Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

High mobility group box 1 protein promotes proliferation,migration and invasion of colon cancer cells

AIM: To investigate the expression of high mobility group box 1 protein (HMGB1) in colon can-cer cells, and to determine its regulatory roles in colon cancer cell proliferation, migration and invasion. METHODS: qPCR and Western blot were used to quantify the mRNA and protein expression levels of HMGB1 in human colon cancer SW620 cells and normal colonic epithelial FHC cells. HMGB1 shRNA was transfected into the SW620 cells to establish the stable HMGB1-downregulating colon cancer cells (shHMGB1 group), and negative control (shNC) group and blank control (blank) group were also set up. The proliferation, migration and invasion of the cells were determined by CCK-8 assay, colony formation experiment and Transwell chamber assays. Western blot was used to determine the protein levels of p-ERK, ERK, c-Myc, matrix metalloproteinase (MMP)-2/9, E-cadherin, N-cadherin, Bcl-2 and Bax. RESULTS: Both of the mRNA and protein levels of HMGB1 in colon cancer cells were higher than those in the normal colonic epithelial cells (P<0.05). HMGB1 gene was successfully knocked down in SW620 cells. Compared with blank group and shNC group, the proliferation, migration and invasion abilities of the cells in shHMGB1 group were significantly inhibited (P<0.05). The protein levels of MMP-2, MMP-9, N-cadherin, c-Myc, Bcl-2 and p-ERK were reduced notably, while the expression of Bax protein was increased (P<0.05) in shHMGB1 group compared with shNC group and blank group.CONCLUSION: HMGB1 effectively promotes the proliferation, migration and invasion of colon cancer cells through ERK/c-Myc signaling pathway.     

Effects of lentivirus-mediated RWDD3 silencing on proliferation and invasion of human glioma U251 cells

AIM: To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells. The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell activity was determined by MTT assay. The colony formation ability was detected by the colony formation assay. The cell proliferation ability was detected by BrdU incorporation assay. The cell invasion and migration were evaluated by Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis.RESULTS: Recombinant lentivirus was successfully transfected into U251 cells. Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G₀/G₁ phase, and the apoptosis was increased in the U251 cells transfected with RWDD3 shRNA(P<0.05).CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells. It may serve as a potential target of gene therapy for glioma.     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Effect of reversing methylation of SARI gene by 5-azacytidine on proliferation and invasion of human breast cancer cells

AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion.     

Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Metformin sensitizes human breast cancer MDA-MB-231 cells to tamo-xifen through down-regulation of c-Myc

AIM: To investigate whether metformin enhances the sensitivity of human breast cancer MDA-MB-231 cells to tamoxifen by down-regulating c-Myc. METHODS: The cell viability, colony formation, apoptosis, and migration and invasion abilities of MDA-MB-231 cells were detected by CCK-8 assay, colony formation experiment, flow cytometry and Transwell assay. The expression level of c-Myc was quantified by Western blot and immunohistochemical analysis. The antitumor effects of metformin and tamoxifen were investigated in vivo in a MDA-MB-231 triple-negative breast cancer xenograft model in the SCID mice. RESULTS: Metformin in combination with tamoxifen exerted synergistic effects on inhibition of the viability, colony formation, migration and invasion, and induced the apoptosis compared with the controls and either agent treatment alone in the MDA-MB-231 cells. The levels of c-Myc was down-regulated in vitro by treatment with metformin and/or tamoxifen (P<0.01). Moreover, metformin or in combination with tamoxifen also reduced the growth of MDA-MB-231 breast cancer tumors in the SCID mice by down-regulation of c-Myc in vivo. CONCLUSION: Metformin in combination with tamoxifen exerts synergistic effects on inhibition of the proliferation, migration, invasion and tumor growth of human triple-negative breast cancer MDA-MB-231 cells by down-regulating c-Myc expression, suggesting that metformin in combination with tamoxifen merits further evaluation as a target.     

