| Background:Colorectal cancer (CRC) is one of the common gastrointestinal malignant tumors in the world that is on the second place in the structure of oncological mortality and the majority of colorectal cancer deaths occur in indeveloped countries. The morbidity for CRC in China is20.6/100000, and the annual rate is rising year by year. Surgical resection is the main treatments for colon cancer,40%-50%of patients experienced postoperative recurrences or metastases, most of them lose the chance to cure.Therefore, studying the mechanisms of CRC and looking for the innovative strategies for effectively treatments become the common focus for oncologists all over the world.Hypoxia is a common phenomenon in the process of solid tumor, and CRC is one of the significant hypoxia of local microenvironment in solid tumors. Thus, understanding the molecular mechanisms of hypoxia in CRC may provide a new strategy for its treatment.Aryl hydrocarbon receptor nuclear translocator (ARNT) is many common obligate gametophyte of dimerization of basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) protein in vivo, including aryl hydrocarbon receptor (AhR), hypoxia-inducible factor (HIF)-1α, HIF-2α, HIF-3α and so on which all have to get the ARNT subunit combination then to participate in many important biological processes related hypoxia. The studies indicate that the AhR may contribute to the development of colon cancer through cell cycle control.The HIF-la could improve hypoxia tolerance of tumor cells and play an important role in promoting angiogenesis and invasion of tumors. Xue X found that HIF-2a activation could promote colorectal cancer progression. But the report from Imamura T is that HIF-2a appeared to restrain cell growth in colon cancer S-W480cells. The function of HIF-3a seems to be be a reverse adjustment factor of hypoxia induced gene expression and relate to the cell apoptosis. Therefore, reduceing ARNT expression, make AhR, HIF-lα,2α and3α lack of corresponding subunits, then what kind of impact on the biological characteristics of colon cancer cells is not clear.The ARNT gene is located on human chromosome one (1q21), and expresses in the nucleus and cytoplasm. ARNT may be associated with the occurrence of human cervical squamous cell carcinoma. Related study has indicated that the ARNT was inversely related to the growth, invasion and metastasis of hepatocellular carcinoma (HCC), and inhibited the growth, invasion and metastasis of HCC. ARNT can promote vascular endothelial cell migration and vascular branch generation, and vascular proliferation could promote the growth of solid tumors such as CRC. Now the role of ARNT gene in biological nature of CRC is not known yet.Lentivector can carry short hairpin RNA(shRNA) expression cassette into host genomics and express shRNA using RNA Pol-Ⅲ promoter. The expressed shRNA is cut into small interferingRNA(siRNA) by an RNase-Ⅲ family nuclease called dicer enzyme and subsequently guide degradation of cognate mRNA. Lentivirus has been shown to mediate RNAi most efficiently and stably based on its remarkable advantages, such as integration of expression cassette into host genomics, powerful transduction efficiency, a wider range of target cells, low immunogenicity, self-inactivated and replication-incompetent after integrating into host genomics. At present, we haven’t found that there were any reports on the physiological function of ARNT in HT29cells.Purpose:To obtain the recombinant lentivirus vector with expressing ARNT-short hairpin RNA (shRNA) that could suppress ARNT expression in human colorectal cancer HT29cells effectively, and to explore the inhibitory effect of ARNT gene silencing on the proliferation, apoptosis in vitro and tumor growth in vivo of human colorectal carcinoma HT29cells. It may offer us new strategy for the treatment of CRC.Methods:1. Design and construction of shRNA vectors targeted ARNTAccording to the purpose gene sequences (Gene ID:405), Four coding regions corresponding to targeting human ARNT in the sequences were selected as siRNA target sequences under the guide of siRNA designing software offered by Thermo. The scrambled siRNA(5’-TTCTCCGAACGTGTCACGT-3’,namely LV-ARNT-RNAi-nc) that was a recognized sequence in many RNA interference experiments is used as a negative control. The negative siRNA control construct is not predicted to target any genes. Construct four lentiviral vectors with expressing ARNT-shRNA and a negative control lentiviral vector and have the sequencing appraisal.2. Packaging