| Long noncoding RNA(IncRNA),as a research hotspot at the molecular level in recent years,is relatively new in the field of tumor biology.Now,the relationship of cancer and LncRNA is poorly understand.The effect mode,regulatory mechanism between cancer and LncRNA are not clear.However,the important role of IncRNA in the development and progression of cancer has been confirmed.LncRNA can serve as oncogene to promote the proliferation and metastasis of cancer cells,but also act as a tumor suppressor gene to inhibit the development of cancer.The study of IncRNA in cancer has been confirmed,and abnormal expression of IncRNAs were detected in various tumors.To this end,this study will study the relationship between LncRNA HNF1A-AS1,BX357664 and colorectal cancer,and provide a basis for thesubsequent treatment of color’ectal cancer.This study divides into two parts according to the different LncRNA.The first is that LncRNA HNF1A-AS1 promotes proliferation and migration of colorectal cancer cells and its mechanism.And the second one is that the LncRNA BX357664 inhibits proliferation and migration of colorectal cancer cells as well as promoting apoptosis.Part 1Objective This study detects the expression of LncRNA HNFIA-AS1 in colorectal cancer tissues and cells.And in order to study the biological functions and mechanisms of proliferation and migration in colorectal cancer cells,the HNF1A-AS1 was knockdowned and overexpressed.Methods(1)Selection of colorectal cancer cell lines:Collecting colorectal cancer tissues and adjacent tissues of 32 patients undergoing colorectal cancer resection in our hospital from June,2015 to June 2017.And then we cultured colorectal cancer cells HCT116,HT29,SW480 and SW620 so that we could use these cell lines to implant subcutaneously in nude mice.HNF1A-AS1 mRNA ware detected in human colorectal cancer tissues,cells and nude mice,and two colorectal cancer cell lines were selected for subsequent experiments.(2)The functional study of knockdown of HNF1A-AS1.For in vitro assay,we knockdown of HNF1A-AS1 in SW480 and SW620 cells,dividing cells into Si-NC group and Si-HNF1A-AS1 group.The knockdown effect was detected by RT-PCR.The proliferation of the two groups id detected by Ki67 staining and MTT.The migration ability of the two groups was detected by transwell method.Apoptosis was detected by Annexin V/FITC.(3)The functional study of overexpression of HNF1A-AS1.For in vitro assay,the plasmid of overexpressing HNF1A-AS1 was constructed and transfected into SW620 cells and SW480 cells,and the cells are divides into plasmid empty group and overexpression group.The GFP FACS and RT-PCR are used for analyzing the expression the HNF1A-AS1.The proliferation of the two groups were detected by Ki67 staining and MTT assay;the migration ability of the two groups was detected by the wound repair assay and transwell assay.(4)In vivo assay were used to detect the effect of HNF1A-AS1 in tumors.First of all,A stable strain of HNF1A-ASI knockdown was constructed by lentiviral transfection.SW480 and SW620 cells were transfected with the packaged lentivirus carrying the HNF1A-AS1 interference fragment,and then screened with G418 to obtain a stable strain.At the same time,a stable strain overexpressing HNF1A-AS1 was constructed by overexpression plasmid transfection,G418 selection,flow sorting.Then,the blank control group,Si-NC group,Si-HNF1A-AS1 group,pEGFP-C1 empty group,and over-expressing Si-HNFIA-ASI group were implanted into the subcutaneous tissues of nude mice to deternine the tumor size in vivo.(5)The mechanism research of HNF1A-AS1 promoting EMT.The luciferase reporter gene determines the targeting relationship between LncRNA HNF1A-AS1 and PIGR.The expressions of PIGR,E-Cadherin and Vimentin in colorectal cancer cells and nude mice were detected by RT-PCR and Western blot.The relationship between PIGR and EMT was then determined by HNF1A-ASI knockdown or overexpressing stable strains.Results(1)The expression of HNF1A-AS1 was up-regulated in colorectal cancer tissues of 32 patients,colorectal cancer cell lines and nude mice.For relative expression of LncRNA HNF1A-AS1,the SW620 cells and SW480 cells were at the lowest and highest,and the proliferation of the four colorectal cancer cell lines in nude mice was not statistically different(P>0.05),so SW480 and SW620 cells were selected for subsequent experiments.