| BackgroundColorectal cancer is the second most common and the third leading cause of cancer-related deaths worldwide,so CRC is a serious harm to human health.CRC patients can be separated in non-symptomatic and symptomatic individuals.Many of the first group are diagnosed based on a positive colonoscopy result following a positive fecal occult blood test.Symptomatic patients often suffer from rectal bleeding,abdominal pain,changes in bowel habits,anemia and occult bleeding.Patients can be admitted as emergency with obstructive symptoms like ileus or with signs of acute abdomen in case of tumor perforation.At this point,most colorectal cancer has been in progress,and the patients have lost the optimal opportunity for treatment.Before surgery,abdominal ultrasound,colonoscopy,thoracic X-ray and CEA blood levels are routinely analyzed.In case of suspected distant metastasis extended CT scans(brain,chest and abdomen)are recommended.In rectal cancer,additional MRI of the pelvis and transrectal endoscopic ultrasound are obligatory.The rectal location has to be evaluated by non-flexible endoscopy as well.Based on the TNM classification,local versus radical,curative versus palliative surgery ranging from local excision to pelvic exenteration has to be performed.Preoperative radio-chemotherapy of rectal carcinomas and postoperative chemotherapy of patients with pT3-4 or N+ stage are part of the treatment options supplementing surgery.Despite tremendous progress in the treatment of CRC in recent decades,the prognosis remains unsatisfactory,especially in advanced-stage tumors with distant metastasis.It is reported that approximately 25%of CRC patients present with synchronous liver metastases at diagnosis,and approximately 50%of CRC patients develop metachronous liver metastases 3 years after treatment.As most CRC patients with liver metastases are not able to undergo surgery,the prognosis is dismal,with a 5-year survival rate less than 10%after the diagnosis of liver metastasis.Carcinoembryonic antigen(CEA)levels may serve as biomarker during follow-up,but cannot be used as reliable screening or diagnostic tool because of a lack in sensitivity and specificity in the early detection of CRC.Rising CEA levels in the follow-up period after surgery may indicate recurrent disease and the patient has to be restaged through computer tomography(CT)scan of the liver.LncRNAs have been found in serum and plasma,which may be the marker of colorectal cancer and have significance for the diagnosis and prognosis of colorectal cancer.In addition,some LncRNAs have been found to affect the sensitivity of colorectal cancer to radiotherapy and chemotherapy and predict the therapeutic effect in theory.Therefore,it is vital to identify the molecular mechanisms and genetic alterations of CRC metastases to develop effective therapies.Recently,integrative genomic studies have revealed that more than 90%of the DNA sequence is actively transcribed,with only 2%of these transcripts encoding protein,while most of the transcripts are referred to as non-coding RNAs(ncRNAs).Genomes encode a wide variety of conserved non-coding RNA transcripts.In addition to classical ’ housekeeping’ RNAs(such as ribosome RNAs,transfer RNAs,and others)and widely-defined microRNAs,long non-coding RNAs(IncRNAs)have recently been identified as one of fraction of untranslated RNA molecules.lncRNA,transcribed by RNA polymerase Ⅱ(RNA pol-Ⅱ),are characterized by lengths of 200 nucleotides to 100kilobases(kb)and by their lack of a significant open reading frame.These mRNA-like molecules are pervasively transcribed and roughly classified as antisense,based on their position relative to the protein-coding genes and exhibit in-cis or in-trans regulatory capabilities for gene expression.Gene expression patterns indicate that these lncRNAs are implicated in diverse biological processes,including nuclear architecture,regulation of gene expression,immune surveillance,or embryonic stem cell pluripotency.Recently,evidence revealing the molecular mechanisms by which these RNA species function has provided some insight into the functional roles they may play in tumorigenesis.Aberrant lncRNA expression participates in carcinogenesis by disrupting major biological processes,such as redirecting chromatin remodeling complexes or inactivating major tumor suppressor genes.The low expression of IncRNA AOC4P is in relation to the poor prognosis in hepatocellular carcinoma(HCC)and LncRNA AOC4P could be as a prognosis factor of HCC which suppresses HCC metastasis by enhancing vimentin degradation and inhibiting EMT.However,the role of LncRNA AOC4P in colorectal cancer remains unclear.One of the most crucial steps in the cancer cell metastatic cascade is the acquisition of invasive capabilities,including destroying cell-cell junctions,degrading the cell matrix,and activating pathways that control the cytoskeletal dynamics of cancer cells.Over the past decade,researchers have identified the key role of epithelial-mesenchymal transition(EMT)-a biological process in which epithelial cells lose their polarity and transition into a mesenchymal phenotype-in cancer cell metastasis.Evidence suggests that EMT enhances cancer cell invasive capabilities,and also contributes to cell growth and survival.The process of EMT results from a spectrum of changes and transitions in response to environmental stimuli,dependent on tissue and signaling context.EMT initiation and progression involves a complicated crosstalk among networks of signaling