and titer of recombinant lentivirusThree kinds of plasmids were extracted, measure the DNA concentration and purity with ultraviolet absorbance spectroscopy. The recombinant lentivirus vectors were produced by co-transfecting293T cells with the lentivirus expression plasmids and packaging plasmids (pHelper1.0and pHelper2.0), according to the specification of Lipofectamine2000. Recombinant lentivirus were harvested at48h post-transfection, centrifuged to remove cell debris, filtered through0.45mm cellulose acetate filters and saved in-80℃. The viral titer was determined by infecting293T cells with serial dilutions of concentrated lentivirus, and then determining the GFP expression of infected cells by fluorescence microscopy96h after infection. 3.Obtain optimal parameters of lentivirus transfecting HT29cells and optimal expression time of fluorescent protein after transfectionThe day before experiment, HT29cells were seeded in12holes of96well plate(cell concentration was5×104/ml,100μl/well).Cell was divided into four groups, each group had three different gradient multiplicity of infection (MOI). At start of the experiment, prepare three different concentrations of lentivirus(1×108,1×107and1×106TU/ml) and take2μl10mg/ml polybrene diluted to400μl. Discard original medium,then complete medium was added into1-6holes(80μl/well), ENi.S. was added into7-12holes (80μl/well). The three different gradients of virus were added into each corresponding holes(10μl/well). Complete medium was added into1-3holes(10μl/well), polybrene diluent was added into4-6and10-12holes(10μl/well), ENi.S was added into7-9holes(10μl/well). Then cell was cultured at37℃under5%CO2. The cell was photoed by fluorescence microscope once a day, understanding the cell fluorescent expression,to determine optimal parameters of lentivirus transfecting HT29cells and optimal expression time of fluorescent protein in HT29cells after transfection. The subsequent experiments adopted the optimal parameters for transfecting HT29cells.4. The test of efficiency of recombinant lentivirus transfected into HT29cellHT29cells were transduced with recombinant lentivirus at MOI of0,10,15,25,50,100and150. After incubation at37℃for72hours, the transduction efficiency was detected by FACSCantoTM II flow cytometry. The subsequent experiments adopted the best MOI.5. Transducing recombinant lentivirus into HT29cells and groupingThe HT29cells were subcultured at1×105cells per well into six-well tissue culture plates. After24h culture, at a MOI of100, the cell at30%-50%confluency was expanded into LV-ARNT-RNAi-1,LV-ARNT-RNAi-2,LV-ARNT-RNAi-3, LV-ARNT-RNAi-4,LV-ARNT-RNAi-nc and parental HT29cell groups, respectively, and at the same time added a working fluid concentration for5μg/μl of polybrene to increase the efficiency of infection. After8h of incubation, the culture medium was replaced with fresh one. The cells were observated and took pictures under inverted fluorescence microscope after72h. The cells that were successful infected by lentivirus were inoculated in96-well plate through the limited dilution, when forming clear clonings, pick the fluorescent clonings to24-well plate for further culture.Lastly, gain five stable transfection cell lines.6. Verify the endogenous targetsBefore extracting RNA or protein,EGFP expression in HT29cells was observed under a fluorescent microscope(original magnification×200). Green fluorescent cells to total proportion was rough count by phase contrast. When the ratio of fluorescent cells was more than90%, the efficiency of infection met the requirements.7. Primers effectiveness appraisalMake up1%agarose gel plate, cDNA samples that were mixed with bromophenol blue were added into electrophoresis holes in turn, the amplicons were separated by agarose gel, and detected by ethidium bromide (EB) staining.8. Prove effectiveness of2-△△Ct method that was used to calculate relative level of ARNT gene expressionAccording to starting quantity of cDNA, divided into0.1,0.5,1,2μl four groups, each group had six holes, half of the holes were added into ARNT primer, the other half were added into Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer, then perform real-time fluorescence quantitative polymerase chain reaction (PCR), repeat3times. Calculate△CT value of ARNT and GAPDH, the linear correlation coefficient r was geted through mapping the log value of the concentration gradient of cDNA to△CT value. According to statistical inference of correlation coefficient in the section2,chapter9,second edition