Up-regulation of GINS1 highlighted a good diagnostic and prognostic potential of survival in three different subtypes of human cancer / A regulação positiva de GINS1 destacou um bom potencial diagnóstico e prognóstico de sobrevivência em três subtipos diferentes de câncer humano

Abstract Cancer is a fatal malignancy and its increasing worldwide prevalence demands the discovery of more sensitive and reliable molecular biomarkers. To investigate the GINS1 expression level and its prognostic value in distinct human cancers using a series of multi-layered in silico approach may help to establish it as a potential shared diagnostic and prognostic biomarker of different cancer subtypes. The GINS1 mRNA, protein expression, and promoter methylation were analyzed using UALCAN and Human Protein Atlas (HPA), while mRNA expression was further validated via GENT2. The potential prognostic values of GINS1 were evaluated through KM plotter. Then, cBioPortal was utilized to examine the GINS1-related genetic mutations and copy number variations (CNVs), while pathway enrichment analysis was performed using DAVID. Moreover, a correlational analysis between GINS1 expression and CD8+ T immune cells and a the construction of gene-drug interaction network was performed using TIMER, CDT, and Cytoscape. The GINS1 was found down-regulated in a single subtypes of human cancer while commonly up-regulated in 23 different other subtypes. The up-regulation of GINS1 was significantly correlated with the poor overall survival (OS) of Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), and Kidney renal clear cell carcinoma (KIRC). The GINS1 was also found up-regulated in LIHC, LUAD, and KIRC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of GINS1 in two diverse pathways, while few interesting correlations were also documented between GINS1 expression and its promoter methylation level, CD8+ T immune cells level, and CNVs. Moreover, we also predicted few drugs that could be used in the treatment of LIHC, LUAD, and KIRC by regulating the GINS1 expression. The expression profiling of GINS1 in the current study has suggested it a novel shared diagnostic and prognostic biomarker of LIHC, LUAD, and KIRC.

Resumo O câncer é uma doença maligna fatal e sua crescente prevalência mundial exige a descoberta de biomarcadores moleculares mais sensíveis e confiáveis. Investigar o nível de expressão de GINS1 e seu valor prognóstico em cânceres humanos distintos, usando uma série de abordagens in silico em várias camadas, pode ajudar a estabelecê-lo como um potencial biomarcador de diagnóstico e prognóstico compartilhado de diferentes subtipos de câncer. O mRNA de GINS1, a expressão da proteína e a metilação do promotor foram analisados ​​usando UALCAN e Human Protein Atlas (HPA), enquanto a expressão de mRNA foi posteriormente validada via GENT2. Os valores prognósticos potenciais de GINS1 foram avaliados por meio do plotter KM. Em seguida, o cBioPortal foi utilizado para examinar as mutações genéticas relacionadas ao GINS1 e as variações do número de cópias (CNVs), enquanto a análise de enriquecimento da via foi realizada usando DAVID. Além disso, uma análise correlacional entre a expressão de GINS1 e células imunes T CD8 + e a construção de uma rede de interação gene-droga foi realizada usando TIMER, CDT e Cytoscape. O GINS1 foi encontrado regulado negativamente em um único subtipo de câncer humano, enquanto comumente regulado positivamente em 23 outros subtipos diferentes. A regulação positiva de GINS1 foi significativamente correlacionada com a sobrevida global pobre (OS) de Carcinoma Hepatocelular de Fígado (LIHC), Adenocarcinoma de Pulmão (LUAD) e Carcinoma de Células Claras Renais de Rim (KIRC). O GINS1 também foi encontrado regulado positivamente em pacientes LIHC, LUAD e KIRC de diferentes características clínico-patológicas. A análise de enriquecimento de vias revelou o envolvimento de GINS1 em duas vias diversas, enquanto poucas correlações interessantes também foram documentadas entre a expressão de GINS1 e seu nível de metilação do promotor, nível de células imunes T CD8 + e CNVs. Além disso, também previmos poucos medicamentos que poderiam ser usados ​​no tratamento de LIHC, LUAD e KIRC, regulando a expressão de GINS1. O perfil de expressão de GINS1 no estudo atual sugeriu que é um novo biomarcador de diagnóstico e prognóstico compartilhado de LIHC, LUAD e KIRC.

Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo / 医学前沿

Nasopharyngeal carcinoma (NPC) is a malignant tumor that mainly occurs in East and Southeast Asia. Although patients benefit from the main NPC treatments (e.g., radiotherapy and concurrent chemotherapy), persistent and recurrent diseases still occur in some NPC patients. Therefore, investigating the pathogenesis of NPC is of great clinical significance. In the present study, replication factor c subunit 4 (RFC4) is a key potential target involved in NPC progression via bioinformatics analysis. Furthermore, the expression and mechanism of RFC4 in NPC were investigated in vitro and in vivo. Our results revealed that RFC4 was more elevated in NPC tumor tissues than in normal tissues. RFC4 knockdown induced G2/M cell cycle arrest and inhibited NPC cell proliferation in vitro and in vivo. Interestingly, HOXA10 was confirmed as a downstream target of RFC4, and the overexpression of HOXA10 attenuated the silencing of RFC4-induced cell proliferation, colony formation inhibition, and cell cycle arrest. For the first time, this study reveals that RFC4 is required for NPC cell proliferation and may play a pivotal role in NPC tumorigenesis.

GDF15 negatively regulates chemosensitivity via TGFBR2-AKT pathway-dependent metabolism in esophageal squamous cell carcinoma / 医学前沿

Treating patients with esophageal squamous cell carcinoma (ESCC) is challenging due to the high chemoresistance. Growth differentiation factor 15 (GDF15) is crucial in the development of various types of tumors and negatively related to the prognosis of ESCC patients according to our previous research. In this study, the link between GDF15 and chemotherapy resistance in ESCC was further explored. The relationship between GDF15 and the chemotherapy response was investigated through in vitro and in vivo studies. ESCC patients with high levels of GDF15 expression showed an inferior chemotherapeutic response. GDF15 improved the tolerance of ESCC cell lines to low-dose cisplatin by regulating AKT phosphorylation via TGFBR2. Through an in vivo study, we further validated that the anti-GDF15 antibody improved the tumor inhibition effect of cisplatin. Metabolomics showed that GDF15 could alter cellular metabolism and enhance the expression of UGT1A. AKT and TGFBR2 inhibition resulted in the reversal of the GDF15-induced expression of UGT1A, indicating that TGFBR2-AKT pathway-dependent metabolic pathways were involved in the resistance of ESCC cells to cisplatin. The present investigation suggests that a high level of GDF15 expression leads to ESCC chemoresistance and that GDF15 can be targeted during chemotherapy, resulting in beneficial therapeutic outcomes.

RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway / 医学前沿

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.

Lnc-TMEM132D-AS1 overexpression reduces sensitivity of non-small cell lung cancer cells to osimertinib / 南方医科大学学报

OBJECTIVE@#To screen the differentially expressed long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells.@*METHODS@#The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR.@*RESULTS@#The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P < 0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P < 0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D-AS1 showed the highest increase of expression after acquired osimertinib resistance (P < 0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P < 0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P < 0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P < 0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway.@*CONCLUSION@#The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinibresistant NSCLC.

Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.

Effect of long non-coding RNA 114227 on gastric cancer cell proliferation and migration / 中南大学学报(医学版)

OBJECTIVES@#Gastric cancer is a common cancer of the digestive system. Long non-coding RNA (lncRNA) plays an important role in the formation and development of gastric cancer. This study aims to investigate the effect of long non-coding lncRNA 114227 on biologic behaviors in gastric cancer cells.@*METHODS@#The experiment was divided into 4 groups: a negative control (NC) group, a lncRNA 114227 small interference (si-lncRNA 114227) group, an empty vector (Vector) group, and an overexpression vector (OE-lncRNA 114227) group. The expressions of lncRNA 114227 in gastric mucosa and gastric cancer tissues, gastric mucosal epithelial cells and different gastric cancer strains were determined by real-time reverse transcription PCR (real-time RT-PCR).The proliferation were detected by CCK-8 assay in gastric cancer cells. The epithelial-mesenchymal transformation (EMT) was utilized by Transwell assay, scratch healing assay, and Western blotting in gastric cancer cells. The effect of lncRNA 114227 on proliferation of gastric cancer cells was detected by tumor bearing experiment in nude mice in vivo.@*RESULTS@#The expression level of lncRNA 114227 in the gastric cancer tissues was significantly lower than that in the gastric mucosa tissues, and in 4 kinds of gastric cancer strains was all significantly lower than that in gastric mucosal epithelial cells (all P<0.01). In vitro, the proliferation and migration abilities of gastric cells were significantly reduced after overexpressing lncRNA 114227, and cell proliferation and migration were enhanced after silencing lncRNA 114227 (all P<0.05). The results of in vivo subcutaneous tumorigenesis in nude mice showed that the tumorigenic volume of the tumor-bearing mice in the OE-lncRNA 114227 group was significantly smaller than that of the Vector group, and the tumorigenic quality was lower than that of the Vector group (P<0.05), indicating that lncRNA 114227 inhibited tumorigenesis.@*CONCLUSIONS@#The expression of lncRNA 114227 is downregulated in gastric cancer gastric cancer tissues and cell lines. LncRNA 114227 may inhibit the proliferation and migration of gastric cancer cells through EMT process.

