| Background and ObjectiveColorectal Cancer(CRC)is a common disease that seriously affects human health and quality of life worldwide.According to the survey of CRC epidemics in 185 countries around the world in 2018,the number of new cases of CRC in these countries were 1800,977,of which 861,663 died and the total mortality rate was about 47.8%.In China,the incidence and mortality of CRC have also been increasing in the past decade,and the 5-year survival rate is low,which seriously threatens the health and life of our citizens.Due to the lack of universal colonoscopy and the poor sensitivity of various screening tests,most patients have developed into advanced stages when diagnosed with C.RC.In recent years,with the improvement of the level of scientific and technical treatment and the introduction of new types of chemotherapy drugs,although the overall prognosis of patients with advanced CRC has been greatly improved,the resistance of tumors to various chemotherapy drugs has become one of important reasons for reducing the survival rate of CRC patients.Therefore,to study the key factors of chemotherapy drug resistance in patients with advanced CRC and to elucidate its mechanism of action,it is very important for the treatment and prognosis of patients with CRC.In recent years,with the development of genetic sequencing technology,humans have deeper understanding of the genome transcriptome of organisms.The exploration and excavation of biological functions of long-chain non-coding RNAs(IncRNAs)is a hot topic in this field.In the past,the scientific community believed that less than 2%of the transcriptional genes in mammalian genomes could encode and translate into proteins.and more than 98%of the genes had no ability to transcribe and translate proteins.However,in-depth research found that these non-coding RNAs can participate in the development of various diseases in the human body by regulating the expression of various genes,especially in the evolution of various types of tumors.The transcription product of Plasmacytoma Variant Translocation 1(PVT1)is homologous to the PVT1 gene of murine,and belongs to the intergenic IncRNA.In the mid-1980s,the PVT1 locus was first discovered in mice.In the established mouse plasmacytoma model,the PVT1 gene frequently undergoes multiple variant translocations.With the improvement of high-throughput sequencing engineering technology,people began to study the structure and chromosomal translocation of PVT1 gene and found its unique biological function.Many studies have found that PVT1 1s highly expressed in a variety of organ tumors and cells,such as gastric cancer,liver cancer,lung cancer,colon cancer,breast cancer,ovarian cancer,etc.,which mainly play a role in proto-oncogenes and promote tumor cell proliferation,invasion.metastasis and chemoresistance.In a variety of tumor tissues,IncRNAs construct a complex and sophisticated regulatory system that can regulate the proliferation,differentiation and apoptosis of various tumor cells at different levels.Therefore,IncRNAs could be used not only as a diagnostic marker and prognostic indicator for related tumors,but also as an effective target for tumor treatment.In recent years,several studies showed that PVT1 exhibits high expression levels in CRC tissues and cells,and its level is closely related to vascular invasion,lymph node metastasis and patient prognosis.The results of relevant clinical studies suggested that the expression level of lncRNA PVT1 may be negatively correlated with the prognosis of CRC patients,and PVT1 gene may promote the proliferation,invasion,metastasis and apoptosis inhibition of CRC cells.Therefore,PVT1 is an important regulatory gene in the process of tumor progression,and it plays a significant molecular biological function both at the level of gene transcription and after transcription.In order to clarify the key role of PVT1 in the chemotherapy resistance of 5-fluorouracil(5-FU)in CRC patients,we first collected the tumor tissue specimens of clinically diagnosed CRC patients and detected the level of PVT1 in CRC tissue by RT-qPCR.The expression level of PVT1 and the clinicopathological parameters were statistically analyzed between chemotherapy effective group and no-effective group in CRC patients.Secondly,the role of PVT 1 expression in CRC cell lines was studied by various molecular biological experiments in’vitro,and the correlation between PVT1 expression and biological behaviors such as cell growth and apoptosis of 5-FU resistant cell lines was investigated.Finally,the correlation between PVT1 and the long-term prognosis of CRC patients was elucidated by detecting PVT1 expression in tumor tissues of CRC patients.We aimed to demonstrate the expression and mechanism of PVT1 in CRC patients through various molecular biology experiments and clinical detection methods,and provide new ideas for early diagnosis,treatment and prognosis of CRC patients.Methods1.Expression of PVT1 gene in CRC tissues and cells.By collecting the tumor tissue specimens of clinically diagnosed CRC patients,according to preoperative clinical evaluation(mainly by preoperative imaging and tumor biomarkers,and compared with tumor biomarkers before and after chemotherapy based on FOLFOX)and intraoperative evaluation of tumor size,The infiltration range and lymph node metastasis were determined as follows:chemotherapy effective group(preoperative FOLFOX chemotherapy base on 5-FU was effective)and chemotherapy no-effective group(preoperative FOLFOX chemotherapy base on 5-FU was not effective).The expression level of PVT1 in chemotherapy effective group and chemotherapy no-effective group CRC tumor tissues was detected by RT－qPCR,and the clinical data of CRC patients were analyzed in detail.Furthermore.the expression of PVT1 in the 5-FU resistant group and the normal group was detected by establishing CRC-related 5-FU resistant cells(HCT－8/5-FU and HCT-116/5-FU).2.Effects of PVT1 gene interference and overexpression on proliferation,apoptosis and expression of drug resistance-related proteins in 5-FU resistant CRC cells.