The Research On The Expression Levels Of Long Non-coding RNA AB007962,RP11-432I5.2 And PGM5-AS1 In Gastrointestinal Cancer,and The Effect And Mechanism Of PGM5-AS1 On Cell Migration

Background and objective:Gastrointestinal cancer is the most common malignant cancer in human digestive system.In 2017,the estimated number of new cases in the United States of stomach,colon and rectal cancer were 28,000,95,000 and 39,000,respectively.In Chinese,it was estimated that there are 679,000 new cases of gastric cancer and 376,000 new cases of colorectal cancer in 2015,and the estimated deaths are 498,000 and 191,000,respectively.The morbidity and mortality of gastric cancer and colorectal cancer are high in human cancers.Gastrointestinal cancer brings immense suffering to families who lost their relatives and brings great economic burden to our society.With the rapid development of surgical techniques,surgical instruments and treatment methods,the survival time of patients have been improved,but the long-term prognosis is still not satisfactory.The main reason is that gastrointestinal cancer is concealed and develops rapidly.It is not discovered and treated until the patient is complicated with gastrointestinal bleeding or obstruction.In addition,postoperative recurrence and metastasis also significantly affected the survival time of patients.As we all know,the occurrence of gastrointestinal cancer is mainly caused by environmental factors and genetic factors.In the process of tumor development,a great number of genes are abnormally expressed and involved in the process.Therefore,exploring the occurrence and development mechanism of gastrointestinal cancer,finding diagnostic and prognostic biomarkers with high sensitivity and specificity,and developing new therapeutic targets seems like particularly important.The human genome sequencing project has given us a new understanding of human genes.Only about 2% of more than 20,000 human genes can encode proteins,while over90% of the remaining transcripts are called non-coding RNA(non-coding RNA,nc RNA).Among them,long non-coding RNA(lnc RNA)gets more attention from researchers in recent years.Lnc RNA is a kind of nc RNA,with length more than 200 nucleotides,has no open reading frame and is transcript from RNA polymerase Ⅱ.According to the position between lnc RNA and their genomic proximity to protein-coding genes,it can be divided into five categories: sense-lnc RNA,antisense-lnc RNA,intergenic lnc RNA,intronic lnc RNA and bidirectional lnc RNA.Lnc RNA is widely present in the nucleus and cytoplasm of eukaryotes and plays an important role in process of human pathophysiology.In addition,recent studies have also found that lnc RNA is involved in variety of malignant cancers and plays a key regulatory role.Among these lnc RNAs,there is no previous study about lnc RNA-AB007962,lnc RNA-PGM5-AS1 and lnc RNA-RP11-432I5.2 at present.In this study,we measured the expression levels of AB007962,PGM5-AS1 and RP11-432I5.2 in gastrointestinal cancer tissues.And explore the relationship between their expression levels and diagnosis,clinicopathological characteristics and prognosis.At the same time,we also attempted to study whether PGM5-AS1 can affect cell proliferation,cell cycle,cell apoptosis and cell migration of colorectal cancer cells.In addition,we also should explore its mechanism to provide new theoretical basis and therapeutic target for colorectal cancer treatment..Method:1.The relative expression level of AB007962 in gastric cancer measured by real-time Polymerase Chain Reaction.(1)We use real-time Polymerase Chain Reaction(RT-PCR)measure the expression level of AB007962 in gastric cancer tissues and normal adjacent tissues(NATs).(2)We use Wilcoxon test to compare the expression levels of AB007962 in gastric cancer tissues and NATs.2.Association between the relative expression levels of AB007962 in gastric cancer tissues and clinicopathologic characteristics,as well as prognosis,of patients with gastric cancer.(1)We use non-parametric test(Manner-Whitney U test and the kruskal-Wallis H test)to analysis relationship between the relative expression levels of AB007962 in cancer tissues and clinicopathologic characteristics of patients with gastric cancer.