| PURPOSE and BACKGROUND:Breast cancer is one of the most common malignancies in women and the most common type of cancer death in women worldwide.Previous studies showed that Ras can induce breast cell canceration,and the resulting tumor is similar to those induced by chemical carcinogens in their developmental kinetics and histopathological profiles.Many scientists believe that blocking the function of the oncogene Ras is a "unique skill" in cancer treatment,because mutations in this gene will drive the development of many different types of cancer.There are three different Ras genes in the human body: H-Ras,K-Ras and N-Ras.Among them,H-Ras is closely related to breast cancer.Studies have shown that H-Ras can be used as a prognostic evaluation factor or treatment target for breast cancer.However,due to different experimental methods,some proteins(genes)may lead to different experimental conclusions,which may affect the application of some mark proteins in diagnosis and treatment.Therefore,further studies on the roles of these proteins(genes)and their mechanisms are of great significance.Peroxisome proliferator-activated receptors gamma(PPARγ),a member of the PPARs superfamily,is a ligand-dependent transcription factor,this superfamily also possesses PPARα and PPARβ/δ.PPARγ can form a heterodimer with retinoid X receptor(RXR),and then combine with a peroxisome proliferator response element(PPRE),an upstream of the target gene promoter,to regulate the transcription of the target gene.PPARβ/δ has been shown to act as an oncogene in breast cancer cells.More and more evidences also show that PPARγ has the role of tumor suppressor gene.Experimental studies have found that activation of RAS can up-regulate the expression of ERK,thereby inhibiting the transcription of PPARγ,suggesting that the decreased expression of PPARγ protein due to the decreased transcription of PPARγ is one of the mechanisms of oncogene RAS causing tumors.Therefore,the induction of breast cancer cell lines by RAS could provide new clues to elucidate the role of PPARγ.Protein tyrosine phosphatase receptor-type F(PTPRF)is a member of the protein tyrosine phosphatase(PTP)family,which catalyzes the dephosphorylation of tyrosine residues.Like many other phosphatases,PTPRF participates in the regulation of cell proliferation and differentiation by targeting its downstream substrates.The transcription level of PTPRF may be regulated by PPARγ,in the promoter region of PTPRF there contains PPRE,a repeat sequence of AGGTCA binding site recognized by PPARγ.Besides onthe changes in protein abundance,scientists have paid more and more attention on the effects of protein post-translational modifications on cancer development,such as ubiquitination and phosphorylation.As a phosphatase,PTPRF participates in the regulation of phosphorylation equilibrium.Studies show that PTPRF is a tumor suppressor gene in liver cancer,non-small cell lung cancer and gastric cancer.PTPRF can inhibit the breast cancer metastasis by activating EGFR signal transduction.On the contrary,another study showed that PTPRF promoted the breast cancer cell metastasis in the mouse model.To date,it is still controversial whether PTPRF is a tumor suppressor gene or a tumor-promoting gene in breast cancer.Therefore,further clarifying the role of PPARγ/PTPRF and its signal transduction pathway in breast cancer will provide a new idea for targeting PPARγ in diagnosis and treatment of breast cancer.Therefore,based on the analysis results of relevant big data,we established a cell line by using the Ras induced rat breast cancer cell line,experimental techniques such as gene transfection and knockout to observe the physiological effects of PPARγ in breast cancer cells,the expression of PTPRF when PPARγ was increased,and to establish the relationship between the expression levels of PPARγ and the proliferation and metastasis of breast cancer cells.Finally,EMSA,ChIP,and luciferase reporting etc were used to verify whether PPARγ regulates PTPRF at the transcriptional level,and were further verified in vivo experiments so as to explore the mechanism of PPARγ’s regulation of PTPRF to inhibit the proliferation and metastasis of breast cancer.METHODS:1.Big data mining: KM plotter software was used to analyze the primary data of 3951 breast cancer patients downloaded from GEO database.Patients were divided into high-expression and low-expression groups according to m RNA expression levels,log-rank and Cox risk regression models were used to calculate the differences between the groups,and a Kaplan-Meier survival analysis chart was drawn.The number of cases,the median m RNA expression level,HR,95% CI and log-rank p-value were calculated and derived from the KM plotter webpage.2.Establishment of cell line: mouse PPARγ full-length c DNA was subcloned into the Eco RI site of pc DNA3.1 expression vector,and transfected into rat breast cancer FE1.2 cells using liposome transfection technology,then cells were screened by G418 to obtain stable expression of PPARγ transfected cell line FE1.2-PPARγhi,and empty vector transfected cell line