Research On The Mechanism That KRas Promotes Pancreatic Cancer Progression By Upregulating CAPN2 MRNA Stability In A M6A Dependent Manner

Backgroud: Pancreatic cancer patients are always with poor prognosis.KRas mutation is the most frequently molecular event that occurs in pancreatic cancer.The Calpains are a family of cysteine proteases that were identified to be associated with a series of cancers including pancreatic cancer.However,the association between KRas and the Calpains is currently unknown.N6-methyladenosine(m6A)is the most abundant m RNA modification,which was widely proved to be involved in the progression of cancer.There is no study on the regulation between KRas and the m6 A.Aim: To find the association between KRas and CAPN2 and its role in the progression of pancreatic cancer.And to explore whether m6 A is involved.Methods: We firstly used calculating of public database and the si RNA knockdown of KRas to find whether the three important subunits of Calpains,which were CAPN1,CAPN2 and CAPNS1,had abnormal expression in the pancreatic cancer and to find the relation between their expression and pancreatic cancer patients' prognosis,and toexplore the relation between them and KRas.Next,we used MTS,colony formation assay,and Western Blot to find whether the knockdown of CAPN2 influenced the phenotype of pancreatic cancer cells.We used m RNA half-life assay and protein half-life assay to find how the KRas influenced the CAPN2 protein expression.The m6A-Seq and RIP were used to identified whether m6 A was occurred in CAPN2 m RNA.We further knocked down the m6A-related factors and used the methodology of Real time RT-q PCR,Western Blot and RIP to find what were the m6 A “writer” and“reader” of CAPN2 m RNA.Moreover,we used si RNA knockdown technique combined with RIP to explore how the KRas regulated the CAPN2 m RNA in a m6 A dependent manner.Results: By using the GEPIA website we found the high CAPN2 expression was positively associated with the poor prognosis of pancreatic cancer patients.The CAPN2 expression was positively correlated with the KRas expression.We further used the CAPN2 si RNA and the PD150606,which was an inhibitor of Calpains,to find that the inhibition of CAPN2 could inhibit the growth of pancreatic cancer cells.The RNA half-life assay and protein half-life assay found that the KRas could promote the stability of CAPN2 m RNA.The m6A-Seq and RIP identified that m6 A was occurred in CAPN2 m RNA.We further screened that METTL16 and YTHDF3 were the m6A“writer” and “reader” of CAPN2 m RNA,respectively,both of which could influence the CAPN2 m RNA stability.Interestingly,we found the knockdown of KRas could reduce the binding of METTL16 to CAPN2 m RNA and lower the m6 A level of CAPN2 m RNA.The knockdown of YTHDF3 could inhibit the growth of pancreatic cancer cells.We also found the knockdown of KRas downregulated the YTHDF3 protein and m RNA.The knockdown of KRas was identified to reduce the stability of YTHDF3 m RNA.Conclusion: KRas could increase the m6 A level of CAPN2 m RNA through the METTL16.The KRas could also upregulate the YTHDF3 to enhance the stability of CAPN2 m RNA and therefore promote the growth of pancreatic cancer cells.