Read-through circular RNA rt-circ-HS promotes hypoxia inducible factor 1α expression and renal carcinoma cell proliferation, migration and invasiveness / 北京大学学报(医学版)

OBJECTIVE@#To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α.@*METHODS@#Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays.@*RESULTS@#We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α.@*CONCLUSION@#The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.

The effect of HOXC10 gene on biological behaviors of glioma cells and mechanism in tumor microenvironment / 中华肿瘤杂志

Objective: To study the effects of Homeobox C10 (HOXC10) on biological characteristics such as migration, invasion and proliferation of glioma cancer cells and to explore the role of HOXC10 gene in glioma microenvironment. Methods: The expression level of HOXC10 in high grade glioma (glioblastoma) and low grade glioma and its effect on patient survival were analyzed by using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) database. Hoxc10-siRNA-1, HOXC10-siRNA-2 and siRNA negative control (NC) were transfected into U251 cells according to the operation instructions of HOXC10-siRNA transfection. 100 ng/ mL recombinant protein chemokine ligand 2 (reCCL2) was added into the transfection group, and was labeled as HOXC10-siRNA-1+ reCCL2 and HOXC10-siRNA-2+ reCCL2 groups. The expressions of HOXC10 mRNA and target protein in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot. The proliferation ability of cells in each group was detected by cell counting kit 8 (CCK8) method. The migration ability of cells was detected by Transwell assay and Nick assay, and cell apoptosis was detected by flow cytometry. The expression of chemokines in each group was detected by multiple factors. Co-incubation assays were performed to determine the role of HOXC10 and chemokine ligand 2 (CCL2) in recruiting and polarizing tumor-associated macrophages (M2-type macrophages). Results: The median expression level of HOXC10 in high grade gliomas was 8.51, higher than 1.00 in low grade gliomas (P<0.001) in TCGA database. The median expression level of HOXC10 in high grade gliomas was 0.83, higher than 0.00 in low grade gliomas (P=0.002) in CGGA database. The 5-year survival rate of patients with high HOXC10 expression in TCGA database was 28.2%, lower than 78.7% of those with low HOXC10 expression (P<0.001), and the 5-year survival rate of patients with high HOXC10 expression in CGGA database was 20.3%, lower than 58.0% of those with low HOXC10 expression (P<0.001). The numbers of cell migration in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (45±3) and (69±4) respectively, lower than (159±3) in NC group (P<0.05). The cell mobility of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group at 48 hours were (15±2)% and (28±4)% respectively, lower than (80±5)% of NC group (P<0.05). The expressions of vimentin in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (141 740.00±34 024.56) and (94 655.00±5 687.97), N-cadherin were (76 810.00±14.14) and (94 254.00±701.45), β-catenin were (75 786.50±789.84) and (107 296.50±9 614.53), lower than (233 768.50±34 114.37), (237 154.50±24 715.50) and (192 449.50±24 178.10) of NC group (P<0.05). The A value of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.44±0.05) and (0.32±0.02) at 96 hours, lower than 0.92±0.12 of NC group (P<0.05). The apoptosis rates of HOXC10-siRNA-1 group and HOXC10 siRNA-2 group were (10.23±1.24)% and (13.81±2.16)%, higher than (4.60±0.07)% of NC group (P<0.05). The expression levels of CCL2 in U251 cells in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (271.63±44.27) and (371.66±50.21), lower than (933.93±29.84) in NC group (P<0.05). The expression levels of CCL5 (234.81±5.95 and 232.62±5.72), CXCL10 (544.13±48.14 and 500.87±15.65) and CXCL11 (215.75±15.30 and 176.18±16.49) in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were higher than those in NC group (9.98±0.71, 470.54±18.84 and 13.55±0.73, respectively, P<0.05). The recruited numbers of CD14(+) THP1 in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (159.33±1.15) and (170.67±1.15), respectively, lower than (360.00±7.81) in NC group (P<0.05), while addition of reCCL2 promoted the recruitment of CD14(+) THP1 cells (287.00±3.61 and 280.67±2.31 in HOXC10-siRNA-1+ reCCL2 group and HOXC10-siRNA-2+ reCCL2 group, respectively, P<0.05). The expressions level of M2-type macrophage-related gene TGF-β in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.30±0.02) and (0.28±0.02), respectively, lower than (1.06±0.10) in NC group (P<0.05). The expressions level of M1-related gene NOS2 in HOXC10-siRNA-1 and HOXC10-siRNA-2 were (11 413.95±1 911.85) and (5 894.00±945.21), respectively, higher than (13.39±4.32) in NC group (P<0.05). Conclusions: The expression of HOXC10 in glioma is high and positively correlated with the poor prognosis of glioma patients. Knockdown of HOXC10 can inhibit the proliferation, migration and metastasis of human glioma U251 cells. HOXC10 may play an immunosuppressive role in glioma microenvironment by promoting the expression of CCL2 and recruiting and polarizing tumor-associated macrophages (M2 macrophages).

