Study Of Single Base Gene Mutant Colorectal Cancer Cells Based On Optical Tweezers Raman Spectroscopy And ~1H NMR Spectroscopy

Activation or aberrant expression of oncogene and/or tumor suppressor genes is one of the main causes of cancer.The Kras gene,as an important member of Ras family has attracted huge attention in cancer field,is considered as a major driver in the development of multiple types of cancer especially colorectal cancer.So the investigation of the biochemistry changes in Kras gene mutation colorectal cells has great meaning in early detection and subsequent treatment of colorectal cancer.In this paper,we combined the optical tweezers Raman spectroscopy and ~1H NMR-based metabonomics with multiple statistical analysis methods to probe the specific changes of biochemical molecules in Kras gene mutation colorectal cells.The main research work is as following:Firstly,DLD-1(Kras mutation)and DKS-8(Kras wild-type),HEK-3(Kras wild-type)and HCT-116(Kras mutation)were used as research objects.We combined optical tweezers Raman spectroscopy and PCA-LDA statistical algorithm were used to achieve the discrimination of colorectal cancer cells with different Kras gene mutation status.The diagnostic sensitivity and specificity for DLD-1(Kras mutant)and DKS-8(Kras wild-type)were 96.5%and 98.6%,respectively.The diagnostic sensitivity and specificity for HEK-3(Kras wild-type)and HCT-116(Kras mutant)were 97.5%and 96.5%respectively.These results suggested that Kras gene mutation can make a great difference in the content of biochemical molecules in the cells.By analyzing the peak intensity and spectrum assignment of cell Raman spectrum,it was found that the concentration of DNA in mutant cells was higher than that in wild-type cells,while the protein and lipids were lower.Secondly,we combined NMR metabonomic with PCA and OPLS-DA statistical analysis methods were used to study the metabolic characteristics of same cells above.NMR-based metabonomics strategy was applied to study the metabolic differences of Kras wild-type and mutation-type,it is helpful to understand the molecular mechanism of Kras gene on cell metabolism.The results showed that:for DKS-8 vs DLD-1 and HEK-3vs HCT-116 cell,the metabolic activity of Kras single base gene mutated cells was significantly enhanced and some characteristic metabolic pathways such as Alanine,aspartate and glutamate metabolism?Aminoacyl-tRNA biosynthesis?D-Glutamine and D-glutamate metabolism?Arginine and proline metabolism.Based on the results of Raman spectra of optical tweezers Raman spectroscopy,we find that Kras single base gene mutation will leads to specific changes of these four metabolic pathways.Due to the changes of characteristic metabolic pathways,the contents of proteins and amino acids were changed,such as phenylalanine,collagen,proline and glycine,which were confirmed by the optical tweezers Raman spectroscopy.The results of our exploratory research show that by using optical tweezers Raman spectroscopy and nuclear magnetic resonance(NMR)based metabonomics,we can find that single base gene mutation in Kras gene segment can cause significant changes in the expression of some vital biochemical molecules in cells and their metabolites.