The Effect And Mechanism Of LMTK3 On The Proliferation Of Endometrioid Adenocarcinoma

Objective: Endometrial cancer is one of the most common malignant tumors of the female reproductive system.According to statistics,although most patients with endometrial cancer can be diagnosed early and have a good prognosis,the number of new cases and deaths is increasing year by year and the trend of younger people is obvious,of which 5% are under the age of 40,and the incidence of endometrial cancer in young women is increasing by nearly 2% every year.According to the pathogenesis and biological behavior characteristics,endometrial cancer is divided into type I and type II.Type I endometrial adenocarcinoma accounts for more than 80% of newly diagnosed endometrial cancer cases.The 2023 NCCN Clinical Practice Guidelines for Uterine Tumors(1st edition)clearly pointed out that regardless of the stage of endometrial cancer,the main surgical treatment is hysterectomy,and the treatment of fertility preservation is only applicable to young,low-grade patients with endometrial adenoma without myometrial invasion and metastasis,with fertility requirements,and without pregnancy and progesterone therapy contraindications.Megestrol,medroxyprogesterone acetate and progesterone intrauterine devices can be used to preserve fertility function.If endometrioid adenocarcinoma persists for 6 to 12 months after progesterone treatment,complete hysterectomy and double adnexal resection plus surgical staging should be performed.The current status of progesterone therapy is relatively low effective rate,high recurrence rate,low pregnancy rate and live birth rate after progesterone therapy.In addition,progesterone treatment will have many adverse reactions,some patients have poor compliance,and even have to terminate treatment because of adverse reactions.Therefore,finding new therapeutic targets and developing efficient targeted drugs with few side effects have become the focus of research on fertility preservation in patients with endometrioid adenocarcinoma at this stage,and the key to the success of fertility preservation therapy.lemur tyrosine kinase 3 is a serine-threonine-tyrosine kinase that has been shown to promote cancer in a variety of tumors,but its role in endometrial cancer has not been reported.Therefore,this study aims to explore the upstream and downstream molecular mechanisms by which LMTK3 promotes the proliferation of endometrioid adenocarcinoma cells.In other words,E2 inhibits mi R-34a-5p to up-regulate LMTK3 by activating GPR30/PTEN/AKT/P53 signaling pathway and inhibiting KAP1 phosphorylation,thereby inhibiting the transcription of PTEN and TP53 and promoting the proliferation of endometrioid adenocarcinoma cells.Methods:Part Ⅰ: Firstly,the expression of LMTK3 gene in normal endometrial and endometrioid adenocarcinoma tissues was analyzed using bioinformatics database,the expression of LMTK3 in endometrial adenocarcinoma and para-cancer tissues was investigated using tissue chip immunohistochemical staining method,and the m RNA or protein expression of LMTK3 in endometrial adenocarcinoma and normal endometrial tissue was detected by RT-q PCR or Western Blot.Then,the effects of LMTK3 on the proliferation of endometrial adenocarcinoma cells were investigated in Ishikawa and HEC-1A by cell viability,cell cycle and apoptosis in vitro cytological tests.Finally,the effects of LMTK3 on the proliferation of endometrial adenocarcinoma cells were investigated by subcutaneous tumor forming experiments in nude mice.Part Ⅱ : Firstly,RT-q PCR was used to explore the expression of mi R-34a-5p in endometrial adenocarcinoma and normal endometrial tissue,and the effects of E2 on the expression of LMTK3 in two endometrial adenocarcinoma cells of Ishikawa and HEC-1A were detected,and then the effects of E2,LMTK3 and the combination of E2 and LMTK3 on the proliferation of endometrial adenocarcinoma cells were further explored.The effects of E2 on the expression of mi R-34a-5p and the effects of E2,the combination of E2 and mi R-34a-5p on the proliferation of endometrioid adenocarcinoma cells,Finally,the effect of mi R-34a-5p on LMTK3 