Effect of chromodomain helicase/ATPase DNA binding protein 1-like gene on the invasion and metastasis of tongue squamous cell carcinoma CAL27 cells / 华西口腔医学杂志

OBJECTIVES@#A study was conducted to investigate the molecular mechanism of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) influencing the invasion and metastasis of tongue squamous cell carcinoma and to provide a new target for clinical inhibition of invasion and metastasis of tongue squamous cell carcinoma.@*METHODS@#Ualcan website was used to analyze the expression of CHD1L in normal epithelial tissue and primary head and neck squamous cell carcinoma and to analyze the effect of lymph node metastasis on the expression of CHD1L in tissues with head and neck squamous cell carcinoma. The relationship between CHD1L expression and the survival rate of patients with head and neck squamous cell carcinoma was tested by the GEPIA website. Western blot was used to quantify the levels of CHD1L protein in human tongue squamous cell carcinoma CAL27 and immortalized human skin keratinocyte cell HaCaT. After knocking down CAL27 in human tongue squamous cell carcinoma cells with an RNA interference plasmid, the cells were designated as SiCHD1L/CAL27 and Scr/CAL27. Western blot was utilized to detect the expression of CHD1L in each group of cells. The change in CAL27 cell proliferation ability was tested by EdU proliferation test after CHD1L knockdown. The change of cell migration ability of each group cells was tested through the wound healing assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) marker E-cadherin and Vimentin protein expression levels.@*RESULTS@#Ualcan database showed that the expression of CHD1L in primary head and neck squamous cell carcinoma tissues was higher than in normal epithelial tissues and in head and neck squamous cell carcinoma tissues with lymph node metastasis. GEPIA website analysis showed that the overall survival rate of patients with head and neck squamous cell carcinoma with high expression of CHD1L was significantly lower than that of patients with low expression. Western blot results showed that CHD1L expression in human tongue squamous carcinoma cells CAL27 was higher than that of human normal skin cells HaCaT. CHD1L expression in SiCHD1L/CAL27 cells was much lower than that in Scr/CAL27 cells. Results of EdU proliferation experiments showed the significant reduction in the cell proliferation ability of the SiCHD1L/CAL27 cells. Results of the wound healing experiments showed the reduction in the migration capacity of the SiCHD1L/CAL27 cells. The expression of E-cadherin increased, whereas that of Vimentin decreased, in SiCHD1L/CAL27 cells.@*CONCLUSIONS@#CHD1L promoted the EMT, proliferation, migration, and invasion ability of tongue squamous cell carcinoma cells.

