Discovery And Functional Investigation Of MiR-520d-5p Inhibiting The Proliferation And Metastasis Of Triple Negative Breast Cancer Cells By Targeting IGSF3

Background: Breast cancer is the most common malignant tumor in women worldwide.In China,breast cancer also continuously accounts for a large portion(about 15%)of all new cancers.Common risk factors for breast cancer include non-genetic and genetic ones.With the development of molecular bioinformatical technology,a large number of studies have explored the biological factors that affect the occurrence,development and prognosis of breast cancer.However,it is still one of the challenges in breast cancer research due to its complex mechanism and numerous influencing factors.Although the molecular typing of breast cancer has brought earth-shaking changes to the treatment of breast cancer,there are still quite a few triple-negative breast cancer cases with poor prognosis,poor effect and lack of means.Therefore,further exploring the specific mechanism of breast cancer proliferation and metastasis and finding its biomarkers are of great significance for the diagnosis and treatment of breast cancer in the future.Micro RNAs(or miRNAs)are non-coding RNAs(about 22 nt in length)that can regulate the silencing and post-transcriptional expression of other genes by inhibiting translation or cleaving complementary target genes.A growing number of studies have shown that abnormally expressed miRNAs are associated with cell proliferation,apoptosis and metastasis in many solid tumors.Among them,some miRNAs have been identified to be associated with breast cancer.In our previous work,we screened serum miRNA expression profiles and found abnormally expressed miRNAs in breast cancer tissues for further screening and verification.Through further screening,five miRNAs(hsa-miR-99 a,hsa-miR-34 c,hsa-miR-520d-5p,hsa-miR-26 b,hsa-miR-206)were found to be down-regulated in breast cancer tissues.Only the expression of miR-520d-5p in breast cancer tissue and adjacent tissue was significantly different(P=0.007),and the difference was more pronounced in triple-negative breast cancer tissue,which suggesting that miR-520d-5p may be a potential triple-negative breast cancer biomarker.In order to investigate the mechanism of miR-520d-5p inhibiting triple-negative breast cancer proliferation and metastasis,we used the database Starbase,miRdb and miRwalk to predict the downstream target genes of miR-520d-5p in the previous experiments.Compared with adjacent tissues,only IGSF3 was significantly overexpressed in breast cancer tissues among the top 5 most likely target genes.The 3’-UTR sequence of IGSF3 m RNA has complementary pairing with the seed sequence of miR-520d-5p,suggesting that IGSF3 may be the downstream target gene of miR-520d-5p.Utill now,there is no report on the mechanism of miR-520d-5p related to the occurrence and development of triple-negative breast cancer.Therefore,this study intends to explore the specific mechanism of Mi R-520d-5p on triple-negative breast cancer proliferation and metastasis through in vitro and in vivo experiments,and provide a new direction for future diagnosis and precise treatment of triple-negative breast cancer.Methods:(1)Follow-up of 60 breast cancer cases: We collected and summarized the clinical and pathological data of 60 female breast cancer patients from July 2016 to May 2018.The mean follow-uptime was 2.9 years.The clinical and pathological data were analyzed and the disease progression and survival of triple-negative and non-triple-negative breast cancer patients were compared.(2)The expression levels of miR-520d-5p in breast cancer tissues and different cell lines: RT-q PCR was used to detect the expression levels of miR-520d-5p in different tissues.In addition,human breast epithelial cell lines(MCF-10A),breast cancer cell lines(MDA-MB-231,MCF-7,T47D)were cultured under appropriate conditions.RT-q PCR was used to detect the expression level of miR-520d-5p in different cells.(3)The effects of up-regulating miR-520d-5p on the proliferation,apoptosis and metastasis of breast cancer cells: Cell transfection was used to up-regulate miR-520d-5p in breast cancer cells;The apoptosis rate of cancer cells was detected by flow cytometry,and the effect on cancer cell viability was tested by CCK8 Assess.The impact on breast cancer cell invasion and migration was tested by transwell invasion test and scratch test.The subcutaneous tumor-bearing nude mice were subjected to miR-520d-5p overexpression intervention by tail vein injection of lentivirus for tumor proliferation curves.Tumor cell apoptosis was detected and analyzed by TUNEL experiment,and HE staining was used to analyze the liver and lung metastasis of breast cancer in nude mice.(4)IGSF3 may be a downstream target gene of miR-520d-5p: By Dual-Luciferase reporter assay,it was preliminarily verified that miR-520d-5p directly inhibited IGSF3 expression by binding to 3’UTR.The biological effects of miR-520d-5p targeting IGSF3 were validated by RNA immunoprecipitation(RIP).The effects of miR-520d-5p overexpression on the expression of IGSF3 protein and IGSF3 m RNA in subcutaneous tumor-bearing tumors of MDA-MB-231 nude mice were analyzed by Western blot and RT-PCR.(5)The effects of down-regulation of IGSF3 on proliferation,apoptosis and metastasis of breast cancer cells: Cell transfection was used to down-regulation of IGSF3 in breast cancer cells by cell transfection,and simultaneous up-regulation of miR-520d-5p and IGSF3 as a supplementary experiment,CCK8 assay was used to evaluate cancer cell viability,Flow cytometry was used to detect the apoptosis rate of cancer cells,and transwell and scratch assays were used to verify the effect on the invasion and migration of breast cancer cells.In the cells with over-expression of MIR-520D-5P,IGSF3 was over-expressed,and the changes in biological behavior were observed.The subcutaneous tumor-bearing nude mice were subjected to IGSF3down-regulation intervention by tail vein injection of lentivirus,for tumor proliferation curves.