Mechanism Of MYCT1 In Influencing The Proliferation And Invasion Of Type Ⅰ Endometrial Cancer By Regulating Preadipocyte Differentiation

Introduction:Endometrial cancer（EC）is the second most common malignancy of the female reproductive system in China and the first in developed countries.In recent years,the incidence of EC has been on the rise in China due to some unhealthy lifestyles including a high-fat,high-heat diet,a low-exercise and etc.Direct spread and invasion into the myometrium are the main metastatic routes of EC and a difficult focus of diagnosis and treatment.In recent years,the targeted therapy and immunotherapy have shown better efficacy in advanced and recurrent metastatic endometrial cancer,but the specific molecular targets of EC are still unclear.Therefore,research into the molecular mechanisms of endometrial cancer could help to identify specific molecular targets to improve the diagnostic and therapeutic levels.Risk factors for EC include persistent estrogen exposure,estrogen replacement therapy without progestin protection,and metabolic abnormalities（e.g.obesity,diabetes）.85%of EC is type I endometrial cancer,also known as estrogen-dependent.Type I endometrial cancer are usually occur in postmenopausal women and associated with obesity.The conversion of androgens to estrogen by aromatase in adipose tissue is thought to be the main source of estrogen in postmenopausal women,and sustained stimulation by estrogen can induce the endometrium cancer.Therefore,it is very important to study the molecular mechanism of EC around estrogen.MYCT1（Myc target 1）is the first gene identified and cloned by our group in laryngeal cancer.It has been shown that MYCT1 functions as an oncogene or suppressor in different tumors,suggesting that it may be involved in tumor development through different signaling pathways.In order to investigate the molecular mechanism of MYCT1 in laryngeal cancer,we constructed Myct1 knockout mice using CRISPR-Cas9 technology and unexpectedly found that the Myct1 knockout mice has abnormal lipid metabolism which indicates that body weight and body fat rate increase in the knockout mice.We hence hypothesized that MYCT1 is associated with lipid metabolism.Previously,we also obtained MYCT1-interacted protein-mass spectrometry profiling by immunoprecipitation against mouse liver tissue and found that acetyl-Coenzyme A acyltransferase 2（Acaa2）is one of potential MYCT1-interated proteins in wild-type mouse liver tissue.ACAA2 is involved in fatty acidβ-oxidation,mainly in mitochondria,participates in fatty acid degradation and elongation by catalyzing the last step of fatty acidβ-oxidation metabolism process,and is an important regulator of lipid metabolism.Obesity occurs as a result of the growth of old adipocytes,and the proliferation and differentiation of new adipocytes.Studies have shown that ACAA2 promotes preadipocyte differentiation in mice and sheep,and functions through fatty acid metabolic pathways in a variety of tumors.We therefore hypothesize that MYCT1 regulates adipocyte differentiation by interacting with ACAA2.Cathepsin D（CTSD）,one of the differentially expressed genes downstream of MYCT1identified by single-cell sequencing in mouse liver,is a direct target gene of estrogen and associated with poor prognosis in breast cancer.Studies have shown that CTSD promotes cancer cell proliferation invasion and metastasis in a variety of tumors including lung,glioma,liver,and renal clear cell carcinoma,but its specific function and exact mechanism in endometrial cancer are still unclear.Therefore,we hypothesized that MYCT1 regulates preadipocyte differentiation by interacting with ACAA2,then affects aromatase production,leading to changes in the conversion of testosterone to estradiol,which in turn directly modulates the expression of the estrogen target gene CTSD through the estrogen receptor,finally affecting the proliferation and invasive abilities of endometrial cancer cells.In this study,we intend to apply relevant molecular biological techniques to detect the regulatory effect of MYCT1 on preadipocyte differentiation,thus affecting the proliferation and invasion function of type I EC and to detect theregulatory effect and molecular mechanism of MYCT1 on downstream target genes,so as to clarify the role of MYCT1 and its related signaling pathways in EC.This study will provide an important basis for further study of MYCT1 as a molecular target for diagnosis and treatment of EC.Materials and methods:1.Materials:Human endometrial carcinoma ishikawa and HEC-1A cell lines,human preadipose cell lines,human type I endometrial carcinoma and paracancer tissues were used in the study.2.Methods:（1）Cell culture:The endometrial carcinoma ishikawa cells,HEC-1A cells and human preadipocytes were cultured for further use.