Analysis of VEGF and SASP factors in vitro and in vivo. (A) VEGF in RERG-transfected and empty-ve... more Analysis of VEGF and SASP factors in vitro and in vivo. (A) VEGF in RERG-transfected and empty-vector-transfected NPC cells (HK1, C666-1) were determined by western blotting (n = 3). (B) mRNA expression of RERG, IL8, IL1β and TNFα in xenografts of nude mice was determined by qRT-PCR (n = 8). GAPDH was used as an internal control. (C) IHC analyses of the expression of VEGF in tumors from nude mice. Original magnification is × 200. Bar represents 50 μm. Data are shown as means ± SD. **: P
Analysis of RAS type GTPase family genes in nasopharyngeal carcinoma primary tumors and nasophary... more Analysis of RAS type GTPase family genes in nasopharyngeal carcinoma primary tumors and nasopharyngeal epithelial tissues. (A, B) Heat map of RAS type GTPase family in NPC (n = 7) and NNE (n = 5) tissues. (A) RAS type GTPase family genes expression alerted using cDNA microarray. (B) Hypermethylated genes of RAS type GTPase family by methylated-DNA capture sequencing. (C) Genes of RAS type GTPase family which were significantly downregulated in cDNA microarray and hypermethylated in methylated-DNA capture sequencing. Methods for methyl-capture sequencing and gene expression array were described in Additional file 4: Supplementary methods. (TIF 2684 kb)
Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high inci... more Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high incidence in southern China. Previous studies have confirmed that taurine shows an anti-cancer effect on a variety of human tumors by inhibiting cell proliferation and inducing apoptosis. However, the underlying molecular mechanism of its anti-cancer effect on NPC is not well understood. To clarify these anti-cancer mechanisms, we performed cell viability and colony formation assays. Apoptotic cells were quantified by flow cytometry. The expression levels of apoptosis-related proteins were evaluated by Western blot. The results showed that taurine markedly inhibited cell proliferation in NPC cells, but only slightly in an immortalized normal nasopharyngeal cell line. Taurine suppressed colony formation and induced apoptosis of NPC cell lines in a dose-dependent manner. Furthermore, taurine increased the active form of caspase-9/3 in a dose-dependent manner. Taurine down-regulated the anti-apoptotic protein Bcl-xL and up-regulated the pro-apoptotic protein Bax and GRP78, a major endoplasmic reticulum (ER) chaperone. These results suggest the involvement of mitochondrial and ER stress signaling in apoptosis. In addition, taurine increased the levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10) and p53, and reduced phosphorylated Akt (protein kinase B). In conclusion, taurine may inhibit cell proliferation and induce apoptosis in NPC through PTEN activation with concomitant Akt inactivation.
Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis th... more Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis through DNA damage. 8-Nitroguanine is a specific marker of inflammation-related carcinogenesis. Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors in southern China and Southeast Asia, and its prognosis has been poor for decades. Previously, we demonstrated that nitrative DNA damage, such as 8-nitroguanine formation, occurs in cancer cells of NPC patients (Ma et al. Int. J. Cancer 2008). In the present study, to investigate the involvement of stem cells in NPC, we performed immunohistochemical analyses to examine nitrative DNA lesions (8-nitroguanine) and several cancer stem / progenitor cell markers (CD44v6, CD24 and ALDH1A1) in nasopharyngeal tissues obtained from chronic nasopharyngitis and NPC patients. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in stroma of NPC than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. 8-Nitroguanine was detected in CD44v6- or ALDH1A1-positive stem cells in NPC tissues. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. Intensive staining of CD44v6 and ALDH1A1 were also detected in NPC cell line HK1 compared to immortalized nasopharyngeal epithelial cells NP460 by a double immunofluorescence study and western blotting assay. In conclusion, CD44v6 and ALDH1A1 could be candidates of cancer stem cell markers for NPC. The present results indicate the possible mechanism by which inflammation causes NPC by inducing inflammatory processes and formation of 8-nitroguanine in CD44v6- and / or ALDH1A1-positive stem cells, and mutant stem cells participate in NPC development.
Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisochinolin) is formed endogenously by a non... more Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisochinolin) is formed endogenously by a nonenzymatic condensation reaction between dopamine and acetaldehyde, after drinking alcohol. Here, we suggest that alcohol-derived salsolinol is a novel causative substance of breast cancer. Recent studies have suggested the possibility of salsolinol formation by the reaction of a neuroendocrine, dopamine, with acetaldehyde accumulated in the mammary glands. We demonstrate that salsolinol induces metal-mediated oxidative DNA damage, including 8-oxodG formation in both isolated and cellular DNA. Interestingly, salsolinol induced significant mammary cell proliferation via estrogen receptor and epidermal growth factor receptor pathways. Our data support the idea that salsolinol plays a role in the initiation and promotion of alcohol-related breast carcinogenesis through oxidative DNA damage and mammary cell proliferation.
Dimethylformamide (DMF) has been suspected to associate with cancers in exposed workers, whereas ... more Dimethylformamide (DMF) has been suspected to associate with cancers in exposed workers, whereas there has been inadequate evidence for carcinogenicity in experimental animals. We demonstrated that H 2 O 2 was generated during the degradation of DMF under aerobic conditions, and that the amount of H 2 O 2 was enhanced by exposure to solar light or by the contamination of trace metal. Experiments using 32 P-5′-end-labeled DNA fragments revealed that the degraded DMF induced DNA damage in the presence of Cu(II). However, purified DMF did not induce DNA damage even in the presence of Cu(II). Addition of purified DMF enhanced DNA damage induced by H 2 O 2 in the presence of Cu(II). The degraded DMF caused Cu(II)-mediated DNA cleavage frequently at thymine and cytosine residues. The similar pattern of site-specific DNA damage was observed with purified DMF and H 2 O 2. Bathocuproine and catalase inhibited the DNA damage, indicating the involvement of Cu(I) and H 2 O 2. A typical free hydroxy radical scavenger showed no inhibitory effect on the DNA damage. Addition of purified DMF enhanced about 3-4-fold 8-oxo-7,8-dihydro-2′-deoxyguanosine formation induced by H 2 O 2 and Cu(II). ESR spectroscopic study demonstrated that carbon-centered radicals and nitrogen-centered radicals were generated in the reaction mixture of DMF, H 2 O 2 , and Cu(II). Inhibitory effects of scavengers on radical formation and DNA damage suggest that carboncentered radicals and/or nitrogen-centered radicals may contribute to the DNA damage. These results suggest that H 2 O 2 generation during DMF degradation is related to the possible carcinogenic activity of DMF.
The soy isoflavones, genistein (5,7,4'-tr... more The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs.
Biochemical and Biophysical Research Communications, 2005
Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxici... more Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5 0-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2 0-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.
Biochemical and Biophysical Research Communications, 2003
Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethyl... more Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu 2þ , but unirradiated ethylbenzene did not. A Cu þ-specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H 2 O 2 were formed in ethylbenzene exposed to sunlight. These results suggest that Cu þ and alkoxyl radical mainly participate in DNA damage, and H 2 O 2 partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2 0-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H 2 O 2 and acetophenone were produced by the sunlight-irradiation of 1phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H 2 O 2 derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.
Background The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer... more Background The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer. TfR is highly expressed in many cancers and is expected to be a promising new target for cancer therapy; however, its role in nasopharyngeal carcinoma (NPC) remains unknown. Methods The TfR levels were investigated in NPC tissues and cell lines using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Knockdown of TFRC using two siRNA to investigate the effects on intracellular iron level and biological functions, including proliferation by CKK-8 assay, colony formation, cell apoptosis and cell cycle by flow cytometry, migration and invasion, and tumor growth in vivo by nude mouse xenografts. RNA sequencing was performed to find possible mechanism after TFRC knockdown on NPC cells and further verify by western blotting. Results TfR is overexpressed in NPC cell lines and tissues. Knockdown of TFRC inhibited cell proliferation concomitant with increased...
