LINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway

BackgroundLINC00941 has been proved to be related to various tumors, but its relationship with laryngocarcinoma remains vague.MethodsLINC00941 expression in laryngocarcinoma tumor and laryngocarcinoma cells was determined by real time‐quantitative polymerase chain reaction (RT‐qPCR). Besides, the five‐year survival of laryngocarcinoma patients with different LINC00941 expression was analyzed with Kaplan–Meier survival analysis, and the clinical characteristics of laryngocarcinoma patients were also recorded. After transfection, cell viability, cell proliferation, apoptosis, cell cycle, migration, and invasion were detected by cell counting kit‐8 (CCK‐8), colony formation, flow cytometry, cell scratch, and Transwell assays, respectively. Glycolysis was assessed by the colorimetric method. Expressions of proliferation‐associated proteins, migration‐associated proteins, glycolysis‐associated proteins, and phosphatidylinositol 3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathway‐associated proteins were detected by Western blot.ResultsIn laryngocarcinoma tumor tissues and cells, LINC00941 was highly expressed. High expression of LINC00941 decreased the 5‐year survival of laryngocarcinoma patients, and it was positively related to lymph node metastasis and clinical stages. LINC00941 overexpression decreased apoptosis but promoted cell viability, proliferation, cell‐cycle progression, migration, and invasion, and glucose consumption and lactate production in laryngocarcinoma cells. Moreover, LINC00941 overexpression elevated expressions of Ki‐67, PCNA, MMP2, N‐Cadherin, HK2, PFKFB4, and PKM, activated the PI3K/AKT/mTOR signal pathway but reduced E‐Cadherin expression, while LINC00941 silencing had the opposite effects. PKM overexpression reversed the effects of LINC00941 silencing on cellular and glycolytic phenotypes.ConclusionLINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma cells by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway.     

Odontogenic ameloblast-associated protein (ODAM) inhibits human colorectal cancer growth by promoting PTEN elevation and inactivating PI3K/AKT signaling

Odontogenic ameloblast-associated protein (ODAM), an acidic matricellular protein, has been implicated in several epithelial neoplasms. However, its biological functions and molecular mechanisms in cancer progression, particular colorectal carcinoma (CRC), remain unknown. Here we demonstrated that ODAM was significantly down-regulated in CRC tissues compared with their normal counterparts. Then, we established that ODAM expression level was closely correlated with CRC development and patient prognosis. The abnormal expression of ODAM dramatically affected CRC cell growth in vitro and in vivo. We further revealed that the inhibitory effects of ODAM on CRC cell growth were associated with PTEN elevation and PI3K/AKT signaling inactivation. Furthermore, we determined that silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing CRC cells. Our study suggests matricellular protein ODAM may serve as a novel prognostic marker and act as a CRC growth suppressor.     

Silencing of lncRNA MIR31HG promotes nasopharyngeal carcinoma cell proliferation and inhibits apoptosis through suppressing the PI3K/AKT signaling pathway

BackgroundMIR31HG has been affirmed to regulate the tumorigenesis of head–neck squamous cell carcinoma (HNSC). This study aims to reveal the function of MIR31HG in nasopharyngeal carcinoma (NPC), which falls into the category of HNSC.MethodsMIR31HG expression pattern in HNSC tissues was predicted by starBase. FISH and qRT‐PCR were employed to detect MIR31HG expression in NPC tissues and to analyze the association between MIR31HG and clinicopathological features. NPC cell viability, colony formation, and apoptosis were measured by MTT assay, colony formation assay, and flow cytometry. The expressions of protein kinase B (AKT), phosphorylated (p)‐AKT, phosphoinositide 3‐kinases (PI3K) and p‐PI3K in NPC cells were analyzed by Western blot. The correlation between MIR31HG expression and AKT1 mRNA expression was analyzed by The Cancer Genome Atlas and starBase.ResultsMIR31HG was highly expressed in HNSC tissues and NPC tissues. Meanwhile, the association between high MIR31HG expression and aggressive clinicopathological traits was significant in NPC patients at tumor stage III‐IV (T3‐T4) and in those with lymph node metastasis 1–2 (N1‐N2). Silencing of MIR31HG suppressed NPC cell viability and colony formation, promoted apoptosis, and decreased the expressions of p‐PI3K, and p‐AKT. 740Y‐P reversed the above effects of si‐MIR31HG on NPC cells. Besides, MIR31HG expression was positively correlated with AKT1 mRNA expression in HNSC patients.ConclusionMIR31HG silencing promotes NPC cell proliferation and inhibits apoptosis through suppressing the PI3K/AKT signaling pathway.     

