The Mechanism Study Of CRNDE/miR-33a-5p/CDK6 Axis In Regulating Liver Cancer Proliferation

Background:Primary liver cancer is the third cause of death in China as a common malignant tumor.Hepatocellular carcinoma(HCC)accounts for about 85%-90% of liver cancer types.The disease is concealed and there are no typical symptoms in the early stage.Most patients have lost the opportunity of radical treatment.Therefore,understanding the molecular mechanism of HCC pathogenesis is great significant for early diagnosis of tumors and reduces the chance of recurrence after surgery.Long non-coding RNA(LncRNA)is a type of RNA with no protein encoding ability of more than 200 nucleotides in length.But with the progress of research,LncRNA regulates gene expression to participate in tumor progression and becomes a potential tumor marker and molecular therapeutic target.CRNDE(Colorectal neoplasia differentially expressed)is a LncRNA that was first found high expressed in cancer tissues of colon cancer patients.It has tissue expression specificity and is highly expressed in breast,testis,bronchi and parotid gland,which promotes tumor proliferation and is closely related to the clinical prognosis of patients.However,the mechanism involve in liver cancer is still unclear.Studies have shown that it may be involved in cell cycle control in liver cancer.CDK6(cyclin-dependent kinase 6,CDK6)is an important protein in cell cycle regulation.The imbalance of CDK6 will affect the cell cycle progression,thereby regulating the proliferation of tumor cells.Through bioinformatics analysis,we found that miR-33a-5p has a target gene site with CRNDE;at the same time,miR-33a-5p also has a binding site with CDK6.In liver cancer,CRNDE may affect the expression of CDK6 by regulating miR-33a-5p,thereby regulating tumor proliferation,which may become a potential target for liver cancer treatment.Objectives:1.To detect the expression of LncRNA CRNDE in hepatocellular carcinoma and to clarify its correlation with clinicopathological features;2.Study on the mechanism of CRNDE/miR-33a-5p/CDK6 axis regulating HCC proliferation;Methods and results:1.The results of the expression of LncRNA CRNDE in 30 pairs of liver cancer and adjacent tissues showed that the expression of CRNDE in liver cancer tissues was significantly higher than that in adjacent tissues.Patients were divided into two group according to the PCR results.In the expression group,it was found that high expression of CRNDE was closely related to tumor TNM stage and presence of microvascular invasion.2.It was found that in hepatocellular carcinoma,the high CRNDE expression patients have the shorter overall survival than the other group through GEPIA data analysis.Using 30 clinical patient data,it was found that LncRNA CRNDE and microvascular infiltration are independent factors of poor prognosis in patients with liver cancer.3.By down-regulation the expression of CRNDE in Huh-7 cells,it was found that down-regulation of CRNDE significantly inhibited the proliferation,migration and invasion of liver cancer cell Huh-7.4.Co-transfection of miR-33a-5p with a dual-luciferase reporter gene vector containing wild-type CRNDE can cause a significant decrease in reporter gene fluorescence intensity using double luciferin detection,and down-regulate the expression of CRNDE in Huh-7 cells.The expression of 33a-5p was significantly increased,indicating that CRNDE targets miR-33a-5p and down-regulates its expression in liver cancer to play a role.To further verify this result,we tested 30 pairs of liver cancer,Examination of adjacent tissues and liver cancer cells revealed that miR-33a-5p was underexpressed in cancer tissues and liver cancer cells.Overexpression of miR-33a-5p inhibited the proliferation of liver cancer cell Huh-7.5.To verify that CRNDE plays a role in HCCvia the CRNDE / miR-33a-5p / CDK6 axis,we found that co-transfection of miR-33a-5p with wild-type CDK6 dual-luciferin reporter vector can lead to a report The fluorescence intensity of the gene significantly decreased,indicating that miR-33a-5p can bind to CDK6.Western Blot analysis revealed that CDK6 expression in cancer tissues was significantly higher than that in adjacent tissues,and the expression of liver cancer cells Huh-7 was also significantly higher than that of normal human liver.Expression in cell L02.And down-regulating CRNDE or over-expressing miR-33a-5p can significantly reduce the expression of CDK6,indicating that CRNDE and miR-33a-5p are upstream of CDK6,and further over-expressing CDK6 found that over-expressing CDK6 can significantly promote the proliferation of Huh-7 cells And its effect can be resisted by miR-33a-5p mimics,indicating that CRNDE can promote the proliferation of liver cancer cells by regulating CDK6.6.To study the role of CRNDE and miR-33a-5p in vivo,we established a xenograft mouse model by injecting sh-CRNDE,miR-33a-5p mimics,and Huh-7 cells transfected with their empty transfection group.The results showed that sh-CRNDE and miR-33a-5p mimics group significantly inhibited tumor growth and reduced tumor volume.Conclusions:1.LncRNA CRNDE is overexpressed in hepatocellular carcinoma tissues,and is significantly associated with tumor TNM staging,presence or absence of microvascular invasion,and is an independent prognostic factor for patients;2.LncRNA CRNDE promotes the proliferation of liver cancer through the CRNDE/miR-33a-5p/CDK6 axis,which may become a new tumor marker and molecular biotherapeutic target for hepatocellular carcinoma.