Novel Tumor Suppressive Mechanisms Of MiR-124 And MiR-139 In Cancers

BackgroudColorectal cancer(CRC)is one of the most common malignant tumors in the digestive tract.Metastasis is the main cause of death in patients with colorectal cancer.Anti-anoikis enhances the survival of cancer cells in the systemic circulatory system,thereby promoting the formation of secondary metastatic tumors in distal organs.micro RNA(miRNA)is an endogenous single strand non-coding small RNA composed of approximately 21-25 nucleotides,which is paired with the 3'UTR base of the target m RNA to inhibit m RNA translation or direct its degradation and inhibit the expression of target gene at the post transcriptional level.Recent studies confirm that abnormal expression of miRNA plays an important role in the development and progression of CRC and other malignant tumors.miRNA-124 is a non-coding small RNA molecule that has been reported to possess multiple tumor suppressor functions.However,the regulation and mechanism of its role in anoikis in tumor cells is still unknown.The classification of molecular subtype in breast cancer has improved the accuracy of clinical prognosis and the effectiveness of treatment strategies.However,due to the high heterogeneity of breast cancer,more biomarkers and functional molecules need to be explored to support accurate diagnosis and treatment.It has been reported that miRNA-139 plays an important role in many malignant tumors.DNA topoisomerase(TOP)exerts a crucial function in DNA replication.It is known that TOP2 a is closely related to the abnormal proliferation of a variety of human malignant tumor cells,and the high level expression of TOP2 a is also an important indicator for the shortening of the patient's lifespan.The major target of anthracycline chemotherapy drugs commonly used in clinic is TOP2 a.However,there are significant differences in the response of the patients with different molecular subtypes of breast cancer.It is particularly critical to analyze the biological contribution,prognostic value and therapeutic significance of TOP2 a in different molecular types of breast cancer patients.Aims1.To observe the effect of miR-124 on anoikis sensitivity in human CRC cells;2.To analyze the molecular mechanism of miR-124 regulating sensitivity of CRC cells to anoikis;3.To observe the effect of ITGA3 on anoikis sensitivity in human CRC cells;4.To determine the clinical significance of ITGA3 expression level in CRC patients;5.To analyze the correlation between the expression level of TOP2 a and prognosis of breast cancer patients with various molecular subtypes;6.To observe the impact of TOP2 a on proliferation in luminal type breast cancer cells;7.To confirm the direct regulation of miRNA-139 on the TOP2 a expression;8.To observe the regulatory effect of miRNA-139 on the proliferation of luminal type breast cancer cells;9.To determine the biological significance of miR-139/TOP2 a regulatory axis on proliferation in luminal type breast cancer cells.Methods1.We established the colorectal cancer cell lines overexpressing miR-124 by using a lentiviral expression system.q RT-PCR was used to detect the expression level,and then trypan blue stain assay and active caspase-3 assay were used to test the effect of miR-124 on anoikis sensitivity of CRC cells in vitro.The tumor cells were injected into the tail vein of nude mice to establish the metastatic tumor model of CRC cells in vivo.The effect of overexpression of miR-124 on metastasis of CRC cells in the body environment was observed using HE staining in lung tissues.2.Bioinformatics analysis was used to determine the novel downstream targets of miR-124 in CRC cells.Luciferase reporter assays were used to determine whether ITGA3 is a putative downstream gene of miR-124.Finally,we used Westenr blot and q RT-PCR to confirm the effect of miR-124 on the ITGA3 expression in CRC cells.3.Stable CRC cell lines expressing ITGA3-sh RNA were established by using the p LKO-sh RNA lentiviral system.Westenr blot was used to detect the expression level,and then trypan blue stain assay and active caspase-3 assay were used to observe the effect of ITGA3 on the anoikis sensitivity of CRC cells in vitro.The tumor cells were injected into the tail vein of nude mice to establish the metastatic tumor model of CRCcells in vivo.The effect of ITGA3-KD on the metastasis of CRC cells in the body environment was observed by HE staining in lung tissues.4.To analyze the correlation between the expression level of ITGA3 m RNA and the prognosis of patients with two colorectal cancer patients using the open database Kaplan-Miemer Plotter from the prognosis analysis of breast cancer,we analyzed and evaluated the TOP2 a expression level and prognosis of breast cancer patients.5.The stable breast cancer cell lines expressing TOP2a-sh RNA were established using the p LKO-sh RNA recombinant lentiviral delivery system.Westenr blot was used to detect the expression level.The effect of TOP2-KD on proliferation in breast cancer cells was observed using MTT assay and plate colony formation assay.6.Bioinformatics analysis was used to determine the novel downstream genes regulated by miR-139.Luciferase reporter assays were used to determine whether TOP2 a is a possible downstream target gene of miR-139.We used Westenr blot and q RT-PCR to calidate the effect of miR-139 on the TOP2 a expression in breast cancer cells.7.We established the luminal type breast cancer cell lines overexpressing miR-139 using recombinant lentiviral system.q RT-PCR was used to detect the expression level.MTT and plate colony formation assays were used to observe the effect of miR-139 on proliferation in breast cancer cells.The METABRIC breast cancer database was used to analyze the relationship between the expression level of miR-139 and the survival rate of breast cancer patients with various molecular subtypes.8.The breast cancer cell lines overexpressing miR-139 were reintroduced into