The Functional Study Of RASSF1A In Colorectal Cancer Cell Line DLD-1

In 2015,the statistics of incidence and mortality of malignant tumors in different regions of China showed that the incidence rate of colorectal cancer in all malignant tumors was third,and the mortality rate was fifth.Abnormal methylation of Cp G island is one of the molecular mechanisms of colorectal cancer.The abnormal methylation of the promoter of tumor suppressor gene results in the inhibition of transcription,which leads to tumorigenesis.RASSF1 A is a tumor suppressor gene,which is often silenced by methylation in colorectal cancer.However,the mechanism of RASSF1 A remains to be further studied.Firstly,the relationship between RASSF1 A promoter methylation and the risk and prognosis of colorectal cancer was studied by meta-analysis.The results showed that RASSF1 A promoter methylation was a risk factor in colorectal cancer(OR=6.35,p<0.001)and indicated a poor clinical prognosis(HR=2.85,p<0.001).Then,the colorectal cancer cells in our laboratory were screened by bisulfite sequencing(BSP),and the DLD-1 cells with RASSF1 A promoter methylation was screened out.The real time PCR was used to verify the silence of RNA expression due to methylation.Then we constructed RASSF1 A overexpression vector and transfected it into DLD-1 cells to restore RASSF1 A expression.PI and anexin-v detection showed that apoptosis of DLD-1 cells was increased,while apoptosis related protein BCL2 was down regulated and PARP cleavage was increased.Yap,downstream of MST/LATS/YAP pathway,was down regulated.Mitochondrial membrane potential,detected by JC-1,showed that mitochondrial membrane potential was decreased,while western blot showed that the expression of PRKN,a protein promoting mitochondrial autophagy,was increased.CCK-8 assay showed that the proliferation of DLD-1 was inhibited,as well as the PI3 K / m TOR and RAS / RAF pathways were down regulated.The expression of proteins related to cell adhesion were also decreased.These results indicate that RASSF1 A can supress cancer by increasing cell apoptosis,promoting mitochondrial autophagy,inhibiting cell proliferation and cell adhesion.In summary,this study will provide theoretical basis for early diagnosis and targeted treatment of colorectal cancer.