Effect of long non-coding RNA-671 on proliferation and invasion of esophageal squamous-cell carcinoma cells

AIM: To detect the expression of long non-coding RNA-671 (lnc671) in esophageal squamous-cell carcinoma cell lines and to investigate the effect of lnc671 on the malignant phenotype of esophageal squamous-cell carcinoma cells. METHODS: The level of lnc671 in the esophageal squamous-cell carcinoma cells was detected by RT-qPCR. Specific lnc671 small interfering RNA (siRNA) used to explore the effects of lnc671 on proliferation, colony formation, invasion and migration abilities of esophageal squamous-cell carcinoma cells. RESULTS: The database of GEPIA analysis showed that increased expression of lnc671 was associated with shorter survival in the patients of esophageal cancer (P<0.05). Compared with normal immortalized esophageal epithelial cells, lnc671 was highly expressed in a variety of esophageal squamous-cell carcinoma cell lines. lnc671 knock-down significantly inhibited the growth, colony formation ability, migration and invasion abilities of esophageal squamous-cell carcinoma cells(P<0.01). CONCLUSION: The expression of lnc671 is increased in various esophageal squamous-cell carcinoma cell lines. Knock-down of lnc671 expression inhibits the malignant phenotype of esophageal squamous-cell carcinoma cells.     

Effects of lupeol and miR-145-5p on proliferation and apoptosis of prostate carcinoma cells

AIM To investigate the effect of lupeol combined with microRNA-145-5p (miR-145-5p) on the proliferation and apoptosis of prostate carcinoma LNCaP cells. METHODS After hsa-miR-145-5p and lupeol were applied to LNCaP cells for 24, 48 and 72 h, the cell viability inhibitory rate was detected by MTT assay. PI single staining plus flow cytometry was used to detect the cycle distribution. The flow cytometry with annexin V/PI double staining and TUNEL experiment were used to detect apoptosis. Transwell method was used to detect cell migration and invasion abilities. Wound healing experiment was used to detect cell migration ability. The cell colony formation assay was used to calculate the colony formation inhibitory rate. RESULTS The cells in cell control group, non-specific control group and solvent group did not show effective LNCaP cell viability inhibition, migration inhibition and invasion inhibition, while the viability, migration and invasion abilities were significantly inhibited, and early apoptosis in vitro were induced in hsa-miR-145-5p group and lupeol group. In particular, the combination (hsa-miR-145-5p+lupeol) group showed more effective proliferation inhibition than the single-drug groups. CONCLUSION Both hsa-miR-145-5p and lupeol inhibit the proliferation, migration and invasion of LNCaP cells, and induce early apoptosis in vitro. Lupeol enhances the proliferation-inhibitory and apoptosis-inducing effects of hsa-miR-145-5p on LNCaP cells.     

Zoledronic acid inhibits proliferation and induces apoptosis in U937 cells

AIM: To study the antiproliferation and proapoptotic effects of zoledronic acid(ZOL) on human acute myeloid leukemia cell line U937. METHODS: The viability of U937 cells was detected by CCK-8 assay. The cell cycle of the U937 cells was analyzed by flow cytometry with PI staining. Apoptotic rate was assessed by flow cytometry with Annexin V-PI and Hoechst 33342 staining. Mitochondrial membrane potential was detected by JC-1 assay. Methylcellulose was used to assess colony formation. The protein levels of p21, Bcl-2 and Bax were determined by Western blot. RESULTS: ZOL inhibited the viability of U937 cells. ZOL induced S-phase cell cycle arrest in the U937 cells. The results of flow cytometry analysis with Annexin V-PI and Hoechst 33342 staining showed that ZOL also induced apoptosis in a dose- and time-dependent manner. Mitochondrial membrane potential assay was also used to verify the apoptosis. The apoptotic rate was consistent with the reduction of mitochondrial membrane potential. Colony formation assay showed that ZOL significantly inhibited the colony formation capacity of the U937 cells. This was achieved by the induction of S-phase cell cycle arrest, and up-regulation of Bax and p21, and down-regulation of Bcl-2. CONCLUSION: ZOL inhibits cell proliferation by regulating the expression of cell cycle-related protein, and induces apoptosis via the mitochondrial apoptotic pathway.     

