Long noncoding RNA LINC00520 prevents the progression of cutaneous squamous cell carcinoma through the inactivation of the PI3K/Akt signaling pathway by downregulating EGFR

Background: Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors. Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC). This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways. Methods: A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples. The A431 cSCC cell line was transfected and assigned different groups. The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively. Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression. This study was ended in September 2017. Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance. The nonparametric Wilcoxon rank sum test was used to analyze skewed data. The enumerated data were analyzed using the chi-square test or Fisher exact test. Results: Data from chip GSE66359 revealed depletion of LINC00520 in cSCC. Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05). Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector+ EGFR vector group showed opposite results (all P < 0.05). Conclusion: Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.

Silencing novel long non-coding RNA FKBP9P1 represses malignant progression and inhibits PI3K/AKT signaling of head and neck squamous cell carcinoma in vitro

Background:Long non-coding RNAs(lncRNAs)play key roles in human cancers.In our previous study,we demonstrated that lncRNA FKBP prolyl isomerase 9 pseudogene 1(FKBP9P1)was highly expressed in head and neck squamous cell cancer(HNSCC)tissues.However,its functional significance remains poorly understood.In the present study,we identify the role and potential molecular biologic mechanisms of FKBP9P1 in HNSCC.Methods:Quantitative real-time polymerase chain reaction was used to detect the expression of FKBP9P1 in HNSCC tissues,matched adjacent normal tissues,human HNSCC cells(FaDu,Cal-27,SCC4,and SCC9),and human immortalized keratinocytes cell HaCaT(normal control).Cal-27 and SCC9 cells were transfected with sh-FKBP9P1-1,sh-FKBP9P1-2,and normal control(sh-NC)lentivirus.Cell counting kit-8 assay,colony formation assay,wound healing assay,and trans-well assay were used to explore the biologic function of FKBP9P1 in HNSCC cells.Furthermore,western blotting was used to determine the mechanism of FKBP9P1 in HNSCC progression.Chi-squared test was performed to assess the clinical significance among FKBP9P1 high-expression and low-expression groups.Survival analyses were performed using the Kaplan-Meier method and assessed using the log-rank test.The comparison between two groups was analyzed by Student t test,and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.Results:FKBP9P1 expression was significantly up-regulated in HNSCC tissues(tumor vs.normal,1.914 vs.0.957,t=7.746,P<0.001)and cell lines(P<0.01 in all HNSCC cell lines).Besides,the median FKBP9P1 expression of HNSCC tissues(1.677)was considered as the threshold.High FKBP9P1 level was correlated with advanced T stage(P=0.022),advanced N stage(P=0.036),advanced clinical stage(P=0.018),and poor prognosis of HNSCC patients(overall survival,P=0.002 and disease-free survival,P<0.001).Knockdown of FKBP9P1 led to marked repression in proliferation,migration,and invasion of HNSCC cells in vitro(P all<0.01).Mechanistically,silencing FKBP9P1 was observed to restrain the PI3K/AKT signaling pathway.Conclusions:Silencing lncRNA FKBP9P1 represses HNSCC progression and inhibits PI3K/AKT(phosphatidylinositol 3 kinase/AKT Serine/Threonine Kinase)signaling in vitro.Therefore,FKBP9P1 could be a potential new target for the diagnosis and treatment of HNSCC patients.

Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma （ESCC）.We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay.The times T1,when the number of living cells reached a plateau and T2,when the number of living cells reached a new plateau after the addition of insulin were found.T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin （MDC）.Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively.Apoptotic ratio and fluorescent intensity were analysed by flow cytometry （FCM） quantitatively.Western blotting analysis was used to estimate the protein expression levels of AKT,mTOR,PI3K,PTEN,autophage related indicator LC3-Ⅱ and autophage related protein Beclin1 changes that occurred in the course of treatment.Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin.Insulin can increase the apoptosis induced by cisplatin.Apoptotic ratio of T1 plateau cells （（32.6±4.3）％） is significantly less than T2 plateau （（47.5±5.6）％）.MDC fluorescent intensity at T1 plateau （104.9±13.2） was significantly higher than intensity at T2 plateau （82.6±10.3）.After cotreatment with insulin,the expression level of LC3-Ⅱ,Beclin1 and PTEN in T2 plateau cells were significantly downregulated,but AKT,mTOR and PI3K expressions significantly upregulated compared with T1 plateau.Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells,which may be attributed to the anticancer effects of cisplatin.

