| Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.In China,the incidence of CRC is gradually increasing,and the morbidity and mortality of CRC are the fifth in all malignancies.Although the pathogenesis of CRC is still unclear,it is considered that the long-term combined effects of environmental factors and genetic characteristics are the main reasons.The changes of epigenetic modifications,such as activation of cancer susceptibility genes,inactivation of tumor suppressor,the dysregulation of signaling pathways,and the regulation of protein-coding genes by noncoding RNA are associated with the development of CRC.Long noncoding RNA(lncRNA)promotes or inhibits the development and progression of CRC via regulating different mechanisms at multiple levels.This study was divided into five parts.In the present study,we investigated the expressions of three lncRNA,including hypoxia-inducible factor 1?-antisense RNA 2(HIF1A-AS2),small ubiquitin-like modifier 1 pseudogene 3(SUMO1P3),and proliferating cell nuclear antigen antisense RNA 1(PCNA-AS1)in CRC tissues and cell lines;analyzed the correlation of HIF1A-AS2,SUMO1P3 and PCNA-AS1 expression with clinicopathological parameters of CRC;discussed the functions of these genes in colorectal cancer cells;and proposed the investigations in the further.Part One Expression of HIF1A-AS2 in CRC and the relationship of HIF1A-AS2 expression with clinicopathological featuresObjective:To investigate the expression of HIF1A-AS2 in CRC tissues and paired adjacent normal tissues,and the relationship between its expression level and clinical pathological features.Methods:Real-time quantitative PCR(qPCR)was used to measure the mRNA expression of HIF1A-AS2 in 57 pairs of CRC tissues and adjacent normal tissues,and to explore the relationship between its expression and clinicopathologic features.Results:HIF1A-AS2 was significantly upregulated in CRC tissues compared with the corresponding adjacent normal samples(p<0.001).Out of the 57 cases,the expression levels of HIF1A-AS2 in 33 samples were higher than that in the normal group,whereas the rest were lower than in the normal group.The relative expression of two groups(—??CT)was 0.832,and the change was 1.781-fold.The expression level of HIF1A-AS2 was positively associated with the histological differentiation grade.High histological differentiation grade tumors expressed high levels of HIF1A-AS2,whereas the low histological differentiation grade tumors had low HIF1A-AS2 expression levels.However,no significant correlation was found between HIF1A-AS2 expression and other factors,including gender,age,tumor invasion,TNM stage,lymph node metastasis,distant metastasis,and pathogenic sites.Conclusions:The expression of HIF1A-AS2 was upregulated in CRC tissues,which was positively associated with the histological differentiation grade.Part Two Functions of HIF1A-AS2 on cell cycle,proliferation,and apoptosis in colon cancer cell linesObjective:To study the functions of HIF1A-AS2 on cell cycle,proliferation,and apoptosis of colon cancer cells in vitro.Methods:The expression levels of HIF1A-AS2 in colon cancer cell lines SW620,HT29,HCT116,and DLD-1,and normal colon epithelial cell line NCM460 were analyzed by qPCR assay.The expression of HIF1A-AS2 was interfered by using siRNA technology in SW620 cells.The effects of HIF1A-AS2 on cell cycle,proliferation,and apoptosis were measured by flow cytometry,Ed U incorporation assay,and Hoechst 33342 staining,respectively.Results:The mRNA expressions of HIF1A-AS2 were significantly higher in colon cancer cell lines than that in normal colon epithelial cell line.The cell cycle was arrested at the G1 phase,and the percentage of EdU-positive cells was decreased significantly by 43.78%in HIF1A-AS2-silenced cells.The results of Hoechst 33342 staining showed that the apoptotic cells in si-HIF1A-AS2group were significantly increased compared with the si-NC-transfected cells.In addition,the apoptotic rate of si-HIF1A-AS2-transfected cells(20.65%)was increased obviously when compared to the si-NC group(7.05%)by flow cytometry analysis(p<0.01).Conclusions:HIF1A-AS2 was significantly upregulated in colon cancer cell lines,and it may promote colon cancer cell proliferation and inhibit apoptosis in vitro.Part Three Expression of SUMO1P3 in CRC and relationship between its expression and clinical pathologic featuresObjective:To detect the