| Cancer is a kind of malignant disease,which is characterized by uncontrolled cell proliferation,resistance to programmed cell death and metastasis through cell invasion or migration to other parts of the body.It is the second largest cause of death in the world,second only to cardiovascular disease.Researchers predict that the number of cancer deaths worldwide will continue to rise in the next decade.Cancer has caused a huge economic burden on the world and has had a significant impact on public health.Among different types of cancer,colorectal cancer has become the third most common cancer type in the world.In Europe and the United States,the mortality rate ranks high,second only to lung cancer.The treatment of colorectal cancer is mainly divided into surgical treatment and non-surgical treatment.Non-surgical treatment also includes chemotherapy and radiotherapy.Different treatment schemes are usually selected according to the patient’s status,location and stage of colorectal cancer,patient’s age and health status.Generally,if early diagnosis and surgical treatment can be carried out for colorectal cancer,the prognosis is mostly good.If patients with colorectal cancer have entered the advanced stage,surgery is no longer the first choice of treatment,usually radiotherapy and chemotherapy.With the significant progress of these treatments,the overall survival rate of colorectal cancer patients has improved,but these treatments also have many side effects.For example,multidrug resistance of chemotherapy drugs will lead to chemotherapy failure,adverse reactions caused by chemotherapy or radiotherapy,common reactions such as chronic diarrhea or intestinal obstruction,which will significantly reduce the quality of life of patients,and individual differences are not sensitive to immunotherapy.The most important point is that these treatments still can not prevent the recurrence and metastasis of colorectal cancer.At present,generally speaking,the incidence rate of colorectal cancer has an increasing trend and will continue to rise.Through global epidemiological investigation and relevant scientific research,multiple risk factors for colorectal cancer have been identified.Including diet,obesity,gender,race,old age,family history of tumor,gene mutation,inflammation and intestinal microbiota disorders.A large amount of evidence has clearly shown that the occurrence of colorectal cancer is a multi-step process,which is the result of gradual accumulation of genetic and epigenetic changes,and finally triggers the transformation of normal colonic epithelial cells into cancer cells.Recent research results show that non coding RNA may promote cancer progression and tumor formation to a large extent.The most thoroughly studied sequences in the human genome are usually protein coding genes,but they only account for 1.5-2.0%of the whole genome.In recent years,non protein coding regions have become a new research field.The genomes of these regions have been proved to have important functions in tissue development and diseases including cancer.LncRNA SNHG4 has been found to be highly expressed in several kinds of malignant tumors,plays an important role in the proliferation and migration of tumor cells,and is closely related to poor prognosis,which promotes the progress of tumor progression.However,there is no relevant research on colorectal cancer.This subject consists of two parts.The first part studied the expression of LncRNA SNHG4 in colorectal cancer tissue,analyzed the relationship between its expression and the clinical syndrome types and pathological characteristics of patients,and also explored its role in the prognosis evaluation of patients.In the second part,we studied the function of LncRNA SNHG4 in colon cancer cells in vivo and in vitro,and further discussed its regulatory value on epithelial mesenchymal transition(EMT).Part Ⅰ Expression and significance of LncRNA SNHG4 in CRCObjective:To investigate the relationship between the relative expression of LncRNA SNHG4 in tumor tissues and adjacent control tissues of colorectal cancer patients and their clinical syndrome types,pathological features and prognosis.Methods:the clinical syndrome types,pathological characteristics and prognosis of 80 patients with colorectal cancer in the second people’s Hospital of Huai’an City were collected.Real time fluorescent quantitative PCR(RT-qPCR)was used to detect the relative expression of LncRNA SNHG4 in tumor tissues and adjacent control tissues.Results:the relative expression of LncRNA SNHG4 in cancer tissues of 66 of 80 patients with colorectal cancer was higher than that in adjacent normal tissues,and the high expression rate was 82.50%.The relative expression of LncRNA SNHG4(△CT)was 8.10±1.41 in CRC tissues and 9.23±1.59 in adjacent control tissues(t=-4.741,P<0.01);The median-△△CT value of LncRNA SNHG4 in cancer tissues and adjacent normal tissues of 80 patients with CRC was 1.676,and the median value of fold change was 2-△△CT=3.195,with an average value of 3.774.The difference was statistically significant(t=8.643,P<0.01).Among the 80 patients,24 were of single syndrome type,which were blood stasis syndrome,Qi and blood deficiency syndrome,damp heat accumulation syndrome and liver and kidney deficiency syndrome respectively.The relative expression of LncRNA