(6)IGSF3 may affect the proliferation and invasion of breast cancer cells by regulating the JAK2-STAT3 pathway: The protein expressions of stat3,p-stat3,jak2 and p-jak2 were detected in MDA-MB-231 cells with up-regulated IGSF3 and down-regulated IGSF3 respectively.The IGSF3-overexpressed breast cancer cells were treated with STAT3 inhibitor.The effect on cancer cell viability was tested by CCK8 Assess,and the impact on breast cancer cell invasion was tested by transwell invasion test.Results:(1)Follow-up data of 60 breast cancer cases: The average follow-up time was2.9 years.The proportion of disease progression in triple-negative breast cancer patients was significantly higher than that in non-triple-negative breast cancer patients,(P <0.05).The OS rate of triple-negative breast cancer patients was also significantly lower,(P <0.05).Among whom,more liver and lung metastases were seen in triple-negative breast cancer patients.(2)The expression of miR-520d-5p in breast cancer tissues and different cell lines:RT-PCR results showed that miR-520d-5p was significantly down-regulated in breast cancer tissues,which was 11.8 times lower than normal adjacent tissues(P <0.05).The expression level of miR-520d-5p in stage III-IV breast cancer tissue was significantly lower than that in stage I-II ones(P <0.05).The expression level of miR-520d-5p in tissue of patients with metastatic breast cancer was significantly lower than that in non-metastatic patients(P <0.05).The expression level of miR-520d-5p in tissue of triple-negative cancer patients was significantly lower than that in non-triple-negative patients(P < 0.05).Compared with human breast epithelial cells(MCF-10A),the expression level of miR-520d-5p in MCF-7,T47 D and MDA-MB-231 cells was significantly down-regulated(P <0.05).And it was most significantly down-regulated in MDA-MB-231 cells.(3)The effects of up-regulating miR-520d-5p on the proliferation,apoptosis and metastasis of triple-negative breast cancer cell MDA-MB-231: CCK8 experiment showed that the cell viability of miR-520d-5p overexpressed MDA-MB-231 cells was significantly reduced compared with the negative and blank controls(P < 0.05).Flow analysis showed increased apoptosis in miR-520d-5p overexpressed MDA-MB-231 cells compared with negative and blank controls.Transwell and scratch experiments showed that the up-regulation of miR-520d-5p could significantly inhibit the invasion and migration of MDA-MB-231 cells compared with the negative control group and blank control group(P < 0.05).Overexpression of miR-520d-5p could significantly reduce tumor volume,and promote the apoptosis of subcutaneous tumor cells in nude mice with breast cancer,and inhibit liver and lung metastasis of tumor cells in nude mice with breast cancer(P < 0.05)(4)IGSF3 may be a downstream target gene of miR-520d-5p:The results of the Dual-Luciferase reporter assay showed that the fluorescence activity was significantly reduced after simultaneous transfection of the mock and wild plasmids;there was no difference in the fluorescence activity after simultaneous transfection of the mock and mutant plasmids(P > 0.05);the expression levels of IGSF3 and IGSF3 m RNA in MDA-MB-231 cells were significantly decreased after transfection of the mock(P <0.05).The results of RIP show that miR-520d-5p can be combined with IGSF3 m RNA by AGO2.Overexpression of miR-520d-5p could significantly reduce the expression levels of IGSF3 protein and IGSF3 m RNA in subcutaneous tumor-bearing tissues of nude mice with breast cancer(P < 0.05).(5)Effects of IGSF3 on proliferation,apoptosis and metastasis of triple-negative breast cancer cell MDA-MB-231: Down-regulation of IGSF3 significantly reduced the proliferation ability of MDA-MB-231 cells(P < 0.05).Compared with the negative control group and blank group,down-regulation of IGSF3 significantly increased the apoptosis rate of breast cancer cells and decreased the invasion and migration of breast cancer cells(P < 0.05).Up-regulation of IGSF3 significantly promoted the proliferation、apoptosis and metastasis of MDA-MB-231 cells(P <0.05).Downregulation of IGSF3 significantly reduced tumor volume in mice.(6)IGSF3 may affect the proliferation and invasion of triple-negative breast cancer cell MDA-MB-231 by regulating the JAK2-STAT3 pathway: the protein expression levels of p-STAT3 and p-JAK2 were significantly increased in MDA-MB-231 cells that up-regulated IGSF3(P < 0.05);The protein expression levels of p-STAT3 and p-JAK2 in MDA-MB-231 cells with down-regulated IGSF3 were significantly down-regulated(P < 0.05).At the same time,there was no significant difference in the protein expression of stat3 and JAK2 between the two groups.The CCK8 experiment showed that the cell viability of IGSF3-overexpressing MDA-MB-231 cells was decreased after STAT3 inhibitor treatment compared with the negative control and blank control(P < 0.05).The results of transwell invasion assay showed that STAT3 inhibitor could significantly inhibit the invasion of IGSF3-overexpressing MDA-MB-231 cells compared with the negative control group and blank control group(P < 0.05).Conclusions: The study demonstrated miR-520d-5p tageted IGSF3 to regulate the proliferation and invasion of triple-negative breast cancer cells by JAK2-STAT3 signaling pathway.