Effects Of SiRNA-mediated Gene FLJ37396 And TTC6 Silencing On The Proliferation And Invasion Of Human Primary Liver Cancer Huh7 Cells

Objective By comparing the difference of the samples gene between primary hepatocellular carcinoma and tumor thrombi in the portal vein,screen the gene FLJ37396 and gene TTC6 with specificity and high content of tumor thrombi.Using RNA interference、 CCK8、Transwell to further explore theirs biological function and the mechanism of action of the progression and metastasis of hepatocellular carcinoma.Methods Combined with clinical,using 10 samples of primary hepatocellular carcinoma with portal vein tumor thrombi by the Illumina sequencing technology.Screening out 10 genes of significant number of expression in the tumor thrombi than the primary foci.Based on the Score、E Value、Identities,selecting the specific target genes FLJ37396 and TTC6 by using NCBI BLAST system.The expression of target genes was detected in a normal cells group L-O2 and four hepatoma carcinoma cells groups hep G2 、 Huh7 、 MHCC97 H 、 HCCLM3 by QPCR technology.Designing and synthesizing the ds RNA of the target genes.Transferring the ds RNA into five groups of cells.Detecting the expression of target genes again by QPCR technology and the proliferation of the cells by CCK8 technology and the invasion of the cells by transwell technology.Then comparing the results before transfection with the results after transfection.Results 1,10 samples of primary hepatocellular carcinoma with portal vein tumor thrombi were sequenced by Illumina sequencing technology.Screening out 10 genes of significant number of expression in the tumor thrombi than the primary foci.The transcripts ID are Novel Tr₅36、Novel Tr₁50、Novel Tr₁258、Novel Tr₁259、Novel Tr₁257 、 Novel Tr₂98 、 Novel Tr₅74 、 Novel Tr₅90 、 Novel Tr₂13 、Novel Tr₁266.2,The 10 transcripts were analyzed by NCBI BLAST system（http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Blast Se arch&LINK_LOC=blasthome）.The following are the protein names of the 10 transcripts:（1）Novel Tr₅36 Transmembrane protein FLJ37396 isoform X11（2）Novel Tr₁50 Homo sapiens uncharacterized LOC105369364（LOC105369364）（3）Novel Tr₁258 Homo sapiens chromosome X open reading frame 49-like（LOC101059915）（4）Homo sapiens chromosome X open reading frame 49-like（LOC101059915）（5）Novel Tr₁257 Homo sapiens chromosome X open reading frame 49-like（LOC101059915）（6）Novel Tr₂98 Homo sapiens neurofibromin 1（NF1）（7）Novel Tr₅74 Homo sapiens gamma-aminobutyric acid type B receptor subunit 1（GABBR1）（8）Novel Tr₅90 Homo sapiens chromosome 3,GRCh38.p7 Primary Assembly（9）Novel Tr₂13 Homo sapiens tetratricopeptide repeat domain 6（TTC6）（10）Novel Tr₁266 Homo sapiens glycerol kinase（GK）.3,According to the coding ability scores（more than 9.5）of the transcripts,length（1000-2500bp）,translation products and other conditions,removing Novel_Tr150（non coding transcript）,Novel_Tr1258（4476bp in length）,Novel_Tr1259（4204bp in length）,Novel_Tr1257（4721bp in length）,Novel_Tr590（length 8031）,Novel_Tr298（translation products as neurofibroma associated protein）,Novel_Tr574（translation products as gamma-aminobutyric acid type B receptor subunit 1）,Novel_Tr1266（translation products as glycerol kinase）.Novel_Tr536 got 14.09 points of coding ability scores and had 1567 bp in length and its translation products were transmembrane protein FLJ37396.Novel_Tr214 got 9.65 points of coding ability scores and had 1057 bp in length and the translation products were tetratricopeptide repeat domain 6（TTC6）.Both of them matched the conditions.Further more,they had an uncertain biological function,and there were a potential impact on proliferation、 migration、 invasion of the cells.Therefore,they were selected for further studying.4,Detecting the expression of two target genes in five groups of cells L-O2、hep G2、Huh7、MHCC97H、HCCLM3 by QPCR technology.We can assume the expression of the gene FLJ37396 in normal liver cells group L-O2 is 1.Then the expression in ordinary hepatoma carcinoma cells group hep G2 is 1.74.The expression in three high metastasis potential hepatoma carcinoma cells groups huh7 、 MHCC97H、HCCLM3 are 55.38,9.32 and 4.31.In this similar way,we can assume the expression of the gene TTC6 in normal liver cells group L-O2 is 1.Then the expression in ordinary hepatoma carcinoma cells group hep G2 is 