Resveratrol inhibits the migration, invasion and epithelial-mesenchymal transition in liver cancer cells through up- miR-186-5p expression / 浙江大学学报·医学版

To investigate the molecular mechanism of resveratrol inhibiting the metastasis of liver cancer . HepG2 and Huh7 cells were treated with different concentrations of resveratrol, and the cell viability was determined by CCK-8 assay to determine the optimal concentration of resveratrol for subsequent experiments. The expressions of miR-186-5p in liver cancer tissues and liver cancer cells were determined by quantitative real-time RT-PCR. The migration and invasion of HepG2 and Huh7 cells were detected by wound healing assay and Transwell assay, and the expression levels of epithelial-mesenchymal transition (EMT) related proteins were determined by Western blotting. Resveratrol with concentration of had no effect on the viability of HepG2 and Huh7 cells, so the concentration of resveratrol in subsequent experiments was 6.25 μmol/L. Resveratrol inhibited the wound healing and invasion of liver cancer cells; increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. The expression of miR-186-5p was significantly down-regulated in liver cancer tissues and cells compared with the adjacent tissues and normal liver cells (both <0.05). Furthermore, resveratrol induced the expression of miR-186-5p in liver cancer cells (both <0.01). Overexpression of miR-186-5p suppressed the migration, invasion and EMT of liver cancer cells. Knockdown of miR-186-5p blocked the inhibition effects of resveratrol on the migration, invasion and EMT of liver cancer cells. Resveratrol could inhibit the metastasis of liver cancer , which might be associated with up-regulating miR-186-5p.

MiR-4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-4443 expression on migration and invasion of breast cancer.@*METHODS@#We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells.@*RESULTS@#The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues (@*CONCLUSIONS@#MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.

Astrocyte elevated gene-1 serves as a target of miR542 to promote glioblastoma proliferation and invasion / 中华医学杂志(英文版)

BACKGROUND@#Epithelial to mesenchymal transition (EMT) is strongly linked with tumor invasion and metastasis, which performs a vital role in carcinogenesis and cancer progression. Emerging evidence suggests that microRNAs (miRNAs) expression are closely associated to EMT by regulating targeted genes. MiR542 has been found to be involved in the EMT program and bound up with various cancers. However, the functions of miR542 and its underlying mechanism in glioblastoma multiforme (GBM) remain largely unknown. In the current study, we investigated the effect of astrocyte elevated gene-1 (AEG-1) on U251 cells aggressiveness, proliferation, apoptosis, and cell cycle.@*METHODS@#The screening of targeted miRNAs was performed, as well as the functional roles and mechanisms of miR542 were explored.@*RESULTS@#MiR542 was selected as the target because of the most significantly differential expression and this high level of expression negatively correlated with cell migration and proliferation, which suggested that miR542 could be a novel tumor suppressor. Moreover, we confirmed that AEG-1 was a direct targeted gene of miR542 by luciferase activity assay, reverse transcription-polymerase chain reaction, and immunoblotting analysis. Furthermore, miR542 suppressed the expression of AEG-1, which upgraded the level of E-cadherin and degraded Vimentin expression contributing to retraining EMT.@*CONCLUSION@#The in vitro findings demonstrated that miR542 inhibited the migration and proliferation of U251 cells and suppressed EMT through targeting AEG-1, indicating that miR542 may be a potential anti-cancer target for GBM.

MiR-128 suppresses metastatic capacity by targeting metadherin in breast cancer cells

BACKGROUND: Breast cancer, the most common cancer in women worldwide, causes the vast majority of cancer-related deaths. Undoubtedly, tumor metastasis and recurrence are responsible for more than 90 percent of these deaths. MicroRNAs are endogenous noncoding RNAs that have been integrated into almost all the physiological and pathological processes, including metastasis. In the present study, the role of miR-128 in breast cancer was investigated. RESULTS: Compared to the corresponding adjacent normal tissue, the expression of miR-128 was significantly suppressed in human breast cancer specimens. More importantly, its expression level was reversely correlated to histological grade of the cancer. Ectopic expression of miR-128 in the aggressive breast cancer cell line MDA-MB-231 could inhibit cell motility and invasive capacity remarkably. Afterwards, Metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric that implicated in various aspects of cancer progression and metastasis, was further identified as a direct target gene of miR-128 and its expression level was up-regulated in clinical samples as expected. Moreover, knockdown of MTDH in MDA-MB-231 cells obviously impaired the migration and invasion capabilities, whereas re-expression of MTDH abrogated the suppressive effect caused by miR-128. CONCLUSIONS: Overall, these findings demonstrate that miR-128 could serve as a novel biomarker for breast cancer metastasis and a potent target for treatment in the future.

