Expression And Effect On Chemotherapy Of LncRNA PVT1 In Colorectal Cancer

Colorectal cancer?CRC?is one of the most common malignant tumors in the digestive tract.At present,about 1.4 million patients were diagnosed with CRC every year in the globe,of which nearly 700,000 die from the disease,CRC is one of the leading causes of cancer-related death worldwide.The overall incidence of CRC is on an upward trend in our country,moreover,the incidence of CRC is the fifth highest among men and the fourth highest among all women in all tumors,and the mortality is the fifth highest both in men and women.In our country,about 376,000 patients were diagnosed with CRC every year,of which nearly 191,000 die from this disease.It is generally accepted that CRC is a series of complex and multi-step pathological processes that occur under the long-term interactions of genetic factors and environmental factors.The occurrence and development of CRC is closely related to abnormalities of many regulatory factors,for instance,the activation of oncogene caused by the gene mutation,tumor suppressor gene inactivation,tumor susceptibility genes Genetic and susceptibility genes such as single nucleotide polymorphisms,as well as tumor-related signal transduction pathway abnormalities and other regulatory abnormalities.In addition,most diseases are also closely related to poor eating habits,obesity and smoking,but the exact pathogenesis is not yet clear.In recent years,a large number of studies have shown that long non-coding RNA?lncRNA?play a role of promoting cancer or tumor suppressor in tumorigenesis,apoptosis regulation,invasion and metastasis in many cancers.Thereby,the in-depth study of LncRNAs will not only enable us to understand the pathogenesis of CRC,but also make it a marker and target of diagnosis and treatment of CRC.Long non-coding RNA PVT1?plasmacytoma variant translocation 1?,as an important carcinogenic lncRNA,is highly expressed in many malignant tumors and suggests a poor prognosis.lncRNA PVT1could promote the tumorigenesis and development of cancers by affecting cell proliferation,migration,invasion and apoptosis.Recently,accumulating vigorous evidence have indicated that lncRNA PVT1 perform a significantly carcinogenic activity or act as an cancer-driven factor in a variety of cancers to promote tumorigenesis in various cancers.LncRNA PVT1 was researched in this present study divided into four parts.Firstly,we investigated the abnormal expression of lncRNA PVT1 in colorectal cancer tissue compared to adjacent normal tissues and the relationship between lncRNA PVT1expression and clinicopathological characteristics of CRC patients.Secondly,we studied the function of lncRNA PVT1 and its effect on chemosensitivity in colorectal cancer cells,which could put forward the direction of further researches.Finally,the prognostic value of lncRNA PVT1 expression in patients with malignant tumors was evaluated.Part 1 The expression and its significance of lncRNA PVT1 in CRC tissueObjective:To study the expression of lncRNA PVT1 in CRC tissue compared to adjacent normal tissues,and reveal the association between lncRNA PVT1 expression level and the clinicopathological characteristics of patients with CRC.Methods:we detected the lncRNA PVT1 relative expression of cancerous tissues in130 patients with CRC by using real-time quantitative polymerase chain reaction?qPCR?,At the same time,we collected the clinicopathological information of patients and demonstrated the relationship between lncRNA PVT1 expression and the clinical pathological features and prognosis of patients with CRC.Results:LncRNA PVT1 was overexpressed in CRC tissues compared to paired-adjacent normal tissues in a cohort of 130 patients with CRC.Of 130 CRC patients,the relative expression of lncRNA PVT1 in 94 cases was higher than that in normal tissues and 36 cases lower than that in normal tissues,and the high expression rate was 72.31%.The relative expression level of lncRNA PVT1??CT?was 10.55±1.64 in CRC patients,and the relative expression level of lncRNA PVT1??CT?was 11.29±1.90 in normal tissues.The difference between tumor group and normal group was statistically significant?t=3.330,p=0.001?.The relative expression level of lncRNA PVT1 in two groups of samples?