the exogenous TOP2 a to restore its expression level.Western blot was used to detect the expression level of TOP2 a in the cells.MTT and plate colony formation assays were use to test the effect of TOP2 a on cancer cells proliferation regulated by miR-139.Results1.Using lentiviral gene expression delivery system,the expression levels of miR-124 in Lovo and SW620 CRC cells were 320 times and 452 times higher than those of the control group,respevtively.The results of trypan blue staining showed that the proportion in the two cell lines with overexpressed miR-124 was 3.12 times and 4.18 times more than that of the control group,respevtively.The results of active caspase-3 assay showed that the relative activity of caspase-3 in the two cell lines with overexpressed miR-124 was 2.51 and 3.12 times more than that of the control group,respevtively.In the in vivo experiment,we found that overexpression of miR-124 in CRC cells sifnificantly decreases the number,volume,and frequency of metastatic tumor nodules in lung tissues.2.Transfection of miR-124 mimics inhibited the luciferase activity of wild type ITGA3-3'UTR.However,the ITGA3-MUT-3'UTR luciferase activity did not be inhibited,indicating that miR-124 directly binds to the 3'UTR of ITGA3.Westenr blot and q RT-PCR showed that overexpression of miR-124 significantly inhibits the ITGA3 expression in both m RNA and protein levels.3.Westenr blot showed the gene silencing effect of ITGA3 using p LKO-sh RNA lentiviral delivery system.The results of trypan blue exclusion test showed that the proportion of cell death in the two stable CRC-sh ITGA3 cell lines was 4.67 and 6.02 times as high as that of the control group,respectively.The data of active caspase-3 assay showed that the relative activity of caspase-3 in the two stable CRC-sh ITGA3 cell lines was 3.98 and 5.12 times as high as that of the control group,respectively.In the in vivo experiment,In the in vivo experiment,we found that knockdown of ITGA3 in CRC cells sifnificantly decreases the number,volume,and frequency of metastatic tumor nodules in lung tissues.4.In the prognostic analysis,using two independent CRC cohorts,we found that the high level of ITGA3 is closely correlated with a poor prognosis in CRC patients.5.The results of Kaplan-Miemer database analysis showed that the high level of TOP2 a expression is negatively correlated with the disease-free survival rate and overall survival rate of breast cancer patients.Further analysis showed that high level expression of TOP2 a is only associated with poor prognosis in patients with luminal type breast cancer,but no relationship with the basal-like and HER2-positive breast cancers.6.The expression of TOP2 a was downregulated using a lentiviral delivery system,and the expression level of TOP2 a in two luminal type breast cancer cell lines(MCF-7 and T47D)was detected by Westenr blot,which was significantly lower than that in the control group.The results of MTT assayt showed that the cell growth rate of the breast cancer cell line expressing TOP2a-sh RNA decreased significantly compared with that in the control group.The results of plate colony formation assay showed that the colony formation ability of the breast cancer cells with knockdown of TOP2 a decreases obviously.7.Transfection of miR-139 mimics inhibited the luciferase activity of wild type TOP2a-3'UTR.However,the TOP2a-MUT-3'UTR luciferase activity had no response to miR-139 mimics,indicating that miR-139 can directly bind to the 3'UTR of TOP2 a.Westenr blot and q RT-PCR showed that overexpression of miR-139 significantly inhibits the TOP2 a expression in both m RNA and protein levels.8.Using lentiviral gene expression delivery system,the expression levels of miR-139 in MCF-7 and T47 D breast cells were 212 times and 98 times higher than those of the control group,respevtively.The results of MTT assayt showed that the cell growth rate of the breast cancer cell line expressing miR-139 decreased significantly compared with that in the control group.The results of plate colony formation assay also showed that the colony formation ability of the breast cancer cells with overexpression of miR-139 decreases obviously.9.When the exogenous TOP2 a was reintroduced into the miR-139 overexpressing cell lines(MCF-7 and T47D),MTT assay showed that the cell proliferation ability of the breast cancer cells are rescued with overexpression of TOP2 a.And the colony formation assay also showed the similar results.Conclusions1.Here,we found that overexpression of miR-124 promotes anoikis of CRC cells in vitro and in vivo.In silico analysis and the experimental evidence supported that ITGA3 is a bona fide target of miR-124.Moreover,we identifies that ITGA3 plays a critical role in the regulation of anoikis sensitivity in CRC cells.Finally,our analysis in TCGA datasets demonstrates that high levels of ITGA3 are closely associated with poor prognosis in CRC patients.Collectively,we establish a functional link between miR-124 and anoikis susceptibility and provide that a miR-124/ITGA3 axis could be a potential target for the treatment of metastatic CRC.2.Here,analyzing the data in a huge cohort of breast cancer patients,we found that Topoisomerase II alpha(TOP2a),an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients,but not in basal like or HER2 positive breast cancer patients.We identified that miR-139,a previous reported anti-metastatic micro RNA targets 3'-untranslated region(3'UTR)of TOP2 a m RNA.Further more,we revealed that the forced expression of miR-139 reduces the TOP2 a expression at both m RNA and protein levels.And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells,while exogenous TOP2 a expression could rescue inhibition of cell proliferation mediated by miR-139.Collectively,our present study demonstrates the miR-139-TOP2 a regulatory axis is important for proliferation in luminal type breast cancer cells.This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment.