| Background and ObjectiveThe high incidence of colorectal cancer is one of the main causes of cancer death in China,which is a serious threat to the health of residents.Most of the patients were in advanced stage,and the prognosis of CRC depended on the pathological stage.Therefore,it is very important to explore the carcinogenic mechanism of colorectal cancer and to find a new therapy for the diagnosis and treatment of colorectal cancer.Long noncoding RNA(lnc RNA)is a RNA molecule,which is longer than 200 nucleotides and can not be translated into protein.Lnc RNAs are involved in all aspects of cellular physiology.Ln c RNAs are important for the formation,progression and metastasis of cancer,and are located in the long arm 25 region of chromosome 1.Many studies have shown that lnc RNA GAS5 expression is down-regulated in a variety of cancers,including breast cancer,prostate cancer,and pancreas.Adenocarcinoma,bladder cancer,lung cancer,gastric cancer and so on,and plays an important role in tumor cell colonization and apoptosis.Mi RNA is reported to be involved in many stages of cancer formation and to play a carcinogenic or suppressive role.It has been shown that the interaction of lnc RNA-miRNA can regulate gene expression.Lnc RNA can act as an endogenous "sponge" to adsorbmiRNA,and regulate gene expression.The abnormal expression of miR-182-5p plays a carcinogenic role in various malignant tumors,including colon cancer.Therefore,we assume that GAS5 can also act on miR-182-5p,but there are few studies on the relationship between GAS5 and miR-182-5p,as well as on the proliferation and apoptosis of colorectal cancer.Therefore,this study starts with GAS5 and miR-182-5p to study the role of GAS5 and miR-182-5p in the proliferation and apoptosis of colorectal cancer cells.MethodsPart Ⅰ: Detection of GAS5 expression in 60 cases of colorectal cancer tissues and normal tissues by RT-PCR.Detection of GAS5 expression in HCT-116 and normal colonic epithelial cell line NCM460 by RT-PCR.GAS5 overexpression vector was constructed to detect the expression of GAS5 in HT-29 and SW480 cells by RT-PCR.To analyze the relationship between the expression level of Gas5 m RNA and clinicopathological features.Part Ⅱ: The over expression vector of Gas5 was constructed and transfected into HT-29 and SW480 cells for 48 h and 72 h respectively.The cell proliferation was analyzed by cell count CCK-8 method.The GAS5 overexpression vector was constructed and transfected into HT-29 and SW480 cells for 48 h,and the apoptosis rate was detected by Annexin V-FITC/PI apoptosis kit.Part Ⅲ: Use bioinformatics online software programs(DIANA tools)to predict potential miRNAs;interactions with GAS5.The levels of Gas5 and miR-182-5p in 80 cases of colorectal cancer were detected by RT-PCR and the correlation was analyzed.The target relationship between miR-182-5p and lnc RNA GAS5 was verified by double luciferase report.The expression of GAS5 and miR-182-5p was detected by RT-PCR after overexpression of GAS5 in HT-29 cells.The expression of GAS5 andmiR-182-5p was detected by RT-PCR after SW480 cells were constructed to silence Gas5.Part Ⅳ: After transfection of miR-182-5p mimics into HT-29 cell lines,CCK-8assay and Annexin V-FITC/PI apoptosis kit were used to detect the proliferation and apoptosis of the cells.After transfection of HT-29 cell line with GAS5 overexpression vector GAS5 and miR-182-5p mimics,the cell proliferation and apoptosis were detected by CCK-8 assay and Annexin V-FITC/PI apoptosis kit.The expression of FOX3 a was detected by RT-PCR and Western Blot after HT-29 cells over-expressed GAS5,SW480 cells silenced GAS5 model.The overexpression vector of GAS5 was transfected into HT-29 cell line respectively.The expression of FOX3 a was detected by RT-PCR and Western Blot.The levels of GAS5 and FOX3 a in 63 cases of colorectal cancer were detected by RT-PCR and the correlation was analyzed.ResultsPart Ⅰ: The relative expression of Gas5 m RNA in colorectal cancer tissues was significantly lower than that in normal colorectal tissues(P < 0.001).The expression of Gas5 m RNA in HCT-116,HT-29,SW480 and Lo Vo cells of CRC cells was lower than that in normal colonic epithelial cell line NCM460,and the difference was significant(P < 0.05).It can be seen that the expression of GAS5 m RNA is obviously increased and the difference is The expression of gas was significantly correlated with lymph node metastasis and TNM stage of colorectal cancer(P < 0.001),but not with age,sex,histological differentiation,depth of invasion and tumor diameter(P >0.001).Part Ⅱ: The CCK-8 results showed that the 24 h OD and 72 h OD values in the pc DNA3.1-GAS5 transfection group were lower than those in the pc DNA3.1-NC group,and there was a significant difference between the 48 h OD value and 72 h ODvalue(p < 0.001).The results of Annexin V-FITC/PI apoptosis detection showed that the apoptotic rate in the pc DNA3.1-GAS5 transfection group was higher than that in the pc DNA3.1-NC group(p < 0.001).Therefore,the overexpression of GAS5 inhibited the proliferation of CRC cells and promoted the apoptosis of CRC cells.Part Ⅲ: It was found that miR-182-5p contained a nucleotide sequence complementary to GAS5.There was a negative correlation between the expression of GAS5 gene and miR-182-5p gene in 80 cases of CRC(r =-0.42,p = 0.001).Double luciferase report assay confirmed that miR-182-5p could specifically bind to GAS5.After pc DNA3.1-GAS5 transfection,the expression of GAS5 gene in HT-29 cells increased significantly,while the expression of miR-182-5p gene decreased significantly.After si-GAS5 silencing,the expression of GAS5 gene in SW480 cells decreased significantly,but the expression of miR-182-5p gene increased significantly.Part Ⅳ:The results of CCK-8 assay showed that the OD value of the transfected group was higher than the OD value of the miR-NC group at every time,and the OD value of the 48 h OD value was significantly different from that of the miR-NC group at 72 h after transfection(p < 0.05).The results of Annexin V-FITC/PI apoptosis detection showed that the apoptosis rate of the mimics transfected group was lower than that of the miR-NC group(p < 0.001).GAS5 inhibited cell proliferation while miR-182-5p promoted cell proliferation and miR-182-5p partially reversed the inhibitory effect of GAS5 on cell proliferation.GAS5 promoted apoptosis,and miR-182-5p partially reversed the apoptotic effect of GAS5.The results of RT-PCR and Western Blot showed that the overexpression of GAS5 promoted the low expression of GAS5 and decreased the expression of FOXO3 a,miR-182-5p could attenuate the effect of GAS5 on FOXO3 a expression.There was a positive correlation between the expression of GAS5 gene and FOXO3 a gene in 63 cases of CRC(r =0.62,p < 0.001).ConclusionsThe expression of GAS5 in colorectal cancer tissue was lower than that in normal tissue and in colorectal cancer cell line was lower than that in normal colorectal cancer cell line.The expression of GAS5 in colorectal adenocarcinoma was correlated with lymph node metastasis and TMN staging,suggesting that our GAS5 might be a precancerous marker in colorectal cancer patients.Functional experiments showed that the overexpression of GAS5 inhibited the proliferation of CRC cells and promoted the apoptosis of CRC cells.The study of mechanism showed that: Gas5 could bind specifically to miR-182-5p and negatively regulate the expression of miR-182-5p.GAS5 overexpression could inhibit cell proliferation and promote cell apoptosis through down-regulation of miR-182-5p,.The mechanism may be that GAS5 regulates FOXO3 a expression by regulating miR-182-5p.Thus,the proliferation and apoptosis of CRC cells were affected. |
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