-??CT?was 0.787,and the median relative expression difference multiples-fold change(2^-??CT)was 1.725 times between in two groups.The maximum difference multiples was 20.836 times and the average value of 2^-??CT was 2.712 times,and the difference between the two groups of samples was statistically significant?t=6.298,p<0.0001?.Furthermore,130 patients with CRC were divided into high expression group?n=65?and low expression group?n=65?according to the median relative expression level of lncRNA PVT1?1.725 times?.The expression level of lncRNA PVT1 was highly correlated with the tumor TNM,the degree of tissue differentiation and the plasma CEA level between in the lncRNA PVT1 high expression group and low expression group,but not with the tumor location,tumor size,tumor pathological type,colorectal polyps and other factors.High expression of lncRNA PVT1 predicts a later tumor stage?X²=10.32,p=0.001?,poorer tissue differentiation?X²=5.482,p=0.019?,and higher plasma CEA level?X²=4.112,p=0.043?.The relationship between lncRNA PVT1 and TNM stage showed that the expression of lncRNA PVT1 was closely related to the status of lymph node metastasis?N1/N2 vs.N0?and and distant metastasis?M1 vs.M0?in CRC patients with statistically significant?X²=9.206,p=0.002,X²=9.113,p=0.003?,but not to tumor T classification?X²=1.105,p=0.314?.The result of prognostic analysis indicated that the 1-year and 3-year DFS of the lncRNA PVT1 low and high expression patients were 93.8%and 81.1%,69.3%and 44.7%,respectively.The median DFS were 44 months in low expression group?95%CI:36.558-51.442?and 26 months in high expression group?95%CI:12.512-39.488?,the difference was statistically significant?X²=5.307,p=0.021,log-rank test?.COX univariate analysis and multivariate analysis showed that TNM staging??/?vs.?/?:RR=6.342,95%CI:2.994-13.433,p<0.001?and the expression level of lncRNA PVT1?high expression group vs.low expression group:RR=3.744,95%CI:1.493-9.392,p=0.005?was closely related to DFS in CRC patients.The high expression of lncRNA PVT1 is a poor prognostic factor in CRC patients.This finding showed that the higher the expression lncRNA PVT,the shorter the DFS.The expression of lncRNA PVT1 and tumor TNM stage were both independent prognostic predictors of DFS in patients with CRC.The ROC curve was constructed using lncRNA PVT1 as a diagnostic marker for CRC.The area under the ROC curve?AUC?was 0.628?95%CI:0.560-0.696,p<0.001?.The diagnostic sensitivity and specificity was 69.2%and 58.5%,respectively.The proportion of patients with high expression lncRNA PVT1?fold change?1.725?in patients with different CRC staging was higher than that of patients with elevated CEA?>5ng/ml?,especially,there was a significant difference in stage I patients?X²=41.717,p<0.0001?.Conclusions:The expression of LncRNA PVT1 in CRC patients was higher than that in normal tissues,and the high expression rate was 72.31%and the mean of expression difference multiples was 2.712 times.LncRNA PVT1 expression is related to the tumor TNM stage,histological differentiation and plasma CEA expression in CRC patients.LncRNA PVT1 over-expression predicts later tumor progression,poorer tissue differentiation,and higher plasma CEA levels.In addition,the higher the level of lncRNA PVT1 expression in CRC patients,the more prone to lymph node metastasis and distant metastasis,whereas lncRNA PVT1 expression has nothing to do with the depth of tumor invasion.High lncRNA PVT1 expression is a poor prognostic factor in patients with CRC,which indicates a shorter DFS time of the patients.COX univariate analysis and multivariate analysis showed that,like TNM staging,the expression level of lncRNA PVT1 was also an independent prognostic predictor of DFS in patients with CRC.The expression of lncRNA PVT1 contributes to the diagnosis of CRC,especially in the early stage of CRC patients.The abnormal expression of lncRNA PVT1 is far higher than the plasma CEA level.This result indicates that abnormal expression of lncRNA PVT1 is earlier than that of plasma CEA,which has the potential value of early diagnosis of CRC.Part 2 Function of lncRNA PVT1 in CRC cells and its effects on the chemosensitivity of CRCObjective:The aim of this study is to investigate expression and function of lncRNA PVT1 in CRC cells and its effects on the chemosensitivity of CRC.Methods:Firstly,we detected the expression of lncRNA PVT1 in colorectal cancer cell line HT-29,RKO,HCT-15,HCT 116,SW480,SW620,LoVo and DLD-1 compared to normal cell line NCM460 using RT-qPCR.Secondly,we investigated the expression and function of lncRNA PVT1 in RKO cell line with the highest relative expression by siRNA interference technology.FACSSAria II flow cytometry,EdU cell proliferation assay,cell scratch assay and CCK-8 cell proliferation/toxicity assay and clone formation assay were used to study the effect of lncRNA PVT1 expression on the biological behavior of RKO colon cancer cells.Thirdly,we studied the effect of silencing lncRNA PVT1expression on the sensitivity of CRC chemotherapy.In addition,the relationship between lncRNA PVT1 expression and the expression of related proteins in the EMT pathway was