member of the DCAFs family.It was found that DTL level were significantly increased in the cancer tissues.DTL was first identified as a down-regulated protein in retinoid-induced NT2 cell differentiation.Subsequently studies revealed that it playd important roles in regulating DNA replication and cell cycle.For example,knocking out DTL in yeast causes a delay in the S phase.In addition,DTL protects cells from DNA damage after S-phase and UV irradiation by regulating the degradation of CDT1,PR-SET7/SET8 and P21.The important roles of DTL in genomic stability suggests that DTL may be involved in tumorigenesis.Previous studies on breast cancer and Ewing's sarcoma have shown that down-regulated DTL attenuates the proliferation and migration of cancer cells.However,systematic studies of the roles and the mechanism of DTL in cancer still need more explored.Programmed cell death 4 is a tumor suppressor gene involved in apoptosis,transformation,invasion and tumor progression.PDCD4 interacted with eukaryotic translation initiation factor 4A(eIF4A)and 4G(eIF4G)to interfere mRNA translation process.It has been found that PDCD4 expression deficiency is closely related to the progression and prognosis of lung,ovarian,colon,kidney and other cancers.Considering the important roles of PDCD4 in cancer progression,the regulation of PDCD4 level in cancer deserves further study.This study mainly discussed the effect of DTL on tumor development through PDCD4 degradation.The results showed that DTL combined with PDCD4 and down-regulated PDCD4 through ubiquitin-degradation,promoting the migration,invasion and proliferation of cancer cells.This study explored the pathway of PDCD4 degradation in vivo,and tried to explain the mechanisms of the effect of DTL on tumor development,providing a theoretical basis for exploring DTL as a therapeutic target for breast cancer.Part ? The expression profile of DTL in tumors[Objects]Bioinformatics methods and the collected breast cancer tissues were used to analyze the DTL gene amplification in the genome,mRNA expression level,and the relationships with survival rates of patients.The aim of this part is to preliminarily explore DTL clinical significance in breast cancer,to provide foundations for further researches on DTL roles in breast cancer.[Methods]1.The expression of DTL in various tumors was analyzed by TCGA.2.GEO public database was used to analyze the expression of DTL in breast cancer.3.KM plotter was used to analyze the prognosis of patients.4.The total protein was extracted from the preserved breast cancer cell lines and epithelial cell lines in the laboratory,and the protein expression levels of DTL was detected by Western blot.5.Breast cancer tissues excised by breast surgery in Qilu Hospital of Shandong University and their corresponding adjacent tissues were collected.Total mRNA was extracted,and the expression levels of DTL gene in breast cancer tissues were detected by qRT-PCR experiment.6.Breast cancer tissues excised by breast surgery in Qilu Hospital of Shandong University and their corresponding adjacent tissues were collected.Total protein was extracted,and Western blot was performed to detect the expression levels of DTL protein in breast cancer tissues.7.Breast cancer tissue chip BR2082a from Alenabio biological company containing 160 breast cancer tissues and 32 normal breast tissues were used to further detect the protein level of DTL in breast cancer tissues through immunohistochemical staining experiment and statistical analysis.