1.36.The expression in three groups huh7、MHCC97H、HCCLM3 are 36.43、5.11、3.85.The expression levels of both target genes are the most obvious in cells group huh7.So the cells group huh7 is selected for the following RNA interference test.5,Detecting the expression of gene FLJ37396 in NC cells group、control group、si RNA-1 group、si RNA-2 group、si RNA-3 group by QPCR technology.We can assume the expression of the gene FLJ37396 in NC cells group is 1.Then the expression in control group is 1.04.The expression in three groups si RNA-1、si RNA-2、si RNA-3 transfected with si RNA are 1.92,0.35 and 0.043.According to the results of the expression of the gene after transfection,the expression of gene in si RNA-1 group was significantly increased than before.The interference effect was not ideal.Target spot 1 failed to knockdown the expression of gene effectively.It might been that the target spot 1 was in a non coding region or si RNA-1 designed unsuitable.However,the gene expression in si RNA-2 group and si RNA-3 group were significantly decreased than before.They were only 0.35 times and 0.043 times respectively of the former.And it achieved the desired effect of interference.Among them,the greatest effect one was target spot 3,which could effectively reduce more than 95% of the expression of gene FLJ37396 in Huh7 cells.So we selected the target spot 3 for the following comparative experiments.6,Detecting the expression of gene TTC6 in NC cells group、control group、si RNA-1 group、si RNA-2 group、si RNA-3 group by QPCR technology.We can assume the expression of the gene TTC6 in NC cells group is 1.Then the expression in control group is 0.65.The expression in three groups si RNA-1、si RNA-2、si RNA-3 transfected with si RNA are 1.29,0.12 and 1.23.According to the results of the gene expression after transfection,the gene expression in si RNA-1 group and si RNA-3 group were slightly increased than before.The interference effect was not ideal.Target spot 1 and target spot 3 failed to knockdown the expression of gene effectively.It might been that the target spot 1 and target spot 3 were in a non coding region or the si RNA of them designed unsuitable.However,the gene expression in si RNA-2 group was significantly decreased than before.It was only 0.12 times of the former.And it could effectively reduce more than 85% of the expression of gene TTC6 in Huh7 cells.So we selected the target spot 2 for the following comparative experiments.7,Detecting the control group、si RNA-NC group、si RNA-FLJ37396 group and si RNA-TTC6 group by CCK-8 test.Recording the OD value of the samples and the control at 450 nm to the eliasa.The first day,the OD value of control group,si RNA-NC,si RNA-FLJ37396 and si RNA-TTC6 was 0.147,0.133,0.142,0.145.The OD value of each group was 0.434,0.391,0.487,0.280 in the second day.The OD value of each group was 0.630,0.602,0.733,0.434 in the third day.According to the OD value,it showed that the proliferation of Huh7 cells significantly inhibited after interfering the gene TTC6.However,it could promote the proliferation of Huh7 cells after interfering the gene FLJ37396.8,Detecting the control group、si RNA-NC group、si RNA-FLJ37396 group and si RNA-TTC6 group by Transwell test.Recording the OD value of the samples and the control at 570 nm to the eliasa.Then calculating the migration and invasion of the huh7 cells according to the OD value.The OD value of control group,si RNA-NC,si RNA-FLJ37396 and si RNA-TTC6 in the transwell migration test was 0.260,0.235,0.173,0.153.The OD value of control group,si RNA-NC,si RNA-FLJ37396 and si RNA-TTC6 in the transwell invasion test was 0.221,0.206,0.170,0.151.According to the OD value,it showed that the migration and invasion of huh7 cells inhibited after interfering the gene TTC6 and gene FLJ37396.Conclusions 1,The transcripts of Novel_Tr536 and Novel_Tr214 got high points of coding ability scores and had a moderate length.Further more,theirs translation products FLJ37396、TTC6 had an uncertain biological function,and there were a potential impact on proliferation、 migration、 invasion of the cells.2,FLJ37396 was a transmembrane protein.A variety of proteins with FLJ structure had been proved that could exert important effects on the growth of different tumor cells.So that FLJ37396 might be related to the metastasis of hepatocellular