Long-noncoding RNA XLOC₀10588 And NAV2-AS5 Regulate The Invasion And Migration Of Colorectal Cancer

Objective: The incidence of colorectal cancer ranks third in malignant tumors in the worldwide,and the mortality rate ranks the second.Colorectal cancer is considered as a significant threat to public health.As one of the most serious public health problems in the world,colorectal cancer is the most frequently diagnosed as malignant tumor.Although the incidence of colorectal cancer has declined over the past few years,nearly 600,000 people die for colorectal cancer each year,therefore,early detection and diagnosis of colorectal cancer is particularly important for high-risk groups.Despite through a lot of research on colorectal cancer we have got a more in-depth understanding of this disease,diagnostic techniques are constantly evolving,treatments are getting more and more effective,however,the overall survival rate of patients with colorectal cancer is still relatively low.High mortality of colorectal cancer is usually attributed to tumor invasion and metastasis,there are a variety of mechanisms which promotesthe invasion and metastasis of tumor cells,epithelial mesenchymal transition?EMT?is a common one to promote tumor cell invasion and migration,which promotes a region of epithelial cells separating from the epithelial tissue and migraing to the other locations.EMT is the basis of tumor metastasis during the development of tumor.To date,improvements in whole-genome and transcriptome sequencing technologies have led to the discovery that the majority of the full mammalian genome can be transcribed;however,most of the transcripts have limited or no protein-coding capacity,and the gene are known as ncRNAs.Distinct from small nc RNAs?such as siRNAs,miRNAs and piRNAs?that have been widely studied,long noncoding RNAs?lncRNAs?are obscure molecules that make up ⁸0% of ncRNAs and have unclear functions at present.LncRNAs,which are transcripts of >200 nucleotides in length,are transcribed by RNA polymerase II?RNAPII?;however,they have few or no open reading frames?ORFs?.Extensive evidence suggests that lnc RNAs serve crucial roles in a wide variety of fundamental biological processes,including embryonic development,epigenetic silencing,transcriptional and translational control,cell growth,cell differentiation,cell migration and tumorigenesis.Additionally,extensive research has provided strong evidence to suggest that lncRNAs serve a functional role in a range of human diseases,including various types of cancer,such as breast,pancreatic,lung,gastric and cervical.Furthermore,it has been verified that numerous lnc RNAs participate in both oncogenic and tumor-suppressing pathways in tumorigenesis.It has been widely recognized that lncRNA plays an important role in tumorigenesis as an oncogene or tumor suppressor gene.Based on the findings of the past decade,some lncRNAs have become biomarkers for diagnosing colorectal cancer,evaluating prognosis,and even predicting the effect of treatment.The abnormal expression of lncRNA plays an irreplaceable role in some functions of colorectal cancer cells,such as proliferation,invasion,apoptosis,metastasis,drug resistance,etc.Therefore,it can be seen that the mechanism of lnc RNA in colorectal cancer is helpful for early diagnosis and the prognosis of patients with colorectal cancer.Methods: In this study,the expression of XLOC₀10588 and NAV2-AS5 in colorectal cancer tissues and adjacent tissues was detected by in-situ hybridization,and the correlation of XLOC₀10588 and NAV2-AS5 with clinicopathological parameters and prognosis of patients was analyzed.Then we routinely cultured normal intestinal epithelial and colorectal cancer cell lines: HIEC,Caco-2,HT29,HCT116,SW480,and SW620 cell lines,and screened for the expression of XLOC₀10588 and NAV2-AS5 in the above cells by q RT-PCR.The HCT116 with low expression of XLOC₀10588 and NAV2-AS5 in colorectal cancer were selected respectively,and the overexpression cell model was constructed by transfection of the expression plasmid.The SW620 with high expression of XLOC₀10588 and NAV2-AS5 in colorectal cancer were selected,and the silencing cell model was constructed by transfection of siRNA.Overexpression and silencing expression efficiency was confirmed by q RT-PCR.After the successful construction of the cell model,scratching and Transwell technique were used to investigate the influence of the expression of XLOC₀10588 and NAV2-AS5 on the invasion and migration of cell lines.To investigate the specific mechanism of XLOC₀10588 and NAV2-AS5 on invasion and migration of colorectal cancer