MicroRNA-663 facilitates the growth, migration and invasion of ovarian cancer cell by inhibiting TUSC2

BACKGROUND: MicroRNAs (miRNAs) have emerged as the critical modulators of the tumorigenesis and tumor progression. METHODS: The levels of miR-663 in ovarian cancer cell lines and clinical tissues were detected using qRT-PCR assays. The Transwell invasion and wound healing assay were conducted to assess the roles of miR-663 in the migration and invasion of ovarian cancer cell in vitro. Rescue assays were carried out to confirm the contribution of tumor suppressor candidate 2 (TUSC2) in the aggressiveness of cancer cell which was regulated by miR-663. RESULTS: The levels of miR-663 were up-regulated in ovarian cancer tissues in comparison with the corresponding normal tissues. Up-regulation of miR-663 increased the proliferation, colony formation, migration and invasion of ovarian cancer SKOV3 cell. Additional, over-expression of miR-663 increased the tumor growth of SKOV3 in xenograft model. Bioinformatics analysis and luciferase reporter assay identified that miR-663 decreased the level of TUSC2 via binding to the 3'-UTR of TUSC2 gene. Finally, the expression of TUSC2 was inversely associated with the level of miR-663 in ovarian carcinoma tissue and over-expression of TUSC2 inhibited the migration and invasion abilities of SKOV3 that was promoted by miR-663. CONCLUSION: Altogether, these results indicate that miR-663 acts as a potential tumor-promoting miRNA through targeting TUSC2 in ovarian cancer.

MicroRNA-382 inhibits cell growth and migration in colorectal cancer by targeting SP1

BACKGROUND: Emerging evidence showed that microRNAs (miRs) play critical roles in human cancers by functioning as either tumor suppressor or oncogene. MIR-382 was found to function as tumor suppressor in certain cancers. However, the role of MIR-382 in colorectal cancer (CRC) is largely unknown. Specificity protein 1 (SP1) is highly expressed in several cancers including CRC and is correlated with poor prognosis, but it is unclear whether or not MIR-382 can regulate the expression of SP1. METHODS: MIR-382 expression level was measured by reverse transcription-quantitative polymerase chain reaction. The connection between MIR-382 and SP1 was validated by luciferase activity reporter assay and western blot assay. Cell counting kit-8 assay and wound-healing assay were conducted to investigate the biological functions of MIR-382 in CRC. RESULTS: In this study, we found MIR-382 expression was downregulated in CRC tissues and cell lines, and the transfection of MIR-382 mimic decreased cell growth and migration. Furthermore, we identified SP1 was a direct target of MIR-382. Overexpression of MIR-382 decreased the expression of SP1, whereas MIR-382 knockdown promoted SP1 expression. We also observed an inversely correlation between MIR-382 and SP1 in CRC tissues. Additionally, we showed that knockdown of SP1 inhibited cell growth and migration and attenuated the effect of MIR-382 inhibitor on cell behaviors. CONCLUSIONS: In conclusion, the present study describes a potential mechanism underlying a MIR-382/SP1 link contributing to CRC development. Thus, MIR-382 may be able to be developed as a novel treatment target for CRC.

Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer

BACKGROUND: Zinc finger RNA binding protein (ZFR) is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer. METHODS: Herein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA) targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system. RESULTS: The expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis. CONCLUSIONS: Knockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

miR-205 promotes proliferation and invasion of laryngeal squamous cell carcinoma by suppressing CDK2AP1 expression

BACKGROUND: The aberrant expression of microRNAs (miRNAs) has been found in various types of cancer. miR-205 was reported to be upregulated in laryngeal squamous cell carcinoma (LSCC) tissues, however, the mechanisms by which miR-205 functions as a regulator of LSCC are largely unknown. RESULTS: In this study, Real-time qPCR and Western blot assay showed that expression of miR-205 was upregulated and expression of cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) was downregulated in LSCC tissues. The expression levels of miR-205 were negatively related to those of CDK2AP1 in LSCC tissues and cell lines. Moreover, we found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the CDK2AP1 expression in LSCC cells. 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide assays and transwell invasion assay were performed to test the proliferation and invasion of LSCC cells. Gelatin zymography was used to detect the activity of MMP2 and MMP9. CDK2AP1, c-Myc and CyclinD1 expression in cells was assessed with Western blotting. We found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the expression of CDK2AP1 in LSCC cells. In addition, miR-205 significantly induced cell proliferation and invasion by suppressing CDK2AP1 expression. Consistent with miR-205 inhibitors, overexpressed CDK2AP1 suppressed the activity of MMP2 and MMP9 and c-Myc and CyclinD1 expression in LSCC cells. CONCLUSION: These findings help us to better elucidate the molecular mechanisms of LSCC progression and provide a new theoretical basis to further investigate miR-205 as a potential biomarker and a promising approach for LSCC treatment.

Prevalencia y características de mutaciones somáticas del gen KRAS en pacientes chilenos con cáncer colorrectal / KRAS gene somatic mutations in Chilean patients with colorectal cancer

Background: The molecular testing of KRAS mutation status in metastatic colorectal cancer patients is mandatory to identify patients eligible for anti-epidermal growth factor receptor monoclonal antibody therapy. Aim: To report the frequency of KRAS gene mutations in Chilean patients with colorectal cancer (CRC). Material and Methods: A cohort of 262 Chilean patients with CRC aged 26 to 90 years (53% males), was studied. KRAS mutation status was analyzed by real-time polymerase chain reaction and correlated with clinicopathological data. Results: Ninety-eight patients (37%) were positive for KRAS mutations. G12D was the most common mutation with a frequency of 36.7%, followed by G12V (25.5%), G13D (17.3%), G12A (7.1%), G12C (6.1%), G12S (5.1%) and G12R (2%). The frequency of the mutation in left, right colon and rectal tumors was 37.8, 32.6 and 44.9%, respectively. Among tumors with mutations, 86.7% were well or moderately differentiated tumors and the rest were poorly differentiated. No significant associations between KRAS gene mutations and other clinicopathological features of the tumor were observed. Conclusions: The frequencies of KRAS mutations reported in this study are similar to frequencies reported for European and North-American populations, lower than in a Spanish study and higher than in a Peruvian study.

Use of microRNAs in directing therapy and evaluating treatment response in colorectal cancer / Utilização dos microRNAs no direcionamento da terapia e na avaliação da resposta ao tratamento do câncer colorretal

Colorectal cancer is the third most common cancer worldwide. Survival and prognosis depend on tumor stage upon diagnosis, and in more than 50% of cases, the tumor has already invaded adjacent tissues or metastasis has occurred. Aiming to improve diagnosis, clinical prognosis and treatment of patients with colorectal cancer, several studies have investigated microRNAs as molecular markers of the disease due to their potential regulatory functions on tumor suppressor genes and oncogenes. This review aimed to summarize the main topics related to the use of microRNAs in diagnosis, clinical prognosis and evaluating treatment response in colorectal cancer.