protein expression and the effect of E2,E2 and mi R-34a-5p on LMTK3 m RNA expression level were detected.Part Ⅲ: Using Genecards database and Fun Rich3.1.3 software,the common genes of LMTK3 and mi R-34a-5p participating signal pathway genes were mined and the signal pathways involved in common genes were predicted.Then,the bioinformatics was used to analyze the major pathways involved in LMTK3.Use STRING to predict whether there is an interaction between LMTK3 and GPR30,determine the downstream target protein GPR30 of LMTK3,then use the above method to uncover the common genes of LMTK3,mi R-34a-5p and GPR30 participating signal pathway genes,use STRING to predict the proteins associated with LMTK3,and use the combination of Genecards and STRING analysis to predict the top 10 proteins associated with GPR30.Combined with the literature,it is speculated that through which pathway LMTK3 promotes the proliferation of endometrioid adenocarcinoma cells.Then,Western Blot was used to detect the effects of E2,LMTK3,E2 and LMTK3,E2 and mi R-34a-5p,as well as the combined effects of E2,LMTK3 and mi R-34a-5p on the expression of key proteins in the above prediction pathway respectively in Ishikawa and HEC-1A.Then,cells are treated with PTEN specific inhibitor VO-Ohpic trihydrate and P53 inactivation agent Pifithrin-α.The effects of LMTK3,the combination of LMTK3 and PTEN,and the combination of LMTK3 and P53 on cell viability is detected through CCK-8 experiment.It was further confirmed that LMTK3 promoted the proliferation of endometrioid adenocarcinoma cells through the above presumed pathway.To further explore the molecular mechanism of LMTK3 gene affecting the expression of tumor suppressor proteins such as PTEN and P53,we explored the effect of LMTK3 on the m RNA expression levels of PTEN and TP53 by RT-q PCR,then observed whether LMTK3 was bound to the promoter of the gene coding region by Ch IP-seq assay,then detected the protein binding of LMTK3 by immunoprecipitation combined with mass spectrometry,and verified the mass spectrometry results by immunoprecipitation assay.Finally,we used KAP1 S824phosphorylation-inducing factor Dox to treat Ishikawa and HEC-1A cells,and observed the effect of overexpression of LMTK3 on KAP1 S824 phosphorylation level.The effects of KAP1 S824 phosphorylation and both overexpression of LMTK3 and KAP1S824 phosphorylation on the transcription levels of PTEN and TP53 genes were observed.Results:Part Ⅰ: Compared with normal endometrial tissue,LMTK3 gene was highly expressed in endometrioid adenocarcinoma(P=1.62E-12<0.001).LMTK3 was highly expressed in endometrioid adenocarcinoma compared with para-cancer tissue,and P<0.001;the m RNA expression of LMTK3 was also highly expressed in endometrioid adenocarcinoma compared with normal endometrial tissue,and P<0.01;the protein expression of LMTK3 was also highly expressed in endometrioid adenocarcinoma compared with normal endometrial tissue,and P<0.001;Knocking down LMTK3 gene inhibited the proliferation of Ishikawa and HEC-1A cells by inhibiting cell viability(P<0.001),enhancing cell cycle arrest in G1 phase(P<0.001),and promoting cell apoptosis(P<0.01).The tumor volume and weight of nude mice in the LMTK3-Ishikawa cell group were smaller(n=4,P<0.001),and the tumor growth inhibition rate was60.27%.The results of immunohistochemical staining showed that Ki67 expression was decreased(P<0.001)and Tunel expression was significantly increased(P<0.001)in the group of LMTK3-Ishikawa cells.Part Ⅱ : Compared with normal endometrial tissue,mi R-34a-5p is downregulated in endometrial adenocarcinoma with P<0.001.In Ishikawa and HEC-1A cells,E2 promoted the expression of LMTK3 protein in a time-dependent manner at a certain concentration.E2 promoted cell viability,and knocked down LMTK3 gene inhibited E2’s role in promoting cell viability.Knocking down LMTK3 gene inhibited cell viability,and adding E2 could save the effect of knocking down LMTK3 gene on cell viability.E2 promoted cell cycle progression,and knocking down LMTK3 gene inhibited E2’s role in promoting cell cycle progression.Knockdown of LMTK3 gene inhibited cell cycle progression,adding