cells,we first detected the mRNA and protein levels of EMT-related markers by qRT-PCR,Western blot and immunofluorescence,understanding that XLOC₀10588 and NAV2-AS5 promote the invasion and migration of colorectal cancer cells through the EMT pathway.Results: 1.LncRNA XLOC₀10588 regulates the invasion and migration of colorectal cancer.?1?Correlation between XLOC₀10588 expression and clinicopathological parameters in patients with colon cancer.We detected the expression of XLOC₀10588 in 111 cases of tumor tissue and 70 cases of tissue adjacent using in situ hybridization assay,respectively.The positive rates of XLOC₀10588 expression were 58.02% and 24.29% respectively.It was proved that XLOC₀10588 was highly expressed in tumor tissues and the high expression rate of cancer tissue is significantly higher than tissue adjacent??2=19.336,P<0.001?.XLOC₀10588 expression level with gender?P=0.030?,the degree of infiltration?P=0.013?and lympH node metastasis?P=0.002?was significantly positive correlation,namely the sex to male,tumor infiltration depth deeper or patients with lymph node metastasis XLOC₀10588 expression levels tend to be higher.We analyzed the relationship between the expression of XLOC₀10588 and the prognosis of patients by Kaplan-Meier method.The results showed that the OS of XLOC₀10588-overexpression patients was significantly shorter than that of patients with low expression of XLOC₀10588?P<0.001?,in tumor size less than or equal to 5 cm?P=0.034?,tumor size greater than 5 cm?P=0.001?,pathological grading for I,II level?P<0.001?,tumor infiltration depth of 1,2 level?P=0.002?,and those with no lympH node metastasis?P<0.001?in patients with XLOC₀10588 high expression of OS significantly shorter than the patients with XLOC₀10588 lower expression.Cox univariate regression analysis found that pathological grade,depth of invasion,lympH node metastasis and XLOC₀10588 expression level correlated with OS.We further analyzed the influence of pathological grade,depth of invasion,lympH node metastasis and XLOC₀10588 expression on OS in Cox multivariate regression model and found that the pathological grade of tumor?P=0.010?and XLOC₀10588?P=0.005?are independent risk factor affecting the clinical prognosis of patients.?2?We used the TCGA database to analyze the bioinformatics of XLOC₀10588.We found that XLOC₀10588 was highly expressed in colon cancer tissues?P<0.001?in 454 colon cancer tissues and 51 paracancerous tissues in the TCGA database.We also analyzed the relationship between XLOC₀10588 expression and TNM grade in patients and found that XLOC₀10588 had a higher degree of tumor infiltration?P<0.001?,lympH node metastasis?P=0.0034?and distant metastasis?P=0.022?.It shows that XLOC₀10588 is highly expressed in colon cancer and closely related to the invasion and migration of colon cancer.?3?XLOC₀10588 is highly expressed in colorectal cancer cell lines.We examined the expression of XLOC₀10588 in different colorectal cancer cell lines?Caco-2,HCT29,HCT116,SW480 and SW620?by q RT-PCR comparing with the expression of XLOC₀10588 in normal intestinal epithelial cell line?HIEC?.The expression of XLOC₀10588 and its relationship with tumor invasion and metastasis were analyzed preliminarily at cytology level.The results of qRT-PCR showed that the expression of XLOC₀10588 in SW620 cells with high invasion ability was significantly different from that in Caco-2,HCT29,HCT116 and SW480 cell lines with low invasion and metastasis?P<0.05?.?4?The invasion and migration ability of colorectal cancer cells changed after XLOC₀10588 were interfered.The silencing of XLOC₀10588 inhibited the invasion and migration of colorectal cancer cells.After the construction of XLOC₀10588 silencing model,the ability of XLOC₀10588 to detect invasion and migration of colorectal cancer cells was detected by scratch test and Transwell assay.The invasion and migration ability of SW620 cells decreased significantly?P<0.05?.XLOC₀10588 overexpression promoted the invasion and migration of colorectal cancer cells.After XLOC₀10588 overexpression model was successfully constructed,the ability of invasion and migration of colorectal cancer cells was examined by scratch assay and Transwell assay.The results showed that XLOC-After overexpression,the invasion and migration of HCT116 cells were significantly enhanced?P<0.05?.?5?XLOC₀10588 affects tumor cell invasion and migration by promoting EMT.After observing the changes of EMT related indexes before and after the silence of XLOC₀10588,the expression changes of E-cadherin,Slug and Vimentin were detected by qRT-PCR,Western blot and immunofluorescence