Effect of ZNF281 on proliferation of hepatocellular carcinoma cells

AIM:To investigate the role of zinc finger protein 281 (ZNF281) in the proliferation of hepatocellular carcinoma (HCC) cells. METHODS:The mRNA expression levels of ZNF281 in peripheral blood mononuclear cells from 80 cases of healthy people and 206 cases of HCC patients were determined by real-time PCR. Statistical analysis were used to illustrate the relationship between the mRNA expression levels of ZNF281 in the peripheral blood mononuclear cells and the clinicopathologic parameters of HCC patients. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of ZNF281 in hepatoma cell lines and immortalized hepatocytes. The silencing of ZNF281 was conducted by transfection of small interfering RNA targeting ZNF281, and then the proliferation of HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was tested by Cell-Light^TM EdU Apollo^®488 In Vitro Imaging Kit. The ability of colony formation of the HCC cells was measured by colony formation assay, and the ability of anchorage-indepen-dent growth was detected by soft agar test. RESULTS:The mRNA expression level of ZNF281 in the peripheral blood mononuclear cells from HCC patients was significantly increased compared with the healthy people, and the high expression level was positively correlated with tumor size, tumor stage and tumor vascular invasion. Concurrently, the expression level of ZNF281 in hepatoma cell lines was significantly higher than that in immortalized hepatocytes. More importantly, the silencing of ZNF281 inhibited the proliferation, DNA synthesis, colony formation and anchorage-independent growth of the HCC cells. CONCLUSION:ZNF281 promotes the proliferation of HCC cells.     

Effect of 3,5-hydroxy-6,7,3’,4’-tetramethoxyflavone isolated from Laggera pterodonta on apoptosis of Hep-2 cells in vitro

AIM: To investigate the effect of 3,5-hydroxy-6,7,3’,4’- tetramethoxyflavone (HTMF) isolated from Laggera pterodonta on Hep-2 cell apoptosis and the underlying mechanism. METHODS: The MTT assay was used to observe the cytotoxicity of HTMF to the normal cells and the inhibitory effect of HTMF on the proliferation of tumor cells. The apoptosis was determined by flow cytometry. Western blotting was used to detect the expression of caspase-3 and caspase-9. RESULTS: HTMF significantly inhibited the growth of Hep-2 cells in dose and time dependent manners. HTMF exhibited weak cytotoxicity to the two normal cell lines Vero and EVC304, while showed low effect of anti-proliferation on HepG2 cells and A549 cells. The increase in apoptosis of Hep-2 cells by HTMF was observed with dose and time dependent manners. Western blotting showed that HTMF time dependently increased the expression of caspase-3 and caspase-9 in Hep-2 cells. CONCLUSION: HTMF has high inhibitory effect on the proliferation of Hep-2 cells by induction of apoptosis in the tumor cells through caspase-9 and caspase-3 activation. However, the cytotoxicity of HTMF to the normal cells is low.     

DHA induced cytotoxicity in NSCLC cells via inhibiting PI3K pathway activation and GLUT2 expression

AIM: To investigate the molecular mechanisms of cytotoxicity induced by dihydroartemisinin (DHA) in non-small cell lung cancer (NSCLC) cells. METHODS: NSCLC cell lines A549 and NCI-H1650 were treated with various concentrations of DHA for indicated time. Subsequently, the effects of DHA on the cell activity, colony formation ability and apoptosis were determined by MTT assay, colony formation assay, Annexin V-FITC/PI staining and flow cytometry, respectively. At the same time, the effects of DHA on glucose, ATP and lactate levels were assessed, and the PI3K pathway activation and glucose transporter 2(GLUT2) expression were detected by Western blot in the A549 cells and NCI-H1650 cells. Overexpression of GLUT2 and Rheb was established in A549 and NCI-H1650 cells by transfection with GST-GLUT2 and GST-Rheb plasmids, respectively, and the effects of DHA on cell activity, apoptosis, glucose level, ATP content and PI3K pathway activation were analyzed in A549 cells and NCI-H1650 cells. The effect of glucose deprivation on the cytotoxicity triggered by DHA in NSCLC cells was also determined. RESULTS: Compared with control group, DHA significantly inhibited cell activity and colony formation ability, and induced remarkable cell apoptosis in the A549 cells and NCI-H1650 cells. At the same time, DHA reduced ATP and lactate contents, and hindered glucose uptake in a time-and dose-dependent manner in A549 cells and NCI-H1650 cells. The activity of PI3K pathway and GLUT2 expression were downregulated, while upregulated GLUT2 expression and activated PI3K pathway reduced the cytotoxicity induced by DHA in NSCLC cells. Glucose deprivation increased DHA-mediated cytotoxicity in NSCLC cells. On the contrary, high levels of glucose inhibited DHA-mediated cytotoxicity in NSCLC cells. CONCLUSION: DHA restrains cell activity and colony formation, and induces apoptosis. DHA induces cytotoxicity via inhibiting PI3K pathway activation and GLUT2 expression, leading to inhibit glycolytic metabolism in NSCLC cells.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.