(2)We use Spearman’s rank correlation test to calculate the correlation between the relative expression levels of AB007962 in cancer tissues and the maximum diameter of cancer.(3)We use the Kaplan-Meier method to calculate overall survival rate and disease-free survival rate of patients with gastric cancer.And use Log-rank test to evaluate difference between the survival rates of the patients with gastric cancer.In addition,univariate analysis and multivariate analysis were performed with Cox proportional hazards model to evaluate whether AB007962 could be used as an independent prognostic factor for gastric cancer.3.The relative expression levels of PGM5-AS1 and RP11-432I5.2 in colorectal cancer measured by RT-PCR.(1)We use RT-PCR measure the expression levels of PGM5-AS1 and RP11-432I5.2 in colorectal cancer tissues and NATs.(2)We use Wilcoxon test to compare the expression levels of PGM5-AS1 and RP11-432I5.2 in colorectal cancer tissues and NATs.4.We construct ROC curve to evaluate the ability of PGM5-AS1 and RP11-432I5.2play as diagnostic biomarkers for colorectal cancer according to their area under the curve(AUC).5.Association between the relative expression levels of PGM5-AS1 and RP11-432I5.2in colorectal cancer tissues and clinicopathologic characteristics,as well as prognosis,of patients with colorectal cancer.(1)We use non-parametric test(Manner-Whitney U test and the kruskal-Wallis H test)to analysis relationship between the relative expression levels of PGM5-AS1 and RP11-432I5.2 in cancer tissues and clinicopathologic characteristics of patients with gastric cancer.(2)We use the Kaplan-Meier method to calculate overall survival rate and disease-free survival rate of patients with gastric cancer.And use Log-rank test to evaluate difference between the survival rates of the patients with gastric cancer.6.Effects of PGM5-AS1 on the cell proliferation and cell migration of colorectal cancer.(1)We use Cell Counting Kit-8(CCK-8)assay to measure the cell proliferation ability of PGM5-AS1 over-expression cell line and Negative control(NC)cell line.And then analysis whether PGM5-AS1 can effect cell proliferation of colorectal cancer.(2)Moreover,we use Transwell assay to measure the cell migration ability of PGM5-AS1 over-expression cell line and NC cell line.Observe whether PGM5-AS1 can effect cell migration of colorectal cancer.7.Effects of PGM5-AS1 on cell apoptosis and cell cycle of colorectal cancer.(1)We use flow cytometry and PI staining to measure the cell apoptosis of PGM5-AS1over-expression cell line and NC cell line and detect whether PGM5-AS1 can regulate cell apoptosis of colorectal cancer.(2)We use flow cytometry and Annexin V-APC/PI staining to measure the cell cycle of PGM5-AS1 over-expression cell line and NC cell line and detect whether PGM5-AS1 can regulate cell cycle of colorectal cancer.8.The regulation of PGM5-AS1 on epithelial mesenchymal transformation(EMT)We use Western Blot to measure the protein expression levels of some EMT marker(E-cadherin,N-cadherin and Snail)and then analysis the influence of PGM5-AS1 on EMT process.Result:1.The expression levels of AB007962 in gastric cancer tissues(1)The result of RT-PCR showed that the expression levels of AB007962 in gastric cancer tissues from 96 patients was significantly decreased compared with NATs(P<0.01).2.The association between the relative expression levels of AB007962 in gastric cancer tissues and clinicopathologic characteristics,as well as prognosis,of patients with gastric cancer.(1)The result of non-parametric test showed that the relative expression of AB007962 was not significantly correlated with the clinicopathologic characteristics of patients,such as age,gender,tumor diameter,invasion depth,lymph node metastasis and TNM stage(P>0.05).However,we found a trend between the relative expression of AB007962 and the diameter of the tumor(P=0.078).Thus,we performed bivariate(Spearman)Correlation analysis and the result indicated that the relative expression level of AB007962 was negatively correlated with the tumor diameter(P=0.023,R2=-0.231).