Research On The Mechanism And Treatment Of Primary Intraocular Malignant Tumors

ObjectivesPrimary intraocular malignant tumors mostly originate from the retina and uveal membrane,which can cause blindness,disability,and death.Retinoblastoma(RB)is the most common pediatric tumor of the eye.Intracranial and distant metastases can be life-threatening.Uveal melanoma(UM)is the most common malignant tumor of the adult eye.It is easy to metastasize and has a poor prognosis.Studies have found that long non-codingRNA(lncRNA)can regulate gene expression at different levels and affect tumorigenesis and development.A large number of clinical studies have confirmed the efficacy of cancer immunotherapy,but rarely involves intraocular malignant tumors.The purpose of this study is to explore the tumorigenesis and development of UM from the perspective of epigenetics,and to find therapeutic targets;to explore tumor-related antigen in RB and efficient treatment strategies from the perspective of immunotherapy.Materials and methodsGD2 expression in RB tissues and cells was detected by immunohistochemistry and flow cytometry.Transcriptome sequencing and immunohistochemistry were conducted to detect the local immune microenvironment and blood-retinal barrier integrity in RB.Lactate dehydrogenase release assay and flow cytometry were used to detect the lysis and apoptosis of RB subjected to dinutuximab combined with NK-92MIh CD16-GFP.Perforin-granzyme B release and CD107a expression in NK-92MIh CD16-GFP were measured by ELISA and flow cytometry to evaluate its cellular cytotoxicity.Real-time quantitative PCR was performed to detect the expression of lncRNA ANRIL in melanomas.The protein expression of INK4a-ARF-INK4b was detected by Western blotting.CCK8,wound healing assay and transwell system were used to test the effects of ANRIL and INK4 gene clusters on the proliferation and migration ability of melanomas.The effect of ANRIL-mediated INK4 gene cluster on the proliferation of UM was tested in mouse xenograft model.ResultsGD2 has a heterogeneously high expression pattern in RB and is positively correlated with tumor stage and Ki67 positive rate.Bioinformatics analysis indicates that the local immune microenvironment of RB is in a state of suppression.Immunohistochemistry reveals immune infiltrates in RB tumor tissues and vitreous bodies.Under the attack of NK-92MI^{h CD16-GFP} combined with dinutuximab,the release of lactate dehydrogenase and apoptosis in RB cells increased significantly;meanwhile the release of perforin-granzyme B and CD107a expression in NK-92MI^{h CD16-GFP} cells increased significantly.LncRNA ANRIL is highly expressed in melanomas,while the INK4 gene cluster are lowly expressed in melanomas.Knockdown of ANRIL can restore the endogenous expression of the tumor suppressor gene cluster INK4a-ARF-INK4b,and then significantly inhibit the proliferation and migration of melanomas.ConclusionsGD2 is heterogeneously highly expressed in RB,which is positively correlated with stage and Ki67 positive rate,suggesting a poor prognosis.Aggressive RB destroys the blood-retinal barrier,accompanied by immune infiltrates.Although the local natural killers may be activated,the whole local immune microenvironment is suppressed.Mediated by GD2-specific monoclonal antibody dinutuximab,NK-92MI^{h CD16-GFP} releases perforin-granzyme B to lyse RB,while avoiding self-damage through upregulating expression of CD107a.The abnormally high expression of lncRNA ANRIL in uveal melanoma and cutaneous melanoma suggests a poor prognosis.Oncogenic ANRIL can down-regulate the expression of the tumor suppressor gene cluster INK4a-ARF-INK4b and other factors to promote the growth and metastasis of melanoma.Our results reveal a novel"domino effect"therapeutic strategy that significantly potentiates anti-melanoma efficiency by reprogramming the aberrant INK4-hub.