| Background In recent years,the incidence and mortality of breast cancer showed a rising trend in our country;among them,about 50%~70% belongs to estrogen-dependent breast cancer.Endocrine treatments have good effect on estrogen-dependent breast cancer,but it but it has a high metastasis rate in the late period,leading to an ineffective treatment.Phosphoglucose isomerase(PGI),a housekeeping gene,plays a ctritical role in both glycolysis and gluconeogenesis,catalyzing the interconversion of glucose-6-phosphate and fructose-6-phosphate in intracellular environment which also behaves extracellularly as a cytokine called autocrine motility factors(AMF).AMF could be used in combinatting with some receptors which may accelerate the progression and metastasis of cancers.The expression of PGI in breast,lung and other gastrointestinal tumors are abnormal,some scholars believe that the abnormal expression of PGI is related to the degree of malignant in tumor.Hypoxia inducible factor-1(HIF-1)is considered as the center regulator of tumors which adapt to hypoxic microenvironment and it also involved in a series of angiogenesis,cell proliferation and adhesion processes.HIF-1 includes HIF-1? and HIF-1?,HIF-1? plays role on the growth and metastasis of tumor.In addition,it also regulates the transcription and expression of HKII,PGI,PKM2 and LDHA gene.Recent studies have shown that PKM2 could reverses the expression of HIF-1?.Does PGI have a similar effect? What is the correlation between PGI and HIF-1?? How does PGI control? There is no report yet.Therefore,the expression of PGI and HIF-1? in breast cancer were detected firstly;and then the MCF-7 cells were transfected by lentivirus for obtaining cells with low expression of PGI;Finally we detected the effects of PGI-si RNA on HIF-1? as well as the proliferation,apoptosis and glycolysis of MCF-7 cells;by the way,we also explored its related mechanisms.Methods Part ? We collected 30 cases of patients with breast carcinoma as well as its corresponding adjacent tissues.The differences of both PGI and HIF-1? in tumor tissues and adjacent tissues were detected by PCR and Immunohistochemistry;the differences of PGI and HIF-1? between MCF-7 cells and MCF-10 A cells were detected by PCR and Western Blot; Part ? The MCF-7 cells were transfected by lentivirus for obtaining cells with low expression of PGI,and the results were detected by immunofluorescence,RT-PCR and Western blot.Then the influence of PGI-si RNA on HIF-1? were detected by RT-PCR and Western blot.The effect of PGI-si RNA on cells were observed by optical microscope.The inhibition of cell proliferation was detected by CCK-8 assay.The changes of cell cycle distribution were examined by flow cytometry.The changes of apoptosis were examined by both Hochest and flow cytometry.Western blot was used to detect the effect of PGI-si RNA on the protein of cell cycle and apoptosis.The effect of PGI-si RNA on the lactic acid production of MCF-7 cells were detected by lactic acid content assay.Finally,the effects on the proteins of glycolytic were detected by Western blot.Results 1.The content of PGI and HIF-1? in breast cancer tissues was higher than that in adjacent tissues.2.The content of PGI and HIF-1? in MCF-7 cells was higher than that in MCF-10 A cell.3.PGI-si RNA could reduce the expression of HIF-1? in MCF-7 cells.4.PGI-si RNA could inhibit the proliferation of MCF-7 cells.5.Cell cycle: PGI-si RNA could arrested cell cycle in G0/G1 phase(P<0.05).The percentage of cells in G0/G1 phase in each group is as follows: PGI-si RNA group(66.12±1.12)%,Control group(43.98±0.45)%,Mock group(45.57±1.16)%.The difference was statistically significant(P <0.01).6.Cell apoptosis: The apoptotic rates of PGI-si RNA group were(11.89±0.65)%,which showed statistically significant difference(P<0.01)compared with the control group(3.45±0.12)% and Mock group(4.35±0.08)%.7.Hochest33258 staining: the result showed that PGI-si RNA appeared to varying degrees of chromatic agglutination,karyopyknosis and a set of typical apoptotic morphological changes in cells.8.Lactic acid assay: The concentration of lactic acid in PGI-si RNA group was(1.133±0.194)mmol/L,which showed statistically significant difference(P < 0.01)compared with the control group(3.801±0.346)mmol/L and Mock group(3.863±0.194)mmol/L,The results suggest that PGI-si RNA can decrease the total lactic acid production of MCF-7 cells.9.Western blot: the results showed that PGI-si RNA could increase the expressions of Bax,Cleaved Caspase-3,while the expressions of Bcl-2,Cyclin D1,CDK4,HKII,PKM2,LDHA,PI3 K,AKT and m TOR proteins were decreased.Conclusions 1.The content of PGI and HIF-1? in breast cancer tissues and cells were high. 2.PGI-si RNA can decrease the expression of HIF-1?,In addition,it can inhibit the glycolysis of MCF-7 cells and play roles in proliferation and apoptosis,and its related regulatory mechanism may be related to the signaling pathway of PI3K/AKT/m TOR which is mediated by HIF-1?. |
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