E2 could save the effect of knockdown of LMTK3 gene on cell cycle progression;E2 inhibited the expression of mi R-34a-5p,and when E2 reached a certain concentration,E2 inhibited the expression of mi R-34a-5p in a concentration-dependent manner.E2 promoted cell viability,increased mi R-34a-5p inhibited the effect of E2 on cell viability,and decreased mi R-34a-5p enhanced the effect of E2 on cell viability.E2 promoted cell cycle progression,increased mi R-34a-5p inhibited the effect of E2 on promoting cell cycle progression,and decreased mi R-34a-5p enhanced the effect of E2 on promoting cell cycle progression.Decreased mi R-34a-5p increased the expression of LMTK3 protein in EEC cells.E2 promoted the expression level of LMTK3 m RNA,increased mi R-34a-5p inhibited E2’s promotion of LMTK3 m RNA expression,and decreased mi R-34a-5p enhanced E2’s promotion of LMTK3 m RNA expression.Part Ⅲ: Bioinformatics analysis found that there were 219 common genes in all genes involved in the signaling pathway of LMTK3 and mi R-34a-5p,and it was predicted that the signaling pathway involved in the common genes was estrogen receptor signaling pathway,LMTK3 was mainly involved in the GPCR pathway and estrogen pathway,and the association between LMTK3 and GPR30 was predicted by STRING.In addition to being an estrogen receptor,GPR30 is also a member of the GPCR family.Finally,GPR30 is identified as the downstream target protein of LMTK3.There are 175 common genes in all genes involved in the signaling pathway of LMTK3,mi R-34a-5p and GPR30,including PTEN,TP53 and AKT,etc.The proteins that associate with LMTK3 by STRING analysis include P53,etc.Meanwhile,the combination of Genecards and STRING analysis shows that,the top 10 proteins associated with GPR30 are P53,PTEN,MDM2,etc.Combined with the literature,it is speculated that E2/mi R-34a-5p regulation of LMTK3 may promote the proliferation of endometrioid adenocarcinoma cells through the GPR30/PTEN/AKT/P53 signaling pathway.Then,in Ishikawa and HEC-1A cells,it was found that E2 promoted the expression of GPR30,P-AKT/AKT and Cyclin D1 proteins,while inhibited the expression of PTEN,P53 and P21;Knocking-down LMTK3 inhibited the expression of GPR30,P-AKT/AKT and Cyclin D1 proteins,while promoted the expression of PTEN,P53 and P21;Knocking-down LMTK3 could reverse the effect of E2;E2 promoted the expression of GPR30,P-AKT/AKT,Cyclin D1 and Ki67 proteins,while inhibited the expression of PTEN and P53;mi R-34a-5p inhibited the expression of LMTK3,GPR30,P-AKT/AKT,Cyclin D1 and Ki67 proteins,and promoted the expression of PTEN and P53;Overexpression of LMTK3 promoted the expression of GPR30,P-AKT/AKT,Cyclin D1 and Ki67 proteins,while inhibited the expression of PTEN and P53;mi R-34a-5p could reverse the effects of E2 or LMTK3.PTEN inhibitor or P53 inactivator reversed the effect of knocking down LMTK3 gene to inhibit cell viability.Knocking down LMTK3 gene promoted the m RNA expression of PTEN and TP53.The results of Ch IP-seq experiment with Ishikawa cells showed that LMTK3 could not bind to the promoter region.The results of immunoprecipitation and mass spectrometry of Ishikawa cells showed that LMTK3 bound to KAP1.The combination of LMTK3 and KAP1 was also found in Ishikawa and HEC-1A cells by immunoprecipitation assay.Overexpression of LMTK3 can reverse the effect of Dox on increasing the phosphorylated expression level of KAP1 S824,and the increased phosphorylated expression level of KAP1 S824 promotes the m RNA expression of PTEN and TP53.Overexpression of LMTK3 further inhibits the m RNA expression of PTEN and TP53 by decreasing the phosphorylated expression level of KAP1 S824.Conclusion:1.LMTK3 is highly expressed in endometrioid adenocarcinoma and promotes the proliferation of endometrioid adenocarcinoma cells.2.E2 promotes the proliferation of endometrioid adenocarcinoma cells by inhibiting mi R-34a-5p and then up-regulating LMTK3 to further activate GPR30/PTEN/AKT/P53 signaling pathway.3.LMTK3 binds to KAP1 through inhibiting KAP1 phosphorylation to decrease the transcription of PTEN and TP53 and further to promote the proliferation of endometrioid adenocarcinoma cells.