| BackgroundBreast cancer is one of the most malignant tumors among female people,accounting for 31%of all malignancies and having the second highest fatality rate.According to the epidemiological data of the World Health Organization in 2022,breast cancer has become the most common malignant cancer around the world accounting for 11.7%of new cases.There were about 300,000 new cases in China last year which was second only to the lung cancer.Breast cancer can be classified into four pathological types:luminal A,luminal B,overexpression of HER 2 and triple-negative type.Radical resection is the most effective therapy.However,postoperative recurrence and metastasis are reasons for the failure of clinical treatments.Therefore,further investigation of molecular regulatory mechanisms of breast cancer recurrence and metastasis might be helpful to provide new targets of the breast cancer clinical treatments.Long non-coding RNA(LncRNA)is a class of RNA with the length over 200 nt.Increasing experimental evidence indicated that different types of tumors usually contained the differential expression profiling of lncRNA,which played an oncogenic or anti-oncogenic role.Our previous experiments showed that LINC00648,was upregulated in breast cancer tissues.Therefore,this study will thoroughly demonstrate the molecular biological mechanisms of LINC00648 in breast cancer progression.Mitochondria is an integral component of the multicellular life which undergoes morphological changes and spatial rearrangements through fission and fusion in order to accommodate biological requirements of eukaryocytes.The dynamic balance of mitochondrial fusion-fission processes is also defined as the mitochondrial dynamics balance.Nowadays,recent studies demonstrated that the mitochondrial dynamics balance was closely related to the biological functions of eukaryocytes.The abnormal expression of proteins which related to mitochondrial dynamics in tumors contributing to the imbalance of mitochondrial dynamics also affect the developments and metastasis of malignant tumors.However,the specific mechanism is still unclear.According to catRAPID database prediction results,a possible interaction between LINC00648 and PGAM 5 is probably existed.Human phosphoglycerate mutase family member 5(PGAM5)is a mitochondrial serine/threonine phosphatase involving in the regulation of various metabolic pathways in human body.Recently,researchers discovered that PGAM5 was related with mitochondrial fission,on MOM,PGAM5 dephosphorylates Drpl at Ser-637 to induce mitochondrial fragmentation,which promoted the epithelialmesenchymal transition(EMT)process.Furthermore,the proliferation and invasion ability of tumor cells would be enhanced.However,the regulatory mechanism of PGAM5 expression in tumor cells is still unknown.Ring finger protein 5(ring finger protein 5,RNF5)is a member of E3 ubiquitin ligase localizing to the endoplasmic reticulum and mitochondrial membrane.RNF5 is recognized to be involved in the regulation of substrate stability and localization through K63 or K48 ubiquitination.Recent studies confirmed that RNF5 could ubiquitinate PGAM5 by interactions,leading to the degradation of PGAM5 and subsequently down-regulating the downstream pathways.In this study,we investigated the role of LINC00648 in the proliferation,migration,and invasion of breast cancer cells through series of experiments in vitro and in vivo.The results confirmed that LINC00648 could cause the imbalance of mitochondrial dynamics of breast cancer cells which promoted breast cancer oncogenic progression by inhibiting RNF5-mediated degradation of PGAM5 ubiquitination.Methods1.To analyze the expression level of LINC00648 in breast cancersTumor and adjacent tissues of breast cancer were collected in clinical samples.Utilizing qRT-PCR,we analyzed the differential expression levels of LINC00648 between breast cancer and adjacent tissues.The GEPIA Database was applied for survival analyzing of breast cancer patients in order to analyze the effect of the expression level of LINC00648 on the prognosis of breast cancer patients.2.To investigate the effects of LINC00648 on proliferation,migration,invasion,and apoptosis processes of breast cancer cellsHuman breast cancer cell lines(MCF-7,T47D,MDA-MB-231)were transfected by lentivirus in vitro in order to construct stable breast cancer cell lines,called Up-regulated LINC00648 and Down-regulated LINC00648.The oncogenic effect of LINC00648 on breast cancer cell lines was analyzed by CCK-8,EdU proliferation assays and Transwell assays.3.To study the effect of LINC00648 on apoptosis in breast cancer cellsThe expression of the apoptosis-related proteins PARP,Cleaved-PARP,Caspase 3,Cleaved-caspase 3 were analyzed by Western-Blot.Apoptosis level was measured by utilizing Tunel assay and flow cytometric technique.4.To investigate the effects of LINC00648 on subcutaneous breast tumor growth and metastasis in vivoMCF-7 cells were calculated and implanted subcutaneously in female nude mice.Subcutaneous tumor size was measured regularly by drawing tumor growth curves.Nude mice were executed 5 weeks later and