The Establishment Of Detection Method Of Long Non-Coding RNA In Serum And Its Application In The Diagnosis Of Colorectal Cancer

BackgroundColorectal cancer is one of the most common malignant tumors in our country and in the world [1],and it is a serious threat to people's physical and mental health.Although patients with early colorectal cancer have high cure rate and 5-year survival rate,most patients with colorectal cancer have been diagnosed at advanced stage due to the lack of effective biomarkers for early colorectal cancer,resulting in the survival rate was only 10% ~ 15%[2].Colorectal carcinogenesis is of a complicated and slow process,which has been considered to be from adenoma or serrated polyp to early cancer and then evolve to advanced colorectal cancer.These progression is involved in the dysregulation of numerous genes and proteins.Carbohydrate antigen(CA)and carcinoembryonic antigen(CEA)can serve as effective biomarkers for the detection of advanced colorectal cancer,but their signature can be hardly used to diagnose early colorectal cancer [3].Therefore,the screening and validation of the expression profiles of colorectal cancer-related genes and proteins are beneficial to improve the early diagnostic rate of colorectal cancer,which will improve the cure rate and prolong the survival time of the patients.Long noncoding RNA(long noncoding RNA,lncRNA)is a single-stranded RNA molecule with the length about 200 ~ 100000 nt and do not code functional protien [4].In recent years,a lot of studies have shown that the recurrence and metastasis of tumor and the prognosis of patients are involved in the dysexpression of lncRNAss[5-7],which expression profiles may serve as novel biomarkers for the diagnosis of malignant tumor,such as HOTAIR [8],ncRuPAR [9],etc.The expressions of several lncRNAs were significantly increased in colorectal cancer tissues and associated with the prognosis of patients,such as CCAT1 [10],HOTAIR [11],H19 [12].The latest data shows that serum lncRNA can serve as an effective and noninvasive biomarker for the diagnosis of malignent tumors,but the expression profiles of lncRNAs in the serum of colorectal cancer patients has been rarely studied [13].Therefore,we take lncRNA HOTAIR as an example to establish the detection method of lncRNA in serum,.Furthmore,the lncRNA microarray was used to screen the dysexpressed lncRNAs in colorectal cancer tissues and followed by qRT-PCR was used to validate the expressions of lncRNAs in the colorectal cancer tissues and serum of patients.The operating characteristic curve(ROC)analysis was used to evaluate the application value of lncRNA expression profiles in the diagnosis of colorectal cancer.PurposeTo establish the detection method of lncRNA in serum and evaluate the different expression of lncRNA in the diagnosis of colorectal cancer.Methods1.LncRNA microarry is used to screen the dysexpressions of lncRNAs in colorectal cancer tissue compared with adjacent normal tissue.LncRNA microarray was used to screen the dysexpression of lncRNAs in three pairs of colorectal cancer tissues and the adjacent normal tissues.Based on the certain principle of screening,seven lncRNAs were considered to be candidate lncRNAs,as well as an other three lncRNAs which had been reported to be overexpressed in colorectal cancer.Therefore,a total of 10 lncRNAs were included as research objects.2.The different expressions of lncRNAs are validated via qRT-PCR.2.1 Tissue RNA was extracted from 25 pairs of colorectal cancer tissues and adjacent normal tissues using TRIzol.Serum RNAs were isolated from the serum of 67 patients with colorectal cancer and 50 healthy subjects using TRIzol LS.2.2 qRT-PCR was used to measure the expressions of HOTAIR and an other nine candidate lncRNAs.The cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser reagent(dalian TaKa Ra company).qPCR was carried out using SYBR Premix Ex Taq ? reagent TaKaRa corporation(dalian).?-actin serves as a reference in tissue,as well as Cel-miR-39 as a reference in serum.2.3.ROC analysis is used to evaluate the accuracy rate of lncRNA expression profiles in the diagnosis of colorectal cancer.2.4 Pearson's test is used to analyze the correlations between TNM stage,lymph node metastasis,CA19-9 values and the expressions of lncRNAs.Results:1.Successfully establish the detection method of lncRNAs(LINC01608,LINC00594,RP11-91J3.3,H19,HOTAIR)in serum.2.HOTAIR is highly expressed in colorectal cancer tissue and in serum of colorectal cancer patients,and it is associated with lymph node metastasis of patients.In 10 paired colorectal cancer and adjacent tissue samples,HOTAIR expression is significantly higher in cancer tissue than corresponding adjacent tissues in 8 cases.The average expression of serum HOTAIR in 47 cases of colorectal cancer patients is higher than that of 40 cases of normal volunteers.Furthermore,serum HOTAIR expression in TNM ? / ? colorectal cancer patients is remarkably higher than that of ?/? period.Compared to the normal volunteers,the expression of serum HOTAIR is also much higher in colorectal cancer patients with or without lymph node metastasis.Moreover,its expression in metastatic colorectal cancer patients is also is significantly higher than those without metastatic colorectal cancer.ROC analysis revealed that HOTAIR may act as an effective biomarker for diagnosis of colon cancer.(3)Screen of the different expressed lncRNAs in colorectal cancer tissues.LncRNA chip screen out the top seven lncRNAs,namely: LINC01608,XIST,LINC00594,RP11-91J3.3,SEMA3B-AS1,RP11-396 o20.2 and RP11-867 g23.13(P < 0.01,lncRNA length <1000 bp,ratio > 5).2 reported lncRNAs(CCAT1 and H19)were also included as positive controls.4.LINC00594 and RP11-91J3.3 are significantly highly expressed in serum of colorectal cancer patients.There is no statistically significant difference between the 25 paired cases of colorectal cancer and adjacent cancer tissue for LINC01608,LINC00594,RP11-91J3.3 and H19.The average expression of LINC00594 and RP11-91J3.3 in serum of 67 cases of colorectal cancer patients is statistically higher than that of 50 normal volunteers in serum samples.ROC analysis suggests it could be potentially used as an effective biomarker for colorectal cancer diagnosis.There is no statistical difference of the expression of LINC01608 and H19 in the two groups.5.The expression of serum LINC00594 and RP11-91J3.3 do not correlates with TNM staging,lymph node metastasis and CA 19-9 value for the colorectal cancer patients.Conclusion1.The detection method of lncRNAs expression were established in serum.2.The expression of serum LINC00594,RP1191J3.3 and HOTAIR may act as new biomarkers for the diagnosis of colon cancer.