(2)There was no statistical difference between the relative expression of AB007962 and the prognosis of patients with gastric cancer(P>0.05).However,we also performed a subgroup analysis and result showed that the relative expression level of AB007962 was significantly correlated with the prognosis of patients with intestinal type(P<0.05).The higher the relative expression levels of AB007962,the better the prognosis of patients have.(3)The result of univariate and multivariate analysis showed that AB007962 can be an independent prognostic factor for gastric cancer patients with intestinal type.3.The expression levels of PGM5-AS1 and RP11-432I5.2 in colorectal cancer tissues(1)We use RT-PCR to measure the expression levels of PGM5-AS1 in cancer tissues and NATs of 103 patients with colorectal cancer.The result indicated that PGM5-AS1 was obviously down-regulated in colorectal cancer tissues compared with NATs(P<0.01).Among them,the relative expression levels of PGM5-AS1 were decrease in95 patients(92.24%)and increase in 8 patients(7.76%).The 2-△CTvalue of cancer tissues and NATs are 0.10±0.32 and 3.72±14.25,respectively.We also construct ROC curve and the result demonstrated that the AUC was 0.859(P<0.001).In addition,sensitivity and specificity was 87.4% and 72.8%,respectively.(2)We use RT-PCR to measure the expression levels of RP11-432I5.2 in cancer tissues and NATs of 77 patients with colorectal cancer.The result indicated that RP11-432I5.2 was down-regulated in colorectal cancer tissues compared with NATs(P<0.01).Among them,the relative expression levels of RP11-432I5.2 were decrease in 56 patients(72.7%)and increase in 21 patients(27.3%).The 2-△CTvalue of cancer tissues and NATs are 0.57±1.63 and 1.62±3.38,respectively.We also construct ROC curve and the result demonstrated that the AUC was 0.702(P<0.001).In addition,sensitivity was 63.6% and specificity was 72.7%,respectively.4.The association between the relative expression levels of PGM5-AS1 and RP11-432I5.2 in cancer tissues and clinicopathologic characteristics,as well as prognosis,of patients with colorectal cancer.(1)The result of non-parametric test showed that the relative expression of PGM5-AS1 and RP11-432I5.2 were not significantly correlated with the clinicopathologic characteristics of patients,such as age,gender,tumor diameter,invasion depth,lymph node metastasis and TNM stage(P>0.05).(2)There was also no statistical difference between the relative expression of PGM5-AS1 and the prognosis of patients with colorectal cancer,as well as RP11-432I5.2(P>0.05).5.Effects of PGM5-AS1 on the cell proliferation and cell migration of colorectal cancer.(1)The result of CCK8 assay indicated that PGM5-AS1 have no ability to effect cell proliferation in colorectal cancer(P>0.05)。(2)The result of Transwell assay indicated that PGM5-AS1 inhibited cell migration in colorectal cancer(P<0.05).6.Effects of PGM5-AS1 on cell apoptosis and cell cycle of colorectal cancer.(1)The result showed that PGM5-AS1 cannot influence cell apoptosis in colorectal cancer.(2)The result showed that PGM5-AS1 cannot influence cell cycle in colorectal cancer.7.The regulation of PGM5-AS1 on epithelial mesenchymal transformation(EMT)The result of Western Blot revealed that overexpression of PGM5-AS1 increase E-cadherin protein expression level and decrease Snail and N-cadherin protein expression level in colorectal cancer.Conclusion:1.The expression levels of AB007962 in gastric cancer tissues was lower than that in NATs,and the relative expression levels of AB007962 was negatively correlated with the maximum diameter of the tumor.Moreover,AB007962 can also be used as an independent prognostic factor for intestinal type gastric cancer patients.2.The expression levels of PGM5-AS1 in colorectal cancer tissues was significantly lower than that in NATs,and PGM5-AS1 could be used as a diagnostic biomarker for colorectal cancer.In addition,PGM5-AS1 can significantly inhibit cell migration of colorectal cancer.PGM5-AS1 can inhibit the progress of EMT by decrease Snail,N-cadherin and increase E-cadherin protein expression.3.The expression levels of RP11-432I5.2 in colorectal cancer tissues was obviously lower than that in NATs,and RP11-432I5.2 might be used as a diagnostic marker for colorectal cancer.