| BackgroundOvarian cancer is the third most common form of disease in the female reproductive system,but has the highest mortality rate.The epithelial ovarian cancer accounts for about 90%and its important clinical features include the hidden incidence,metastasis,high drug resistance and high recurrence,which pose a serious threat to the health of women.Due to the lack of early symptoms,the majority of patients are at the late stage of cancer,and their survival rate and uality of life were greatly affected.So far the clinical treatment of ovarian cancer still faces lots of challenge.Therefore,to explore the mechanism of recurrence,metastasis and drug resistance of ovarian cancer and the new target of early diagnosis and treatment are hot areas of ovarian cancer research.Cancer stem cells(CSCs)are the few tumor cells with self-renewal capacity.The most essential characteristics of CSCs include infinite proliferation,self-renewal,and high tumorigenicity.CSCs plays an important role in the occurrence,invasion and metastasis,recurrence and chemotherapy resistance.Based on the above theories,it is a novel strategy for ovarian cancer treatment to explore the mechanism of development,metastasis and drug resistance from the perspective of ovarian cancer stem cells.Spermatogenesis and oogenesis transcription factor 2 is an important member of the basic helix-loop-helixb(HLH)family and is widely expressed in normal human tissues such as ovary,breast and colon.Sohlh2 expression in several tumor cell lines was significantly lower than that of the normal tissue respectively.Our previous experiments showed that the reduced expression of Sohlh2 in ovarian cancer was negatively correlated with the stage and grade of the tumor.Overexpression of Sohlh2 can inhibit the proliferation,migration and invasion of ovarian cancer cells,and the growth,metastasis of subcutaneous transplanted tumor of nude mice by down-regulating the expression of cyclinDl,mmp9.The above results suggest that Sohlh2,as a tumor suppressor,is involved in the regulation of development of ovarian cancer.Whether it participates in the regulation of the proliferation and differentiation of ovarian cancer stem cells has not been reported.The aberrant activation of Wnt/?-catenin signaling pathway is an important mechanism for the development of ovarian cancer.It has been confirmed that the activation of Wnt/?-catenin signaling pathway in ovarian cancer stem cells could significantly promote the proliferation of CSCs.?-catenin is the key molecule of this signaling pathway.It could be increased in the cytoplasm by the abnormal activated regulation factors.?-catenin nuclear accumulation leads to more combination of?-catenin and TCF/LEF,which promotes the expression of downstream target gene C-myc,cyclinDl and MMP9,and played an important regulatory role in the tumor development,recurrence and metastasis.In our study,ovarian cancer stem cells were enriched in CSC culture medium.Using the methods of gene transfection,RNA interference,FACS,real-time quantitative PCR,Western Blot,immunofluorescence staining,subcutaneous tumor of nude mice,and so on,we detected the role of Sohlh2 in the proliferation and differentiation of ovarian cancer stem cells and the regulation of Wnt/?-catenin signaling pathway in CSCs.Methods1.To detect the effects of Sohlh2 on the differentiation of epithelial ovarian cancer stem cells(1)The effects of Sohlh2 on the sternness of epithelial ovarian cancer stem cells.Gene transfection and RNA interference technique were performed to establish stable Sohlh2 overexpression or knockdown ovarian cancer cell line(HO8910 and Skov3).H08910 cells were divided into four groups ? H08910 Con group;?HO8910 P-Sohlh2 group;?HO8910 CSC Con group;?HO8910 CSC P-Sohlh2 group;Skov3 cells were divided into four groups?Skov3 Si-Con group;?Skov3 Si-Sohlh2 group;?Skov3 CSC Si-Con group;?CSC Si-Sohlh2 group.Effects of Sohlh2 on the differentiation of ovarian cancer stem cells was tested by stem cell clone formation,FACS and cisplatin resistance.(2)To detect the effect of Sohlh2 on the expression of molecular markers of epithelial ovarian stem cellsUsing immunofluorescence,Real-time fluorescence quantitative PCR and Western Blot technique,stem cell markers CD44,SOX2 expression at mRNA and protein levels were analyzed in four groups of HO8910 cells(Con,P-Sohlh2,CSC Con,CSC P-Sohlh2)and four groups of Skov3 cells(Si-Con,Si-Sohlh2,CSC Si-Con,CSC Si-Sohlh2).2.To detect the effects of Sohlh2 on the growth of subcutaneous tumor in nude miceThe effect of Sohlh2 on the differentiation of ovarian cancer stem cells in vivo was detected by subcutaneous tumor of nude mice.The HO8910 cells(CSC Con,CSC P-Sohlh2)were injected into different subcutaneous parts of the same nude mouse respectively.The growth of subcutaneous tumor was observed regularly and the size of tumor mass was recorded.The tumor weight was checked at the end of the experiment.Immunohistochemical staining and Western Blot analysis were used to detect the expression of CD44 and SOX2 in subcutaneous tumors.3.To explore the mechanism of Sohlh2 on CSC differentiation in ovarian cancer(1)The effects of sohlh2 on the expression of ?