| BackgroundColon cancer is one of the most common malignant tumors in the gastrointestinal tract,ranking third in the global incidence of cancer.With the improvement of living standard,the incidence and mortality of colon cancer in China are increasing year by year,and the age of onset is obviously advanced.At present,surgical resection assisting with chemotherapy and radiotherapy are the most common ways to treat colon cancer,but about half of the patients have postoperative recurrence and metastasis,which are the main causes of death in patients with colon cancer.Therefore,elucidating the molecular mechanism of colon cancer recurrence and metastasis is crucial to improve the prognosis of colon cancer patients.There are a small number of cancer stem cells(CSCs)with self-renewal and multidifferentiation potentials in tumor tissues,which are considered to play key roles in the process of tumor recurrence and metastasis and are the main cause of tumor growth,metastasis and recurrence.It has been confirmed that there are CSCs in colon cancer tissues.Therefore,targeting CSCs is crucial in the treatment of colon cancer.Sohlh2 is a member of the superfamily of basic helix-loop-helix(bhlh)transcription factors,which can bind to the target gene promoters to regulate target gene expression,and participate in cellular behaviors such as cell proliferation,differentiation and migration.It has been confirmed that abnormal expression of bhlh transcription factors is associated with numerous tumorigenesis.Previous experimental results have confirmed that sohlh2 is a novel tumor suppressor.Sohlh2 inhibits proliferation,migration and invasion of breast and colon cancer cells.Preliminary results showed that sohlh2 expression in colon cancer stem cells was significantly down-regulated,suggesting that sohlh2 might be involved in the regulation of proliferation of colon cancer stem cells.In this study,we investigated the role and mechanism of sohlh2 in the amplification of colon cancer stem cells.Wnt/?-catenin signaling pathway regulates cell proliferation and stem cell self-renewal,and its abnormal activation plays a very important role in the development of colon cancer.?-catenin is a key molecule in the Wnt signaling pathway,which can enter the nucleus and bind to TCF/LEF transcription factors to promote the expression of downstream target genes cyclin D1 and c-myc,and contribute to the growth of colon cancer and the maintenance of CSCs.The increase of ?-catenin expression predicts the poor prognosis of colon cancer patients,and interfering with ?-catenin expression can inhibit the progress of colon cancerLong non-coding RNA(LncRNA)plays a key role in tumor invasion and metastasis LncRNA-H19 is one of the earliest reported LncRNAs,which is highly expressed in a variety of tumors and can be used as competitive endogenous RNA to bind miR-141,up-regulate the expression of ?-catenin,activate the downstream signaling pathway and promote the invasion and metastasis of colon cancer cells.Previous laboratory studies confirmed that sohlh2 inhibited Wnt/?-catenin signaling pathway in colon cancer.LncRNA high-throughput sequencing results showed that overexpression of sohlh2 downregulated LncRNA-H19 expression.Therefore,we hypothesized that sohlh2 may be involved in the regulation of expansion of colon cancer stem cells by downregulating LncRNA-H19 transcription and inhibiting Wnt/?-catenin signaling pathway.Methods1.Isolation,culture and identification of colon cancer stem cellsIn the low adhesion petri dishes,human colon cancer cells HCT116,and mouse colon cancer cells CT26 were cultured in serum free medium.After 5-7 days of culture,cell spheres were collected for passage,cryopreservation and resuscitation.Immunofluorescence staining and FACS were performed to identify whether the cells enriched in serum-free medium were colon cancer stem cells2.To explore the effects of Sohlh2 on the expansion of colon cancer stem cellsHCT116 and CT26 cells were cultured in vitro.The sohlh2 overexpression and sohlh2 knock down stable cell lines were conducted by lentivirus transfection.CSC spheres were collected by the serum-free suspension culture method.The effects of sohlh2 on colon cancer stem cell amplification were detected by clonal sphere formation,soft agar colony formation,CCK-8,immunofluorescence,and FACS experimental methods3.To detect the effects of Sohlh2 on CSC marker expressionThe effects of sohlh2 on Oct4,sox2,CD44,CD 133 and Lgr5 expression in colon cancer stem cells were examined by q-PCR and Western-blot assays4.To explore the effect of sohlh2 on subcutaneous tumorigenesis of colon cancer stem cells in vivoEnriched colon cancer stem cells in HCT116 and CT26 cells with ectopic expression and corresponding control cells were injected into the skin of nude mice and BALB/c mice respectively to measure the tumor size regularly and plot the growth curve.After 4 weeks,the subcutaneous tumor was removed,weighed,photographed,and fixed by 4%paraformaldehyde.Immunohistochemical staining was used to detect the expression of Lgr5 and CD 133.The effect of sohlh2 on subcutaneous tumorigenesis of colon cancer stem cells was investigated in vivo.5.To explore whether sohlh2 inhibits Wnt/?