FE1.2-Vector.The mouse PTPRF full-length c DNA was subcloned into the Xho I and Eco RI sites of the retroviral MSCV2.2 plasmid,and transfected into rat breast cancer FE1.2 cells using retroviral transfection technology,and then G418 was used for screening,the obtained polyclonal cell line,namely G418 resistant cells,was then subcloned by limiting dilution method,and finally a single clone with stable and high expression of PTPRF was obtained,named FE1.2-PTPRFhi.3.RT-q PCR and Western Blot: RT-q PCR and Western Blot were exploited to detect the m RNA and protein expressions of PPARγ and PTPRF in the clinical breast cancer and adjacent tissue samples.4.Cell proliferation experiment: The cell growth curve was drawn based on the cell count,and the effect of PPARγ on cell proliferation was detected by soft agar assay.The effect of PPARγ on cell migration was detected by scratch method,and the effect of PPARγ on the invasion ability of breast cancer cells was detected by thetranswell assay.5.EMSA: An 80 bp oligonucleotide containing the same PPRE(AGGTCA)was synthesized as a probe in the Ptprf promoter region,and combined the nuclear extract of the cell to be tested with the [γ-32P]ATP-labeled oligonucleotide incubate,and a gel migration test(EMSA)was performed to detect whether PPARγ was binded to the Ptprf promoter.6.ChIP: PPARγ antibodies and IgG from the same species were used to perform chromatin immunoprecipitation(ChIP)experiments,and specific primers were used for the Ptprf promoter to perform RT-q PCR to verify the difference between PPARγ and Ptprf promoter interaction.In order to explore how PPARγ up-regulates the expression of PTPRF,we further used ChIP experiments to detect the binding of RNA polymerase II(pol II)to the Ptprf promoter sequence in FE1.2-PPARγhi and FE1.2-Vector cell lines.Finally,the degree of histone modification of the Ptprf promoter was analyzed by ChIP experiment to reveal the mechanism of PPARγ regulating the transcription activity of the Ptprf promoter.7.Luciferase reporter experiment: The Ptprf promoter sequence was constructed into a dual fluorescent reporter gene vector.By co-transfecting the reporter gene with PPARγ or its empty vector into FE1.2 cells,after 48 hours,the two types of fluorescence were detected by a multifunctional enzyme marking instrument to determine whether PPARγ can increase the transcriptional activity of the Ptprf promoter.8.PTPRF knockout stable cell lines were constructed in FE1.2-Vector and FE1.2-PPARγhi cell lines respectively,then the growth of the cells,and the relationship between PPARγ and breast cancer cell growth after knocking out the Ptprf gene were detected.In order to confirm the regulatory effect of PPARγ on PTPRF,PPARγ antagonist GW9662 and PTP inhibitor NSC-87877 were added to the culture system.After counting the cells for 4 consecutive days,the growth curve of each group of cells was drawn,and Western blotting was performed on PTPRF,to determine the biological behavior of cells after PPARγ/PTPRF was inhibited.9.Mouse tumor-bearing experiment: FE1.2-Vector and FE1.2-PPARγhi cells were injected into the nipple fat pads of 5 mice in each group.The third group(5 mice)was injected with FE1.2-Vector cells,and the PPARγ agonist RG(1 mg/kg)was given every other day for the next two weeks.The mice were sacrificed on the 21 st day,breast tumor tissue and lung tissue were excised,measured and photographed,and the inhibitory effects of PPARγ on breast cancer growth and metastasis in vivo were detected.RESULTS:1.Correlation between PPARγ and PTPRF in breast cancer patientsKM plotter software analysis showed that the survival time of breast cancer patients with high PPARG m RNA expression was longer than low PPARG m RNA expression;and also the survival time of breast cancer patients with high expression of PTPRF m RNA was longer than low expression of PTPRF m RNA.2.Expressions of PPARG and PTPRF m RNA were detected in human breast cancer tissuesThe mean expression levels of PPARG and PTPRF m RNA in the breast cancer paracancerous tissues were much higher than those in the cancer tissues,and correlation analysis showed that the expression levels of PPARG and PTPRF m RNA in the paracancerous tissues of the same patients were also much higher than those in the cancer tissues.3.Correlation between Ras and PPARγThe growths of rat breast cancer cell lines FE1.2 and FE1.3 induced by v-Ha-Ras were very different.Western blot result showed that the expression of PPARγ in FE1.3 cells was significantly higher than that in FE1.2 cells,and PPARγ was nearlcy not expressed in FE1.2 cells.PPARγ and PPARδ were uniquely expressed in Fe1.2 and Fe1.3 cells,respectively.4.The effect of overexpression of PPARγ on the proliferation,metastasis and migration of breast cancer cells.The cell growth curve showed that compared with the control group,the FE1.2-PPARγhi group cells showed obvious delayed proliferation and slow growth.The results of soft agar assay showed that compared with the control group,the clone formation rate of the FE1.2-PPARγhi group was reduced by about 70%.Cell scratch test