| BackgroundThe mortality rate of renal carcinoma ranks second in malignant tumors of the urinary system.A status report of global tumor morbidity and mortality in 2020 showed that the morbidity of renal carcinoma accounted for 2.2%of the new cancer cases,and the mortality of renal carcinoma accounted for 1.8%of the dead cancer patients.Thus,the mortality and morbidity of renal carcinoma were higher.In China,the morbidity of renal carcinoma has increased year by year in recent years,increasing at an average annual rate of 1.5%,and the morbidity is higher in men than women.The kidney is a retroperitoneal organ,and it is difficult to be found in the early stage.Once diagnosed,it has reached the middle and advanced stages,and the five-year survival rate of metastatic renal carcinoma in the middle and advanced stage is only 10%.In addition,radiotherapy and chemotherapy can not inhibit the progression of renal carcinoma,and the effects of cytokine therapy and molecular targeted therapy are also not ideal.Hence,clarification of the mechanism of the genesis and progression of renal carcinoma can not only provide theoretical basis for clinical treatment of renal carcinoma,but also provide new targets for early diagnosis and treatment of renal carcinoma.Klotho is not only an anti-aging protein but also an important tumor suppressor gene.It has a variety of renal protection and anti-renal cancer functions.Klotho is highly expressed in renal tubular epithelial cells,and low Klotho expression in renal cancer tissues is closely related to the poor prognosis of renal cancer.DNA methylation can regulate the expression of Klotho,which is closely related to the occurrence of diseases such as cancer,aging,dementia and other diseases.Relevant literature has confirmed that DNA methyltransferase 1 and 3a inhibit Klotho expression in the kidney by up-regulating methylation of Klotho promoter.DNMT1 and DNMT3a are highly expressed in renal cancer tissues.Knockdown of DNMT1/DNMT3a in renal carcinoma and overexpression of Klotho may effectively inhibit the malignant behavior of renal carcinoma.Our previous experimental results showed that the expression of DNMT3a was the highest in renal carcinoma,so it was selected as one of the research targets.Sohlh2 is a key member of the basic-loop-helix transcription factor family.It can bind to the conserved E-box sequence in the promoter region of the target gene and regulate the expression of the target gene.Furthermore,it is involved in the regulation of various cell behaviors such as cell proliferation,migration,invasion and metastasis.We have previously confirmed that sohlh2 is a new tumor suppressor gene that can significantly inhibit the proliferation,migration,invasion and metastasis of breast cancer,ovarian cancer cells.Our previous experimental results showed that sohlh2 and Klotho expression were positively correlated in renal cancer tissues,indicating that sohlh2 may play a tumor-suppressive role by up-regulating Klotho expression in renal cancer.In addition,sohlh2 could significantly inhibit the transcription of DNMT3 a.In summary,we came up with a hypothesis that sohlh2 up-regulated Klotho by down-regulating the expression of DNMT3a,and functioned as a tumor suppressor in renal carcinoma.Methods1.To explore the expression of sohlh2 in renal carcinoma tissuesSohlh2 expression was detected in 85 cases of clinical renal cancer specimens,including 5 cases of adjacent noncancerous tissues,6 cases of grading 1 renal cancer tissues,66 cases of grading 2 renal cancer tissues,and 8 cases of grading 3 renal cancer tissues.Immunohistochemistry was used to analyze the correlation between sohlh2,the grading,and clinical features of renal carcinoma patients2.To explore the role of sohlh2 in proliferation,EMT,migration,and invasion of renal carcinoma cells in vitroThree human-derived renal cancer cell lines(ACHN,A498,786-0)were selected to construct stable transgenic cell lines with sohlh2 silencing and sohlh2 overexpression by lentiviral transfection,which were divided into the following groups:A:ACHN con B:ACHN sohlh2 C:786-O con D:786-O sohlh2 E:A498 si-Con F:A498 si-Sohlh2-1/2.Colony formation assay,CCK-8 proliferation assay,qPCR,western-blot,wound healing assay,Transwell migration assay and Matrigel invasion assay were performed to explore the role of sohlh2 in the proliferation,EMT,migration and invasion of renal carcinoma cells in vitro.3.To explore the role of sohlh2 in subcutaneous tumor growth and tumor metastasis in nude mice by in vivo experimentsTwelve nude mice were purchased,and six in each group.Every nude mouse was injected subcutaneously with 2×106 stable sohlh2 overexpression or sohlh2 silencing ACHN cells.After 8 weeks(the tumor volume ≤1,000 mm3),mice were euthanized,and the subcutaneous tumors were removed,photographed and weighed.qPCR,western-blot and immunohistochemical analysis were performed to explore the correlation of sohlh2 with DNMT3a and Klotho.Another 10 male nude mice were divided into two groups and raised at Specific Pathogen Free(SPF)laboratory animal room.5×105 cells were injected via tail veins.Four weeks later,mice should be euthanized,and livers and lungs were taken out.The effects of sohlh2 on tumor metastasis were detected by the counting of metastases and HE staining.4.To explore whether sohlh2 exerts tumor-suppressive effect through DNMT3a/KlothoFirstly,qPCR and western blot were used to detect the expression of sohlh2,DNMT3a and Klotho in sohlh2 overexpression and sohlh2 silencing