Functional And Regulatory Mechanisms Of Highly Expressed Antisense Non-Coding RNA In The INK4 Locus In Renal Cell Carcinoma And Inhibition Of Retinoic Acid Chalcone On Renal Cell Carcinoma

Background and objective:Antisense noncoding RNA in the INK4 locus(ANRIL),a kind of Lnc RNAs,is originally found in the patients suffering familial melanoma.After that,accumulating evidences have identified the effect of ANRIL on substantial cancer progressions.ANRIL is encoded in the chromosome 9p21 region and has been proved to modulate its neighbor,cyclin-dependent kinase inhibitors(CDKN)2A/2B which act as tumor suppressors,through epigenetic mechanisms.More recently,a study has indicated that CDKN2 B mutation might be a novel cause of familial RCC.Therefore,we hypothesized that ANRIL might be involved in development of RCC.Considering that little is known on the role of ANRIL in RCC progression,uncovering the regulatory role of ANRIL in RCC is greatly significant.Retinoic acid and other retinoids(RAs)are used in the prevention and treatment of dermatological diseases.Retinoic acid has been shown to be promising candidate in the treatment of cancer as well.Retinoic acids are known to effect in vitro proliferation,differentiation,and apoptosis of normal and abnormal cells of several cancers.It includes colon,prostate,lung,and leukemia.There are reports that all trans-retinoic acid(ATRA)and 9-cis RA also influence the morphological differentiation,proliferation,and gene expression of neuroblastoma and astrocytoma cells.Recurrent malignant cerebral gliomas have been treated with ATRA and 13-cis RA.Retinoids are known to have anti-proliferation,anti-migration,and anti-invasive activity against human malignant gliomas,suggesting that retinoids are suitable anticancer agents to inhibit progression of tumors.In the present study the effect of retinoic acid amide having more bioavailability compared to the parent compound on human liver cancer apoptosis was investigated.Methods:1.The expression of ANRIL in clinical tumor tissues and 4 kinds of RCC cell lines were evaluated by RT-PCR;2.After transfection,the cell viability,colony number,apoptosis,migration and invasion were all assessed.Then,the expression of proteins related to apoptosis,epithelial to mesenchymal transition(EMT)and β-catenin signaling pathway were all assessed;3.The effect of IWR-endo(β-catenin inhibitor)on cell viability,migration and invasion as well as β-catenin expression were also evaluated;4.In order to investigate the effect of RAC on inhibition of cell proliferation and apoptosis of renal carcinoma cells.MTT assay and flow cytometry analysis were used to determine cell proliferation and apoptosis along with cell cycle examination;5.Western blot analysis and immunohistochemistry were used for the detection of expression levels of Notch1 and Jagged1 in renal cell carcinoma(RCC)and normal kidney tissues.Results:1.ANRIL was highly expressed in RCC tissues and RCC cell lines;2.ANRIL significantly promoted cell proliferation,migration,invasion and EMT but inhibited cell apoptosis;3.The expression levels of β-catenin,Ki-67,glycogen synthase kinase 3β(GSK-3β),phosphorylated GSK-3β,T cell transcription factor 4(TCF-4)and leukemia enhancer factor 1(LEF-1)were all markedly up-regulated by ANRIL;4.The effect of ARNIL silence was opposite to ANRIL overexpression.The effect of ARNIL on proliferation,migration and invasion of RCC cells could be reversed by IWR-endo;5.It’s revealed that a significant inhibition of cell proliferation,G2/M phase cell cycle arrest and cell apoptosis at 30 μM concentration of RAC after 72 h.In ACHN and 769-P cells,the population in G2/M phase was increased to 45.27,and 54.23%respectively on treatment with 30 μM RAC for 72 h.In 769-P and ACHN renal carcinoma cells treatment with 30 μM RAC caused 69.71 and 59.27% of the cells to undergo apoptosis compared to 5.23 and 4.93% respectively in control cells.6.The positive staining rates of Notch1 and Jagged1 in renal carcinoma tissues were 95.3 and 93.0% compared to normal kidney tissues 36.4 and 42.4% respectively.Treatment of renal carcinoma tissues caused a significant decrease in staining rates of Notch1 and Jagged1 after 96 h.Conclusion:1.ANRIL,highly expressed in RCC,acted as a carcinogen in RCC cells through activation of β-catenin pathway;2.RAC can be a potent agent in the treatment of renal cell carcinoma.