showed that the migration ability of the FE1.2-Vector group during cell damage repair was significantly higher than that of the FE1.2-PPARγhi group.Transwell cell invasion experiment showed that compared with the control group,the invasiveness of the FE1.2-PPARγhi group was reduced by about 60%.5.PPARγ up-regulates the expression of PTPRFRT-q PCR showed that Ptprf m RNA expression was lower in FE1.2-Vector cells,while higher in FE1.2-PPARγhi cells.Western blotting showed that the expression of PTPRF in FE1.2-PPARγhi cells was significantly higher than that in FE1.2-Vector cells.The cell counting method showed that,compared with the control group,the proliferation ability of FE1.2-PTPRFhi cells was significantly reduced.That is,PTPRF was negatively correlated with the proliferation of breast cancer cells,while positively correlated with the expression of PPARγ.6.The mechanism of PPARγ regulating PTPRF gene expressionEMSA experiment results showed that FE1.2 cells did not show obvious bands,while FE1.3 cells showed PPARγ1 and PPARγ2 bands at same time,but these two bands disappeared under the competition of excessive cold oligonucleotides,and the addition of PPARγ antibody produced a supershift band in the FE1.3 cell protein,and the PPARγ1 and PPARγ2 bands became lighter.The subsequent ChIP experiment showed that there was a larger amount of Ptprf promoter sequence in the eluate of the PPARγ antibody.In the FE1.2-PPARγhi group,RNA polymerase II(Pol II)binds more Ptprf promoter sequences than in the FE1.2-Vector group.H3K4me3,which can activate the transcriptional activity of the promoter,accumulates more on the Ptprf promoter in the FE1.2-PPARγhi group.The results of the luciferase report experiment showed that the fluorescence of luciferase co-transformed with PPARγ increased by 3 times.7.Inhibition of PTPRF expression and observation of the effect of PPARγ on the proliferation of breast cancer cellsWe found that after knocking out the Ptprf gene,no matter what the expression level of PPARγ,breast cancer cells did not show any slow growth trend.8.The effect of PPARγ antagonists and PTP inhibitors on the proliferation of breast cancer cellsReverse experiment results showed that in the presence of PPARγ antagonist GW9662 and PTP inhibitor NSC-87877,the growth rate of PPARγhi cells was significantly increased.For PTPRFhi cells,when NSC-87877 was present,the cell growth rate increased.When GW9662 was present,there was no obvious change in the growth rate of cells.9.Detection of the inhibitory effect of PPARγ on breast cancer in NOD/SCID mouse tumor-bearing modelThe results of the measurement of transplanted tumor volume in mice showed that compared with control mice in the FE1.2-Vector group,the tumor volume in the FE1.2-Vector+RG group was reduced by about 30%,and the tumor volume in the FE1.2-PPARγhi group was reduced up to 60%.The results of lung tumor metastases showed that the number and size of lung tumor metastases in the FE1.2-PPARγhi group were significantly lower than those in the FE1.2-Vector control group.The constructed stable PTPRF knockout cell line and the control cell line were injected into the mouse model,with RG stimulation and non-stimulation,then the tumors in the mice were taken out,and the tumor volume was calculated.The results showed that whether in FE1.2-PPARγhi cell line that highly expressed PPARγ or in the FE1.2-Vector that induced PPARγ through RG,once the expression of PTPRF was knocked out,the tumor growth rate was no longer inhibited.CONCLUSION:1.By using KM Plotter software to analyze big data and clinical tissue samples to detect the expression of PTPRF and PPARγ,our results showed that the expressions of PPARγ and PTPRF were correlated in breast cancer.2.In the breast cancer cell lines obtained by injection of retrovirus containing Ras,PTPRF expression was enhanced after PPARγ transfection in FE1.2 cells.After the PTPRF gene was knocked out,the proliferation of breast cancer cells was not affected regardless of the expression level of PPARγ.These results indicated that PPARγ was the upstream of PTPRF and inhibited the proliferation of breast cancer cells by up-regulating PTPRF.3.The specific binding of PTPRF promoter to PPARγ protein was verified by EMSA and ChIP assay.The ChIP assay also verified that the binding of PPARγ to the PTPRF promoter facilitated the aggregation of RNA polymerase to the PTPRF promoter,thus promoting transcription,inducing the 3-dimethylation of H3K4 in which the promoter was located,and enhancing the transcriptional activity of the promoter.These results indicated that PPARγ was a specific binding protein of PTPRF.4.In vivo experiment in NOD/SCID mouse tumor-bearing model further confirmed that PTPRF is a downstream gene of PPARγ,PPARγ not only inhibits the growth of transplanted tumors,but also reduces the metastasis of remote organs by inhibiting cell migration and invasion.The above results suggest that increasing PPARγ can promote the transcription of PTPRF to inhibit the proliferation and metastasis of breast cancer. |
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