The Research Of Initating NIH3T3 Cell Line Malignant Transformation Through Transfected Mutated MtDNA Of Colorectal Carcinoma

BackgroundThe morbidity and mortality of colorectal carcinoma(CRC), one of mostcommon malignant gastrointestinal tumors, were gradually increasing from the 1990sof twentieth century and now are similar all over the world. The mobidity of CRCremains the forth in that of malignant tumor in the world, and the same in our country.CRC is also a kind of malignant tumor associated with various genes, factors andphases. The research on some paper shows that CRC occur is depend on not onlyinside nuclear genetic material but also outside mtDNA. MtDNA is an uniqueextra-nuclear genetic material in cell, and have self-replicating DNA genome.There are few reports on the variation of mtDNA in colorectal carcinoma in ourcountry research field, and there are also have different opinions overseas. It reportedby Savre-Train that big segment of 2583bp was detected absent by rate of 1ï¼…, but didnot detect the mutation of base and mtDNA genetic instability in cancer of colon cellline of Caco-2. It was reported by Heerdt that there were existence geneticpolymorphism, but no mutation or genetic instability was detected in mtDNA D-loopof cancer of colon cell. It was also reported by Habano that there were mutation by 7/45(16ï¼…) including 3 cases with frame shift mutation that code ND₁ and ND₅, 2with missense mutation, 15Kbp deletion without any absence of big segment, andD-loop area instability of mtDNA were reported in 22 cases in the further researchesin 45 pairs cancer of colon and normal. The above researches proved there existsvariation of mtDNA in colorectal carcinoma cell, but pointed out neither actionmechanism of occurrence and development in colorectal carcinoma nor interiorrelationships between mtDNA and oncogene or anti-oncogene related to theoccurrence of colorectal carcinoma. Human mtDAN genome is circle double-strandDNA. Because the full-length mtDNA is only more than 16Kbp, and its sequence iscompletely clarified, it is easy to get the sequence of mtDNA just through designingreasonable primer, amplifying PCR and sequencing. So it provides convenience forresearch. MtDNA involves of replication origin of heavy chain and other importantsequence, which is considered as the hot point of current researches.Our research groups have analyzed the mtDNA variation of the colorectalcarcinoma cells; the colorectal carcinoma tissue; the colorectal carcinoma normaltissue besides and the peripheral white blood cell of colorectal carcinoma patients bythe technique of the Polymerase chain action (PCR) and silver stained single strandconformation polymorphism(SSCP). And the characteristic and rule of variationcontain the point mutation; depletion and the DNA unstability. Our research groupsalso constructed the different eukaryotic cell expression recombinant plasmids suchas pcDNA3.1 (+)-Sw480,pcDNA3.1 (+)-Lovo and pcDNA3.1 (+)-NHWBC. In orderto prove the relationship between mtDNA mutation and the formation of colorectalcarcinoma, We transfected mutated mtDNA of human colorectal carcinoma cell lineinto NIH3T3 cell line, both the alteration behaviors and the integration of mtDNAhave been observed of different NIH3T3 group cells. We also do further study thechanges of biological behaviors of between different NIH3T3 group cells. The study aims to rudiment find out the relationship between mtDNA mutation andcarcinogenesis in colorectal carcinoma.Methods:1. Through the XhoI and BamHI double enzyme or BarnHI single enzymedegested the pcDNA3.1(+)-Sw480 mtDNA,pcDNA3.1(+)-Lovo mtDNA andpcDNA3.1(+)-NHWBC mtDNA different eukaryotic cell expression recombinantplasmids, then purify and sequence the recombinant plasmids in ABI377 seqencermade in America, lastly contrasted with the mtDNA genebank data to analyze thedifferent recombinant plasmids.2. While transfenting mutated mtDNA of human colorectal carcinoma cell lineinto NIH3T3 cell line using lipofection 2000^TM and screening positive cell by G418,validates the integration of mtDNA in transfection cells nuclear. We also have doneon the alteration behaviors and do further study the changes of biological behaviors ofthe different NIH3T3 group cells.3. We vaccinated the stabilized positive cloning cell line into the subcutaneoulyof the BALB/C hairless mouse, so that we can observe the effective and process oftumorigenesis.Results:1. The pcDNA3.1(+)-Sw480 mtDNA,pcDNA3.1(+)-Lovo mtDNA andpcDNA3.1(+)-NHWBC mtDNA different eukaryotic cell expression recombinantplasmids had been done that can be use to transfect into the NIH3T3 cell line.2. Transfected the recombinant plasmids into NIH3T3 ceils and screenedpositive cloning cell using G418, We can get three positive clone transfected cell linesand amplified then.3. Three kinds of non-radioactive DIG labellde mtDNA probes were preparedby PCR.The chromosomes in NIH3T3 cells can be integrated by mtDNA probes by analyzed in fluorescence in situ hybridization(FISH).4. There are distinguished changes in chromosome karyotype NIH3T3 cell lineof the different group cells.5. There are have differences in apoptosis between the different transfectiongroup cells.6. There are respectively 11, 8, 8 variation identified in the three transfectedNIH3T3 cell lines.There are respectively 7, 7, 7 polymorphism mutation points. Andthere are respectively 4, 1, 1 mutation points. We found that of mutation in NIH3T3cell mtDNA transfected the pcDNA3.1 (+)-Sw480 mtDNA, eukaryotic cell expressionrecombinant were more than pcDNA3.1(+)-NHWBC mtDNA and emptypcDNA3.1(+) mtDNA.7. Through the statistics analysis of the optical density OD₅₇₀ of different groupcells between different time, P＜0.05, We can conclude that there are have differencesin the proliferation level between groups after transfection.8. The percent number of soft agarose colony forming rates of between threepositive clone transfection cell lines and no transfection NIH3T3 have significancedistinction.9. We vaccinated 1×10⁶ the stabilized positive cloning cell line into thesubcutaneouly of the BALB/Chairless mouse, but they can not initiate the tumor.Conclusions:1. The mutated mtDNA can influence the mtDNA mutational andpolymorphism mutation points. And the mutated mtDNA may affect the changes ofbiological behaviors of intransfected NIH3T3 cell line such as the chromosomekaryotype and the apoptosis and the proliferation level through intergrating thecolorectal cancinoma cells mutated mtDNA to its nuclear. But it is going to do furtherresearches to know wheather it may provocate or block the MAPK cell signal transduction path after intergration.2. The carcinoma occurred which is associated with various genes, factors andphases. Tranfecting individual gene into the NIH3T3 cell line can not initate thetumors enoughly.