expression of SUMO1P3 in CRC tissues and paired adjacent normal tissues,and to investigate the relationship between its expression level and clinical pathological features.Methods:qPCR was used to detect the expression level of SUMO1P3 in 57 pairs of CRC tissues and the adjacent normal tissues,and to establish the relationship between its expression and clinicopathologic features.Results:SUMO1P3was markedly upregulated in CRC samples(p<0.001).Out of the 57 cases,the expressions of SUMO1P3 in 37 samples were higher than that in the normal group,whereas the rest were lower than in the normal group.The relative expression of two groups(—??CT)was 1.463,and the change was 2.757-fold.However,the expression level of SUMO1P3 was not significantly associated with the clinical pathological features.Finally,we observed the feasibility of SUMO1P3 as a diagnostic maker of CRC.The ROC curve(AUC)was 0.677(95%credibility interval,0.576~0.778,p<0.001).Moreover,the sensitivity,specificity,cutoff point value(?C_t),and Youden index were 0.912,0.474,1.526,and 0.386,respectively.Conclusions:The expression of SUMO1P3 was upregulated in CRC.The SUMO1P3 may function as a potential diagnostic biomarker of CRC.Part Four Effects of SUMO1P3 on cell cycle,proliferation and apoptosis in colon cancer cellObjective:To study the functions of SUMO1P3 on cell cycle,proliferation and apoptosis of colon cancer cells in vitro.Methods:The expression levels of SUMO1P3 in colon cancer cell lines SW620,HT29,HCT116,DLD-1 and normal colon epithelial cell line NCM460 were measured by qPCR.The expression of SUMO1P3 was interfered by using siRNA technology in SW620 cell line.We explored the functions of SUMO1P3 on cell cycle,proliferation,and apoptosis by flow cytometry,EdU incorporation assay,and Hoechst 33342 staining,respectively.Results:The expressions of SUMO1P3 were significantly higher in colon cancer cell lines than that in normal colon epithelial cell line.SUMO1P3 knockdown led to significant increase in G1 phase of colon cells and decrease in EdU-positive cells.Hoechst 33342 staining showed that the apoptotic rate in si-SUMO1P3 group was significantly increased compared to the si-NC group.Flow cytometry assay also demonstrated that the apoptotic cells in si-SUMO1P3-transfected group(22.65%)were increased obviously compared to si-NC-transfected cells(7.05%;p<0.001).Conclusions:SUMO1P3 was highly expressed in colon cancer cell lines,and it may promote colon cancer cell proliferation and inhibit apoptosis in vitro.Part Five Expression of PCNA-AS1 in CRC and relationship between its expression and clinical pathologic featuresObjective:To detect the expression of PCNA-AS1 in CRC tissues and paired adjacent normal tissues,and to research the relationship between its expression level and clinical pathological features.Methods:Using qPCR,we detected the expression level of PCNA-AS1 in 57 pairs of CRC tissues and the adjacent normal tissues,and to establish the relationship between PCNA-AS1 expression and clinicopathologic features.Moreover,we investigated the expression of PCNA-AS1 in colon cancer cell lines and normal colonic epithelial cell line.Results:Compared to the adjacent normal tissues,PCNA-AS1 was upregulated in CRC samples(p<0.001).Out of the 57 cases,the expression levels of PCNA-AS1 in 48 samples were higher than in the normal group,whereas the rest were lower than in the normal group.The relative expression of two groups(—??CT)was1.819,and the change was 3.529-fold.The PCNA-AS1 expression levels were posilitively correlated with tumor invasion and TNM stage,whereas not associated with other factors,including gender,age,lymph node metastasis,distant metastasis,pathogenic sites,and histological differentiation.Then,we observed the feasibility of PCNA-AS1 as a diagnostic maker of CRC.The ROC curve(AUC)was 0.824(95%credibility interval,0.750~0.899;p<0.001).Moreover,the sensitivity,specificity,cutoff point value(?C_t),and Youden index were 0.632,0.86,4.164,and 0.491,respectively.Finally,we verified the significant upregulation of PCNA-AS1 in colon cancer cell lines HCT116,HT29,SW620,and SW480 in contrasted with normal colonic epithelial cell line NCM460.Conclusions:The expression of PCNA-AS1 was significantly upregulated in CRC tissues and cell lines.The PCNA-AS1 may function as a potential tumor biomarker for diagnosing CRC. |
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