The knockdown of MALAT1 inhibits the proliferation,invasion and migration of hemangioma endothelial cells by regulating MiR-206 / VEGFA axis

AimLncRNA MALAT1 is involved in regulation of angiogenesis, however, its expression and mechanism in infantile hemangioma (IH) are less reported. The study aimed to investigate MALAT1 in IH and to reveal the potential mechanism of MALAT1 acting on IH.MethodsIsolated form IH tissue, human CD31⁺ hemangioma endothelial cells (HemECs) were cultured and sorted by magnetic-activated cell sorting (MACS). Quantitative real-time (qRT)-PCR was performed to detect the expressions of MALAT1, miR-206 and VEGFA. The correlations among MALAT1, miR-206 and VEGFA were confirmed by bioinformatics analysis and dual-luciferase reporter assay. The effects of MALAT1, miR-206 and VEGFA on cell proliferation were detected by cell counting kit-8 (CCK-8) and cell colony formation assay. Flow cytometry, wound scratch, Transwell and Tube formation assay were performed to determine cell apoptosis, migration, invasion and vasoformation, respectively. Apoptosis-related proteins were determined by Western blot.ResultsThe results showed that MALAT1 and VEGFA were high-expressed and miR-206 was low-expressed in IH tissues. SiMALAT1 negatively affected the cell proliferation, migration, invasion and vasoformation of HemECs and promoted apoptosis of HemECs. Moreover, Bcl-2 expression was significantly inhibited and the expressions of Bax and c cleaved-3 were greatly promoted. MALAT1 directly targeted and inhibited the expression of miR-206, and VEGFA was predicted to be the target gene for miR-206. SiMALAT1 suppressed the cell proliferation, migration, invasion and vasoformation of HemECs through modulating miR-206/VEGFA axis.ConclusionKnock-down of MALAT1 inhibits the growth of HemECs through regulating miR-206/VEGFA axis, indicating that MALAT1 is a potential therapeutic mechanism for the treatment of IH.     

Recent insights in the PI3K/Akt pathway as a promising therapeutic target in combination with EGFR-targeting agents to treat head and neck squamous cell carcinoma

Resistance to therapies targeting the epidermal growth factor receptor (EGFR), such as cetuximab, remains a major roadblock in the search for effective therapeutic strategies in head and neck squamous cell carcinoma (HNSCC). Due to its close interaction with the EGFR pathway, redundant or compensatory activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been proposed as a major driver of resistance to EGFR inhibitors. Understanding the role of each of the main proteins involved in this pathway is utterly important to develop rational combination strategies able to circumvent resistance. Therefore, the current work reviewed the role of PI3K/Akt pathway proteins, including Ras, PI3K, tumor suppressor phosphatase and tensing homolog, Akt and mammalian target of rapamycin in resistance to anti-EGFR treatment in HNSCC. In addition, we summarize PI3K/Akt pathway inhibitors that are currently under (pre)clinical investigation with focus on overcoming resistance to EGFR inhibitors. In conclusion, genomic alterations in and/or overexpression of one or more of these proteins are common in both human papillomavirus (HPV)-positive and HPV-negative HNSCC tumors. Therefore, downstream effectors of the PI3K/Akt pathway serve as promising drug targets in the search for novel therapeutic strategies that are able to overcome resistance to anti-EGFR treatment. Co-targeting EGFR and the PI3K/Akt pathway can lead to synergistic drug interactions, possibly restoring sensitivity to EGFR inhibitors and hereby improving clinical efficacy. Better understanding of the predictive value of PI3K/Akt pathway alterations is needed to allow the identification of patient populations that might benefit most from these combination strategies.     

MicroRNA-21调控口腔鳞癌细胞顺铂耐药及其作用机制

目的:探讨micro RNA-21(mi RNA-21)在人口腔鳞癌顺铂耐药细胞中的表达及其作用机制。方法 :采用实时荧光定量PCR(RT-PCR)法检测mi RNA-21在口腔鳞癌细胞株Tca8113及顺铂耐药细胞株Tca8113/DDP的表达;采用体外转染法将mi RNA-21模拟物(mimics)或抑制物(inhibitor)分别转染Tca8113和Tca8113/DDP细胞,采用RT-PCR检测转染前后细胞中mi RNA-21的表达情况;采用CCK8实验检测转染前后细胞对顺铂敏感性的变化;并用Western blot检测转染前后细胞中PTEN的表达变化。采用双荧光素酶报告基因验证mi RNA-21是否作用于PTEN基因的3’-UTR区预测靶位。结果:mi RNA-21在Tca8113/DDP细胞中的表达水平是Tca8113细胞的(8.26±1.37)倍(P<0.01)。Tca8113细胞转染mi RNA-21 mimics后,细胞中mi RNA-21表达水平是转染前的(10.51±2.18)倍(P<0.01),顺铂对Tca8113细胞增殖的抑制率较转染前明显下降(P<0.05),细胞内PTEN表达水平较转染前明显下降(P<0.05)。Tca8113/DDP细胞在转染mi RNA-21 inhibitor后,细胞中mi RNA-21表达水平是转染前的(0.32±0.14)倍(P<0.01),顺铂对Tca8113/DDP细胞增殖的抑制率与转染前比较明显增加(P<0.05),细胞内PTEN表达水平较转染前明显增高(P<0.05)。经双荧光素酶报告基因验证PTEN是mi RNA-21的靶基因。结论:mi RNA-21在口腔鳞癌顺铂耐药细胞Tca8113/DDP中异常高表达,下调其表达可部分逆转细胞耐药性,mi RNA-21可能通过作用于PTEN参与口腔鳞癌细胞顺铂耐药的发生和发展。     