-catenin signaling pathway by regulating LncRNA-H19 transcriptionIn colon cancer stem cells and subcutaneous tumor tissues,the effects of sohlh2 on LncRNA-H19,miR-141,?-catenin,c-myc,cyclin D1 expression were detected by q-PCR and Western-blot assays.To explore whether sohlh2 can directly regulate the transcription of LncRNA-H19,ChIP and luciferase report assays were used.LncRNA-H19 overexpression plasmid was transfected into sohlh2 overexpression cell lines to explore whether LncRNA-H19 mediated the effects of sohlh2 on CSC marker expression and activation of Wnt/?-catenin signaling pathway6.To explore the correlation between sohlh2 and Lgr5 and CD133 in clinical colon cancer samplesUsing colon cancer tissue chip,the correlations between sohlh2 and Lgr5,CD 133 expression in colon cancer were analyzed by immunohistochemical stainingResults1.Isolation,culture and identification of colon cancer stem cellsUsing serum-free suspension culture,CSC clone spheres were successfully enriched in human and mouse colon cancer cell lines.CSC clone spheres could be passaged,frozen,and resuscitated.Through q-PCR,immunofluorescence,and FACS assays,it was determined that colon cancer stem cells were successfully enriched through serum-free suspension culture,and expressed CSC markers,Lgr5 and CD 133.2.Sohlh2 inhibits the expansion of colon cancer stem cellsThe results of clone sphere formation and soft agar colony formation experiments showed that the size and number of CSC spheres in the sohlh2 group were significantly smaller than that of the control group,while the size and number of CSC spheres of the knock-down sohlh2 group were significantly larger than that of the control group(p<0.01).The results of immunofluorescence staining showed that overexpression of sohlh2 inhibited the expression of surface markers of colon cancer stem cells,Lgr5 and CD 133,whereas the knockdown of sohlh2 promoted the expression of Lgr5 and CD133.The results of CCK-8 showed that overexpression of sohlh2 inhibited the expansion of colon cancer stem cells,and the results of knockdown sohlh2 were opposite.The results of immunofluorescence staining showed that sohlh2 overexpression inhibited the expression of Lgr5 and CD133,while knockdown of sohlh2,the expression of Lgr5 and CD133 was upregulated.The results of FACS analysis showed that the proportion of Lgr5/CD133 double positive colon cancer stem cells was significantly lower in the sohlh2 group than that of the control group,while the proportion of Lgr5/CD133 double positive colon cancer stem cells in the sohlh2 knockdown group was significantly higher than that in the control group(p<0.01).3.Sohlh2 inhibits the expression of Oct4,sox2,CD44,CD133 and Lgr5 in colon cancer stem cellsThe results of q-PCR and Western-blot showed that sohlh2 overexpression down-regulated the expression of colon CSC markers,Oct4,sox2,CD44,CD133 and Lgr5,while sohlh2 knockdown,the above gene expressions were significantly upregulated compared with the control group4.Sohlh2 inhibits the growth of subcutaneous tumors from colon cancer stem cells in vivoThe results of subcutaneous tumorigenesis in nude and BALB/c mice showed that sohlh2 inhibited the growth of subcutaneous tumors from colon cancer stem cells.The results of immunohistochemical staining showed that the expression of Lgr5 and CD 133 in the sohlh2 group was significantly lower than that in the control group.5.Sohlh2 inhibits Wnt/?-catenin signaling by regulating LncRNA-H19 transcriptionThe results of q-PCR and Western-blot showed that sohlh2 downregulated the expression of ?-catenin and its target genes c-myc,cyclin D1 in colon cancer stem cells and in nude mouse subcutaneous tumor tissues,while knockdown of sohlh2 had the opposite effects.Overexpression of sohlh2 decreased LncRNA-H19 mRNA level and increased miR-141 mRNA level,while knockdown of sohlh2 had the opposite effect.The results of ChIP and luciferase report assays confirmed that sohlh2 could bind to the promoter region of LncRNA-H19 and inhibit its transcription activity,while in sohlh2 knockdown,the results were opposite.After transfection of LncRNA-H19 overexpression plasmid in sohlh2 HCT116 and sohlh2 CT26 cells,the results of q-PCR and Western-blot showed that LncRNA-H19 could partially block the inhibition of sohlh2 on the expression of CSC markers,?-catenin and its target genes in colon cancer stem cells6.Sohlh2 was negatively correlated with the expression of Lgr5 and CD133 in clinical colon cancer samplesThe results of immunohistochemical staining showed that the expression of sohlh2 was negatively correlated with the expression of Lgr5 and CD 133 in colon cancer tissues.Conclusion1.Sohlh2 inhibits the expansion of colon cancer stem cells2.Sohlh2 inhibits the downstream signaling pathway of ?-catenin by downregulating LncRNA-H 19 transcription. |
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