subcutaneous tumors were dissected in order to be photographed and weighed.Immunohistochemistry staining assays were applied for demonstrating the effect of LINC00648 on the implanted subcutaneous tumors.Different series of MCF-7 cells were calculated and implanted subcutaneously in female nude mice.After 5 weeks,the growth and metastasis of breast cancer cells were observed by bioluminescence imaging system.Subsequently,HE staining method was utilized to study the effect of LINC00648 on breast cancer metastasis activity.5.To investigate the effect of LINC00648 on mitochondrial fission in breast cancer cellsMito-Tracker Red CMXRos was utilized to visualize the mitochondria in order to investigate the effect of LINC00648 on mitochondrial fission and ROS producing.6.To clarify the mechanism of LINC00648 in regulating mitochondrial fission and cancer progression in breast cancer cells via RNF-mediated ubiquitinated degradation progress of PGAM5Western-Blot and immunohistochemical staining assay were applied for investigating the effect of LINC00648 on PGAM 5 expression in breast cancer cells and tumor tissues of nude mice.The distribution of LINC00648 and PGAM5 in breast cancer cells was observed by fluorescence hybridization in situ(FISH)and immunofluorescence technique.Co-IP assay was applied to investigate whether LINC00648 affected the interaction of RNF5-PGAM 5 and PGAM 5 ubiquitination modification level.Using CCK-8,EdU proliferation,Transwell assay and Mito-Tracker staining assays were utilized to investigate whether PGAM 5 mediated the ubiquitinated degradation progress of PGAM5 and the role of LINC00648 in the regulation of mitochondrial division and breast cancer progression in breast cancer cells.Results1.LINC00648 is highly expressed in breast cancer tissues which negatively correlates with prognosisThe results of qRT-PCR showed that the expression of LINC00648 in breast tumor tissues was significantly higher than that in adjacent tissues.Database analysis showed that LINC00648 was negatively correlated with the prognosis of breast cancer patients.2.LINC00648 promotes proliferation,migration and invasion of breast cancer cellsThe results of CCK-8 and EdU proliferation experiments showed that overexpression of LINC00648 promoted the proliferation of breast cancer cells,and knocking down LINC00648 inhibited the proliferation process.The results of Trans well experiments showed that overexpression of LINC00648 significantly promoted the migration and invasion of breast cancer cells,while knocking out LINC00648 inhibited these processes.3.LINC00648 suppresses apoptosis of breast cancer cellsThe results of Western-Blot showed that compared with the control group,the expression level of LINC00648 overexpressed apoptosis-related protein was significantly reduced,while LINC00648 silencing increased the expression level of apoptosis-related protein.The results of FACS and Tunel staining showed that the proportion of apoptotic cells was significantly lower by overexpression of LINC00648 than that in the control group,while the proportion of apoptosis in breast cancer cells was significantly increased due to deletion of LINC00648.4.LINC00648 overexpression promotes the growth and metastasis of breast tumor in nude miceThe results of subcutaneous tumorigenesis in nude mice showed that overexpression of LINC00648 promoted the growth of subcutaneous breast tumors.It was observed by the IHC that nude mice implanted with LINC00648 breast cancer cells obtained stronger metastasis ability than that in the control group.The results showed that LINC00648 elevated the growth and metastasis level of breast tumors in vivo.5.LINC00648 promotes mitochondrial fission in breast cancer cellsThe results of Mito-Tracker staining showed that cells overexpressed LINC00648 had obvious mitochondria division phenomenon compared with the control group,while cells down-regulated LINC00648 led to significant lengthening of mitochondrial elements.The result of FACS showed that LINC00648 promote the production of ROS.6.LINC00648 promotes mitochondrial fission and breast cancer progression by inhibiting RNF5-mediated ubiquitination degradation of PGAM5The results of Western-Blot and immunohistochemical staining showed that comparing with the control group,the expression level of PGAM5 in the LINC00648 overexpression group was significantly increased,while the expression level of PGAM5 was significantly reduced by knocking down LINC00648.The results of CCK-8,EdU assay,Transwell assay and Mito-Tracker staining showed that knocking down PGAM5 could inhibit the promotion of LINC00648 on proliferation,migration,invasion and mitochondrial fission of breast cancer cell.Co-IP experiments showed that LINC00648 inhibited the interaction between RNF5 and PGAM5,so that the ubiquitination level of PGAM5 decreased.Conclusion1.LINC00648 promotes mitochondrial fission,proliferation,migration and invasion ability while inhibits apoptosis level in breast cancer cells.2.LINC00648 inhibits the degradation of PGAM5 by decreasing the ubiquitination modification of PGAM5 by RNF5,which exerts cancer-promoting effects. |