The m6A methyltransferase METTL14 inhibits the proliferation,migration, and invasion of gastric cancer by regulating the PI3K/AKT/mTOR signaling pathway

Background N6‐methyladenosine (m6A) modification may participate in the regulation of occurrence and development of tumors. However, the m6A level and the potential regulatory mechanism of m6A in gastric cancer (GC) remain uncertain.MethodsRNA m6A quantification assay was conducted to detect the m6A level in GC tissues and cell lines. Methyltransferase‐like 14 (METTL14) expression in GC tissues was explored by bioinformatics and immunohistochemistry. Then, the function of METTL14 in GC cells was examined by CCK‐8, colony formation assay, wound healing assay, and Transwell assay. Besides, Western blotting was conducted to probe the PI3K/AKT/mTOR pathway and the epithelial‐mesenchymal transformation (EMT) pathway‐related gene expression.ResultsThe m6A modification level was decreased in GC and METTL14 was a key regulator resulting in m6A disorder in GC. METTL14 was downregulated in GC by analyzing both clinical samples and bioinformatics. METTL14 overexpression suppressed GC cell proliferation and aggression by deactivating the PI3K/AKT/mTOR pathway and the EMT pathway, respectively.ConclusionsOur findings indicate that METTL14 partakes in the biological process of GC as a tumor suppressor and may be an emerging biomarker in GC.     

钩吻总碱通过JAK2/STAT3/Survivin通路调控人舌癌细胞株Tca8113增殖、凋亡的作用探讨

目的观察钩吻总碱对人舌癌细胞株Tca8113增殖、凋亡的作用,并探讨其对酪氨酸蛋白激酶2(JAK2)/信号传导及转录激活因子3(STAT3)/Survivin通路的调控作用。方法取人舌癌细胞株Tca8113,培养至对数期,分装至6孔板中,分为对照组、A组、B组和C组,每组设置5个复孔。A组、B组和C组分别加入25μmol/L、50μmol/L、100μmol/L浓度的钩吻总碱处理(20μl),对照组加入等量PBS缓冲液。以倒置显微镜观察72 h后各组细胞形态;以MTT法检测24 h、48 h、72 h后细胞增殖抑制率;以流式细胞仪检测72 h后细胞凋亡率;以RT-PCR检测72 h后JAK2、STAT3、Survivin mRNA相对表达量;以WB检测72 h后IL-6蛋白、JAK2、STAT3、Survivin蛋白表达并计算p-JAK2/JAK2、p-STAT3/STAT3、p-Survivin/Survivin。结果72 h后,倒置显微镜下观察结果显示对照组细胞紧密,轮廓清晰,贴壁生长状态良好;A组细胞有浮起、变圆,B组和C组可见不同程度细胞壁皱缩和细胞碎片,其中C组变化最为明显,B组次之,A组变化最轻;各组24 h、48 h、72 h后增殖抑制率比较差异均有统计学意义(P<0.05),各组增殖抑制率均随时间延长显著增长,且呈时间依赖性和剂量依赖性;各组72 h后细胞凋亡率比较差异有统计学意义(P<0.05),各组凋亡率均随剂量升高显著增高;各组72 h后JAK2、STAT3、Survivin mRNA相对表达量比较差异均无统计学意义(P>0.05);各组p-JAK2/JAK2、p-STAT3/STAT3、p-Survivin/Survivin比较差异均有统计学意义(P<0.05),每两组间比较也可见差异具有统计学意义(P<0.05),其中对照组均最高,A组均次之,B组均稍低,C组均最低;各组IL-6蛋白表达比较差异无统计学意义(P>0.05)。结论钩吻总碱能够呈剂量依赖性抑制人舌癌细胞株Tca8113增殖、促进凋亡,推测与直接调控JAK2/STAT3/Survivin通路,抑制JAK2、STAT3、Survivin蛋白磷酸化有关。     

Circ_016719 plays a critical role in neuron cell apoptosis induced by I/R via targeting miR-29c/Map2k6

BackgroundStroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown.MethodsMice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B–I, LC3B-II, Beclin 1, and p62 expression.ResultsSignificantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B–I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719.ConclusionsOur results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.