Effect Of SiRNA On Neutrophil Gelatinase-associated Lipocalin (NGAL) Gene Silence In Colon Cancer Cells

BackgroundColorectal cancer is the most common malignant tumor in the world,occupying the third in the cancer morbidity and fourth in morbidity in man and the second in the cancer morbidity and third in morbidity in women respectively.The incidence of colorectal cancer ranks third and the mortality ranks second in malignant tumors of the United States.In 2015,there were 376,300 new cases of colorectal cancer in our country,with a death cases of 19.1 million.The morbidity and mortality ranked the fifth in most regions of China.The 5-year survival rate of patients without metastasis was up to 90%,whereas the patients with metastasis was only 13%.The liver is the most dominant site of CRC metastases with an incidence of 15%-20% at diagnosis.More than half of the CRC patients developed liver metastases,which were the common cause of death.Surgery remains the only curative treatment.After radical resection of liver metastases,approximately 35–40% of patients could survive for 5 years.The five-year survival rate was only 1% for patients with unresectable colorectal cancer liver metastases,and the overall survival(OS)was only 30 months.Neutrophil gelatinase-associated lipocalin(NGAL)is a novel member of the lipocalin family.In recent years,the researcher had found that NGAL was involved in tumor proliferation,invasion and metastasis through influencing the activity of matrix metalloproteinase-9(MMP-9),iron-transport systems and the epithelio-mesenchymal transition(EMT).There is increasing researches which revealed that NGAL was over-expression in digestive system neoplasm such as esophageal cancer,gastric cancer,colorectal cancer,and pancreatic cancer,and involved in their occurrence and development.The effect of siRNA on Neutrophil Gelatinase-associated Lipocalin(NGAL)gene silence in colon cancer cells has not been reported at home by now.In this research,NGAL siRNA encapsulated by ribo FECT CP Transfection Kit(TK),which was then used to transfected human colon cancer cell lines.The level of NGAL m RNA and protein expression were detected,so as to explore the role of NGAL in the colon cancer cell proliferation,migration and apoptosis.This study may also search for the evidence for RNAi in the treatment of advanced colorectal cancer.ObjectiveTo explore the impact of NGAL knockdown by siRNA on proliferation,migration and apoptosis of colon cancer cell,and search for the evidence for RNAi in the treatment of advanced colorectal cancer.MethodsNGAL siRNA encapsulated by ribo FECT ? CP Transfection Kit(TK),which was then used to transfected human colon cancer cell lines HT-29.The level of NGAL m RNA and protein expression were detected by the real-time quantitative PCR(RT-QPCR)and Enzyme Linked Immunosorbent Assay(ELISA)respectively,so as to explore the role of NGAL in the HT-29 cell proliferation,migration and apoptosis.ResultsSeventy-two hours after transfection,the results of RT-QPCR analysis showed that the level of NGAL m RNA expression in TK group was 0.123,significantly lower than the control group(1.000)(p?0.05).The ELISA examination revealed that the degree of NGAL protein expression in TK group[(0.328±0.055)?g/L] was significantly lower than in the control group[(1.071±0.143)?g/L],two days after transfection(t=8.205,p=0.001).Apoptosis detection showed that the apoptosis rate in TK group was significantly higher than in control group 48 hours after transfection[(18.87±1.65)%vs.(11.93±1.90)%,t=-4.752,p=0.009].Migration assay suggested that the 24-hour migration rate of TK group was significantly lower than that of control group[(23.35±6.35)% vs.100%,t=11.930,p<0.01],meanwhile the Proliferation rate of TK group was slightly lower than that of control group[(90.00±5.30)%vs.100%,t=1.983,p=0.118].ConclusionNGAL siRNA could down-regulate the expression of NGAL,which was significantly associated with the low migration and the high level of apoptosis of HT-29.RNAi may be used in the treatment of advanced colorectal cancer.