| Renal cancer is a relatively common malignant tumor of the urinary system,in which clear cell renal cell carcinoma(cc RCC)is the main type,and its incidence is on the rise.Surgical treatment for early-stage cc RCC is effective,but the effect of late-stage is poor.Although targeted drug therapy and immunotherapy have therapeutic effects on advanced cc RCC,drug resistance is high.Most cc RCC patients will die from tumor metastasis.In recent years,with the rapid development of gene chips and sequencing technologies,significant progress has been made in the study of cc RCC at the genetic level,which has led to a deeper understanding of the molecular mechanisms of cc RCC.However,the role in the therapeutic field has been minimal.Many studies have shown that long non-coding RNAs have been involved in human biological processes in recent years.The object of this study was the antisense Lnc RNA of MMP2,named MMP2-AS1.Firstly,the expression of MMP2-ASl in cc RCC was examined.The effects of MMP2-AS1 on the proliferation,migration and invasion of cc RCC cells were investigated by cell function assays.Secondly,the regulatory mechanism between MMP2 and MMP2-ASl was investigated,which provided new ideas for the pathogenesis,diagnosis and treatment of cc RCC.Part one The expression and biological behavior of long non-coding RNA MMP2-AS1 in renal cell carcinomaObjectives:1.To investigate the expression of MMP2-AS1 in cc RCC tissues and cell lines.2.To examine its effect on the malignant biological behavior of cc RCC cells.Methods:1.Quantitative real-time PCR(q RT-PCR)was used to detect the expression of MMP2-AS1 in cc RCC tissues and cell lines.2.MTS and clone formation assay determined the effect of MMP2-AS1 on the proliferation ability of cc RCC cells,the effect of MMP2-AS1 on the migration ability of cc RCC cells was measured by scratch assay,the effect of MMP2-AS1 on the invasion ability of cc RCC cells was detected by Transwell invasion assay.Result:1.MMP2-AS1 was highly expressed in cc RCC tissues and cell lines.2.Knockdown of MMP2-AS1 inhibited the proliferation,migration and invasive ability of cc RCC cells.Overexpression of MMP2-AS1 promoted the proliferation,migration and invasive ability of cc RCC cells.Conclusion:MMP2-AS1 was significantly overexpressed in cc RCC,MMP2-AS1 promoted cc RCC cells’ proliferation,migration and invasion ability,and MMP2-AS1 exerted oncogenic effects in cc RCC.Part two The molecular mechanism of MMP2-AS1/miR-34c-5p/MMP2 axis in renal cell carcinomaObjectives: Based on the study in the previous part,we analyzed the expression changes and biological roles of MMP2-AS1 in cc RCC.This part will investigate the correlation and regulatory role between MMP2 and MMP2-AS1.Methods:1.q RT-PCR was used to examine MMP2 expression in cc RCC,and the effect of MMP2-AS1 on MMP2 expression level.2.The nucleus and cytoplasm of cc RCC cell lines ACHN and 786-O were isolated by applying the Paris kit.The nucleoplasmic localization of MMP2-AS1 in these two cell lines was examined by q RT-PCR.3.Bioinformatics tools were used to predict potential MMP2-AS1/miR-34c-5p/MMP2 interactions.The association between the three was confirmed by dual-luciferase reporter gene and RNA immunoprecipitation(RIP)assays.4.miR-34c-5p expression in cc RCC was detected by q RT-PCR.Analysis of the correlation between MMP2-AS1,miR-34c-5p and MMP2 in cc RCC.5.MTS and clone formation assay measured the effects of miR-34c-5p on the proliferation ability of cc RCC cells,the effects of miR-34c-5p on the migration ability of cc RCC cells were measured by scratch assay,and the effects of miR-34c-5p on the invasion ability of cc RCC cells were measured by Transwell invasion assay.Result:1.The expression of MMP2 was significantly higher in cc RCC,the expression of MMP2-AS1 and MMP2 were positively correlated,and MMP2-AS1 actively regulated the expression of MMP2.2.MMP2-AS1 was mainly located in the cytoplasm of cc RCC cells.MMP2-AS1/miR-34c-5p/MMP2 interacted with each other.3 miR-34c-5p is lowly expressed in cc RCC and inhibits the ability of cc RCC cells to migrate and invade.Conclusion:miR-34c-5p is lowly expressed in cc RCC,and overexpression of miR-34c-5p inhibits migration and invasion of cc RCC.MMP2-AS1 is highly expressed in cc RCC,and its abnormally elevated expression can act as ce RNA to adsorb miR-34c-5p,thus promoting MMP2 expression.Part three The effect of MMP2-AS1/miR-34c-5p cotransfection on the biological behavior of cc RCC cellsObjectives: In the previous part,we investigated the correlation and regulatory mechanism of MMP2 and MMP2-ASl.This part will explore the co-role of miR-34c-5p and MMP2-AS1 in cc RCC.Method:MTS and clone formation assays were used to identify the effect of MMP2-AS1 cotransfection with miR-34c-5p on the proliferation ability of cc RCC cells.Scratch assays were used to establish the effect of cotransfection on the migration ability of cc RCC cells.Transwell invasion assays were used to identify the effect of cotransfection on the invasive ability of cc RCC cells.Result:Response experiments confirmed that MMP2-AS1 affects the MMP2 expression through the ce RNA mechanism,thus promoting the migration and invasive ability of cc RCC cells.Conclusion:MMP2-AS1 and miR-34c-5p collectively regulate the biological behavior of cc RCC cells.Abnormally raised expression of MMP2-AS1 can act as a ce RNA to adsorb miR-34c-5p,thereby promoting the expression of MMP2 and ultimately leading to the malignant progression of cc RCC.The above findings suggest that MMP2-AS1 plays a crucial role in the proliferation,invasion,and migration,providing a new theoretical basis and therapeutic target for cc RCC. |
