| BackgroundUveal melanoma(UM)originates from melanocytes in the stroma and usually occurs in the posterior pole of the eye,it is the most common primary malignant tumor in the eyes of adults.The life expectancy of UM patients is mainly affected by distant metastasis.The prognosis will be extremely poor if the metastasis is detected in UM patients,usually with an average survival range of 5-8 months.Unfortunately,almost 50%UM patients have subclinical metastases at the first time of diagnosis.The traditional therapeutic method is ophthalmectomy,however,this method has a big trauma and the life quality of UM patients is seriously affected.There are several treatment methods for UM in clinic nowadays,the main alternatives include enucleation,proton beam radiotherapy,transpupillary thermotherapy and plaque radiotherapy.Although the progress and availability of alternative therapeutic models,the survival rates of UM patients are unchanged in nearly 30 years.Burgeoning literatures have indicated that high-mobility group AT-hookl(HMGA1)protein acts as an oncogene in the tumorigenesis and progression of various human cancers,which may be served as a new and effective molecular target of clinical tumor treatment.HMGA was first discovered in bovine thymocytes in 1973 and named for its rapid migration in polyacrylamide gel electrophoresis(PAGE).HMGA1 protein is the crucial contributor in the assembly of transcriptional factors and cofactors to form enhanceosomes.Previous studies found that high mobility group protein family is an important biological regulatory factor of tumor malignant phenotype,including HMGA1,which can initial tumorigenesis and tumor progression in different human malignancies.The mechanism of HMGA1 is involved in regulating downstream pathway,which related to tumor proliferation,invasion,angiogenesis and inflammation reaction.Therefore,we may block the tumor activation through controlling the HMGA1-dependent pathway.In previous studies,we found that HMGA1 was overexpressed in the tumor tissues of patients with UM.which was related to distant metastasis of UM tumor and shortened survival time.HMGA1 is highly expressed in poorly differentiated human tumors,however,poorly expressed or absent in normal tissues and benign tumors.Hence,we assumed that HMGA1 intervention could inhibit the growth,metastasis,invasion and other functions of tumor cells,and the treatment of HMGA1 inhibition has relatively high safety to human body.MicroRNA(miRNA)is small noncoding RNA that plays a critical role in basic biological and pathological processes.MiRNA is composed of 20-24 nucleotides,it is widely expressed in mounts of tissues and cells,and participates in several basic cellular functions,such as apoptosis,proliferation and differentiation.Under physiological conditions,they play roles in essential processes,such as angiogenesis,vessel wound healing,atherosclerotic and vascular aging.Recently years,abnormal expression level of miR-222 has been extensively studied in many human tumors,including lung cancer,breast cancer,cervical cancer,liver cancer,glioma and multiple myeloma.Some studies reported that HMGA1 could promote cell proliferation by targeting miR-222 in lung cancer and cervical cancer,leading to repression of PPP2R2A expression and activation of Akt signaling.The underlying mechanism of HMGA1 and miR-222 in UM has not yet been fully understood Therefore,the purpose of this study is to illustrate the mechanism of HMGA1 and miR-222 on UM,then provides data and potential molecular targets for UM clinical treatmentPrevious studies have reported that the PI3K/Akt/MMP-9 pathway is a distinct downstream pathway of HMGA1,which is vital for tumor initiation and progression in the microenvironment.Matrix metalloproteinase(MMP)is an important enzyme to regulate the invasion and migration of tumor cells.Matrix metallopeptidase-9(MMP-9)degrades the basement membranes and exposes cryptic peptide epitopes in the extracellular matrix,associating with tumor dissemination.Therefore,it is essential to suppress the MMP-9 expression in UM tumor through PI3K/Akt/MMP-9 pathway or its upstream signal HMGA1,MMP-9 inhibition would be a promising strategy to increase the anti-tumor treatment efficacy.However,no literature has reported the regulatory mechanism of HMGA1 and miR-222 in UM tumor cells.Therefore,we intend to verify the