SNHG4 among the groups showed that the relative expression of LncRNA SNHG4 in patients with blood stasis syndrome was the highest and significantly higher than that in patients with other three types(all P<0.05).There was a significant positive correlation between LncRNA SNHG4 and the score of blood stasis syndrome in 10 patients with blood stasis syndrome(r=0.696,p=0.025);There was also a significant positive correlation between LncRNA SNHG4 and the score of blood stasis syndrome in 26 patients whose main syndrome was blood stasis syndrome(r=0.811,P<0.01).According to the median fold change value of relative expression of LncRNA SNHG4(3.195),the patients were divided into high expression group and low expression group,with 40 people in each group.The expression of lncrna snhg4 has a certain correlation with the blood stasis syndrome classification of patients,and the relative expression is positively correlated with the blood stasis syndrome score of patients.The expression level of LncRNA SNHG4was highly correlated with tumor T stage,lymph node metastasis status,TNM stage,plasma CEA level and tissue differentiation,but not with other general factors of the patients.The high expression of LncRNA SNHG4 suggests that patients have a later TNM stage(x2=11.314,p=0.001),deeper tumor invasion(x2=9.825,p=0.002),more prone to lymph node metastasis(x2=24.444,p=0.000),and higher plasma CEA value(x2=18.152,p=0.000).Survival analysis showed that the 5-year OS in the high expression group of LncRNA SNHG4 was significantly lower than that in the low expression group(58.8%vs 81.8%),and the difference was statistically significant(x2=4.876,p=0.027).Cox model univariate analysis showed that sex,age,tumor location,tumor diameter and plasma CEA level were not related to OS in CRC patients,but TNM stage,tumor invasion level,LncRNA SNHG4 expression level and tumor tissue differentiation were closely related to OS in CRC patients.Cox multivariate analysis showed that TNM stage(stage III/Ⅳ vs Ⅰ/Ⅱ)(HR=3.702,95%CI:1.096-12.502,p=0.035)was an independent factor affecting the overall survival of colorectal cancer patients.Conclusion:the relative expression of LncRNA SNHG4 in colorectal cancer tissues was higher than that in adjacent control tissues,with a high expression rate of 82.50%,and the average value of 2-△△CT was 3.774 times.The expression of LncRNA SNHG4 has a certain correlation with the blood stasis syndrome classification of patients,and the relative expression is positively correlated with the blood stasis syndrome score of patients.Abnormal expression of LncRNA SNHG4 is correlated with tumor invasion level(T),lymph node metastasis status(n),TNM stage,plasma CEA value,pathological malignancy in colorectal cancer patients.Its high expression indicates deeper tumor invasion level,more lymph node metastasis,later tumor stage,worse tissue differentiation,higher plasma CEA level.High expression of LncRNA SNHG4 is one of the adverse prognostic factors in colorectal cancer patients.High expression of LncRNA SNHG4predicts shorter survival,but it is not an independent risk factor.TNM stage is an independent prognostic factor in the overall survival of colorectal cancer patients.Part Ⅱ LncRNASNHG4 promotes colon cancer cell proliferation and migration in vitro and in vivo and regulates EMT pathwayObjective:To investigate the expression and biological function of LncRNA SNHG4 in colorectal cancer cells.Methods:The expression of LncRNA SNHG4 in SW480,HCT116,LoVo,HT29 and human normal colonic epithelial cells(FHC)was detected by RT-qPCR;We focused on HCT116 cells with the highest relative expression of LncRNA SNHG4,and down regulated the expression of LncRNA SNHG4 through siRNA interference.We used CCK8 cell proliferation test,plate colony formation test,Transwell migration test and zebrafish xenotransplantation test to study the effect of LncRNA SNHG4 expression on the proliferation and migration of HCT116 colon cancer cells,The expression levels of EMT related genes and proteins were further detected.Results:The results of RT qPCR showed that the relative expression of LncRNA SNHG4 in SW480,HCT116,LoVo and HT29 cells was significantly higher than that in normal intestinal epithelial cells(FHC)(P<0.05).After down regulating the expression of LncRNA SNHG4 in HCT116 cells by gene silencing technique,the proliferation of tumor cells was significantly inhibited(P<0.01);In addition,downregulation of LncRNA SNHG4 expression significantly reduced the migration ability of tumor cells(t=15.142,p=0.005).The results of zebrafish xenotransplantation model also showed that after down regulating the expression of LncRNA SNHG4 in HCT116 cells,cell proliferation and migration decreased significantly(P<0.05).Silencing the expression of LncRNA SNHG4 in HCT116 cells can up regulate the mRNAand protein expression of E-cadherin,the epithelial marker of EMT pathway,and reduce the mRNA and protein expression of N-cadherin.Conclusion:the expression of LncRNA SNHG4 in HCT116 cells was the highest among the four colon cancer cells detected in this study.In vivo and in vitro experiments confirmed that down regulating the expression of LncRNA SNHG4 could reduce the proliferation and migration of HCT116 cells.In addition,the expression of LncRNA SNHG4 was involved in the regulation of EMT pathway. |