(1)Interfering with HCT-8/5-FU and HCT-116/5-FU.and overexpressing HCT-8 and HCT-116 cells using interference and stable gene silencing techniques;(2)Using CCK-8 to detect the proliferation and activity of CRC cells;(3)Flow cytometry to detect and evaluate apoptosis;(4)Western-blotting was used to detect the expression of drug resistance-related proteins,such as Bcl-2,P-gp,mTOR and MRP1.3.Preliminary study of PVT1 regulating Akt signaling pathway involved in CRC 5-FU chemoresistance(1)Western-blotting was used to evaluate the expression levels of Akt and p-Akt in HCT-8,HCT-8/5-FU,HCT-116 and HCT-116/5-FU cells;(2)The expression levels of Akt and p-Akt in HCT－8 and HCT-116 cells after PVT1 overexpression were detected by Western-blotting;(3)HCT-8/5-FU and HCT-116/5-FU cells were treated with LY294002 inhibitor,and Akt and p-Akt expression were detected and evaluated by Western-blotting.4.The correlation between PVT1 expression and short-term prognosis in patients with CRC.By collecting tumor tissue and paired adjacent paracancerous tissue specimens and detailed clinical data of clinical and pathologically confirmed C.RC patient.RT-qPCR was used to detect the expression of PVT1 in CRC patients and normal adjacent tissues,and to analyze the correlation between PVT1 expression and clinical features.The expression levels of PVT1 in different tissues were analyzed by one-way ANOVA and LSD post hoc test,and the relationship between PVT1 expression and categorical variables was analyzed using Pearson χ2 test.The long-term survival curve of CRC patients was mapped by Kaplan-Meier and its significance was analyzed.Results1.PVT1 is highly expressed in chemotherapy no-effective group CRC tissues and 5-FU resistant CRC cells.(1)The expression level of PVT1 in CRC tumor tissues of chemotherapy no-effective group was significantly higher than that in chemotherapy effective group(P<0.001);(2)The expression levels of PVT1 gene in HCT-8/5-FU and HCT-116/5-FU were significantly higher than those of HCT-8 and HCT-116(P<0.001).2.Overexpression of PVT1 gene promotes CRC tumor progression andchemoresistance(1)After the PVT1 gene was stably interfered,the proliferation of HCT-8/5-FU/siPVT1 and HCT-116/5-FU/siPVT1 cells was significantly inhibited(P<0.001).In contrast,after overexpressing the PVT1 gene,the growth rate of HCT-8/oePVT1 and HCT-116/oePVT1 cell lines was significantly higher than that of the control group(P<0.001);(2)After interfering with PVT1 expression,5-FU was used to interfer with 5-FU resistant HCT-8 and HCT-116 cells,and the number of apoptotic cells increased significantly(P<0.001).When over-expressing PVT1 gene,and then 5-FU was used to intervene of HCT-8 and HCT-116 cells,the number of apoptotic cells was significantly reduced(P<0.001);(3)In HCT-8/oePVT1 and HCT-116/oePVT1 cells,the expression levels of resistance-related proteins mTOR.MRP1,P-gp and Bcl-2 were significantly increased in PVT1 overexpression group(P<0.001).3.PVT1 participates in CRC 5-FU chemoresistance by up-regulating Akt signaling pathway(1)The expression levels of Akt in HCT-8/5-FU and HCT-116/5-FU cells were not different from those of HCT-8 and HCT-116 cells.,whereas the expression of P-Akt in HCT’8/5-FU and HCT-116/5-FU cells was significantly higher than that in HCT-8 and HCT-116 cells;(2)The expression level of P-Akt was significantly up-regulated after over-expression of PVT1 in HCT-8 and HCT-116 cells:(3)The expression of Akt and p-Akt in HCT-8/5-FU and HCT-116/5-FU cells was significant increased.There was no significant change in Akt protein after LY294002 treatment,but p-Akt protein was down-regulated significantly(P<0.001),there was also no significant change in PVT1 gene expression.4.PVT1 expression is negatively correlated with the long-term prognosis of patients with CRC(1)The expression level of PVTl in CRC tumor tissues was significantly higher than that in matched paracancerous tissues(P<0.001),and PVT1 expression was not related to gender,age and tumor location.However,PVT1 expression levels were significantly associated with tumor differentiation,depth of invasion,TNM stage,and lymph node metastasis(P<0.001);(2)Kaplan-Meier analysis showed that DFS(Log-rank test P<0.001)and OS(Log-rank test P<0.001)were significantly higher in patients with CRC who had high PVT1 expression(>6.75)in both early and late CRC patients.Significantly lower than the PVT1 low expression group(<6.75).Conclusion1.PVT1 is highly expressed in CRC tumor tissues and may be involved in the molecular mechanism of CRC chemotherapy drug resistance.With the in-depth study of the PVT1 gene,it may become an important target for CRC assessment and treatment of chemotherapy resistance and prognosis;2.PVT1 plays a role in promoting cell resistance in 5-FU resistant CRC cells.Interfering with PVT1 can inhibit the proliferation of 5-FU resistant CRC and promote apoptosis,while CRC cells proliferate after over-expression of PVT1.The ability is significantly increased and the apoptosis is significantly reduced;3.PVT1 can promote the progression of CRC tumors and induce tumor resistance to 5-FU chemotherapy.Up-regulation of mTOR by activation of PI3K/Akt signaling pathway is an important mechanism for inducing 5-FU CRC resistance;4.High expression of PVT1 can help predict early recurrence or metastasis after radical resection of CRC,so the level of PVT1 in tumor tissue can be used for individualized treatment options of CRC patients,thereby improving the long-term survival time of patients. |
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