| Backgrounds and objectivesColorectal carcinoma(CRC)is one of the most common malignancies and remains the third leading cause of cancer-related death worldwide,and about one million of people are diagnosed with this fatal disease each year.In our country,with the increase of aging population and carcinogens,there is an upward trend in incidence of CRC.Although much progress has been made,the early diagnosis,high recurrence and poor survival rate of CRC remain problems to be solved.Therefore,it is of great importance to clarify the molecular mechanisms of CRC and to discover more effective methods for the diagnosis and treatment.B cell CLL/lymphoma 6 member B(BCL6B),a Bcl6-homologous gene,is located on chromosome 17p13.1,it is expressed ubiquitously in normal human tissues while is frequently mutated or deleted in human carcinomas;BCL6B is silenced or down-regulated in a variety of tumors due to its promoter DNA hypermethylation,including gastric cancer,hepatocellular carcinoma and CRC,and the methylation level of its promoter region is positively correlated with tumor malignancy degree and poor prognosis;in CRC,the reduced expression of BCL6 B is associated with tumor TNM stage and lymph node metastasis,and restoration of BCL6 B expression suppresses CRC growth by activating p53 signaling.All of these suggest that BCL6 B may be a tumor suppressor.However,the function of BCL6 B in tumor development is largely unknown.In this study,we investigated the expression,as well as the biological role and the underlying molecular mechanisms of BCL6 B in CRC cell lines SW480 and Lo Vo.Our work provided substantial evidence for elucidating the role and the mechanism of BCL6 B in CRC development and provided a new clue for the diagnosis and treatment of CRC.Methods1.Detection the endogenous expression of BC6 B in human normal intestinal epithelial cell line and CRC cell lines: RT-PCR,q PCR and Western blot were used to detect the expression of BC6 B in human normal intestinal epithelial cell line FHC and two CRC cell lines SW480 and Lo Vo.2.Amplification and identification of pc DNA3.1-BCL6B: The recombinant plasmids carrying the coding sequence of human BCL6 B gene(pc DNA3.1-BCL6B)and the control recombinant plasmids(pc DNA3.1)were transformed into E.coli DH5? bacteria by calcium chloride,11 respectively,then cultivated in LB medium for amplification.Finally,recombinant plasmids were extracted,quantified by spectrophotometry and stored at-20?C.pc DNA3.1-BCL6 B was used to transfect CRC cells lines SW480 and Lo Vo while pc DNA3.1 was used as a negative control,and the transfection efficiency of pc DNA3.1-BCL6 B was identified by RT-PCR and Western blot.3.The role of BCL6 B on proliferation of SW480 and Lo Vo cells: after transfecting SW480 and Lo Vo cells with pc DNA3.1-BCL6 B,the MTT assay and colony formation assay were used to detect cell proliferation.4.The role of BCL6 B on cell cycle and apoptosis of SW480 and Lo Vo cells: flow cytometry(FCM)assay was employed to detect cell cycle and apoptosis of SW480 and Lo Vo cells after transfection with pc DNA3.1-BCL6 B.5.The role of BCL6 B on migration and invasion of SW480 and Lo Vo cells: wound closure assay and Transwell assay were used to detect migration and invasion of SW480 and Lo Vo cells after transfection with pc DNA3.1-BCL6 B.6.The effect of BCL6 B on the PI3K/AKT signaling pathway in SW480 and Lo Vo cells: Western blot was used to detect the expression of p-AKT in SW480 and Lo Vo cells treated with pc DNA3.1-BCL6 B,and RT-PCR and Western blot were used to detect the levels of Cyclin D1,E-cadherin and MMP-9,which are the downstream targets of PI3K/AKT signaling pathway.7.The effect of the PI3K/AKT signaling pathway on BCL6 B inhibited proliferation and migration of Lo Vo cells: after Lo Vo cells were treated with pc DNA3.1-BCL6 B and PI3 K inhibitor LY294002,the MTT assay and the Transwell assay were applied to detect cell proliferation and migration,and Western blot was used to detect the expression of p-AKT,Cyclin D1,E-cadherin and MMP-9.Results1.q RT-PCR results showed the relative expression of BCL6 B m RNA in FHC,SW480 and Lo Vo cells were 0.93 ± 0.60,0.04 ± 0.05 and 0.06 ± 0.04,respectively;Western blot results showed the relative gray values of BCL6 B protein in FHC,SW480 and Lo Vo cells were 0.74 ± 0.20,0.06 ± 0.04 and 0.10 ± 0.03,respectively.Compared with normal intestinal epithelial cell line FHC,the expression of BCL6 B was notably decreased in CRC cell lines SW480 and Lo Vo.2.After transfection with pc DNA3.1-BCL6 B for 48 h,the relative gray values of BCL6 B m RNA and protein in SW480 cells were 16.7 times(P<0.01)and 10.5 times(P<0.01)as much those of its control group,respectively;the relative gray