O câncer colorretal é o terceiro tipo de câncer mais comum em todo o mundo. A sobrevivência e o prognóstico dependem do estágio do tumor no diagnóstico, momento em que, em mais de 50% dos casos, o tumor já invadiu tecidos adjacentes ou ocorreu metástase. Objetivando-se melhorar o diagnóstico, o prognóstico clínico e o tratamento de pacientes com câncer colorretal, vários estudos investigaram microRNAs como marcadores moleculares da doença, devido à sua função reguladora potencial sobre genes supressores de tumor e oncogenes. Esta revisão procura resumir os principais tópicos relacionados ao uso de microRNAs no diagnóstico, na determinação do prognóstico clínico e na avaliação de resposta ao tratamento do câncer colorretal.

DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.

Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

PURPOSE: Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. MATERIALS AND METHODS: We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. RESULTS: MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. CONCLUSION: We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

MiR-152 suppresses the proliferation and invasion of NSCLC cells by inhibiting FGF2

MicroRNAs (miRNAs) regulate the proliferation and metastasis of cancer cells. Here, we showed that miR-152 was downregulated in non-small-cell lung cancer (NSCLC) tissues and cell lines. Overexpression of miR-152 suppressed cell proliferation and colony formation and also limited migration and invasion. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-152. FGF2 knockdown suppressed cell proliferation, colony formation, migration and invasion, whereas FGF2 overexpression partially reversed the suppressive effect of miR-152. Furthermore, the presence of miR-152 was inversely correlated with FGF2 in NSCLC tissues. Overall, this study demonstrated that miR-152 suppressed the proliferation and invasion of NSCLC cells by downregulating FGF2. These findings provide novel insights with potential therapeutic applications for the treatment of NSCLC.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Analysis of breast cancer progression using principal component analysis and clustering.

We develop a new technique to analyse microarray data which uses a combination of principal components analysis and consensus ensemble k-clustering to find robust clusters and gene markers in the data. We apply our method to a public microarray breast cancer dataset which has expression levels of genes in normal samples as well as in three pathological stages of disease; namely, atypical ductal hyperplasia or ADH, ductal carcinoma in situ or DCIS and invasive ductal carcinoma or IDC. Our method averages over clustering techniques and data perturbation to find stable, robust clusters and gene markers. We identify the clusters and their pathways with distinct subtypes of breast cancer (Luminal,Basal and Her2+). We confirm that the cancer phenotype develops early (in early hyperplasia or ADH stage) and find from our analysis that each subtype progresses from ADH to DCIS to IDC along its own specific pathway, as if each was a distinct disease.

Effect of TSLC1 gene on proliferation, invasion and apoptosis of human hepatocellular carcinoma cell line HepG2 / 华中科技大学学报（医学）（英德文版）

The recombinant plasmid pCI-TSLC1 carrying TSLC1 gene was stably transfected into human hepatocellular carcinoma cell line HepG2. Cell proliferation was analyzed by MTT assay. The ability of migration was determined by transwell and FACSort flow cytometry was used to detect the cell cycle distribution and apoptosis. Western blotting revealed that H4 expressed higher amounts of TSLC1 protein than H15 and H0 did. The growth of TSLC1-transfected cells was significantly suppressed in vitro, and the ability of migration was reduced as well. The re-expression of TSLC1 could induce cell apoptosis. It was concluded that TSLC1 strongly inhibited the growth and ability of migration of HepG2 cell line in vitro and also induced apoptosis, suggesting that TSLC1 could reduce the tumorigenicity of human hepatocellular carcinoma cell line HepG2 in vitro, which provided a basis for further exploring the roles of TSLC1 in hepatocellular cellular carcinoma.

Mecanismo de infección y transformación neoplásica producido por virus papiloma humano en el epitelio cervical / Infection mechanism and neoplastic transformation produced by human papilloma virus in cervical epithelium

Se presenta una revisión bibliográfica del mecanismo de infección y transformación neoplásica producida por el virus papiloma humano, de alto riesgo oncogénico, en el epitelio cervical. Se expone la interacción cápside receptor, internalización celular, expresión de genes tempranos, integración del genoma viral al de la célula huésped y algunos mecanismos vinculados a la proliferación y desarrollo neoplásico.