| Background:Endometrial carcinoma(EC)as a member of cancers,mostly occurs in perimenopausal and postmenopausal women,and the incidence has continued to increase in the past two decades and is showing a younger trend.Study shows that there are 60,000 new cases of endometrial cancer and 11,000 deaths each year in the United States alone.The incidence of endometrial cancer is second only to cervical cancer in China,and it ranks second among malignant tumors of the female reproductive system.It is currently believed that endometrial cancer can be divided into type I(estrogen-dependent)and type II(non-estrogen-dependent).Type Ⅰ usually has a good prognosis,while type II has a high risk of recurrence and metastasis,and a poor prognosis.Although the overall survival rate and prognosis of endometrial cancer are good,the prognosis of advanced patients with metastasis or recurrence is still poor,and more effective targeted therapy is still needed.However,the current research on endometrial cancer is still not deep enough,and its pathogenesis and therapeutic targets still need to be explored continuously.MicroRNA(miRNA)has appeared in the public as a new type of tumor treatment target.miRNA is a short-stranded non-coding RNA which mainly affects the stability and translation of mRNA by binding to the 3’UTR region of target mRNA.More and more evidences show that miRNAs play a key role in tumorigenesis and tumor development.MiR1290 has been proven to be a tumor-promoting miRNA in various malignant tumors such as laryngeal squamous cell carcinoma and lung small cell carcinoma.In addition,the expression of miR1290 is regulated by estrogen,and miR1290 increases when the level of estrogen increases.But so far,there is no research on the expression of miR1290 in endometrial cancer.The online database predicts that the target gene of miR1290 is NLRC3.NLRC3(NOD3)is a relatively new and less researched member of the NLR family and can be used as a tumor suppressor molecule in colon cancer.However,there is no research on the expression of NLRC3 in endometrial cancer.Purpose:1.Explore the expression of miR1290 in endometrial cancer tissues,adjacent tissues and normal endometrial tissues and the relationship between miR1290 and pathological grades.2.Study the change of proliferation,invasion and migration and epithelial-mesenchymal transition(EMT)of endometrial cancer cell lines after miR1290 lentivirus knockdown miR1290 expression in vitro.3.Find out the effect of knocking down miR1290 on the pathways of endometrial cancer cell lines.4.Explore the effect of knocking-down miR1290 on proliferation ability of endometrial cancer cell lines in nude mice.5.Explore the possible miR1290 downstream molecule NLRC3.Results:1.A total of 37 cases of endometrioid adenocarcinoma(including 10 cases of paired adjacent tissues)and 23 cases of normal endometrial tissue were obtained.Using RT-PCR technology to detect the expression of miR1290 in each specimen.The results showed that the expression of miR1290 in endometrial cancer tissues was 1.75 times that of adjacent tissues(P<0.05).At the same time,the expression of miR1290 in cancer tissues was 1.5 times that of normal tissues(P<0.05);The expression of miR1290 in the secretory endometrial tissue was 1.7 times that in the proliferative tissues(P<0.01),while the expression of miR1290 in the mid-poorly differentiated endometrial cancer group was 1.3 times than well-differentiated group(P>0.05).2.Two cell lines KLE and ISHIKAWA were transfected with miR1290 inhibitor lentivirus to construct a stable miR1290 low-expressing cell line,and the knockdown efficiency was verified by RT-PCR technology.After successful knockdown,CCK8,clone formation experiment,scratch experiment,Transwell invasion,migration experiment and Western Blot experiment were used to verify the changes in cell growth and proliferation,invasion and migration and EMT ability in vitro.In the KLE cell line,the CCK8 experiment and the clone formation experiment showed that knocking down miR1290 promoted cell proliferation(P<0.05),and the same result was also shown in the ISHIKAWA cell line(P<0.05);In wound healing experiment,knocking down miR1290 significantly inhibited the healing of scratches(P<0.05),indicating that the migration ability of KLE cells and ISHIKAWA cells was reduced;In Transwell migration and invasion experiment,the decreased expression of miR1290 reduced the number of cells crossing through the chamber membrane(P<0.05),indicating the attenuation of migration and invasion ability of KLE and ISHIKAWA cells is weakened;Western blot experiment was used to compare the changes of EMT indicators,and the results showed that the expression of E-cadherin(having protective effect)was increased in KLE and ISHIKAWA cells in Sh-miR1290 group,while the expression of N-cadherin,vimentin,Snail and Slug decreased in Sh-miR1290 group,indicating that the reduction of miR1290 inhibited the epithelial-mesenchymal transition of endometrial cancer cells.3.Knockdown of miR1290 in KLE cells can lead to decreased expression of PI3K,PAKT/AKT(P<0.05).Knockdown of miR1290 in ISHIKAWA cells can cause the expression of Wnt and β-catenin to decrease(P<0.05).It indicates that miR1290 may act through the PI3K/AKT pathway in KLE,while it may act through the Wnt/β-catenin pathway in ISHIKAWA.4.Injecting KLE/Sh-miR1290 group and control group into the subcutaneous of 9 nude mice,and on the 28th day after inoculation,the tumor was completely removed.The tumor weight of in Sh-miR1290 group is increased compared with the control group(P<0.05).RT-PCR was performed on tumor specimens to detect the expression of miR1290,and the results showed that the expression of miR1290 in the experimental groups was reduced(P<0.05).5.Target Scan Human software predicted that NLRC3 is the target gene of miR1290.Western blot results of 24 cases of endometrioid adenocarcinoma and 10 cases of normal endometrial tissue showed that the expression of NLRC3 in endometrial cancer was 0.6 times that of normal uterine tissue.The expression of miR1290 is negatively correlated with the expression of NLRC3,the correlation coefficient r2=0.0811,indicating that the correlation between miR1290 and NLRC3 is poor.And there was no difference in NLRC3 expression between miR1290 knockdown group and control group in KLE and ISHIKAWA cell lines.Conclusion:1.The expression of miR1290 in endometrial cancer is significantly higher than that in the adjacent tissues and normal endometrial tissues,the expression in the secretory phase of the endometrium is higher than that in the proliferative phase,and the expression in the medium-poorly differentiated group is higher than that in endometrial cancer in the well-differentiated group.2.Knocking down the expression of miR1290 can promote the proliferation of endometrial cancer cells in vivo and in vitro,but it can significantly inhibit the invasion and migration ability of cells and the epithelial-mesenchymal transition ability in vitro.3.It may work through the PI3K/AKT pathway and the Wnt/β-catenin pathway.4.The expression of NLRC3 in endometrial cancer tumors was significantly lower than that in normal endometrial tissues,but changing the expression of miR1290 did not affect the expression of NLRC3,suggesting that miR1290 may promote tumor progression through other targets,and in-depth study are still needed to find out the right molecular target. |
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