| Objective To study the role of long chain noncoding RNA GCLC-1(lnc-GCLC-1)in the malignant transformation of human normal liver cell induced by microcystin-LR(MCLR).Methods(1)Real-time quantitative PCR(q RT-PCR)was used to detect the expression of lnc-GCLC-1 in hepatocytes and liver tissues.Nuclear and cytoplasmic components were separated by nuclear/Cytosol Fractionation kit,and the expression of lnc-GCLC-1 in nucleus and cytoplasm was detected by q RT-PCR.(2)The lentivirus expression vector of lnc-GCLC-1 silencing and negative control was constructed,and the best short hairpin RNA(sh RNA)was selected by transient transfection.To Package lentivirus and infect human liver cell lines L02,and use puromycin to screen stable transfected cell line.In order to detect the transfection effect of the transfected cell line,the green fluorescence was observed by fluorescence microscope,and q RT-PCR was used to identify the transfected cell line.(3)The human liver cell lines L02 was set as normal control group(CON group),sh RNA negative control transfection group(sh-NC group),lnc-GCLC-1 silencing group(sh-lnc-GCLC-1 group)and their corresponding MCLR exposure groups(MCLR group,sh-NC + MCLR group and sh-lnc-GCLC-1 + MCLR group,respectively),cultured conventionally and exposed to 10 nmol/L MCLR continuously until the 25 passages.The cells treated and passaged for 10,15 and 25 passages were labeled as P10,P15,P25,respectively.The expression of lnc-GCLC-1 in MCLR induced malignant hepatocytes was detected by q RT-PCR.The ability of cell proliferation was detected by CCK-8 assay.The ability of cell serum-independent growth was assessed by serum-dependent growth test.Cell cycle and apoptosis were detected by flow cytometry.The ability of cell clone formation was detected by colony-formation assay.The anchorage independent growth of cells was evaluated by soft agar assay.The ability of cell migration in vitro was detected by scratch wound-healing motility and Transwell migration assay.The ability of cell invasion in vitro was evaluated by Transwell invasion assay.The ability of cell tumorigenesis in vivo was detected by tumorigenicity assay in nude mice.Results1.The expression of lnc-GCLC-1 in the cells exposed to MCLR for 15 days,30 days and 45 days was significantly lower than that of the corresponding unexposed cells(P<0.05).The expression of lnc-GCLC-1 in HCC cell line was lower than that of normal liver cell lines(P<0.05).The expression of lnc-GCLC-1 in cytoplasm was slightly higher than that of nucleus.The expression of lnc-GCLC-1 in liver cancer tissues was significantly lower than that of normal liver tissues(P = 0.000797).2.The agarose gel electrophoresis of the recombinant plasmid after digestion showed a fragment of about 8 138 bp,which was consistent with the expected result.The positive plasmids identified by electrophoresis after digestion were sequenced,and the results were consistent with the design.After transiently transfected with sh RNA-2 and sh RNA-4,the lnc-GCLC-1expressions were lower than that of sh-NC(P<0.05),but sh RNA-4 decreased more significantly,so sh RNA-4 was the best sh RNA,and was labeled as sh-lnc-GCLC-1.After the target cells were infected by lentivirus,almost all of them had fluorescence after screening with 0.5 μg/ml puromycin for 2 weeks,and the expression level of lnc-GCLC-1 in sh-lnc-GCLC-1 transfected cells was significantly lower than that in sh-NC group(P<0.05).3.The expression of lnc-GCLC-1 in the normal control group,sh-NC group and sh-lnc-GCLC-1 group,which was treated by MCLR,respectively,was lower than that of the corresponding untreated group(P<0.05)。4.The result of CCK-8 assays in the 15 th passage cells showed that from the second day,the proliferation rate of sh-lnc-GCLC-1 + MCLR group was significantly faster than those of sh-lnc-GCLC-1 group,MCLR group and sh-NC + MCLR group(P<0.05);On the 4th day,the growth rate of each treated group was significantly faster than that of the corresponding non-treated group(P<0.05),and the growth rate of sh-lnc-GCLC-1 group was faster than those of CON group and sh-NC group(P<0.05).For the 25 th passage cells,the proliferation rate of each group began to show difference from the first day(P<0.05);The proliferation rate of each exposed group was superior than that of the corresponding unexposed group(P<0.05);The proliferation rate of sh-lnc-GCLC-1 groups were superior than those of CON group and sh-NC group(P<0.05),and the proliferation rate of sh-lnc-GCLC-1 + MCLR group were superior than those of MCLR group and sh-NC + MCLR group(P<0.05).Compared with the 15 th passage cells,the difference of proliferation rate among the 25 th passage cells appeared earlier,the proliferation rate of each group was faster than that of the same group in the 15 th passage cells.5.The result of serum-dependent growth tests in the 25 th passage cells showed that after culturing for 24 h,the growth rate of L02 cells under the culture conditions of 0.5%,1.5% and 4.5% FBS was significantly different in each group(P<0.05);The proliferation rate of each exposed group was superior than that of the corresponding unexposed group(P<0.05);The proliferation rate of sh-lnc-GCLC-1 group under the culture conditions of 1.5% and 