(2)After siRNA interference knocked down HNF1A-AS1 in SW480 and SW620 cells,the knockdown efficiency was detected by RT-PCR.There was statistical difference between the Si-NC group and Si-HNF1A-AS1 group(P<0.05),indicating that HNF1A-AS1 knockdown was successful in SW480 and SW620 cells.At the same time,there was no statistically significant difference in cell proliferation,migration and apoptosis between the blank control group and the Si-NC group(P>0.05).Compared with the blank control group and the Si-NC group,the proliferation of Si-HNF1A-AS1 cells was weakened,the migration ability was decreased,and apoptosis was increased.(3)The recombinant plasmid HNF1A-AS1-pEGFP-CI that overexpressing HNF1A-AS1 was constructed,and then transfected into SW480 and SW60 cells.The expression efficiency of HNF1A-AS1 was detected by GFP analysis and RT-PCR.The plasmid transfection efficiency was research 70-80%.There was no significant difference in the mRNA expression level of HNF1A-AS1 between the pEGFP-C1 empty group and the overexpression plasmid group(P<0.05),indicating that the recombinant plasmid overexpressing HNF1A-AS1 was successfully constructed.At the same time,there was no statistically significant difference in cell proliferation and migration between the blank control group and the pEGFP-C1 empty group(P>0.05).Compared with the blank control group and the pEGFP-C1 empty group,the overexpression plasmid group HNF1A-AS1-pEGFP-C1 enhanced proliferation and migration ability.(4)The SW480 and SW620 cells were infected with a lentivirus carrying the HNF1A-AS1 interference fragment,and a stable strain was obtained by G418 selection.After the recombinant plasmid overexpressing HNF1A-AS1 was successfully constructed,SW480 and SW620 cells were transfected,and G418 was screened and then sorted by FACS to obtain stably strains.After the tumor formation in nude nice,the tumor size of the blank group,Si-NC group and pEGFP-C1 empty group was comparable,but the tumor of Si-HNF1A-AS1 group was significantly smaller than that of the blank group and Si-NC group,while the tumor of HNF1A-AS1 group was overexpressed.The volume is significantly larger.The weekly tumor volume test showed that the tumorigenic ability of the five groups of cells did not differ statistically in the first four weeks,but with the prolongation of cell injection time,Si-HNF1A-AS1 group,overexpression group and blank group and Si-The NC group showed significant differences(P<0.05).(5)The luciferase reporter gene assay indicates that PIGR is the target of HNF1A-AS1 gene.The results of Western blot analysis of PIGR,E-cadherin and Vimentin protein showed that the gray value of the protein between the blank group and the Si-NC group and the pEGFP-C1 empty group was equivalent.The PIGR and Vimentin of HNF1A-AS1 knockdown cells decreased,and E-cadherin increased.The PIGR and Vimentin of the HNF1A-AS1 overexpression group increased,and the E-cadherin decreased.The results of RT-PCR were consistent and the difference was statistically significant.Conclusion The expression of LncRNA HNF1A-AS1 is increased in colorectal cancer tissues,colorectal cancer cell lines and nude mice.And in vitro assay,the expression level of HNF1A-AS1 was successfully knocked down or increased by SiRNA interference technology and recombinant plasmid transfection technology.The biological function of HNF1A-AS1 in colorectal cancer cells was verified.HNF1A-AS1 could promote proliferation and migration and inhibit apoptosis.In order to further study the functions of HNF1A-AS1,we successfully constructed stable strains that knocked down and overexpressed HNF1A-AS1,and subcutaneously implanted in nude mice,and verified the effect of HNF1A-AS1 on tumors in vivo.As for the mechanism study,some experiments were performed.By knocking down and overexpressing HNF1A-AS1,it was verified that HNF1A-AS1 can promote epithelial-mesenchymal transition of colorectal cancer through PIGR.Part 2Objective The first part studies the function of LncRNA HNF1A-AS1 to promote proliferation,migration and inhibition of apoptosis.To this end,the effect of LncRNA on tumors will be described by studying the biological