miR-21调控PTEN/AKT信号通路对SCC9细胞凋亡的影响

目的:观察舌鳞状细胞癌患者血清miR-21变化水平,利用SCC9细胞探讨miR-21调控染色体10上缺失的磷酸酶和张力蛋白同源物/蛋白激酶B(PTEN/AKT)信号通路影响细胞凋亡的作用机制。方法:选取中国人民解放军联勤保障部队第九O九医院收治的舌鳞状细胞癌患者31例和健康体检者31例分别作为观察组和对照组。实时荧光定量PCR(qPCR)检测两组受试者血清miR-21相对表达量。体外培养舌鳞状SCC9细胞,分为miR-21 NC组、miR-21 mimics组和miR-21 inhibitors组并用相应的质粒进行转染,采用qPCR检测miR-21相对表达量,免疫蛋白印迹法(Western blotting)检测PTEN、AKT和p-AKT蛋白的相对表达量,流式细胞术检测细胞凋亡情况。结果:观察组血清miR-21相对表达量高于对照组(P=0.000)。与miR-21 NC组比较,miR-21 mimics组miR-21相对表达量、p-AKT蛋白相对表达量均升高(均P<0.05),PTEN蛋白、细胞凋亡率均降低(P<0.05);miR-21 inhibitors组miR-21相对表达量、p-AKT蛋白相对表达量均降低(均P<0.05),PTEN蛋白、细胞凋亡率均升高(P<0.05)。结论:舌鳞状细胞癌患者血清miR-21相对表达量高于正常人,下调miR-21可能通过抑制PTEN/AKT信号通路促进癌细胞的凋亡。

鱼藤素抑制舌鳞癌细胞增殖及诱导裸鼠移植瘤细胞凋亡机制的研究

目的:探索鱼藤素(DEG)对舌鳞癌(TSCC)的体外及体内抗癌作用及其作用机制。方法:CCK-8实验检测DEG对舌鳞癌细胞系CAL27细胞的增殖的影响,流式细胞术检测细胞凋亡及活性氧(ROS)水平。构建裸鼠移植瘤模型并分组处理,测量瘤体体积、质量及肿瘤抑制率;TUNEL检测细胞凋亡;Western blot实验检测PI3K/AKT信号通路相关蛋白表达。结果:DEG对CAL27细胞具有增殖抑制和诱导凋亡作用;DEG能够通过上调CAL27细胞中的ROS水平,抑制PI3K/AKT信号通路;体内实验证明DEG能够以浓度依赖性的方式抑制裸鼠移植瘤的生长,相同浓度下抑制效果强于顺铂,30 mg/kg的DEG对瘤体的抑制率高达72.6%。结论:DEG对舌鳞癌细胞具有抑制增殖及诱导凋亡作用,其机制与调节肿瘤细胞中ROS以及PI3K/AKP信号通路有关。

热疗联合Id-1基因沉默对舌鳞癌CAL-27细胞增殖及侵袭的影响

目的:探讨热疗联合Id-1基因沉默对舌鳞癌细胞增殖及侵袭的影响及其机制。方法:将培养的舌鳞癌细胞株CAL-27分为4组。(1)沉默Id-1组(Id-1-siRNA)—用siRNA基因转染法沉默舌鳞癌CAL-27细胞中Id-1的表达,RT-PCR法检测其Id-1的mRNA表达变化;(2)热疗组(HT)—细胞置于42.5℃恒温培养箱内40min;(3)联合组(HT+Id-1-siRNA)—siRNA基因转染法沉默细胞内Id-1的表达后进行42.5℃、40 min的热疗处理;(4)空白对照组—常规培养细胞。通过CCK8和Transwell侵袭实验,分别检测CAL-27细胞的增殖能力和侵袭能力变化; Western免疫印迹法检测CAL-27细胞中PI3K、磷酸化Akt(p-Akt)蛋白表达变化。采用SPSS19.0软件包对数据进行统计学分析。结果:siRNA能成功沉默CAL-27细胞内Id-1的表达,沉默Id-1组与空白对照组相比,Id-1的mRNA表达水平下降81.6%。沉默Id-1和热疗均可抑制CAL-27细胞的增殖和侵袭能力,下调PI3K、p-Akt蛋白表达(P<0.05),热疗联合沉默Id-1作用于CAL-27细胞后的结果更为显著(P<0.01)。结论:热疗协同沉默Id-1可显著抑制人舌鳞癌细胞的增殖及侵袭能力,其作用是通过PI3K/Akt信号通路来实现。