values of BCL6 B m RNA and protein in Lo Vo cells were 12.7 times(P<0.01)and 7.3 times(P<0.05)as much those of its control group,respectively;suggesting pc DNA3.1-BCL6 B had a relative high efficiency of transfection and expression in CRC SW480 and Lo Vo cells,which can be used for our further research.3.BCL6 B inhibited proliferation of SW480 and Lo Vo cells.1)MTT assay showed: The difference of OD values between the BCL6 B group and the control group of SW480 and Lo Vo cells was not obvious at 1d and 2d(P>0.05).At 3d,the OD values of the BCL6B-SW480 group and BCL6B-Lo Vo group were 65.6%(P<0.05)and 70.7%(P<0.05)of that of the control group,respectively;At 4d,the OD values of the BCL6B-SW480 group and BCL6B-Lo Vo group were 61.2%(P<0.05)and 52.7%(P<0.01)of that of the control group,respectively.2)Colony formation assay showed: The colony numbers of the BCL6B-SW480 group and BCL6B-Lo Vo group were 20.3%(P<0.01)and 42.2%(P<0.05)of that of the control group,respectively.4.BCL6 B induced cell cycle arrest and promoted apoptosis of SW480 and Lo Vo cells.FCM analysis showed: The percentage of cells in G1 phase of the BCL6B-SW480 group and BCL6B-Lo Vo group were 1.63 times(P<0.01)and 1.67 times(P<0.01)of that of the control group,respectively.And the percentage of cells in S phase of the BCL6B-SW480 group and BCL6B-Lo Vo group were 61.1%(P<0.01)and 67.9%(P<0.05)of that of the control group,respectively.Meanwhile,the apoptotic rates were increased by 0.75 times(P<0.05)and 1.2 times(P<0.01)in the BCL6B-SW480 group and BCL6B-Lo Vo group,respectively.5.BCL6 B suppressed migration and invasion of SW480 and Lo Vo cells.1)Wound closure assay showed: At 48 h,the wound closure rates of the BCL6B-SW480 group and BCL6B-Lo Vo group were 61.1%(P<0.05)and 55.6%(P<0.05)of that of the control group,respectively;At 72 h,the wound closure rates of the BCL6B-SW480 group and BCL6B-Lo Vo group were 68.8%(P<0.05)and 63.9%(P<0.05)of that of the control group,respectively.2)Transwell assay showed: In the BCL6 B group,the invasive number of SW480 and Lo Vo cells were 177 ± 40 and 143 ± 31,respectively;in the control group,the invasive number of SW480 and Lo Vo cells were 69 ± 37 and 48 ± 24,respectively.The number of invasive cells in the BCL6 B group was much less than that of the control group,the difference has statistical significance(P<0.05).6.In pc DNA3.1-BCL6B-transfected SW480 and Lo Vo cells: 1)The phosphorylation level of AKT was decreased by 67.7%(P<0.05)and 45.2%(P<0.05),respectively.2)The m RNA levels of Cyclin D1,E-cadherin and MMP-9,which are the downstream target genes of the PI3K/AKT signaling pathway,were 63.3%(P<0.05),2.54 times(P<0.05)and 29.4%(P<0.05)of those in the control group in SW480 cells,respectively;the m RNA levels of these three genes were 32.2%(P<0.01),3.32 times(P<0.05)and 51.8%(P<0.05)of those in the control group in Lo Vo cells,respectively;3)Consistent with the above results,the protein levels of Cyclin D1,E-cadherin and MMP-9 were 54.4%(P<0.05),2.05 times(P<0.05)and 50.6%(P<0.05)of those in the control group in SW480 cells,15 respectively;the protein levels of these three genes were 18.9%(P<0.01),2.04 times(P<0.05)and 46.4%(P<0.01)of those in the control group in Lo Vo cells,respectively.These results suggested BCL6 B can upregulate E-cadherin expression and down-regulate Cyclin D1 and MMP-9 expression through inhibition of the PI3K/AKT pathway in CRC cells lines SW480 and Lo Vo,and therefore inhibit the proliferation and migration of CRC cells.7.The suppressive effect of BCL6 B on viability and migration of Lo Vo cells was notably aggravated by PI3K/AKT inhibitor LY294002,which were evaluated by MTT assay(P<0.05)and Transwell assay(P<0.05),respectively.At the same time,the increased expression of E-caherin and the decreased expression of p-AKT,Cyclin D1 and MMP-9 induced by BCL6 B in Lo Vo cells were enhanced by LY294002(P<0.05).These data suggested the inhibition of the PI3K/AKT pathway is involved in BCL6B-induced proliferation and migration suppression of CRC cells.Conclusion1.BCL6 B expression in CRC cell lines SW480 and Lo Vo was much lower than that in human normal intestinal epithelial cell line FHC.2.BCL6 B promotes apoptosis and suppresses proliferation,migration and invasion of SW480 and Lo Vo cells.3.BCL6 B inhibits the PI3K/AKT signaling pathway and upregulates E-cadherin expression and downregulates Cyclin D1 and MMP-9 expression in SW480 and Lo Vo cells.4.The inhibition of the PI3K/AKT signaling pathway by BCL6 B is involved in the roles of BCL6 B on CRC Lo Vo cells,including suppressing cell proliferation and migration,upregulating E-cadherin and downregulating Cyclin D1 and MMP-9. |
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