pathways and transcriptional regulators.The hallmark of EMT is the downregulation of E-cadherin,a major epithelial marker,which is regulated by a group of EMT transcription factors.These factors,including the SNAIL,TWIST,and ZEB families,play important roles in both embryogenesis and tumorous settings.Importantly,regulation of these EMT-related transcription factors is associated with invasiveness,metastasis,and poor prognosis of CRC,emphasizing the importance of EMT in tumor progression and metastasis.It has not been reported that lncRNA AOC4P effects the metastasis of colorectal cancer by regulating the epithelial-mesenchymal transition(EMT)process of colorectal cancer cells.ObjectiveIn this study,we would detect the expression of LncRNA AOC4P in colorectal cancer tissue,and then explore the relationship with clinical characteristics.At the same time we would study its effect and mechanism which contribute to the proliferation,migration and invasion of colorectal cancer cells.So that it could provide a theoretic foundation for the clinical research of colorectal cancer markers and related drug targets.Methods1.The expression of LncRNA AOC4P was detected in colorectal cancer tissues and adjacent normal tissues by real-time fluorescent quantitative PCR.Then the correlation analysis between lncRNA AOC4P expression and clinical features or survival time of patients suffered from colorectal cancer was done.2.The expression of CCC-HIE-2 and four kinds of colorectal cancer cell lines(SW480,HCT166,HT29,SW620)was detected by real-time fluorescent quantitative PCR to further make sure the difference of lncRNA AOC4P expression between them.The empty plasmid and lncRNA AOC4P plasmid were transfected into colorectal cancer cell.lines.Then the changes.of proliferation ability of colorectal cancer cell lines acquiring the overexpression of lncRNA AOC4P were measured by CCK8 test.In addition,transwell assay was used to detect the migration and invasion ability of colorectal cancer cell lines after transfection being done.3.Flow cytometry was used to detect the difference of the apoptosis and cell cycle of colorectal cancer cell lines with overexpression of lncRNA AOC4P.The expression of EMT-related proteins such as E-cadherin,N-cadherin and Vimentin in colorectal cancer cell lines was detected by Western blot.Results:1.The expression of LncRNA AOC4P and the correlation with clinical features and survival time(1)The results of real-time fluorescent quantitative PCR showed that the expression of lncRNAAOC4P in colorectal cancer tissues was significantly lower than that in normal tissues adjacent to the cancer(P<0.05).(2)The results of real-time fluorescent quantitative PCR showed that the expression of lncRNA AOC4P in I and II colorectal cancer was significantly higher than that in Ⅲ and Ⅳ colorectal cancer(P<0.05).(3)The results of real-time fluorescent quantitative PCR showed that the expression of lncRNA AOC4P with distant metastasis was significantly lower than that without metastasis(P<0.05).(4)The results of Kaplan-Meier survival curve showed that patients with high expression of LncRNA AOC4P had a longer survival time than those with low expression of LncRNA AOC4P(log-rank test,P<0.05).The COX regression showed that LncRNA AOC4P is an independent prognostic factor for overall survial in patients with colorectal cancer.2.The effect of LncRNA AOC4P on proliferation,migration and invasion of colorectal cancer cells(1)The results of real-time fluorescent quantitative PCR showed that the expression of LncRNA AOC4P in four kinds of colorectal cancer cell lines(SW480,HCT166,HT29,SW620)was lower than that in normal mucosa cell line(CCC-HIE-2)(P<0.05),and that of the two colorectal cancer cell lines SW480 and HT29 were relatively lower which were used in the following test.(2)Plasmids were tranfected into SW480 and HT29 with high efficiency and the stable cell lines were constructed successfully which was confirmed by real-time fluorescent quantitative PCR.Comparing with the colorectal cancer cell lines handled with empty vector,the growth activity was significantly inhibited(P<0.05),and their migration capability and invasion ability was also obviously weakened(P<0.05).3.The effect of LncRNA AOC4P on EMT,apoptosis and cell cycle of the colorectal cancer cells(1)The results of the flow cytometry showed that cell apoptosis ratio of colorectal cancer cell lines overexpressing LncRNA AOC4P was higher than colorectal cancer cell lines transfected by empty vector(P<0.05).However,there was no significant difference in cell cycle between them(P>0.05).(2)Western blot results showd that the expression of Vimentin and N-cadherin in colorectal cancer cell lines overexpressing LncRNA AOC4P was lower than that with empty vector(P<0.05),and the expression of E-cadherin was significantly increased(P<0.05).Conclusion1.The expression of LncRNA AOC4P is decreased in colorectal cancer tissues,and which is related to clinical characteristics of colorectal cancer.It is an independent prognostic factor for overall survial in patients with colorectal cancer.2.Overexpression of LncRNA AOC4P in colorectal cancer cell lines can prominently suppress cell proliferation,migration and invasion.3.Overexpression of LncRNA AOC4P inhibits cell growth by promoting colorectal cancer cell apoptosis and supresses colorectal cancer cell migration and invasion by reversing the EMT process.4.LncRNA AOC4P could be as a new biomarker of colorectal cancer and a new target for molecular biological therapy. |