investigated.Results:The expression level of lncRNA PVT1 in RKO,HT-29,SW480 and HCT116 cells was higher than that in normal intestinal epithelial cells NCM460 using RT-qPCR,and the highest expression level of RKO was 5.174 times tnan that of NCM460.The expression level of HCT15 and LOVO cells was lower than that of NCM460,The difference was statistically significant,while SW620 and DLD-1 cells lncRNA PVT1expression level and NCM460 no significant difference.The knockdown of lncRNA PVT1in RKO cells by gene silencing resulted in an increase of apoptosis with statistical significance?t=3.653,p=0.0035?,and led to cell arrest in G0/1 phase?p<0.001?.EdU cell proliferation assay confirmed that down-regulation of lncRNA PVT1 significantly inhibited the proliferation of tumor cells.The number of EdU-positive cells in si-RNA group was 44.1%compared with 71.4%in si-NC group?t=15.40,p<0.001?.In addition,down-regulation of lncRNA PVT1 expression significantly reduced tumor cell migration?p<0.001?and colony formation?p=0.0035?.CCK-8 cytotoxicity assay showed that down-regulation of lncRNA PVT1 can reduce the IC50 of 5-FU and Oxaliplatin on RKO cells,respectively,and increase the sensitivity of the two chemotherapeutic drugs by 1.82and 2.29 folds respectively.Further EdU cell proliferation experiments confirmed that silencing lncRNA PVT1 expression was increased of 5-FU and Oxaliplatin on the killing of RKO tumor cells,the difference was statistically significant?p<0.001 and p=0.0357,respectively?.Furthermore,down-regulation of lncRNA PVT1 significantly increased the inhibitory effect of the 5-FU chemotherapy drug on RKO cell clone formation,that is,decreased the tumorigenic potential of tumor cells and increased the anti-tumor activity of5-FU.In addition,silencing lncRNA PVT1 of RKO cells can lead to up-regulating the mRNA and protein expression of EMT pathway epithelial markers E-cadherin and?-catentin,while down-regulating the mRNA and protein expression of the interstitial marker Vimentin.Conclusions:This study showed that the expression lncRNA PVT1 of RKO cells is the highest in 8 types CRC cell lines.Down-regulation of lncRNA PVT1 expression can promote apoptosis,arrest the cell cycle,reduce the proliferation,migration and clone formation of RKO tumor cells.Meanwhile,down-regulation of lncRNA PVT1 can reduce the IC50 of 5-FU and Oxaliplatin on RKO cells,increase chemosensitivity of RKO cells to both drugs and inhibit tumor growth.Moreover,the expression of lncRNA PVT1 is involved in the regulation of the EMT pathway and related to the chemotherapy resistance of cancers.Thereby,the intervention on lncRNA PVT1 expression is expected to provide a potential therapeutic target for CRC therapy.Part 3 Prognostic significance of PVT1 in cancers-meta-analysisBackground:Plasmacytoma variant translocation 1?PVT1?,an oncogenic long noncoding RNA located in a recognized cancer-risk gene region-8q24,is significantly overexpressed in various cancers.Many studies have found that high expression of PVT1is correlated with poor prognosis.Methods:This meta-analysis is performed by searching electronic databases Pubmed,Web of Science,Chinese National Knowledge Infrastructure,WanFang,and ChongQing VIP for eligible papers on the prognostic impact and clinicopathological characteristics of PVT1 expression in cancer from inception to January 31,2017.The hazard ratio?HR?and odds ratio?OR?with 95%confidence intervals?CI?were computed to estimate the pooled effect PVT1 on prognosis of cancers using Stata 12.0 version software.Results:13 studies were finally included in this review including 1559 patients.The pooled result indicated that overexpressed PVT1 predicts a poorer prognosis of cancerous patients for overall survival?HR=1.91,95%CI:1.61-2.26,p<0.001?and disease-free survival?HR=1.90,95%CI:1.46-2.48,p<0.001?or recurrence-free survival?HR=1.77,95%CI:1.24-2.52,p=0.002?or progression-free survival?HR=2.84,95%CI:1.67-4.82,p<0.001?.The high expression of PVT1 is closely associated with TNM stage?III/IV vs.I/II:OR=3.19,95%CI:2.43-4.18,p<0.001?.Additionally,the significant correlation between PVT1 expression and TNM stage is found in T classification?T3/4 vs.T1/2:OR=6.48,95%CI:2.93-14.31,p<0.001?and lymph node metastasis?present vs.absent:OR=2.56,95%CI:1.36-4.80,p=0.003?,but not in distant metastasis of patients with cancers?yes vs.no:OR=2.50,95%CI:0.72-8.66,p=0.15?.Furthermore,the cancerous patients with high PVT1 expression had a worse histological differentiation than those with low PVT1 expression?undifferentiated/poorly vs.moderately/well:OR=1.48,95%CI:1.02-2.14,p=0.039?.Conclusions:PVT1 could serve as a potent predictor of prognosis in different types of cancers including CRC.