Expression Level Of WTX Gene In Colon Cancer And Its Influence To Cell Growth Of Colon Cancer Cell Strain Lovo

Objective1. To study expression level and location of tumor suppressor gene WTX in fresh colon cancer tissues, peficancerous tissues and normal colon tissues. The relationship between gene expression level and clinical and pathological features of colon cancer were analysed and compared in order to study the relevance between abnormal gene expression of WTX and colon cancer.2. To study methylation level of WTX gene in fresh colon cancer tissues, peficancerous tissues, normal colon tissues and colon cancer cell strain Lovo.3. To establish lentiviral vector of WTX and transfect WTX into colon cancer cell line Lovo in vitro. Detecting expression of WTX in transcription and protein level and its influence to cell proliferation and apoptosis.Methods1. Fresh colon cancer tissues, peficancerous tissues and normal colon tissues of50colon cancer patients who had been accepted radical surgery and pathological confirmed were collected. These50specimens were divided into3groups including group of cancer tissues (group C), group of peficancerous tissues (group CB) and normal colon tissues (group N). RNA of these tissues was extracted and qPCR was used to detect transcription level of WTX in different tissues. Total proteins of these tissues were extracted and Western blot was used to detect protein level of WTX in different tissues. Immunohistochemistry was used to detect location of WTX in different tissues and immunohistochemistry score (IHCS) was analyzed. The relationship between WTX expression level and clinical and pathological features of group C were analysed and compared. 2. DNA of all specimens an colon cancer cell strain Lovo were extracted and modified by sodium hydrogensulfite. We utilized Wizard purified resin to purifiy DNA.4methylation specific PCR (MSP) specific primers were designed for detecting methylation level of WTX in all specimens and Lovo cells by MSP.3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was constructed by Gateway Technology (Invitrogen Company). The positive cloning plasmid was picked for positive cloning sequencing. The lentiviral vector and lentiviral packaging plasmid were co-trasfected into293FT cells. Lentivirus titer was caculated after virus supernatant was obtained by centrifugation. Lovo cells were devided into3groups including blank cells (group Cell), blank vector (group Blank Vector) and WTX vector (group WTX) and were trsducted by lentivirus. Fluorescence microscope was used to observe transducting efficiency at48-72h after transducting.We utilized qPCR and Western blot to detect expression of WTX in transcription and protein level of each cell groups. Flow cytometry was used to detect cell apoptosis level and cell cycle. Regent CCK8was used to detect cell inhibition ratio and draw cell growth curve.Results1. All specimens have WTX expression. Expression of WTX in transcription and protein level detected by qPCR and Western blot showed that mean expression level of WTX in group C was lower than group N and CB, mean expression level of WTX in group CB was lower than group N. The difference was statistically significant (p<0.05). Results of immunohistochemistry showed that expression of WTX was located at cytoplasm and little at cytomembrane. IHCS of group C was lower than group N and CB (p<0.05). IHCS of group N and CB had no obvious difference. IHCS of group C has no correlation with sex (r=-0.168,p=0.245), age (r=-0.170,p=0.238) and tumor position (r=0.022, p=0.879). IHCS of group C has positive correlation with tumor differentiation degree (the higher level of tumor differentiation, the higher of IHCS, r=0.711, p<0.01), and has negative correlation with tumor size (the bigger of tumor size, the lower of IHCS, r=-0.510,p<0.01), and has negative correlation with tumor vessel infiltration (when there is tumor vessel infiltration, the lower of IHCS, r=-0.804,p<0.01), and has negative correlation with tumor nerve infiltration (when there is tumor nerve infiltration, the lower of IHCS, r=-0.785, p<0.01), and has negative correlation with tumor TNM stages (the higher level of TNM stages, the lower of IHCS, r=-0.948,p<0.01).2. MSP detecting of tissues showed that positive rate of WTX methylation in group C was higher than group CB and N (p<0.05), but there was no WTX methylation found in colon cancer cell strain Lovo.3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed by Gateway Technology and identified perfectly correct. Lentivirus was successfully packaged and titer was accorded with the requirement of experiment. Fluorescence of group WTX and Blank Vector were weak at48-72h after transducting WTX into Lovo cells. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established by picking clone several times and cell culture. Then we proceeded transient transfection of WTX plasmid and contract blank plasmid. Fluorescence can be observed at48-72h after transient transfecting and transfection efficiency of group WTX and Blank Vector was about35%and70%respectively. Results of qPCR and Western blot showed that expression level rised after transfecting. Results of flow cytometry showed that excessive expression of WTX had no obvious influence to cell apoptosis level of Lovo cells. Results of CCK8test showed that with time increasing, cell population of group WTX reduced but fluorescence increased compared with group cell and group Blank Vector in96-well plates. Fluorescence cell population of group WTX was lesser than group Blank Vector. Excessive expression of WTX could obviously suppress cell proliferation. Cell cycle detection indicates that Lovo cells were blocked in G0/G1phase.Conclusions 1. Lower expression level of tumor suppressor gene WTX in colon cancer is closely ralated to grade malignancy and infiltration of surrounding tissues. Loss of WTX expression maybe a important reason of occurring of colon cancer.2. Although positive rate of WTX methylation in group C was higher than group CB and N, but WTX methylation was negative in colon cancer cell strain Lovo. More MSP should be done to detect whether methylation is exist in other colon cancer cell strains in order to clear whether methylation is a reason of lower expression of WTX in colon cancer tissues.3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed but cell trasducting efficiency was low. Long CDS region of WTX may influence eGFP expression, and this may be the reason that why transducting efficiency was low. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established successfully. Transient transfection of WTX plasmid lead higher WTX expression in transcription and protein level. Transfection of WTX could obviously suppress cell proliferation but had no influence to cell apoptosis.