Effects of miR-9-5p on the migration, invasion and epithelial-mesenchymal transition process in cervical squamous cell carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells / 中华肿瘤杂志

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway / 中华肿瘤杂志

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

Knockdown of ACC1 promotes migration of esophageal cancer cell / 中华肿瘤杂志

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

Expression level of miR-199b in human colorectal cancer tissues and its correlation with clinicopathological features and prognosis of patients / 中华肿瘤杂志

Objective: To investigate the relationship between miR-199b and clinicopathologic features and prognosis of patients with colorectal cancer. Methods: Cancer tissues and adjacent normal tissues of 202 patients with colorectal cancer treated in Cancer Hospital of Chinese Academy of Medical Sciences from March to December 2011 were collected. Reverse transcription-quantitative real-time polymerase chain reaction was used to detect the expression level of miR-199b in colorectal cancer tissues and corresponding adjacent normal tissues. Kaplan-Meier method and Log rank test were used for survival analysis, and receiver operating characteristic (ROC) curve was used to evaluate the prognostic value of miR-199b in colorectal cancer patients. Results: The relative expression level of miR-199b in colorectal cancer tissues (-7.88±0.11) was lower than that in adjacent normal tissues (-6.49±0.12, P<0.001). The expression level of miR-199b in colorectal cancer tissues with lymph node metastasis (-7.51±0.14) was higher than that in colorectal cancer tissues without lymph node metastasis (-8.23±0.17, P<0.001). The relative expression levels of miR-199b in stage Ⅰ/Ⅱ, Ⅲ and Ⅳ colorectal cancer tissues were gradually increased, which were -8.26±0.17, -7.70±0.16 and -6.57±0.27, respectively, and the difference was statistically significant (P<0.001). The 5-year survival rates of patients with high and low expressions of miR-199b were 75.6% and 84.6%(P=0.045) respectively. ROC curve showed that when miR-199b was -7.965, the area under the curve was 0.578 (95% CI: 0.468, 0.688). Conclusion: The high expression of miR-199b in colorectal cancer tissues is associated with late TNM stage, lymph node metastasis and poor prognosis in colorectal cancer patients, and miR-199b may be used as a potential marker for postoperative progress and prognosis in colorectal cancer patients.

H19 recruited N 6 -methyladenosine (m 6 A) reader YTHDF1 to promote SCARB1 translation and facilitate angiogenesis in gastric cancer / 中华医学杂志(英文版)

