Cucurbitacin B Inhibits Cell Proliferation By Regulating X-inactive Specific Transcript/miR-29b Expression In Tongue Cancer

Cucurbitacin B Inhibits Cell Proliferation by Regulating X-InactiveSpecific Transcript/ miR-29 b Expression in Tongue Cancer Purpose:Cucurbitacin B（CuB）is a natural product that has anti-tumor effects on a variety of cancers.Many long non-codingRNA（LncRNA）are abnormally expressed in TSCC.XIST is a lncRNA that regulates X chromosome inactivation and plays a very important role in the occurrence and development of many cancers.To study the expression of long non-codingRNA（LncRNA）,we performedRNA sequencing（RNA-seq）and quantitative PCR（q PCR）.The results showed that the expression of XIST in CAL27 and SCC9 tongue squamous cell carcinoma（TSCC）cells decreased significantly after CuB treatment.In addition,our results show that the expression of XIST increased in human tongue carcinoma.In this study,CuB treatment not only induced apoptosis but also inhibited the growth and invasion of SCC9 cells.Interestingly,knockout of XIST gene can inhibit cell proliferation and induce apoptosis in vitro.In addition,the decreased expression of XIST also inhibited cell migration and invasion.MicroRNA29b（miR-29b）is identified as the direct target of XIST.It has been reported that miR-29 b has a regulatory effect on p53 protein.Our result suggests that the overexpression of miR-29 b induces apoptosis through p53 protein.Cluster regular interval short palindromic repeat sequence（CRISPR）/ CRISPR-associated protein 9（CRISPR/Cas9）system verified the role of XIST gene knockout in tumorigenesis in vivo.To sum up,these results show that CuB plays a significant anticancer activity through the expression of XIST/miR-29 b.Methods:1.The cytotoxicity of cucurbitacin B on SCC9 was detected by CCK 8 method.2.After CAL27 and SCC9 were treated with CuB,Annexin-FITC/PI staining and flow cytometry was used to detect the apoptosis of tongue squamous cell carcinoma,and scratch test and Transwell were used to detect the changes of migration and invasion of tongue squamous cell carcinoma.3.After SCC9 was treated with CuB,the differential expression of LncRNA in SCC9 cells was analyzed byRNA-seq,theRNA expression of XIST and miR-29 b was detected by real-time quantitative PCR,and the expression of p53 and E-cadherin protein was detected by Western blot.4.Cancer tissue samples and healthy tongue tissue samples from patients with clinical tongue squamous cell carcinoma were collected,and theRNA expression levels of XIST and miR-29 b in tissues were detected by real-time quantitative PCR.5.The plasmids pc DNA3.1-XIST overexpressing XIST and small interferenceRNA（siRNA）si XIST,miR-29b-3p mimics and miR-29b-3p inhibitor knocking down XIST were synthesized and transfected into tongue squamous cell carcinoma cell line SCC9 with liposome 2000.The growth of tongue squamous cell carcinoma was detected by CCK 8 method,and the apoptosis of tongue squamous cell carcinoma was detected by Annexin-FITC/PI staining and flow cytometry.Scratch test and Transwell were used to detect the changes in migration and invasion ability of tongue squamous cell carcinoma cells.Real-time quantitative PCR was used to detect the changes in miRNA expression after the change in XIST expression level.P53 and E-Cadherin protein expression was detected by the Western blot method.6.The stable SCC9 cell line with XIST/miR-29b-3p binding site knockout was constructed by CRISPR/Cas9 technique,and the stable SCC9 cell line with overexpression of XIST was constructed.The SCC9 cell line with overexpression,normal expression,and knockout XIST was injected into the axillary subcutaneous of nude mice to construct the model of heterotopic transplant tumor.The inhibitory effect and mechanism of CuB on tongue squamous cell carcinoma in vivo were analyzed by intraperitoneal injection of PBS and CuB.The expression level of XIST in tumor tissue was detected by real-time quantitative PCR.Result:1.CuB can promote the apoptosis of tongue squamous cell carcinoma SCC9 and inhibit its growth and invasion.2.CuB treatment,there were 90 up-regulated and 87 down-regulated lncRNA,including BBC3,STK11 IP,XIST,FDFT1 and PRR14.QPCR results showed that the expression of XIST in CAL27 and SCC9 cells decreased after CuB treatment.3.The expression of XIST in cancer tissues of patients with tongue squamous cell carcinoma was higher than that in paracancerous tissues,and the expression of miR-29b-3p in tumor tissues decreased.4.Overexpression of XIST can reduce the apoptosis of tongue squamous cell carcinoma and stimulate the proliferation,migration and invasion of tongue squamous cell carcinoma,while knocking down XIST has the opposite result.5.Overexpression of miR-29b-3p can promote apoptosis and inhibit the growth and invasion of tongue squamous cell carcinoma,while knocking down miR-29b-3p can promote its growth.6.CuB treatment inhibited the growth of tongue squamous cell carcinoma in vivo.QPCR results showed that CuB treatment decreased the expression of XIST.7.Compared with the normal expression group,the tumor growth inhibited in XISTKO group.In addition,the treatment with CuB in the XIST overexpression group inhibited tumor growth.Conclusion:Cucurbitacin B can down-regulate the expression of XIST in tongue squamous cell carcinoma tissues and cells.XIST acts as a miR-29b-3p sponge and makes miR-29b-3p inhibit the proliferation,migration,and invasion of tongue cancer cells and promote the apoptosis of tongue cancer cells by regulating the expression of p53 and E-cadherin.