interaction between HMGA1 and miR-222 in UM,and whether the PI3K/Akt/MMP-9 pathway can regulate the function of UM cells,it is important for providing experimental data and potential biologic therapeutic targets for early clinical diagnosis,prognosis and clinical treatment.Part ? Overexpression of HMGA1 in uveal melanoma cellsObjective:Investigate the expression of HMGA1 in UM cell lines including C918 and MUM-2B.HMGA1 belongs to the high mobility group protein family and it is an important protein in regulating malignant phenotype of tumors.The mechanism of HMGA1 mainly involves the regulation of downstream genes,which are involved in a series of processes including tumor proliferation,invasion,angiogenesis and inflammatory response,so blocking the corresponding signaling pathway of HMGA1 is likely to block the tumor activity.Our previous study analyzed the HMGA1 expression in tumor tissues of 89 UM patients,the results showed that HMGA1 overexpressed in UM tissues and correlated with distant metastasis and shortened median survival of patients.The purpose of first part was to detect the expression of HMGA1 in UM cell lines C918 and MUM-2B,laying a foundation for exploring the regulatory mechanism of HMGA1 in UM.Methods:RNA was extracted using TRIzol reagent from C918 and MUM-2B cells.HMGA1 expression was detected by Real-time quantitative polymerase chain reaction(RT-PCR).Results:HMGA1 expression was significantly increased in C918 and MUM-2B cells compared with the control group by RT-PCR(P<0.05).Conclusion:HMGA1 expression was significantly increased in two UM cell lines,indicating that HMGA1 may play an important role in the UM occurrence and progression.Part ? HMGA1 regulation on PI3K/Akt/MMP-9 pathway in uveal melanomaObjective:HMGA1 overexpression has been found:in UM cells in our previous study,and the overexpression of HMGA1 is associated with distant metastasis and shortened median survival of tumor cells in UM patients.However,what is the regulatory mechanism of HMGA1 in UM?This issue has not been resolved.In this part,we aimed to study the specific mechanism of HMGA1 on the regulation of UM cell function.To verify the regulatory effect of HMGA1 on the downstream pathway PI3K/Akt/MMP-9 as an upstream molecular signal.Methods:Lentivirus(LV)-HMGAl-RNAi was used to down-regulate the HMGA1 expression in two different human UM cell lines C918 and MUM-2B.Negative control virus was applied as negative control(NC)group.The transfection efficiency of lentivirus labeled with Enhanced Green Fluorescent Protein(EGFP)was checked by fluorescence microscope.The cell culture medium was changed and puromycin(2?g/mL)was added for two weeks of screening to obtain UM cell.After transfection,RNA and total protein of UM cells were extracted,then the expression of HMGA1 and PI3K/Akt/MMP-9 in LV-HMGA1-RNAi and NC were detected by RT-PCR and Western blot,respectively.Results:Fluorescence microscope represented the transfection rate of lentivirus was more than 90%in C918,and MUM-2B was about 80%.Total RNA and protein were extracted 72 hours after transfection.RT-PCR results showed that HMGA1 and MMP-9 expressions in C918 and MUM-2B cells were significantly down-regulated in LV-HMGA1 group comparing with the NC group(P<0.05).Western blot showed the HMGA1,p-PI3K,p-Akt and MMP-9 expressions in C918 and MUM-2B were significantly decreased in LV-HMGA1 group,which was consistent with RT-PCR results(P<0.05).Conclusion:High level of HMGA1 expression in UM is a high-risk factor for tumor progression,HMGA1 could promote tumor proliferation and metastasis by activating the downstream PI3K/Akt/MMP-9 pathway.HMGA1 expression was down-regulated by lentivirus,both RT-PCR and Western blot results showed that HMGA1 inhibition could effectively down-regulate the PI3K/Akt/MMP-9 pathway.Hence,HMGA1 plays an essential role in the regulation of UM proliferation,invasion and migration,revealing the mechanism of HMGA1 might provide a new strategy for UM treatment.Part ? The effect of microRNA-222 on the biological function of uveal melanoma cellsObjective:Investigate the impact of miRNA-222 expression level on UM cells proliferation,apoptosis and migration.Methods:MiR-222-3p mimics,miR-222-3p inhibitor and their corresponding negative controls(NC)were successfully constructed and transfected into two UM cell lines,including C918 and MUM-2B.The