干扰素γ对舌鳞癌细胞增殖影响的研究

目的:观察干扰素γ对舌鳞癌细胞增殖的影响。方法:采用不同浓度的干扰素γ对舌鳞癌细胞处理,观察肿瘤细胞的增殖情况;基因芯片分析干扰素γ处理后基因表达的变化,并采用实时定量PCR验证;western blot检测AKT信号通路的表达情况。结果:采用不同浓度的干扰素γ处理CAL27细胞后,肿瘤细胞的生长明显加快,肿瘤生长72、96h后,细胞数量有显著性差异(P<0.05)。基因芯片结果显示PIK3C2A的表达提高2.5倍以上。western blot结果证实干扰素γ处理细胞后,AKT信号通路被激活。结论:干扰素γ处理舌鳞癌细胞后能加快肿瘤细胞的生长,激活AKT信号通路,其不适用直接对舌鳞癌进行治疗。

过表达YAP基因通过PI3 K/AKT/mTOR信号通路促进舌鳞癌增殖

目的基于PI3K/AKT/mTOR信号通路,探究过表达YAP基因对舌鳞癌细胞增殖和凋亡的分子学调控机制.方法构建稳定过表达YAP基因的舌鳞癌CAL27和SCC25细胞系.采用CCK-8和流式细胞术分析过表达YAP基因对细胞增殖、周期和凋亡的影响.蛋白免疫印迹法分析过表达YAP基因对细胞内CDK4、CDK6、p21、BAX及对PI3K/AKT/mTOR信号通路的影响.结果CAL27细胞和SCC25细胞中过表达YAP组YAP mRNA较对照组升高(t=-19.29,P<0.001;t=-5.44,P<0.001),且过表达YAP组的YAP蛋白表达量较对照组增加(t=-31.28,P<0.001;t=-42.84,P<0.001);过表达YAP组的G1期细胞比例减少,过表达YAP组S期细胞比例较对照组升高;过表达YAP组的早期凋亡和晚期凋亡细胞都减少;过表达YAP组细胞中CDK4、CDK6、BCL-2、PARP等蛋白表达升高,p21、BAX及caspase3蛋白的表达减少;过表达YAP基因可使CAL27和SCC25细胞内AKT(tCAL27=-30.60,P<0.001;tSCC25=-16.77,P<0.001)、p-AKT(tCAL27=-10.23,P<0.001;tSCC25=-10.67,P<0.001),mTOR(tCAL27=-13.93,P<0.001;tSCC25=-9.57,P<0.001)、p-mTOR(tCAL27=-17.17,P<0.001;tSCC25=-8.51,P<0.001)表达量较对照组增高.结论过表达YAP基因可以通过PI3K/AKT/mTOR信号通路促进CAL27和SCC25细胞增殖并抑制细胞凋亡.

干扰素诱导的跨膜蛋白1在宫颈鳞癌中的表达及其生物学作用

目的:探讨干扰素诱导的跨膜蛋白1(IFITM1)在宫颈鳞癌中的表达及其生物学作用。方法:通过免疫组化、RT-PCR和Western blot检测宫颈鳞癌组织和癌旁组织中IFITM1的表达。使用靶向IFITM1的si RNA(si-IFITM1组)和高表达IFITM1基因的重组pcDNA3.1质粒(pcDNA3.1-si-IFITM1组)转染Si Ha细胞下调或上调IFITM1的表达。通过伤口愈合实验、Transwells迁移实验和基质胶侵袭实验检测细胞迁移和侵袭能力。细胞计数试剂盒-8 (CCK-8)检测细胞增殖;流式细胞术测定细胞凋亡。Western blot检测PTEN、PI3K和AKT的表达。通过对BALB/c Nude裸鼠接种Si Ha细胞来考察IFITM1在体外对肿瘤生长的影响。结果:癌组织中IFITM1的表达水平明显高于癌旁组织(P<0.05)。与对照组比较,si-IFITM1组的迁移和侵袭能力明显增强,而pcDNA3.1-si-IFITM1组明显降低(P<0.05)。细胞转染48 h和72 h后,与对照组比较,si-IFITM1组的细胞增殖明显增强,而pcDNA3.1-si-IFITM1组明显减弱(P<0.05)。与对照组比较,si-IFITM1组的细胞凋亡率明显降低,而pcDNA3.1-si-IFITM1组明显升高(P<0.05)。与对照组比较,si-IFITM1组的PTEN被下调,而PI3K和AKT被上调(P<0.05);pcDNA3.1-si-IFITM1组的PTEN的被上调,而PI3K和AKT被下调(P<0.05)。与对照组比较,si-IFITM1组裸鼠的肿瘤体积显著增大,而pcDNA3.1-si-IFITM1组显著减小(P<0.05)。结论:IFITM1过表达抑制人宫颈鳞癌细胞Si Ha的生长和转移能力,并在体外抑制肿瘤的形成,从而发挥抗癌作用。IFITM1可能通过调控PTEN/PI3K/AKT信号通路发挥抗癌作用。