of 《medical statistics》(Chief editor is Sun ZQ), if alpha>0.05, it meaned that the amplification efficiency of target gene was consistent with housekeeper gene, and the relative quantitative of ARNT gene expression could be computed by2-△△Ct method.9. Detecting the interference effects of different targetsThe ARNT mRNA and protein expression were examined by real-time quantitative PCR and western blotting, respectively, screening out effective silence targets. Total cellular RNA was isolated using RNAiso Plus, PrimeScript(?) RT reagent Kit was used to create cDNA for further analyses. Quantitative real-time PCR assays were carried out using SYBR(?) Premix EX TaqTM and7500realtime PCR amplification equipment.PCR parameters were as follows:95℃for30S, then95℃for5S,60℃for34S for40cycles. The2-△△Ct method was used for relative quantitative comparison among samples.The expression level of ARNT protein was detected by Western-blot analysis: The whole-cell protein extracts from six groups of cells were prepared with radio-immunoprecipitation assay (RIP A) lysis buffer in accordance with the manufacturer’s instructions. Protein concentrations were determined using an BCA protein concentration measurement kit (enhanced). Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were incubated in blocking buffer for1h at room temperature, followed by hybridization with anti-ARNT antibody and anti-β-actin antibody at4℃overnight. The membranes underwent hybridization with a horseradish peroxidase-conjugated secondary antibody rabbit IgG for1h at room temperature.Signals were detected by chemiluminescence using ECL luminous fluid and scanned by Quantity One software.The expression of ARNT protein was determined by normalization of the gray value of these genes to that of the control housekeeping gene (β-actin). Using blank control group as a sample for reference, calculate the rest relative level of each protein. The recombinant lentivirus vector with the highest interference efficiency was selected for use in subsequent experiments. Independent experiments were repeated three times for each sample.10. Cell proliferation was determined by CCK-8assayCells were seeded in96-well culture plates in culture medium at an optimal density (1×104cells per well) for the three stable transfection cell lines (parental HT29, LV-ARNT-RNAi-nc and LV-ARNT-RNAi-3),each group was6holes. CCK-8assay was used to measure cell proliferation.11. Flow cytometric analysis of apoptosis The cell was divided into blank control,LV-ARNT-RNAi-nc and LV-ARNT-RNAi-3groups. Cell apoptosis was assayed using the Annexin-V-PE/7-AAD apoptosis assay kit under the manufacturer’s instructions.Cells were analyzed using a BD FACSCanto TMⅡ flow cytometer.12. Nude mice xenograft modelA heterotopic (subcutaneous) human CRC xenograft nude mouse model was used to evaluate the influence of HT29cell growing after reduceing ARNT expression in vivo. Mice were randomly divided into two groups, namely A and B, each experimental group consisted of six mice. Within the groups were randomly divided into two groups (each group of three):the left backs of nude mice (A1-A3) were injected with ARNT-RNAi-3cells and the right backs were injected with ARNT-RNAi-nc cells, the left backs of nude mice (A4-A6) were injected with ARNT-RNAi-nc cells and the right backs were injected with ARNT-RNAi-3cells. The left backs of nude mice (B1-B3) were injected with parental HT29cells and the right backs were injected with ARNT-RNAi-nc cells, the left backs of nude mice (B4-B6) were injected with ARNT-RNAi-nc cells and the right backs were injected with parental HT29cells. Under aseptic conditions, about2×106logarithmically growing cells in0.1ml cold phosphatebuffered saline were injected subcutaneously into the dorsal scapular department of nude mice. After four weeks observation, the mice were sacrificed by cervical dislocation, tumors were stripped and the tumor weight and volume were measured by specialist blind method.Results:1. The measurement of sequences of positive cloningsGene sequencing results showed ARNT-shRNA sequences cloned into GV115plasmids for ARNT RNAi were completely in accordance with designed shRNA sequences. The plasmids that had right cloning were extracted, then measured plasmid DNA of restructured lentivirus, A260/A280values were1.8-2.0, the results explained that the purity and integrity of plasmids DNA were very high.2. Volumes and titers of recombinant lentivirus The volumes and titers of the recombinant lentivirus condensed supernatant