expression level of miR-222-3p in two UM cells was down-regulated by siRNA,then analyze the differences of proliferation,apoptosis and migration features in two cell groups.Annexin V-FITC apoptosis assay was used to analyze the apoptosis status;CCK-8 kit was applied to check the viability and proliferation of UM cell;We used wound healing assay and Transwell chamber to analyze UM cells migration ability.The regulatory role of miR-222 in UM cells was analyzed through the experiments above.Results:MiR-222-3p mimics,miR-222-3p inhibitor and its corresponding NC group were transfected into C918 and MUM-2B cells by transfection reagent,the total RNA was extracted.The expression level of miR-222-3p in UM cells was detected by RT-PCR to evaluate the transfection.RT-PCR results showed that the expression level of miR-222 was significantly up-regulated or down-regulated in C918 and MUM-2B cells after transfection with miR-222 mimics or miR-222 inhibitor in UM cells,suggesting the transfection is eligible for the further experiment.The effect of miR-222 on UM cell apoptosis,proliferation and migration were analyzed(P<0.05).C918 induced by miR-222 mimics showed less annexin V-positive and PI-positive cells comparing to the NC group,on the contrary,C918 cells interfered by miR-222 inhibitor showed much more annexin V-positive cells.Similar results were found in MUM-2B.The proliferation of UM cells was measured by CCK-8 assay.The UM cells treated with miR-222 showed a higher 450nm absorbance and miR-222 inhibitor-induced C918 represented a lower value of absorbance(P<0.05).UM cell migration treated with LV-HMGA1 and NC was performed monitored by the Transwell chamber and wound healing assay.Migration rate of C918 and MUM-2B was enhanced with miR-222 mimics and attenuated with miR-222 inhibitor(P<0.05).These assays were analyzed and calculated using Image J.Conclusion:MiR-222 could promote the proliferation and migration of C918 and MUM-2B cells,conversely,it could inhibit the UM apoptosis,indicating that silencing miR-222 might represent an intriguing approach for therapeutic studies.Part IV Modulation of miR-222 enhances PI3K/Akt/MMP-9 pathway mediated by HMGA1 in UM cellsObjective:In the previous section,we found that miR-222 could inhibit apoptosis,promote proliferation and migration of two UM cell lines including C918 and MUM-2B,indicating that miR-222 played a role in promoting the growth and progression of UM tumors.In this part,we aimed to explore the relationship between miR-222 and HMGA1 in UM,the regulation of miR-222 on the downstream PI3K/Akt/MMP-9 pathway.Methods:After the HMGAl expression in C918 and MUM-2B cells was down-regulated by lentivirus,the level of miR-222 in HMGA1 down-regulated group and NC group was detected by RT-PCR as well.MiR-222-3p mimics,miR-222-3p inhibitor and their corresponding NC groups were constructed and transfected into C918 and MUM-2B cells,respectively.48h after transfection,proteins were extracted for Western blot to check the HMGA1 and PI3K/Akt/MMP-9 pathway signal expressions.The target between miR-222 and HMGA1 was predicted by the biological website database.Results:We found the interaction between miR-222 and HMGA1 through biological website database miRTarBase(http://mirtarbase.mbc.nctu.edu.tw/php/index.php).MiR-222 expression levels in C918 and MUM-2B cells were detected by RT-PCR,and it was found that in the HMGA1 down-regulated group,miR-222 levels in C918 and MUM-2B cells were significantly decreased(P<0.05).Western blot results showed that the expressions of HMGA1,p-PI3K,p-Akt and MMP-9 in C918 and MUM-2B cells were significantly up-regulated in the UM cell group transfected with miR-222-3p mimics,and decreased in the miR-222-3p inhibitor group(P<0.05).However,there was no significant difference in the total proteins of PI3K and Akt in the miR-222-3p mimics group and inhibitor group.We speculated that phosphorylated PI3K and Akt(p-PI3K,p-Akt)may be the activation form of PI3K and Akt.Therefore,there were no significant differences in the levels of total PI3K and total Akt,while the expressions of p-PI3K and p-Akt were significantly changed.Conclusion:Our data revealed that there is an interaction between HMGA1 and miR-222 in UM,miR-222 plays a role in promoting the proliferation and migration of UM tumor cells,and regulates UM cells by PI3K/Akt/MMP-9 pathway.Part V Effect