method,and the EMT process was suppressed after the silence of XLOC₀10588?P<0.05?.The changes of E-cadherin,Slug and Vimentin expression after over-expression of XLOC₀10588 were observed.It was found that XLOC₀10588 overexpression promoted EMT?P<0.05?.2.LncRNA NAV2-AS5 regulates the invasion and migration of colorectal cancer.?1?Correlation between NAV2-AS5 expression and clinicopathological parameters in patients with colon cancer.We detected the expression of NAV2-AS5 in 138 cases of tumor tissue and 102 cases of tissue adjacent using in situ hybridization assay,respectively.The positive rates of NAV2-AS5 expression were 63.04% and 22.54% respectively.It was proved that NAV2-AS5 was highly expressed in tumor tissues and the high expression rate of cancer tissue is significantly higher than tissue adjacent??2=32.640,P<0.001?.The expression level of NAV2-AS5 was positively correlated with the degree of infiltration?P=0.020?,that is,the expression level of NAV2-AS5 was always higher in patients with deeper tumor infiltration.We analyzed the relationship between the expression of NAV2-AS5 and the prognosis of patients by Kaplan-Meier method.The results showed that the OS of NAV2-AS5-overexpression patients was significantly shorter than that of patients with low expression of NAV2-AS5?P=0.019?,in tumor size greater than 5 cm?P=0.007?and pathological grading for I,II level?P=0.015?patients with NAV2-AS5 high expression of OS significantly shorter than the patients with NAV2-AS5 lower expression.Cox univariate regression analysis found that tumor size,pathological grading,infiltration depth,lympH node metastasis and NAV2-AS5 expression level were related to OS.We further adopt Cox multivariate regression model analysis of the tumor size and pathology classification,infiltration depth,lymph node metastasis and NAV2-AS5 expression level influence on patients with OS,found that tumor size?P=0.022?,tumor pathological grade?P=0.010?and NAV2-AS5 expression levels?P=0.045?are the independent risk factors of patients with clinical prognosis.?2?NAV2-AS5 is highly expressed in colorectal cancer cell lines.We examined the expression of NAV2-AS5 in different colorectal cancer cell lines?Caco-2,HCT29,HCT116,SW480 and SW620?by q RT-PCR comparing with the expression of NAV2-AS5 in normal intestinal epithelial cell line?HIEC?.The expression of NAV2-AS5 and its relationship with tumor invasion and metastasis were analyzed preliminarily at cytology level.The results of qRT-PCR showed that the expression of NAV2-AS5 in SW620 cells with high invasion ability was significantly different from that in Caco-2,HCT29,HCT116 and SW480 cell lines with low invasion and metastasis?P<0.05?.?3?The invasion and migration ability of colorectal cancer cells changed after NAV2-AS5 were interfered.The silencing of NAV2-AS5 inhibited the invasion and migration of colorectal cancer cells.After the construction of NAV2-AS5 silencing model,the ability of NAV2-AS5 to detect invasion and migration of colorectal cancer cells was detected by scratch test and Transwell assay,the invasion and migration ability of SW620 cells decreased significantly?P<0.05?.NAV2-AS5 overexpression promoted the invasion and migration of colorectal cancer cells.After NAV2-AS5 overexpression model was successfully constructed,the ability of invasion and migration of colorectal cancer cells was examined by scratch assay and Transwell assay.The results showed that after overexpression of NAV2-AS5,the invasion and migration of HCT116 cells were significantly enhanced?P<0.05?.?4?NAV2-AS5 affects tumor cell invasion and migration by promoting EMT.After observing the changes of EMT related indexes before and after the silence of NAV2-AS5,the expression changes of E-cadherin,N-cadherin,Slug,Twist2 and Vimentin were detected by qRT-PCR,Western blot and immunofluorescence method,and the EMT process was suppressed after the silence of NAV2-AS5?P<0.05?.The changes of E-cadherin,N-cadherin,Slug,Twist2 and Vimentin expression after over-expression of NAV2-AS5 were observed.It was found that NAV2-AS5 overexpression promoted EMT?P<0.05?.Conclusion: 1.The high expression of XLOC₀10588 in colon cancer tissue is associated with poor prognosis and it is an independent risk factor affecting prognosis.2.XLOC₀10588 can promote the invasion and migration of colorectal cancer by promoting tumor cell EMT.3.The high expression of NAV2-AS5 in colon cancer tissue is associated with poor prognosis and it is an independent risk factor affecting prognosis.4.NAV2-AS5 can promote the invasion and migration of colorectal cancer by promoting tumor cell EMT.