carcinoma.But the encoding sequences of this protein had no clear information in the official and the encoding protein was not conclusive in human genome transcription.It meant that the complete sequences of the gene of this protein were not to confirm in human genome.3,TTC6（tetratricopeptide repeat domain 6）contained TPR structure,which was a unique domain composed entirely of helical structures.TPR protein was a common medium in the interaction between different proteins and it was a kind of important molecular chaperone in the genomes of high grade organisms.In addition,heat shock protein was another important class of molecular chaperones.A large number of studies show that TPR protein bound to HSP70/90 to regulate the conformation,activity,intracellular localization and hydrolysis of the protein.It could exert important effects on the cycle,apoptosis,activity of the cells through this way.So TTC6 might be related to the development and metastasis of hepatocellular carcinoma.4,According to the results of five groups of cells samples by QPCR test,the expression of FLJ37396 and TTC6 in ordinary hepatoma carcinoma cells group hep G2 was close to the normal liver cells group L-O2.However,the expression increased significantly in three high metastasis potential hepatoma carcinoma cells groups MHCC97H、huh7、HCCLM3.These results suggested that FLJ37396 and TTC6 may been related to the progression and metastasis of hepatocellular carcinoma and the formation of portal vein tumor thrombi.The results were in accord with the initial conjecture.5,The transfection rate of Huh7 cells was different at different concentrations of si RNA.After transfecting with different concentrations,the transfection rate was the highest at 30 n M of the si RNA concentration.And the transfection rate was above 95%.6,Gene FLJ37396 and TTC6 were respectively provided with a plurality of different RNA interference target spots.Both of them could find effective interference target spots.They could effectively reduce more than 90% of the expression of both genes after the interfering.However,the gene expression was increased after interfering at some target spots.It might been that some target spots were in a non coding region or the si RNA of them designed unsuitable.This indicated the complexity of RNA interference and gene silencing.7,The results of the CCK-8 test showed that the proliferation of Huh7 cells significantly inhibited after interfering the gene TTC6.It further indicated that the gene TTC6 might promote the proliferation of hepatocellular carcinoma cells.On the other hand,it could promote the proliferation of Huh7 cells after interfering the gene FLJ37396.Thus gene FLJ37396 might inhibit the proliferation of hepatocellular carcinoma cells or enhance the invasion of hepatocellular carcinoma cells through the other ways.8,The results of the Transwell test showed that the migration and invasion of huh7 cells inhibited after interfering the gene TTC6 and gene FLJ37396.It further indicated that the gene TTC6 and gene FLJ37396 promote the migration and invasion of hepatocellular carcinoma cells.All in all,this study collected 10 samples of portal vein tumor thrombi and primary hepatocellular carcinoma.Screening out 10 genes of significant number of expression in the tumor thrombi than the primary foci by the Illumina sequencing technology.According to NCBI BLAST system,we selected gene FLJ37396 and TTC6 which met the relevant requirements.Detecting the expression of two target genes in a normal cells group L-O2 and four hepatoma carcinoma cells groups hep G2、Huh7、MHCC97H、HCCLM3 by QPCR technology.The expression levels of both target genes are the most obvious in cells group huh7.Disrupting the expression of two target genes through si RNA.Detecting the proliferation of the cells by CCK8 technology and the invasion of the cells by transwell technology.The results showed that gene TTC6 promote the proliferation of hepatocellular carcinoma cells and gene TTC6 and gene FLJ37396 promote the migration and invasion of hepatocellular carcinoma cells.These two genes might be related to the metastasis of primary hepatocellular carcinoma.It might provide a new target for the prevention and treatment of metastasis of primary hepatocellular carcinoma through inhibiting these corresponding genes or proteins.