regulation of LncRNA BX357664 on colorectal cancer.In this study,LncRNA BX357664 expression was detected in colorectal cancer tissues and cells,and BX357664 was used to study the proliferation,migration,apoptosis,cycle and EMT regulation of colorectal cancer cells by constructing BX357664 plasmid.Methods Collecting colorectal cancer tissues and adjacent tissues of patients undergoing colorectal cancer surgery.And we cultured colorectal cancer cells COLO205,HT29,HCTI 16 and human intestinal epithelial cells HIEC-6 which serves as control.And then we used four cell lines to detect the mRNA expression of HNF1A-ASI,Therefore,two colorectal cancer cell lines were selected for subsequent experiments.Next,the recombinant plasmid expressing BX35764 was constructed and transfected into HCT116 cells and HT29 cells.The cells were divided into blank control group,plasmid empty group and BX357664 overexpression group.The overexpression effect was detected by RT-PCR.The proliferation of the three groups was detected by colony formation assay,MTT assay and cell cycle assay.The migration ability of the three groups was detected by wound repair assay,migration assay and invasion assay.The expression of cell cycle-associated protein and EMT-related protein was detected by Western blot.In addition,apoptosis detection kit detects apoptosis of three groups of cells.RESULTS The expression level of LncRNA BX357664 mRNA was down-regulated in colorectal cancer tissues and colorectal cancer cell lines.HCT116 and HT29 cells were selected for subsequent experiments.Next,the recombinant plasmid expressing BX357664 was successfully constructed,and the mRNA expression level of BX357664 was increased after transfection of HCT116 cells and HT29 cells.The clone formation experiment,MTT assay and cell cycle assay showed no statistical difference between the blank control group and the plasmid empty group(P>0.05),but overexpressed BX357664 plasmid group compared with the blank control group and the plasmid empty group.The ability to clone formation was reduced,cell growth was slowed,and the cycle was blocked.These differences were statistically significant(P<0.05).At the same time,the results of cell scratch test,migration test and invasion experiment showed that compared with the blank control and plasmid empty load,the migration ability,invasion ability and damage repair ability of the overexpressed BX357664 plasmid group were weak,and the difference was statistically significant(P<0.05).).Western blot showed that the expression of cell cycle-associated proteins Cyclin B1,CDC25C and Cyclin D1 in BX357664 plasmid group decreased,while epithelial marker E-Cadherin increased,and interstitial marker N-Cadherin decreased,the difference was statistically significant(P<0.05).At the same time,the apoptosis detection kit showed that overexpression of BX357664 plasmid increased the expression of Caspase3 and Caspase9,but there was no significant difference in the expression of Caspase8,indicating that overexpression of BX357664 promoted apoptosis of colorectal cancer cells through exogenous pathway.Conclusions The mRNA expression level of BX357664 in colorectal cancer tissues and colorectal cancer cell lines COLO205,HCT116 and HT29 is down-regulated.The recombinant plasmid overexpressing BX357664 was successfully constructed.BX357664 inhibited the proliferation and migration of colorectal cancer cells and EMT,and promoted the apoptosis of colorectal cancer cells.This study is divided into two parts.LncRNA HNF1A-AS1 promotes proliferation and migration of colorectal cancer cells and inhibits apoptosis.LncRNA BX357664 inhibits proliferation and migration of colorectal cancer cells and promotes apoptosis.And the EMT mechanism has been elaborated.Two different Lnc RNAs have the opposite effect on colorectal cancer cells,consistent with previous studies.And LncRNA has a bidirectional charcateristic,which promotes and inhibits the development of colorectal cancer cells.Studying two different LncRNAs can further explain the role of LncRNA in tumors,and provide new targets and new therapeutic ideas for clinical treatment of colorectal cancer,and provide a basis for clinical treatment and medical reference. |
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