The Mechanism Of Long Non-coding RNA LINC00339 In Doxorubicin-induced Cardiomyocyte Apoptosis

BackgroundDoxorubicin(Dox),is one of the most widely used and successful anti-tumor drugs.Studies have shown that doxorubicin-induced car-diac pathology is similar to dilated cardiomyopathy(DCM).The abnormally expressed long non-coding RNA(lncRNA)has been considered as one of the key sources of heart disease.However,the role of lncRNA in doxorubicin(DOX)-induced cardiotoxicity remains largely unknown.Therefore,we established a DOX-induced cardiomyocyte injury model and explored the role and mechanism of IncRNA in doxorubicin(DOX)-induced cardiotoxicity in vitroMethodsPart1:Preparation of primary cultured cardiomyocytes(PC),calculation of survival rate of primary cardiomyocytes,observation of cell morphology and viability analysis and calculation of cell purity(identification)Patr2:The role of long-chain non-coding RNA LINC00339 in doxorubicin-induced cardiomyocyte apoptosis1.CCK-8 method was used to detect the effect of doxorubicin on cardiomyocyte proliferation activity.2.The effect of doxorubicin on the expression of Caspase 3 protein in cardiomyocytes was detected by Western blot3.Q-PCR method was used to detect the expression of LINC00339 in cells after adriamycin stimulated cardiomyocytes.4.Q-PCR method was used to detect the expression of LINC00339 in cardiomyocytes after knockdown of LINC003395.CCK-8 method was used to detect the effect of knockdown of LINC00339 on cardiomyocyte proliferation activity.6.Flow cytometry and Tunel staining wcere used to detect the effect of knockdown of LINC00339 on doxorubicin-induced cardiomyocyte apoptosisPart3:The mechanism of action of long-chain non-coding RNA LINC00339 in doxorubicin-induced cardiomyocyte apoptosis.1.Using Q-PCR method to detect the expression of miR-484 in cells after doxorubicin stimulation of cardiomyocytes.2.Using the StarBase v3.0 database to predict the target of miR-484.3.To detect the regulation of miR-484 on LINC00339 by Dual luciferase reporter gene assay.4.The effect of overexpression of miR-484 and miR-484 inhibitors on the expression of LINC00339 in cardiomyocytes was detected by Q-PCR.5.The effect of knockdown of LINC00339 on the expression of miR-484 in cardiomyocytes stimulated by doxorubicin was detected by Q-PCR.6.Flow cytometry and Tunel staining were used to detect the effect of miR-484 on adriamycin-induced cardiomyocyte apoptosis.ResultsPart 1:The survival rate of primary cardiomyocytes was(93.88±3.08)%.The primary cardiomyocytes had good morphology,spontaneous pulsation and strong contraction.The purity of cardiomyocytes was(91.83±1.92)%.The primary cardiomyocyte experiment was successfully prepared and meet the requirements of subsequent experiments.Part 2:1.Doxorubicin inhibits the proliferative activity of cardiomyocytes.2.Adriamycin promotes the expression of Caspasc 3 protein,a protein related to cardiomyocyte apoptosis.3.LINC00339 is highly expressed in doxorubicin-stimulated primary cardiomyocytes and rat H9c2 cardiomyocytes.4.Small interfering RNAs si-LINC00339 1 and si-LINC00339 2 can significantly reduce the mRNA level of LINC00339 in primary cardiomyocytes and rat H9c2 cardiomyocytes.5.The knockdown of LINC00339 restored the decrease in cardiomyocyte activity induced by doxorubicin.6.Knockdown of LINC00339 can inhibit doxorubicin-induced cardiomyocyte apoptosis.Part 3:1.MiR-484 is under-expressed in doxorubicin-stimulated primary cardiomyocytes and rat H9c2 cardiomyocytes.2.LINC00339 can be used as a predictive target for miR-484.3.MiR-484 has a direct inhibitory effect on LINC00339 at the post-transcriptional level.4.Overexpression of miR-484 can reduce the expression of LINC00339 in cardiomyocytes,and miR-484 inhibitor can up-regulate the expression of LINC00339 in cardiomyocytes.5.Knockdown of LINC00339 can up-regulate the expression of miR-484 in cardiomyocytes stimulated by doxorubicin,while co-transfection with miR-484 inhibitor significantly decreases the expression of miR-484.6.Knockdown of LINC00339 can inhibit doxorubicin-induced cardiomyocyte apoptosis,and co-transfection with miR-484 inhibitor will reverse this effect and aggravate cardiomyocyte apoptosis.Conclusion1.LINC00339 is highly expressed in doxorubicin-stimulated primary cardiomyocytes and rat H9c2 cardiomyocytes.2.Knockdown of LINC00339 restores the decrease in cardiomyocyte activity induced by doxorubicin and inhibits doxorubicin-induced cardiomyocyte apoptosis.3.miR-484 is under-expressed in doxorubicin-stimulated primary cardiomyocytes and rat H9c2 cardiomyocytes.4.LINC00339 directly binds to miR-484 and regulates doxorubicin-induced cardiomyocyte apoptosis by targeting miR-484.