The Effects And Mechanism Research Of Grp94 And MiR-3652 In The Human Breast Cancer Cell MCF-7

Endocrine therapy is widely used in the treatment of estrogen receptor positive breast cancer.But recently more and more patients have been found to be resistant to the endocrine drugs.Therefore,we are focusing on the research of molecular targeting agents.Numerous studies have showed that endoplasmic reticulum stress(ERS)is closely related to the apoptosis of cancer cells.Agents targeting ERS have emerged as the potential antitumor drugs.The ERS in breast cancer cells has not been fully investigated.Glucose regulated protein 94(Grp94)is the biomark of ERS.It has been reported that Grp94 is highly expressed in many cancers,such as breast cancer.Its expression level is related to the tumorigenesis and development,as well as the poor prognosis.In this research,we investigated the relationships between the interference to Grp94 and the apoptosis induced by ERS in the ER+ breast cancer cell MCF-7 and the mechanism of it.These results provide a new idea for the application of Grp94 to cure the breast cancer.Meanwhile,we found a microRNA,named miR-3652,located in the 5' exon of Grp94 gene.We also did some experiments to study the effects of miR-3652 over-expression on the biological characteristics of MCF-7.PART 1: THE MECHANISM RESEARCH OF THE APOPTOSIS INDUCED BY ERS IN THE HUMAN BREAST CANCER CELL MCF-7 INTERFERENCE TO GRP94Objective: To explore the relationships between the glucose regulated protein94(Grp94)interference and the apoptosis induced by endoplasmic reticulum stress(ERS)in human breast cancer cell MCF-7 and investigate the mechanism of it.Methods: Tunicamycin(Tm)was choosed to treat the human breast cancer cell line MCF-7 to build the ERS model in vitro.The siRNA sequence targeting Grp94 were synthesized and transferred into MCF-7.The effect of si-Grp94 on the apoptosis of MCF-7 after the treatment with Tm was evaluated by flow cytometry(FCM).The expression levels of ERS and unfolded protein response(UPR)indicators were detected by Western Blotting.Result: ERS model was built in vitro successfully.Compared with the control group,the apoptosis rate of si-Grp94 group detected by FCM was increased after the Tm treatment.Western blotting results showed that knockdown of Grp94 increased the expression of Bip,IRE-1 and p-JNK,and reduced the Bcl-2 protein level under ERS condition.Conclusion: The ERS status and apoptosis rate of si-Grp94 group was strengthened under the ERS condition in MCF-7.The pro-apoptosis effect may functioned via IRE1-JNK-BCL-2 pathway.PART 2: EFFECTS OF MIR-3652 OVER-EXPRESSION ON BIOLOGICAL BEHAVIOR IN THE HUMAN BREAST CANCERCELL MCF-7Objective: To explore the effect of miR-3652 over-expression on biological behavior in the human breast cancer cell MCF-7.Methods: The expression of miR-3652 was detected in a panel of breast cancer cell lines to select the suitable cells for the follow-up studies.The change of miR-3652 expression was detected in Grp94 knockdown cells by Real-time PCR to clarify the relationship between miR-3652 and Grp94 in the transcription level.Designed and synthesized the miR-3652 mimics and detected the efficiency of miR-3652 mimics by Real-time PCR.Transfected the mimics into MCF-7 and detect the variation of cell cycle,apoptosis and proliferation via FCM and MTT.Result: The lowest expression level of miR-3652 is in MCF cells.The expression of miR-3652 was decreased after the knockdown of Grp94(P <0.01).MiR-3652 mimics can increase the mRNA level of miR-3652 and promote the proliferation of MCF-7 atfter 48h(P<0.05);No significant difference was found when it comes to cell cycle and apoptosis.Conclusion: The expression of miR-3652 is in accordance with its host gene Grp94.The two genes may belong to the same transcription unit and miR-3652 may regulate by the promoter of Grp94.Over-expression of miR-3652 promoted the proliferation of MCF-7 but had no impact on cell cycle or cell apoptosis.