（2）Induced differentiation of human preadipocytes:The differentiation of human preadipocytes were induced by using human adipogenic induction differentiation medium.（3）Oil red O staining:The method was used to detect lipid accumulation in adipocytes.（4）Conditioned culture:The method was used to detect the effect of estradiol secreted by adipocytes on the proliferation and invasion of endometrial cancer cells.（5）Transient gene transfection:MYCT1 overexpressed vector,si MYCT1,si ACAA2,si CTSD and corresponding control group were transfected into the corresponding endometrial carcinoma and preadipocytes for subsequent gene function study.（6）CCK8 assay:The method was used to detect the number of endometrial cancer living cells.（7）Colony formation experiment:The method was used to detect the colony formation ability of endometrial cancer cells.（8）Transwell invasion experiment:The method was used to detect the invasion ability of endometrial cancer cells.（9）Immunohistochemistry:The method was used to detect the protein expression levels of MYCT1 and CTSD in endometrial carcinoma typeⅠand paracancer tissue.（10）Co-immunoprecipitation（Co-IP）:The method was used to detect the interaction between MYCT1 and ACAA2 in preadipocytes.（11）Immunofluorescence:The method was used to detect the co-localization of MYCT1 and ACAA2 in preadipocytes.（12）Chromatin immunoprecipitation（Ch IP）:The method was used to detect the binding ability of ERαto CTSD promoter region.（13）Enzyme-linked immunosorbent assay（ELISA）:The method was used to detect the content of estradiol in the supernatant of adipocyte culture.（14）Real-time quantitative fluorescent PCR（q-PCR）:The method was used to detect the m RNA levels of the MTCT1,ACAA2 and CTSD genes in adipocytes and endometrial cancer cells,respectively.（15）Western Blot:The method was used to detect the protein levels of the MTCT1,ACAA2,CTSD and COXⅣgenes in adipocytes and endometrial cancer cells,respectively.（16）Statistical analysis:The paired t test,independent sample t test,One-way ANOVA,Chi-square test and Spearman correlation analysis were used to analyze the experimental data.Results:1.Effects of MYCT1 on proliferation and differentiation of preadipocytes（1）Oil red O staining results displayed that on the 4th day of induction differentiation,red lipid droplets began to appear in cells and with the passage of time,fat drops gradually increased.90%of human preadipocytes were differentiated into the typical mature adipocytes on the 21st day of induction differentiation,showing rich cytoplasm and a large number of lipid droplets stained as red color,which were distributed around the nucleus and formed a"ring like"structure.Lipid droplets were red after oil red O staining,indicating the successful induction of differentiation.On the 8th and 14th day of induction differentiation,the Oil red O staining results showed that the content of lipid droplets in the si MYCT1 treatment group was significantly higher than that in the control group,while the content of lipid droplets in the overexpressed MYCT1 group was significantly lower than that in the control group.（2）Western blot result showed that on the 14th day of induction,compared with the control groups,the expression levels of PPARγand C/EBPαwere significantly decreased in the overexpressed MYCT1 group（P<0.05）,whereas PPARγand C/EBPαexpressions were significantly increased in MYCT1 knockdown group（P<0.05）.（3）The results of CCK8 and clonal formation experiments showed that there were no significant changes in adipose proliferation abilities in MYCT1 overexpression and knockdown groups compared the controls（P>0.05）.2.Effects of MYCT1 on the proliferation and invasion of endometrial carcinoma by inhibiting preadipocyte differentiation（1）Compared with the control groups,the estrogen content and the ratio of E₂/T in adipocyte culture medium were significantly decreased and increased when MYCT1 was overexpressed and knocked down,respectively（P<0.05）.（2）The results of CCK8 and clone formation experiments showed the proliferative ability of ishikawa cells but not HEC-1A cells in adipocyte conditioned culture in MYCT1overexpression and knockdown groups were significantly decreased and increased compared to the control groups,respectively（P<0.05）.