| Colorectal Cancer(CRC)is one of the most common malignant tumors in humans,with morbidity and mortality ranking the third in all malignant tumors.In the past 10 years,CRC has been increasing year by year in China,which seriously affects people’s physical and mental health.For patients with Local Advanced Rectal Cancer(LARC),preoperative neoadjuvant chemoradiotherapy(NCRT)combined with radical surgery has become the standard treatment.Studies have shown that the above treatment regimen has obvious advantages in tumor downsizing and downstaging,improvement of anal preservation rate and reduction of local recurrence of rectal cancer.However,some patients with chemoradiotherapy resistance caused treatment failure,disease recurrence,and lead to poor prognosis.Therefore,to explore the sensitivity mechanism of rectal cancer to chemoradiotherapy and find new therapeutic targets that are resistant to chemoradiotherapy to enhance the sensitivity of chemoradiotherapy,so that patients can obtain greater treatment benefits.Long non-coding RNA(LncRNA)is a group of RNAs with a length over 200 nucleotides and no protein coding function,which can regulate the expression of related genes at various levels such as pre-transcription,transcription and post-transcription,and thus play an important biological function.In recent years,more and more studies have shown that LncRNA is closely related to the occurrence,development,drug resistance,recurrence and metastasis of colorectal cancer,and can be used as a potential molecular marker and therapeutic target for the diagnosis and prognosis of colorectal cancer patients.Existing studies have revealed the abnormal expression levels of some LncRNAs in colorectal cancer,and some of them have been reported in in-depth studies.However,the biological functions and molecular mechanisms of most lncRNAs in the occurrence and development of colorectal cancer remain unclear.Linc00909 is a novel LncRNA molecule,which has been shown to be an important oncogenic LncRNA and is highly expressed in colorectal cancer tissues and suggests a poor prognosis.However,whether it is involved in the regulation of NCRT sensitivity in colorectal cancer has not been reported.This study first analyzed the expression of Linc00909 in colorectal cancer and its correlation with prognosis,and preliminarily explored the influence of changing the expression of Linc00909 on the sensitivity to chemoradiotherapy and its possible molecular mechanism in vivo and in vitro.The research is divided into the following two parts:PART ONEThe expression and its clinical significance of Linc00909 in Colorectal Cancer nd chemoradiotherapy tissue Projective To investigate the expression difference of Linc00909 between NCRT-resistant and sensitive colorectal cancer tissues,as well as between colorectal cancer tissues and paracancerous tissues,and to analyze whether the expression of this LncRNA is correlated with postoperative survival prognosis and sensitivity to chemoradiotherapy of patients.In order to prove whether it can be used to predict the sensitivity of chemoradiotherapy and to evaluate patients molecular markers of prognosis provide a theoretical basis.Methods The patients who received preoperative neoadjuvant chemoradiotherapy and radical resection in the Union Hospital of Fujian Medical University from January2017 to December 2017 were collected,and 30 cases of NCRT-resistant and sensitive tissue samples(19 cases of NCRT-resistant tissue and 11 cases of NCRT-sensitive tissue)obtained by surgical resection were collected.The expression of Linc00909 between the two groups was detected by RT-qPCR assay.A total of 196 pairs of colorectal cancer tissues and adjacent adjacent tissues were collected from Union Hospital Affiliated to Fujian Medical University from January 2017 to December2017.RT-qPCR was used to detect the expression level of Linc00909 between the two groups,and the patients were followed up to obtain the corresponding clinical information and follow-up information,and the correlation between Linc00909 and postoperative survival prognosis and clinicopathological information was statistically analyzed.The relative expression level of Linc00909 is expressed by ΔCt,that is,the smaller the ΔCt,the higher the relative expression level.Results RT-qPCR results showed that the expression level of Linc00909 was significantly up-regulated in NCRT-resistant tissues(ΔCt=7.59±0.37)compared with NCRT-sensitive