-catenin,C-myc,and cyclinDl in the HO8910 cells and Skov3 cells were detected by Western Blot analysis.(2)Immunohistochemistry and Western Blot technique were used to detect the regulation of sohlh2 on the expression of P-catenin,C-myc and cyclinDl at the protein level in the subcutaneous tumor of nude mice.(3)After 24h of addition of Wnt/p-catenin signal pathway agonist(B4818)and antagonist(A8217),the protein expression levels of C-myc,CD44 and SOX2 were analyzed by Western Blot.4.To detect the correlation of Sohlh2,SOX2,?-catenin and C-myc in ovarian cancer tissuesThe correlation of Sohlh2,SOX2,?-catenin,C-myc expression were analyzed in the ovarian cancer tissue microarray by immunohistochemical staining.Results1.(1)The results of clone formation of stem cells showed,the number and size of CSC colonies in CSC P-Sohlh2 group were significantly smaller than that of CSC Con group;while the number and size of CSC colonies in CSC Si-Sohlh2 group were significantly larger than the CSC Si-Con group.The results of FACS analysis showed the percentage of CSCs in CSC P-Sohlh2 group was significantly lower than that in CSC Con group.MTT results showed that after 48h treatment at different concentrations of cisplatin,the OD value of HO8910 P-Sohlh2 group was significantly less than that of Con group(P<0.05),and the OD value of Skov3 Si-Sohlh2 group was greater than that of Si-Con group(P<0.05).(2)Real-time quantitative PCR results showed that the mRNA levels of CD44,SOX2 and OCT4 in the HO8910 P-Sohlh2 group were significantly lower than those in the Con group;Compared with the CSC Con group,the nRNA levels of CD44,SOX2 and OCT4 in HO8910 CSC P-Sohlh2 group were also significantly reduced.In the Sohlh2 knockdown Skov3 cells,the mRNA levels of CD44,SOX2 and OCT4 in the Si-Sohlh2 group and CSC Si-Sohlh2 group were significantly higher than those in the Control group,respectively.The results of immunofluorescent staining showed that the fluorescent intensity of CD44 of the HO8910 CSC P-Sohlh2 group was less than that of CSC Con group.While the fluorescent intensity of Skov3 CSC Si-Sohlh2 group was stronger than that of CSC Si-Con group.The results of Western Blot analysis showed that compared with the Con group,the protein level of CD44 and SOX2 in the HO8910 P-Sohlh2 group was decreased,and the protein levels of CD44 and SOX2 in the HO8910 CSC P-Sohlh2group were also decreased compared with the CSC Con cells.Sohlh2 knockdown in Skov3 cells got the opposite results.2.The effect of sohlh2 on the differentiation of ovarian CSCs was further confirmed by the experiment of subcutaneous tumor in nude mice.Sohlh2 overexpression in ovarian CSCs inhibited the growth of subcutaneous tumor in nude mice.The results of Western Blot and immunohistochemistry staining showed that the protein levels of CD44 and SOX2 in the tumor tissues of CSC P-Sohlh2 group was significantly decreased.The above results suggest that Sohlh2 could inhibit the proliferation of ovarian CSCs and promotes CSC differentiation in vivo.3.Overexpression of Sohlh2 downregulated the protein expression of the target genes of the Wnt/?-catenin signaling pathway,C-myc and cyclinDl,in ovarian cancer cells lines and subcutaneous tumor tissues in nude mice.On the contrary,the expression of C-myc and cyclinDl was significantly increased at the protein level after the knockdown of Sohlh2.Add Wnt/p-catenin signaling pathway agonist(B4818)or antagonist(A8217)into cultured ovarian CSCs,the results of Western Blot showed that the regulation of Sohlh2 to the expression of C-myc,CD44 and SOX2 genes was mediated by Wnt/?-catenin signaling pathway.4.The immunohistochemical results of ovarian cancer tissue showed that the expression of Sohlh2 in was negatively correlated with the expression of SOX2,?-catenin and C-myc.Conclusions1.Sohlh2 promotes the differentiation of ovarian cancer stem cells;2.The roles of Sohlh2 in ovarian cancer stem cells is mediated by inhibiting the activation of Wnt/?-catenin signaling pathway. |
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