were as follows:120μl,1.0E+9TU/ml (LV-ARNT-RN-Ai-1);200μl,6.0E+8TU/ml (LV-ARNT-RNAi-2);200μl,6.0E+8TU/ml (LV-ARNT-RNAi-3);160μl,8.0E+8TU/ml (LV-ARNT-RNAi-4);120μl,1.0E+9TU/ml (LV-ARNT-RNAi-nc). The results showed that there were a large number of plasmids into293T cells and the virus packaging were successful.3.Obtain the best transfection parameterPolybrene mildly increased transfection efficiency of lentivirus transfecting HT29cells. Using infection enhanced liquid (ENi.S.) not obviously increased the transfection efficiency. After transfecting lentivirus for48h, only a few cells could express fluorescent protein, and a lot of fluorescent cells were observed on third day. The fluorescence intensity and the number of fluorescent cells had no obvious difference between72h and96h. Considering cell growth cycle, the72h after cell transfected by lentivirus was the best time for observation.4. Obtain the best MOI valueWhen MOI was0,10,15,25,50,100and150, the transfection efficiency in turn was0,25.80,35.90,53.50,64.59,75.90and78.5%. With the increasing of MOI, the transfection efficiency significantly increased slowly, considering the dosage of lentivirus, the MOI=100was considered as the best transfection conditions.5. Endogenous target validationEGFP expression in HT29cells was observed under a fluorescent microscope. The ratio of green fluorescent cells was more than90%, the infection efficiency met the requirements.6. The results of primer testElectrophoresis results showed that the molecular size of amplicon was consistent with the theoretical value and nonspecific banding was not found,it suggested that the primers were effective.7.The results on effectiveness of2-△△Ct method that was used to calculate relative level of gene expression r=0.0451, according to the t value formula of test statistic,t value was0.0638, v=2, check t boundary value table, a>0.05, then accept Ho, it meaned that the amplification efficiency of target gene was consistent with housekeeper gene,and the relative quantitative of ARNT gene expression could be computed by2-△△Ct method.8. Selection of the most effective ARNT-specific siRNA expression vectorFour groups of recombinant lentivirus that could express ARNT-shRNA were successfully constructed.One of the groups(LV-ARNT-RNAi-3)could significantly suppress ARNT expression in HT29cells, the mRNA expression fell by70%(P<0.05) and the protein expression fell by60%(P<0.05), respectively, compared with the control group.9. Examination of cell proliferationCell proliferation was significantly promoted by LV-ARNT-RNAi-3in HT29cells compared with LV-ARNT-RNAi-nc or parental HT29cell groups(P<0.05), there were no significant difference between LV-ARNT-RNAi-nc and parental cell groups (P>0.05).Test of Between-Subjects Effects:Between different groups(F=116.235, P=0.000),Between different time points(F=607.075,P=0.000).ANOVA:4hours, F=0.441,P=0.651;1day,F=37.500,P=0.000;2days,F=56.244,P=0.000;3days, F=32.701, P=0.000.10. Examination of apoptosis cellsApoptosis in LV-ARNT-RNAi-3group was higher than parental HT29cell group(P=0.000) and lower than LV-ARNT-RNAi-nc group (P=0.003).11. LV-ARNT-RNAi-3lentivirus promoted tumor growth in vivoA nude mouse in group A only survived for three days after inoculation. Five days later, tumors in the injection sites of nude mice were observed visually.The volume and quality of tumors in LV-ARNT-RNAi-3group were significantly bigger compared with LV-ARNT-RNA-nc group in group A,respectively(t=2.872,P=0.038;t=3.069,P=0.033), and no obvious variances were found between the parental HT29cell group and LV-ARNT-RNAi-nc group in group B,respectively(t=0.998, P=0.342;t=0.503,P=0.626).Between group A and B:There was no significant difference in sum of bilateral tumor volume and quality(t=-0.564, P=0.587; t=-0.090, P=0.930); no obvious difference in nude mice quality was found when the nude mice were injected with carcinoma cell suspension (t=-0.000,P=1.000), and no obvious difference also was found when the nude mice were sacrificed (t=-1.640, P=0.165).Conclusions:The experimental results show that:LV-ARNT-RNAi-3can efficiently inhibit ARNT expression in HT29cells. Knockdown of ARNT gene could promote the proliferation, inhibit the apoptosis when HT29cells were cultured in vitro normoxia circumstance.Down-regulation of ARNT expression could promote transplantation tumor growth of human colon cacer HT29cells. ARNT gene in human colon cancer HT29cells is tumor suppressor gene. |
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