[Results]1.The copy number of DTL gene in the genome of breast cancer patients was significantly increased.2.Through the analysis of TCGA database,it was found that the expression of DTL gene was increased in vanous tissues.3.By analyzing GEO database,it was found that the expression of DTL gene was increased in breast cancer.4.High expression of DTL negatively correlated with the survival rates of breast cancer patients.5.Western blot analysis was performed on breast cancer cell lines and breast epithelial cell lines.The results showed that the protein expression level of DTL in breast cancer cell lines was up-regulated.6.qRT-PCR was performed to detect DTL mRNA expression level in the collected breast cancer tissues and their corresponding adjacent tissues,demonstrating that the expression of DTL in the breast cancer tissues was up-regulated at the transcriptional level.7.Western blot analysis was performed to detect DTL expression in collected breast cancer tissues and their corresponding adjacent tissues,demonstrating that the protein level of DTL was up-regulated in breast cancer tissues.8.Immunohistochemical staining was performed on breast cancer tissues and normal breast tissues on the tissue chip with DTL antibody,and quantitative analysis was conducted with Image Pro Plus.The results further proved that the expression of DTL protein in breast cancer tissues was up-regulated.[Conclusions]1.The results of public database analysis showed that DTL was up-regulated in breast cancer tissues compared with normal breast tissue.2.High expression of DTL is negatively correlated with prognosis of patients.3.The expression of DTL in collected tissue samples and cell lines was analyzed,and the results further verified that the expression of DTL was highly expressed in tumor tissues.Part ? DTL promotes tumor proliferation,invasion and migration abilities[Objects]According to the analysis results of DTL on expression and prognosis in part ?,the effects of DTL on the proliferation,migration and invasion of breast cancer were studied using in vitro cell experiment and nude mouse metastasis model.[Methods]1.DTL overexpression plasmid pLVX-DTL was constructed,and the plasmid pLVX with lentivirus packaging plasmid psPAX and PMD2.G were co-transfect into 293T cells.MCF7,MDA-MB-468,and BT549 were used to construct stable DTL overexpression cell lines and the corresponding control cell lines.The expression levels of DTL in stably transfected cell lines were detected by qRT-PCR and Western blot.2.Lentivirus vector pLKO-TetOn-Puro was used to construct DTL knockdown plasmids,and named shDTL#1,shDTL#2 and shDTL#3 respectively.Random sequence was used as blank control and named as Scramble.These constructed plasmids were co-transfected into 293T with lentivirus packaging plasmid psPAX and PMD2.G respectively.The proportion is pLKO:psPAX:PMD2.G=4:3:1.MDA-MB-231 was used to construct stable DTL knockdown cell lines and control cell lines.The expression levels of DTL in stably transfected cell lines were detected by qRT-PCR and Western blot.3.MTT and colony formation experiments were used to detect the proliferation ability of breast cancer cell lines.4.Western blot was used to detect the influence of DTL on EMT.5.Transwell and Matrigel assays were used to detect the migration and invasion abilities of cancer cells.6.Using the stable overexpression and knockdown cells of DTL,nude mouse metastasis model was established.The number of metastatic foci in lung tissues was detected by HE staining,and the in vivo experiment proved that DTL could inhibit the metastasis of breast cancer cells.[Results]1.pLVX-DTL plasmid was successfully constructed,and DTL overexpression cell lines MCF7(MCF7-pLVX,MCF7-DTL)and BT549(BT549-pLVX,BT549-DTL)were established by lentiviral transfection.qRT-PCR and Western blot were used to confirm the construction of the stable DTL overexpressed cell lines.2.Plasmids shDTL#1,shDTL#2 and shDTL#1 were successfully constructed,and DTL knockdown cell lines MDA-MB-231(named 231-shDTL#1,231-shDTL#2 and 231-shDTL#3,respectively)were established through lentiviral infection.It was confirmed by qRT-PCR and Western blot that the DTL knockdown cell lines were successfully established.3.DTL overexpression enhanced the proliferation ability of breast cancer cells.4.DTL overexpression improved the invasion and migration ability of breast cancer cells.5.The subcutaneous tumor formation experiment in nude mice showed that DTL silencing inhibited the proliferation of breast cancer.The metastatic tumor experiment in nude mice showed that the overexpression of DTL promoted the metastasis of breast cancer cells in vivo,while the DTL knockdown inhibited the metastasis of breast cancer.