The Research Of Transfecting Mutated MtDNA Of Colorectal Carcinoma Cell Line Into NIH3T3 Cell Line And LST Cell Line

Colorectal carcinoma(CRC) is a kind of malignant tumor associated with various genes,factors and phases.The research on this paper shows that there exist cancer suppressor factors outside chromosome and mtDNA plays a role in keeping cell's tumor characteristics^[1]. There are few reports on the variation of mtDNA in colorectal carcinoma , with different opinions.It was reported by Savre-Train^[2] that big segment of 2583bp was detected absent by rate of 1% ,but did not detect the mutation of base and mtDNA genetic instability in cancer of colon cell line of Caco-2. It was reported by Heerdt^[3] that there were existense genetic polymorphism ,but no mutation or genetic instability was detected in mtDNA D-loop of cancer of colon cell. It was also reported by Habano^[46] . that there were mutation by 7/45(16%) including 3 cases with frame shift mutation that code ND1and ND5,2 with missense mutation, 15Kbp deletion without any absence of big segment, and D-loop area instability of mtDNA were reported in 22 cases in the further researches in 45 pairs cancer of colon and normal.The above researches proved there exists variation of mtDNA in colorectal carcinoma cell, but pointed outneither action mechanism of occurrence and development in colorectal carcinoma nor interior relationships between mtDNA and oncogene or anti-oncogene related to the occurrence of colorectal carcinoma. Human mtDNA genome are circle, double-strand DNA . Because the full-length mtDNA is only more than 16 Kb, and its sequence is completely clarified,it is easy to get the sequence of mtDNA just through designing reasonable primer ,amplifying PCR and sequencing .So it provides convenience for research . mtDNA involves coding region and discoding region(D-loop) and mtDNA D-loop including origin of rephlication of heavy chain and other important sequence ,which is considered as hot point of current researches. Our project team analyzes the variations of mtDNA in colorecatal carcinoma including mutation, absence ,and DNA instability using PCR techniques.In previous study, we found among the three colonial tumor cell lines(SW480, LoVo,HT29), there were 10, 9, 8 mutations were identified .And we detected the same point mutations in different colorectal carcinoma cell lines, such as T to C mutation at npl6224 and np 16311 in SW480 and LoVo , C to T mutation at npll4, np 498,and np 16234 in SW480 and HT29. The same mutations found in different colorectal carcinoma cell lines were associated with typical point mutation. In order to prove the relationship between mtDNA mutation and the formation of colorectal carcinoma, we transfected mutated mtDNA of human colorectal carcinoma cell line into NM3T3 cell line and LST cell line , lots of observations have been done on the alteration behaviors of intransfected cell and the integration of mtDNA in intransfected cells nuclear and further study the changes of biological behaviors of intransfected cells. The study aims to rudimently find out the relationship between mtDNA mutation and carcinogenesis in colorectal carcinoma. Methods:1 .Cloned the mutated D-loop region gene of mtDNA in colorectal carcinomacell lines. Constructed the pcDNA3. 1(+) — mtDNA eukaryotic cell expression recombinant2. While transfecting mutated mtDNA of human colorectal carcinoma cell line into NIH3T3 cell line and LST cell line using lipofectamine 2000 and screening masculine cloning cell by G418(500ug/L), validates the integration of mtDNA in intransfected cells nuclear and observations have been done on the alteration behaviors of intransfected cell and further study the changes of biological behaviors of intransfected cells.Result:1.There different eukaryotic cell expression recombinant such as pcDNA3.1(+)-SW48,pcDNA3.1(+)-LoVo, pcDNA3.1(+)-NHWB eukaryotic cell expression recombinant are successfully constructed.2. Transfected the recombinant into NIH3T3 cells and screened masculine cloning cell using G418(500ug/L) , got four masculine clone transfected cell lines and amplified then.3. There were respectively 9,11,8 ,4 mutations identified in the four transfected NIH3T3 cell lines. And there were respectively 3,4,3,2 polymorphism mutation points. We found that of mutation points in NIH3T3 cell mtDNA transfected the pcDNA3.1(+)-SW480 mtDNA , pcDNA3.1(+)-LoVo , pcDNA3.1(+)-NHWB eukaryotic cell expression recombinant were more than empty pcDNA3.1(+).4. There's no obvious differences in apoptosis between groups after transfection.5. Target gene and Neo gene can be amplified in nuclear genome of transfected cells.Conclusions:1. After transfected the mutated mtDNA of colorectal carcinoma, the mtDNAD-loop region of the transfected cells displays new mutation points.2. The external source pieces of the mutated mtDNA can integrate to uclear genome after transfection.3. There's no differences in apoptosis between combinations after transfected the mutation of mtDNA in NIH3T3 and LST cells4. The mutated mtDNA may affect the action mechanism of occurrence and development in colorectal carcinoma through affecting its mtDNA mutation or integrating exogenetic mtDNA to its nuclear which may cause the abnormal expression of oncogene or anti-oncogene.