stable renal cancer cell lines.DNMT3a overexpression plasmid or Klotho silencing plasmid was transfected into sohlh2 overexpression cell line,DNMT3a inhibitor or Klotho overexpression plasmid was added or transfected into the sohlh2 silencing cell line.Western-blot,CCK-8,Transwell and Matrigel assays were performed to detect whether sohlh2 played a role through DNMT3a/Klotho.Finally,the mRNA and protein levels were used to detect whether Klotho has a regulatory effect on sohlh25.To explore the correlation of sohlh2,Klotho and DNMT3a in renal carcinoma tissues85 clinical renal cancer tissues were selected to analyze the correlation between sohlh2,DNMT3a and Klotho expression by immunohistochemical stainingResults1.Sohlh2 is lowly expressed in renal carcinoma tissuesThe results of immunohistochemistry showed that the expression of sohlh2 in renal carcinoma was significantly lower than that in adjacent tissues.At the same time,the analysis of sohlh2 and clinical features showed that sohlh2 was negatively correlated with the grading of renal carcinoma,but there was no significant difference between sohlh2 with age and sex.2.Sohlh2 inhibits renal carcinoma cell proliferation,EMT,migration and invasionThe results of colony formation assay and CCK-8 proliferation assay showed that overexpression of sohlh2 could inhibit the proliferation of renal cancer cells,and knockdown of sohlh2 could promote the proliferation.The results of qPCR and western-blot showed that overexpression of sohlh2 inhibited the metastasis of renal carcinoma.Sohlh2 overexpression increased the expression of epithelial cell markers(E-cadherin,ZO-1,Claudin)but slowed down the expression of mesenchymal cell markers(ZEB1,Fibronectin,Vimentin).The result of sohlh2 silencing group was contrary to the above.Wounding healing assay showed that sohlh2 overexpression decreased the closure of wound compared with the control group,and sohlh2 silencing gained the contrary result.It was also verified by Transwell migration assay.These data indicated that sohlh2 inhibited the migration of renal carcinoma cells in vitroThe result of invasion assay showed that overexpression of sohlh2 decreased the number of ACHN and 786-O cells passing through the matrigel,while sohlh2 silencing promoted the capacities of invasion3.1 Sohlh2 inhibits the growth of subcutaneously implanted tumor in nude miceFor the model of subcutaneous tumor formation,compared with the control group,the tumor volume and weight of sohlh2 overexpression group were smaller,indicating that the proliferation rate of tumor cells was lower and sohlh2 inhibited the growth of tumor.Western-blot and immunohistochemical staining results showed that sohlh2 overexpression inhibited the expression of Ki67 compared with the control group.qPCR results also showed that the expression of DNMT3a in sohlh2 overexpression group was significantly lower than that in control group,while Klotho expression was increased.The results of western blot and immunohistochemistry were consistent with qPCR’.3.2 Sohlh2 inhibits EMT and metastasis of renal carcinoma in vivoThe results of qPCR,western-blot and immunohistochemical staining showed that sohlh2 overexpression inhibited the expression of mesenchymal markers in EMT.The results of the counting of metastases in liver and lung showed that sohlh2 overexpression decreased the number of metastases in livers and lungs compared to the ACHN control group,and the statistical analysis had significant differences.Moreover,these results were verified by the HE staining again.These results suggest that sohlh2 can inhibit the metastasis of renal carcinoma.4.Sohlh2 exerts tumor-suppressive effects by regulating the expression of DNMT3a and KlothoqPCR and western-blot results showed that the mRNA and protein of sohlh2 had no change,indicating that DNMT3a did not regulate the expression of sohlh2,but the reduction of Klotho suggested that Klotho was regulated by DNMT3a.In addition,it showed that DNMT3a could block the effect of sohlh2 on Klotho expression.CCK-8 assay indicated that Klotho knockdown could partially block the inhibition of sohlh2 on the proliferation of renal carcinoma cells.Transwell migration and Matrigel invasion assays showed that Klotho could mediate sohlh2 to inhibit the migration and invasion of renal carcinoma cells.Then,we further study whether Klotho plays a regulatory role on sohlh2,qPCR and western-blot showed that Klotho overexpression did not regulate the expression of sohlh2,and the conclusion of the Klotho knockdown group was consistent with the above.These data strongly confirmed that sohlh2 played a role in tumor inhibition by regulating the expression of DNMT3a and Klotho.5.Sohlh2 was positively correlated with Klotho and negatively correlated with DNMT3aImmunohistochemical staining results showed that the expression of Klotho was lower and DNMT3a expression was higher when sohlh2 expression was lowly in renal carcinoma tissues.But Klotho was significantly overexpressed in sohlh2 overexpressed renal cancer tissues,and DNMT3a was significantly underexpressed.Image analysis and statistical analysis results showed that sohlh2 was positively correlated with Klotho and negatively correlated with DNMT3a.Conclusion1.Sohlh2 inhibits the proliferation,EMT,migration,invasion and metastasis of renal carcinoma cells.2.Sohlh2 up-regulates Klotho by down-regulating the expression of DNMT3a,and functions as a tumor suppressor in renal carcinoma. |
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