[Conclusions]1.DTL promoted the proliferation abilities of breast cancer cells.2.DTL promoted EMT,migration,invasion and metastasis abilities of breast cancer cells in vitro.3.DTL promoted the proliferation,invasion and metastasis of breast cancer cells in vivo.Part ? DTL promotes tumor development through PDCD4 degradation[Objects]The second part of the in vivo and in vitro experiment results showed that in breast cancer,DTL promoted breast cancer cell proliferation,migration and invasion abilities,as well as EMT.But the underlying mechanism in breast cancer still remains unclear.In this part,we mainly studies the molecular mechanism of DTL in breast cancer.[Methods]1.Detection and identification of DTL interacting proteins by protein gel electrophoresis and mass spectrometry.2.Immunoprecipitation method was used to detect the interaction between DTL and PDCD4.DTL antibody and PDCD4 antibody were used for immunoprecipitation in 293T cells.3.The interaction between DTL and PDCD4 were further detected by immunofluorescence in BT549 and MDA-MB-468 cell lines.4.pLVX-DTL and pLVX plasmids were transfected into 293T cells respectively,and the mRNA and protein expression levels of PDCD4 were detected by qRT-PCR and Western blot.plko-shDTL#1,plko-shDTL#2 and plko-shDTL#3 were transfected into 293T cells and treated with doxycycline.mRNA and protein expression levels of PDCD4 were detected by qRT,PCR and Western blot.5.Immunohistochemical was conducted on the tissue chip to detect the correlation between DTL and PDCD4 expression level.6.Cells were treated by MG132 with or without CHX,and PDCD4 protein expression level was detected by Western blot.7.293T cells were transfected with pLVX-DTL and pLVX plasmid respectively.After 48h,293T cells were treated with CHX.PDCD4 protein expression level was detected by Western blot per 30 mm.8.Flag-DTL,Myc-PDCD4 and HA-ubiquitin were co-transfected into 293T cells,and Myc antibody was immunoprecipitated,followed by Western blotting to detect PDCD4 ubiquitination level.9.Plasmids plko-shPDCD4#1,plko-shPDCD#2 and plko-shPDCD#3 were constructed and transfected into 293T cell lines with lentivirus packaging plasmids psPAX and PMD2.G respectively,at a ratio of pLKO:psPAX:PMD2.G=4:3:1.On the basis of 231-shDTL#1 cell line,PDCD4 silencing cell lines were constructed.Double silencing cell lines 231-shDTL#1-shPDCD4#1,231-shDTL#1-shPDCD4#2 and 231-shDTL#1-shPDCD4#3 were constructed successfully.10.The colony formation ability of double silencing cell lines was detected by MTT assay.The migration ability of double silencing cell lines was detected by wound healing,Transwell and Matrigel assays were used to determine the migration and invasion abilities.[Results]1.Mass spectrometry results showed that PDCD4 may bind with DTL.2.The co-immunoprecipitation experiment confirmed that PDCD4 and DTL interacted with each other.3.Structural simulation and co-immunoprecipitation experiment showed that DTL binds to PDCD4 through WD40 domain.4.PDCD4 interacted with CUL4A.Silencing DTL weakened the interaction between PDCD4 and CUL4A.5.DTL overexpression led to a decrease of PDCD4 protein level.Silencing DTL led to an increase of PDCD4 protein level.The mRNA level of PDCD4 was not influenced by DTL.6.There is a negative correlation between DTL and PDCD4 protein levels in breast cancer tissues.7.MG132 treatment blocked the degradation of PDCD4 caused by DTL overexpression.CHX and MG 132 treatment demonstrated that ubiquitin system was important to PDCD4 expression.8.DTL shortend the half-time of PDCD4.9.The Ser67 of PDCD4 was important in the degradation of PDCD4.10.DTL promoted the ubiquitin level of PDCD4.11.Silencing PDCD4 recovered the deficiencies of cells in proliferation,migration and invasion caused by DTL knockdown.12.The JNK pathway was activated by DTL.JNK inhibitor JNK-IN-8 treatment inhibited the promotion effect of DTL on breast cancer cells.[Conlusions]1.DTL interacted with PDCD4.2.DTL degraded PDCD4 via ubiquitin-proteasome system.3.Silencing PDCD4 recovered the deficiencies of cells caused by silencing DTL. |
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