BACKGROUND@#Angiogenesis is described as a complex process in which new microvessels sprout from endothelial cells of existing vasculature. This study aimed to determine whether long non-coding RNA (lncRNA) H19 induced the angiogenesis of gastric cancer (GC) and its possible mechanism.@*METHODS@#Gene expression level was determined by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8, transwell, 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay, and human umbilical vein endothelial cells (HUVECs) angiogenesis assay as well as Matrigel plug assay were conducted to study the proliferation, migration, and angiogenesis of GC in vitro and in vivo . The binding protein of H19 was found by RNA pull-down and RNA Immunoprecipitation (RIP). High-throughput sequencing was performed and next Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to analyze the genes that are under H19 regulation. Methylated RIP (me-RIP) assay was used to investigate the sites and abundance among target mRNA. The transcription factor acted as upstream of H19 was determined through chromatin immunoprecipitation (ChIP) and luciferase assay.@*RESULTS@#In this study, we found that hypoxia-induced factor (HIF)-1α could bind to the promoter region of H19, leading to H19 overexpression. High expression of H19 was correlated with angiogenesis in GC, and H19 knocking down could inhibit cell proliferation, migration and angiogenesis. Mechanistically, the oncogenic role of H19 was achieved by binding with the N 6 -methyladenosine (m 6 A) reader YTH domain-containing family protein 1 (YTHDF1), which could recognize the m 6 A site on the 3'-untransated regions (3'-UTR) of scavenger receptor class B member 1 (SCARB1) mRNA, resulting in over-translation of SCARB1 and thus promoting the proliferation, migration, and angiogenesis of GC cells.@*CONCLUSION@#HIF-1α induced overexpression of H19 via binding with the promoter of H19, and H19 promoted GC cells proliferation, migration and angiogenesis through YTHDF1/SCARB1, which might be a beneficial target for antiangiogenic therapy for GC.

lncRNA AC005224.4/miR-140-3p/SNAI2 regulating axis facilitates the invasion and metastasis of ovarian cancer through epithelial-mesenchymal transition / 中华医学杂志(英文版)

BACKGROUND@#Ovarian cancer is one of the most widespread malignant diseases of the female reproductive system worldwide. The plurality of ovarian cancer is diagnosed with metastasis in the abdominal cavity. Epithelial-mesenchymal transition (EMT) exerts a vital role in tumor cell metastasis. However, it remains unclear whether long non-coding RNA (lncRNA) are implicated in EMT and influence ovarian cancer cell invasion and metastasis. This study was designed to investigate the impacts of lncRNA AC005224.4 on ovarian cancer.@*METHODS@#LncRNA AC005224.4, miR-140-3p, and snail family transcriptional repressor 2 ( SNAI2 ) expression levels in ovarian cancer and normal ovarian tissues were determined using real-time quantitative polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and Transwell (migration and invasion) assays were conducted to measure SKOV3 and CAOV-3 cell proliferation and metastasis. E-cadherin, N-cadherin, Snail, and Vimentin contents were detected using Western blot. Nude mouse xenograft assay was utilized to validate AC005224.4 effects in vivo . Dual-luciferase reporter gene assay confirmed the targeted relationship between miR-140-3p and AC005224.4 or SNAI2 .@*RESULTS@#AC005224.4 and SNAI2 upregulation and miR-140-3p downregulation were observed in ovarian cancer tissues and cells. Silencing of AC005224.4 observably moderated SKOV3 and CAOV-3 cell proliferation, migration, invasion, and EMT process in vitro and impaired the tumorigenesis in vivo . miR-140-3p was a target of AC005224.4 and its reduced expression level was mediated by AC005224.4. miR-140-3p mimics decreased the proliferation, migration, and invasion of ovarian cancer cells. SNAI2 was identified as a novel target of miR-140-3p and its expression level was promoted by either AC005224.4 overexpression or miR-140-3p knockdown. Overexpression of SNAI2 also facilitated ovarian cancer cell viability and metastasis.@*CONCLUSION@#AC005224.4 was confirmed as an oncogene via sponging miR-140-3p and promoted SNAI2 expression, contributing to better understanding of ovarian cancer pathogenesis and shedding light on exploiting the novel lncRNA-directed therapy against ovarian cancer.

MiR-30e-5p overexpression promotes proliferation and migration of colorectal cancer cells by activating the CXCL12 axis via downregulating PTEN / 南方医科大学学报

OBJECTIVE@#To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects.@*METHODS@#Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice.@*RESULTS@#Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P < 0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P < 0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P < 0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P < 0.05), and co-transfection with LV-PTEN obviously reversed these changes (P < 0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P < 0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P < 0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis.@*CONCLUSION@#Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.

Lactate-induced up-regulation of PLEKHA4 promotes proliferation and apoptosis of human glioma cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism.@*METHODS@#GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway.@*RESULTS@#The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01).@*CONCLUSION@#PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.