4.5% FBS was superior than that of sh-NC group(P<0.05).The proliferation rate of sh-lnc-GCLC-1 + MCLR group were superior than those of MCLR group and sh-NC + MCLR group(P<0.05).After culturing for 48 h,72 h and 96 h,the proliferation rate of L02 cells under the culture conditions of 0.5%,1.5% and4.5% FBS appeared similar regularity.6.The result of cell-cycle assays in the 25 th passage cells showed that the proportion of G0-G1 phase in each exposure group was lower than that in the corresponding non-exposure group(P<0.05);The percentage of G2-M phase in MCLR group and sh-NC + MCLR group were higher than that of the corresponding non-exposure group(P<0.05);The proportion of S phase in sh-lnc-GCLC-1 + MCLR group was higher than that of the sh-lnc-GCLC-1group(P<0.05);The silencing of lnc-GCLC-1 in unexposed cells had no significant effect on cell cycle distribution(P>0.05);Compared with MCLR group and sh-NC + MCLR group,the proportions of G0-G1 phase and G2-M phase in sh-lnc-GCLC-1 + MCLR group decreased(P<0.05),and the proportion of S phase increased(P<0.05).7.The result of cell apoptosis assays in the 25 th passage cells showed that the apoptosis rate of each exposed group was lower than that of the corresponding non-exposed group(P<0.05);The apoptosis rate of lnc-GCLC-1silencing group without being exposed to MCLR did not decrease in L02 cells(P>0.05);However,the apoptosis rate of sh-lnc-GCLC-1 + MCLR group was lower than those of MCLR group and sh-NC + MCLR group(P<0.05).8.The result of colony-formation assays in the 25 th passage cells showed that the colony formation rate of each exposed group was higher than that of the corresponding unexposed group(P<0.05).The colony formation rate of sh-lnc-GCLC-1 groups were higher than those of CON group and sh-NC group(P<0.05).The colony formation rate of sh-lnc-GCLC-1 + MCLR group were higher than those of MCLR group and sh-NC + MCLR group(P<0.05).9.The result of soft agar assays showed that no colony formation was found in each group at P10.Start from P15,colonies could be formed in agar.By the time of P25,each group could form distinct colonies in agar,and the colony formation rate of each exposed group was higher than that of the corresponding unexposed group(P<0.05);The colony formation rate of sh-lnc-GCLC-1 group were higher than those of CON group and sh-NC group(P<0.05),and the colony formation rate of sh-lnc-GCLC-1 + MCLR group were significantly higher than those of MCLR group and sh-NC + MCLR group(P<0.05).10.The result of scratch wound-healing motility assays in the 25 th passage cells showed that the migration area of each exposed group was significantly larger than that of the corresponding non-exposed group(P< 0.05);The migration area of sh-lnc-GCLC-1 group were significantly larger than those of CON group and sh-NC group(P<0.05);In addition,the migration area of sh-lnc-GCLC-1 + MCLR group were significantly larger than those of MCLR group and sh-NC + MCLR group(P<0.05);What’s more,the results of Transwell migration assays were similar to those of scratch wound-healing motility assays.11.The result of Transwell invasion assays in the 25 th passage cells displayed that the number of cells penetrating the chambers in each exposed group was significantly higher than that in the control group(P< 0.05);The number of cells penetrating the chambers in sh-lnc-GCLC-1 group were not significantly larger than those of CON group and sh-NC group(P<0.05);However,the number of cells penetrating the chambers in sh-lnc-GCLC-1 +MCLR group were obviously larger than those of MCLR group and sh-NC +MCLR group(P<0.05).12.The result of tumorigenicity assay in the 25 th passage cells displayed that mungbean-sized nodules were observed in each group four days after subcutaneous inoculation in the neck of nude mice.On the 10 th day,the subcutaneous nodule of CON and sh-NC groups began to decrease slowly,but the growth rate of tumor in other groups did not slow down.From the 13 th day,the growth rate of tumors in each group began to show a significant difference(P<0.05),and the tumor growth rate in each treated group was significantly faster than that in the corresponding untreated group(P<0.05).Furthermore,the tumor growth rate of sh-lnc-GCLC-1 + MCLR group was superior than that of MCLR group and sh-NC + MCLR group(P<0.05).What’s more,the tumor weight of each exposed group was larger than that in the corresponding non-exposed group(P<0.05),and the tumor weight of sh-lnc-GCLC-1 + MCLR group was larger than that of MCLR group and sh-NC + MCLR group(P<0.05).Conclusions1.By using lentivirus transfection method,human liver cell line L02 with stable silencing of lnc-GCLC-1 is successfully constructed for the first time.2.Chronic exposure to MCLR at low dose can induce down-regulation of lnc-GCLC-1 expression,inhibit apoptosis,affect cell cycle,enhance cell proliferation,migration,invasion and tumor formation in vivo,and induce malignant transformation of cells.Silencing of lnc-GCLC-1 promotes MCLR-induced apoptosis,cell cycle progression and malignant trait phenotype,suggesting that lnc-GCLC-1 silencing promotes the malignant transformation of liver cell lines induced by microcystin-LR. |
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