（3）Transwell invasion result showed that compared with the control group,the invasion ability of ishikawa cells but not that of HEC-1A cells in adipocyte conditioned culture in MYCT1 overexpression and knockdown MYCT1 groups were significantly decreased and increased compared with the control groups,respectively（P<0.05）.3.Effect and clinical significance of MYCT1 in obese patients with type I endometrial carcinomaImmunohistochemical result showed that the protein level of MYCT1 significantly decreased in type I endometrial cancer tissues of obese patients compared with paracancer tissues（P<0.05）.Statistical analysis of clinical data showed that MYCT1 expression level in type I endometrial cancer tissues of obese patients was significantly negatively correlated with the depth of tumor invasion and clinical stage（R=-0.217,R=-0.638,P<0.05）.4.MYCT1 interacts with ACAA2 in preadipocytesCo-IP and immunofluorescence detection results showed that MYCT1 and ACAA2interacted in human preadipose cells and were located in the same regions of the cells.5.CTSD is a direct target gene of estrogen in endometrial carcinoma cells（1）Real-time PCR and Western blot results showed that CTSD m RNA levels in ishikawa cells but not HEC-1A cells were significantly higher and lower in the estrogen and its receptor inhibitor groups than the controls,respectively（P<0.05）.（2）Ch IP result showed that ERαcould bind the CTSD promoter region,and significantly promoted the recruitment of ERαto the CTSD promoter region in ishikawa cells after the addition of estrogen（P<0.05）.6.Detection and clinical significance of CTSD expression in patients with typeⅠendometrial carcinomaImmunohistochemical result showed that CTSD protein level was significantly increased in typeⅠendometrial carcinoma tissues compared with paracancer tissues（P<0.05）.Statistical analysis of clinical data showed that CTSD protein level in type I endometrial carcinoma tissues was significantly positively correlated with the depth of tumor invasion and clinical stage（R=0.634,R=0.843,P<0.05）.7.MYCT1 regulates preadipocyte differentiation by interacting with ACAA2（1）Oil red O staining showed that MYCT1 knockdown promoted preadipocyte differentiation compared with the control group at the 8th and 14th day of induction differentiation,whereas ACAA2 knockdown inhibited preadipocyte differentiation and restored the promoting effect of knockdown MYCT1 on preadipocyte differentiation（P<0.05）.（2）Western blot results showed that on the 14th day of induction,both the protein levels of PPARγand C/EBPαwere significantly up-regulated and down-regulated in MYCT1 and ACAA2 knockdown groups compared with control groups,respectively（P<0.05）.Meanwhile,ACAA2 knockdown significantly restored the effects of MYCT1knockdown on PPARγand C/EBPαprotein expression levels compared with control groups（P<0.05）.8.Effects of MYCT1 on proliferation and invasion of type I endometrial carcinoma by regulating CTSD（1）Real-time PCR and Western blot results showed that compared with control groups,both m RNA and protein levels of the CTSD gene significantly increased in ishikawa cells treated with MYCT1 knockdown conditioned culture（P<0.05）.（2）The results of CCK8 and clone formation experiments showed that compared with control groups,CTSD knockdown significantly inhibited the proliferation of ishikawa cells and restored the proliferation of ishikawa cells treated with MYCT1 knockdown conditioned culture（P<0.05）.（3）Transwell assay result showed that compared with the control group,CTSD knockdown significantly inhibited the invasion of ishikawa cells and restored the invasion of ishikawa cells treated with MYCT1 knockdown conditioned culture（P<0.05）.Conclusions:1.The low expression of MYCT1 in obese patients with type I endometrial cancer was significantly negatively correlated with the depth of tumor invasion and clinical stage of the patients,suggesting that MYCT1 may play a potential role as a tumor suppressor gene in endometrial cancer with abnormal fat metabolism.2.MYCT1 inhibits the differentiation of human preadipocytes and thus estrogen levels by interacting with ACAA2 via PPARγ/C/EBPα-mediated signaling pathway.3.In endometrial carcinoma ishikawa cells,ERαdirectly binds the CTSD promoter region and promotes its transcriptional activity.4.CTSD protein level in typeⅠendometrial carcinoma is high and significantly positively correlated with the depth of tumor invasion and clinical stage of the patients,suggesting that CTSD plays a potential role as an oncogene in endometrial cancer.5.MYCT1 inhibits the proliferation and invasion of endometrial carcinoma ishikawa cells through ER/CTSD-mediated signaling pathway by regulating estrogen produced in adipocytes.