of HMGA1 on biological behavior of uveal melanoma cells in nude miceObjective:Lentivirus(LV)-HMGAl-RNAi was used to down-regulate the HMGA1 expression in C918 cells,and the suspension of C918 cells was injected into nude mice for tumor formation experiment.The impact of HMGA1 on the biological behavior of UM tumor was observed.The modulation of PI3K/Akt/MMP-9 pathway by HMGA1 expression in tumor tissues was analyzed by immunohistochemistry(IHC)and Western blot.Methods:To confirm the effect of HMGA1 on UM tumorigenesis in vivo,C918 cells treated by LV-HMGA1 and LV-NC lentivirus were injected subcutaneously into the posterior flank of nude mice.The changes of tumor volume and weight were recorded every two days from one week after injection,and tumor growth curve was made.After 28 days,the protein was extracted from the tumor tissue,and the expression levels of HMGA1 and PI3K/Akt/MMP-9 signaling pathway were detected by Western blot.At the same time,part of the tumor tissue was collected and the HMGA1 expression was analyzed by IHC.Results:The average tumor size was significantly smaller in LV-HMGA1 groups than in the NC groups,and the individual growth velocity of tumor cells in the LV-HMGA1 group was obviously decelerated comparing with that of their control group(P<0.05).Western blot and IHC showed that the PI3K,p-PI3K,p-Akt and MMP-9 expressions were decreased in HMGA1 down-regulated group(P<0.05).Conclusion:HMGA1 plays an important role in promoting tumor growth and proliferation.Lentivirus was used to construct C918 cells with reduced HMGA1 expression and injected into the nude mice,and then the tumor growth was significantly inhibited.Hence,we hypothesize that HMGA1 may regulate tumor proliferation through the PI3K/Akt/MMP-9 pathway.ConclusionHMGA1 is an important oncogene in the uveal melanoma development and progression,its overexpression level is related to UM cell proliferation,migration and other functions.Our previous study demonstrated that HMGA1 overexpression is associated with worse prognosis of UM patients analyzed by IHC of 89 tumor samples.Besides,the UM cell proliferation,migration and invasion have been significantly inhibited when the HMGA1 expression was suppressed by siRNA.It is reported that HMGA1 is highly expressed in human low-differentiation tumors,while poorly expressed or absent in normal tissues and benign tumors.Hence,HMGA1 plays an essential role in the regulation of UM cells,however,the underlying mechanism of HMGA1 in the UM tumorigenesis is still poorly understood.Based on our previous study,two different UM cell lines including C918 and MUM-2B were transfected with lentivirus,which could down-regulate the expression of HMGA1 in UM cells.The results showed that HMGA1 could promote the proliferation and metastasis of UM tumor by activating the PI3K/Akt/MMP-9 signaling pathway.Under physiological conditions,miR-222 plays an important regulatory role in angiogenesis,wound healing,vascular aging and other processes to maintain body homeostasis.MiRNA-222 has been reported in the progression of several malignancies;including cervical cancer and lung cancer.We used the miRTarBase(http://mirtarbase.mbc.nctu.edu.tw/php/index.P hp)to confirm the relation between miR-222-3p and its putative binding sequence of HMGA1.MiR-222-3p mimics and miR-222-3p inhibitor were applied to interfere the miR-222 expression in UM cells,the results represented that the proliferation and migration abilities of C918 and MUM-2B were significantly increased in the miR-222-3p mimics group,with decreasing apoptosis.In contrast,the apoptosis of UM cells was increased in the miR-222-3p inhibitor group,and the proliferation and migration ability were inhibited.We identified that HMGA1 could promote the progression of UM through PI3K/Akt/MMP-9 signaling pathway and positively regulated miR-222 in vitro and in vivo.Current novel work establishes a link between HMGA1 and PI3K/Akt/MMP-9 pathway,suggesting HMGA1 plays an important role in UM cell proliferation and migration.Moreover,current results indicated that oncogenic miR-222 could be positively mediated by HMGA1,which could be considered as diagnostic and therapeutic biomarkers for UM.Accordingly,we hope our findings merit further investigation of targeting HMGA1-related gene for the clinical treatment of UM. |
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