Sohlh2 Suppresses Colon Cancer Cell Proliferation, Migration, Invasion And Metastasis Via Inhibiting Wnt/β-catenin Pathway

Background and Significance Colon cancer is one of the most common malignant cancer wordwide.In the developed countries, like America, the incidence and mortality of colon cancer is ranked in the third status. In recent years, our economic has developed a lot, and communication with Western countries in culture kept increasing, and lifestyle and habits happened changing, which leads to obvious growing of the incidence of colon cancer.Colon cancer develops by synergistic effect of various factors, which decides troubles in prevention.However, the progress is relative slow, the cure rate in early stage is very high, and 5 years survival rate surpasses 90%.In the base of that, implement study for colon cancer to explore more information on mechanisms, to seek for more and better screening and diagnosis methods, has necessary significance for early diagnosis, timely therapy, improvement survival.Activation of Wnt/β-catenin signaling promotes cell proliferation and growth, which plays critical roles in the regulation of initiation and progress of various cancers, including colon cancer. Over 90% colon cancer patients have mutations in the key regulator genes of the pathway. These mutations can contribute to β-catenin nuclear accumulation. β-catenin entering nuclear combines with TCF/LEF transcription factors, which causes expression of target genes, such as c-myc,cyclinDl. The final objects are to promote cell proliferation and control characteristics of the malignant phenotype.Sohlh2 (Spermatogenesis and oogenesis basic helix-loop-helix (bHLH) transcription factor 2) is a member of bHLH transcription factor, regulating reproduction cells’differentiation in mouse. Our study shows that, Sohlh2 expresses at a high level in human normal tissues, but a little in some tumors. Sohlh2 inhibits ovarian cancer cell proliferation by upregulation of p21 and downregulation of cyclinDl. Sohlh2 inhibits human ovarian cancer cell invasion and metastasis by transcriptional inactivation of MMP9.The role of Sohlh2 in colon cancer has not investigated.Our study is to explore the expression and role of Sohlh2 in colon cancer. MTT, colony formation assay, BrdU, FACS, wound healing, Transwell, Matrigel, mouse xenograft model and experimental metastasis, real-time quantitative PCR, Western Blot, immunohistochemistry, immunofluorescence and transfections are performed to explore the effects of Sohlh2 in colon on proliferation, migration, invasion and metastasis and its mechanism.Our results imply that Sohlh2 might be a new target in diagnosis and therapy of colon cancer.Methods1. To detect Sohlh2 expression in human normal colon tissues and colon cancer tissues.Examples from clinical colon cancer patients in different grades were performed immunohistochemistry staining to observe and analyze Sohlh2 expression in human normal colon tissues and different grades’ colon cancer tissues.2. To detect the role of Sohlh2 on proliferation in vitroUsing transfection, LoVo cells were divided into four groups:Con group; Sohlh2 overexpression group(P-Sohlh2 group); si-Con group; si-Sohlh2 group. The first two groups were transfected with plasmids without or with Sohlh2 cDNA.800μg/mlG418 were needed to screen for two weeks. Similarly, the last two groups were screen with 3μg/ml pd for two weeks after transfected control siRNA or Sohlh2 siRNA. MTT,Colony formation assay,BrdU,FACS were performed to explore proliferation of four groups.3. To exam the function of Sohlh2 for colon cancer on migration, invasion in vitroUsing wound healing,Transwell and Matrigel exam the difference between four groups in the capacity of migration,invasion.4. To explore the effect of Sohlh2 in LoVo in vivoFour group cells were subcutaneously injected into both sides of the dorsal thigh of five male BALB/C nude mice in each group. Tumor size was monitored twice a week for 4 weeks and tumor weight was measured at the end of the experiment. Tumors were fixed in formalin and embedded in paraffin, and 5μm sections were stained with immunohistochemistry.Cells were injected into tail veins of 6-8 week-old male BALB/c nude mice. Eight weeks after injection, mice were euthanized; the lungs and livers were removed. Lungs and livers were fixed in formalin and embedded in paraffin, and 5μm sections were stained with H&E.5. To explore the regulation of Sohlh2 for Wnt/β-catenin signaling.(1)Performing real-time quantitative PCR to detect expression of c-myc, cyclinD1, mmp9, lgr5 in mRNA level. Western Blot and immunofluorescence were done to exam expression of c-myc,cyclin D1 in protein level.(2)To decide the relations of Sohlh2 and β-catenin by Western Blot, immunohischemistry and immunofluorescence.(3)Licl activates Wnt/β-catenin signaling pathway. Con group and Sohlh2 over-expression group added Licl lOmM.Cells were harvested after 24 hours. Employing real-time quantitative PCR, immunofluorescence and Western Blot was to detect expression of c-myc, cyclinD1, mmp9 and lgr5 just qPCR.And BrdU was also performed to reflect proliferation conditions.Results1. Immunohistochemistry results revealed that the expression of Sohlh2 in colon cancer was lower than in normal colon tissues,the difference was significant (P<0.05),and reduced in worse differentiated grade. Sohlh2 immunostaining was both in nuclear and cytoplasm.2. Sohlh2 overexpression inhibited the proliferation of LoVo cells over a 4 day period. Conversely, Sohlh2 ablation by siRNA in LoVo cells promoted cell proliferation. Sohlh2 overexpression in LoVo cells led to a significant decrease in the number of colonies after fourteen days culture, while Sohlh2 knockdown significantly increased the number of colonies. Moreover, BrdU assay revealed that Sohlh2 overexpression dramatically attenuated the LoVo cell proliferation, as evidenced by less BrdU-positive cells than that of vector control. Sohlh2 knockdown resulted in more BrdU-positive cells than that of vector control. These results indicate that Sohlh2 suppresses the proliferation of colon cancer cells.We tested whether overexpression of Sohlh2 resulted in cell cycle arrest by flow cytometry analysis. Sohlh2 overexpression increased the fraction of cells in the G0/G1 phase compared with control cells.in a reciprocal experiment; Sohlh2 knockdown decreased the proportion of cells in G0/G1 phase.3. The results showed that Sohlh2 overexpression decreased the migrated distance.The numbers of migration and invasive cells of Sohlh2 overexpressing LoVo cells were significantly reduced compared with those in the control group. We found that induction of Sohlh2 expression led to a decrease in the cell migration and invasiveness. In contrast, Sohlh2 reduction in LoVo cells increased the migrated distance, the numbers of migration and invasive cells of Sohlh2 knockdown LoVo cells were significantly increased compared with those in the control group.4. Sohlh2 overexpression significantly inhibited tumor growth in the course of the Mouse xenograft model experiment. Consistent with this finding, the tumor weight in P-Sohlh2-injected mice was significantly less than that in pEGFP-injected mice. Sohlh2 overexpression resulted in reduction in the lung metastasis.5. Sohlh2 overexpression inhibited the expression of c-myc,cyclinD1,in the level of mRNA or protein. The expression of β-catenin in nuclear and Sohlh2 showed a contrary tendency.Furthermore,Sohlh2 suppressed the activation of Wnt/β-catenin resulting from Licl.Conclusion1. Sohlh2 inhibits cell proliferation,migration,invasion and metastasis of colon cancer.2. The suppression of Sohlh2 for colon cancer is performed by inhibiting Wnt/β-catenin signaling pathway.