NKD1 promotes glucose uptake in colon cancer cells by activating YWHAE transcription / 南方医科大学学报

OBJECTIVE@#Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation.@*METHODS@#HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1-/- cells), and SW620-nkd1-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells.@*RESULTS@#In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P < 0.05).@*CONCLUSION@#NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.

Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells.@*METHODS@#Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated.@*RESULTS@#Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing.@*CONCLUSION@#RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

HSDL2 overexpression promotes rectal cancer progression by regulating cancer cell cycle and promoting cell proliferation / 南方医科大学学报

OBJECTIVE@#To analyze the expression of hydroxysteroid dehydrogenase like 2 (HSDL2) in rectal cancer tissues and the effect of changes in HSDL2 expression level on proliferation of rectal cancer cells.@*METHODS@#Clinical data and tissue samples of 90 patients with rectal cancer admitted to our hospital from January 2020 to June 2022 were collected from the prospective clinical database and biological specimen database. The expression level of HSDL2 in rectal cancer and adjacent tissues was detected by immunohistochemistry, and based on the median level of HSDL2 expression, the patients were divided into high expression group (n=45) and low expression group (n=45) for analysis the correlation between HSDL2 expression level and the clinicopathological parameters. GO and KEGG enrichment analyses were performed to explore the role of HSDL2 in rectal cancer progression. The effects of changes in HSDL2 expression levels on rectal cancer cell proliferation, cell cycle and protein expressions were investigated in SW480 cells with lentivirus-mediated HSDL2 silencing or HSDL2 overexpression using CCK-8 assay, flow cytometry and Western blotting.@*RESULTS@#The expressions of HSDL2 and Ki67 were significantly higher in rectal cancer tissues than in the adjacent tissues (P < 0.05). Spearman correlation analysis showed that the expression of HSDL2 protein was positively correlated with Ki67, CEA and CA19-9 expressions (P < 0.01). The rectal cancer patients with high HSDL2 expressions had significantly higher likelihood of having CEA ≥5 μg/L, CA19-9 ≥37 kU/L, T3-4 stage, and N2-3 stage than those with a low HSDL2 expression (P < 0.05). GO and KEGG analysis showed that HSDL2 was mainly enriched in DNA replication and cell cycle. In SW480 cells, HSDL2 overexpression significantly promoted cell proliferation, increased cell percentage in S phase, and enhanced the expression levels of CDK6 and cyclinD1 (P < 0.05), and HSDL2 silencing produced the opposite effects (P < 0.05).@*CONCLUSION@#The high expression of HSDL2 in rectal cancer participates in malignant progression of the tumor by promoting the proliferation and cell cycle progress of the cancer cells.

Study on F9 gene expression downregulation and its clinical value in hepatocellular carcinoma / 中华肝脏病杂志

Objective: To analyze the expression levels of the F9 gene and F9 protein in hepatocellular carcinoma by combining multiple gene chip data, real-time fluorescence quantitative PCR (RT qPCR), and immunohistochemistry. Additionally, explore their correlation with the occurrence and development of hepatocellular carcinoma, as well as with various clinical indicators and prognosis. Methods: The mRNA microarray dataset from the GEO database was analyzed to identify the F9 gene with significant expression differences associated with hepatocellular carcinoma. Liver cancer and adjacent tissues were collected from 18 cases of hepatocellular carcinoma. RT-qPCR method was used to detect the F9 gene expression level. Immunohistochemistry was used to detect the F9 protein level. Combined with the TCGA database information, the correlation between F9 gene expression level and prognostic and clinicopathological parameters was analyzed. The biological function of F9 co-expressed genes associated with hepatocellular carcinoma was analyzed by the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Statistical analysis was performed using Graphpad Prism software. Results: Meta-analysis results showed that the expression of the F9 gene was lower in HCC tissues than in non-cancerous tissues. Immunohistochemistry results were basically consistent with those of RT-qPCR. The data obtained from TCGA showed that the F9 gene had lower expression values in stages III-IV, T3-T4, and patients with vascular invasion. A total of 127 genes were selected for bioinformatics analysis as co-expressed genes of F9, which were highly enriched in redox processes and metabolic pathways. Conclusion: This study validates that the F9 gene and F9 protein are lower in HCC. The down-regulation of the F9 gene predicts adverse outcomes, which may provide a new therapeutic target for HCC.