tissues(ΔCt=10.02±0.73)(P<0.01).Moreover,the expression level in cancer tissues(ΔCt=8.78±0.16)was significantly higher than that in paracancerous tissues(ΔCt=10.64±0.12)(P<0.001).Correlation analysis of clinicopathological information showed that the expression level of Linc00909 was positively correlated with p M stage.The results of survival analysis showed that the expression level of Linc00909 was negatively correlated with overall survival and disease-free survival.Cox regression analysis showed that nerve invasion(HR=3.109,95%CI:1.245-7.764,P=0.015)and the expression level of Linc00909(HR=5.067,95%CI:1.629-15.765,P=0.005)were independently associated with OS.The expression level of LINC00909(HR=5.335,95%CI: 2.467-7.764,P < 0.001)was independently associated with patient DFS.Conclusion Linc00909 was highly expressed in NCRT-resistant tissues.The expression level was also up-regulated in CRC cancer tissues.The expression level of Linc00909 was negatively correlated with OS and DFS,and could be used as an independent risk factor affecting OS and DFS in patients.Linc00909 has the potential to be used as a molecular marker to predict the sensitivity to chemoradiotherapy in patients and to evaluate the prognosis of patients.PART TWOLinc00909 affects the sensitivity of chemoradiotherapy by enhancing the stem cell-like properties of colorectal cancer cells Projective To explore the influence of overexpression of Linc00909 gene on malignant biological behavior of colorectal cancer cells,and to preliminarily explore the possible molecular mechanism of its influence on the sensitivity of colorectal cancer cells to chemoradiotherapy.Methods The colorectal cancer cell lines SW620 and DLD1 were transfected with overexpressing Linc00909 lentiviral vector to establish a stable transfected cell line with overexpressing Linc00909;RT-qPCR was used to detect the expression of Linc00909 gene;CCK-8 and Plate cloning method were used to detect the effect of proliferation;Flow cytometry was used to analyze its effect on cell apoptosis and cell cycle;Stem cell spheroidization test detected the effect of overexpression of Linc00909 on cell spheroidizing ability;The sensitivity of CRC cells to 5-FU(5-Fluorouracil,5-fluorouracil)and RT after overexpression of Linc00909 was measured by CCK-8 method and flow cytometry..Then,the effect of overexpression of Linc00909 gene on tumor formation in colorectal cancer cells was evaluated by Subcutaneous transplantation tumor experiment in nude mice.Lastly,the miRNA and its downstream target genes that may interact with Linc00909 were predicted by bioinformatics analysis.RT-qPCR and Western-blot experiments were used to preliminarily explore the molecular mechanism of Linc00909 in chemoradiotherapy sensitivity of colorectal cancer cells in the form of ce RNA.Results RT-qPCR results showed that the expression of Linc00909 was significantly up-regulated in the overexpression cell group(P<0.01)compared with the empty lentivirus group(Vector)and the control group(Control)cells.After overexpression of Linc00909,colorectal cancer cell proliferation ability,clone formation ability,anti-apoptosis ability,cell formation ability and resistance to 5-FU and RT were all significantly enhanced;it can induce cell cycle transformation of colorectal cancer to promote cell proliferation;and can promote the growth of colorectal cancer cells in nude mice.Bioinformatics analysis showed that miR-625-5p may have a binding site with Linc00909.The results of RT—qPCR revealed that the expression of miR-625-5p in colorectal cancer cell lines SW620 and DLD1 was dowm-regulated after over expression Linc00909.SOX2 was predicted by bioinformatics analysis to be one of the possible target genes of miR-625-5p in colorectal cancer.The results of RT-qPCR and Western-blot showed that the expression of SOX2 in colorectal cancer cell lines SW620 and DLD1 was significantly increased after over expression of Linc00909.Conclusion Linc00909 can affect colorectal cancer cell proliferation,clone formation,apoptosis,cycle regulation,spheroidization process and sensitivity to 5-FU and RT;and overexpression of Linc00909 gene can promote the growth of colorectal cancer cells in nude mice.Linc00909 may affect the sensitivity of colorectal cancer cells to chemoradiotherapy by regulating the expression of downstream target gene SOX2 through the adsorption of miR-625-5p.Linc00909 can be used as a potential therapeutic target for colorectal cancer. |
PDF Full Text Request