Analysis of VEGF and SASP factors in vitro and in vivo. (A) VEGF in RERG-transfected and empty-ve... more Analysis of VEGF and SASP factors in vitro and in vivo. (A) VEGF in RERG-transfected and empty-vector-transfected NPC cells (HK1, C666-1) were determined by western blotting (n = 3). (B) mRNA expression of RERG, IL8, IL1β and TNFα in xenografts of nude mice was determined by qRT-PCR (n = 8). GAPDH was used as an internal control. (C) IHC analyses of the expression of VEGF in tumors from nude mice. Original magnification is × 200. Bar represents 50 μm. Data are shown as means ± SD. **: P
Analysis of RAS type GTPase family genes in nasopharyngeal carcinoma primary tumors and nasophary... more Analysis of RAS type GTPase family genes in nasopharyngeal carcinoma primary tumors and nasopharyngeal epithelial tissues. (A, B) Heat map of RAS type GTPase family in NPC (n = 7) and NNE (n = 5) tissues. (A) RAS type GTPase family genes expression alerted using cDNA microarray. (B) Hypermethylated genes of RAS type GTPase family by methylated-DNA capture sequencing. (C) Genes of RAS type GTPase family which were significantly downregulated in cDNA microarray and hypermethylated in methylated-DNA capture sequencing. Methods for methyl-capture sequencing and gene expression array were described in Additional file 4: Supplementary methods. (TIF 2684 kb)
Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high inci... more Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high incidence in southern China. Previous studies have confirmed that taurine shows an anti-cancer effect on a variety of human tumors by inhibiting cell proliferation and inducing apoptosis. However, the underlying molecular mechanism of its anti-cancer effect on NPC is not well understood. To clarify these anti-cancer mechanisms, we performed cell viability and colony formation assays. Apoptotic cells were quantified by flow cytometry. The expression levels of apoptosis-related proteins were evaluated by Western blot. The results showed that taurine markedly inhibited cell proliferation in NPC cells, but only slightly in an immortalized normal nasopharyngeal cell line. Taurine suppressed colony formation and induced apoptosis of NPC cell lines in a dose-dependent manner. Furthermore, taurine increased the active form of caspase-9/3 in a dose-dependent manner. Taurine down-regulated the anti-apoptotic protein Bcl-xL and up-regulated the pro-apoptotic protein Bax and GRP78, a major endoplasmic reticulum (ER) chaperone. These results suggest the involvement of mitochondrial and ER stress signaling in apoptosis. In addition, taurine increased the levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10) and p53, and reduced phosphorylated Akt (protein kinase B). In conclusion, taurine may inhibit cell proliferation and induce apoptosis in NPC through PTEN activation with concomitant Akt inactivation.
Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis th... more Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis through DNA damage. 8-Nitroguanine is a specific marker of inflammation-related carcinogenesis. Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors in southern China and Southeast Asia, and its prognosis has been poor for decades. Previously, we demonstrated that nitrative DNA damage, such as 8-nitroguanine formation, occurs in cancer cells of NPC patients (Ma et al. Int. J. Cancer 2008). In the present study, to investigate the involvement of stem cells in NPC, we performed immunohistochemical analyses to examine nitrative DNA lesions (8-nitroguanine) and several cancer stem / progenitor cell markers (CD44v6, CD24 and ALDH1A1) in nasopharyngeal tissues obtained from chronic nasopharyngitis and NPC patients. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in stroma of NPC than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. 8-Nitroguanine was detected in CD44v6- or ALDH1A1-positive stem cells in NPC tissues. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. Intensive staining of CD44v6 and ALDH1A1 were also detected in NPC cell line HK1 compared to immortalized nasopharyngeal epithelial cells NP460 by a double immunofluorescence study and western blotting assay. In conclusion, CD44v6 and ALDH1A1 could be candidates of cancer stem cell markers for NPC. The present results indicate the possible mechanism by which inflammation causes NPC by inducing inflammatory processes and formation of 8-nitroguanine in CD44v6- and / or ALDH1A1-positive stem cells, and mutant stem cells participate in NPC development.
Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisochinolin) is formed endogenously by a non... more Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisochinolin) is formed endogenously by a nonenzymatic condensation reaction between dopamine and acetaldehyde, after drinking alcohol. Here, we suggest that alcohol-derived salsolinol is a novel causative substance of breast cancer. Recent studies have suggested the possibility of salsolinol formation by the reaction of a neuroendocrine, dopamine, with acetaldehyde accumulated in the mammary glands. We demonstrate that salsolinol induces metal-mediated oxidative DNA damage, including 8-oxodG formation in both isolated and cellular DNA. Interestingly, salsolinol induced significant mammary cell proliferation via estrogen receptor and epidermal growth factor receptor pathways. Our data support the idea that salsolinol plays a role in the initiation and promotion of alcohol-related breast carcinogenesis through oxidative DNA damage and mammary cell proliferation.
Dimethylformamide (DMF) has been suspected to associate with cancers in exposed workers, whereas ... more Dimethylformamide (DMF) has been suspected to associate with cancers in exposed workers, whereas there has been inadequate evidence for carcinogenicity in experimental animals. We demonstrated that H 2 O 2 was generated during the degradation of DMF under aerobic conditions, and that the amount of H 2 O 2 was enhanced by exposure to solar light or by the contamination of trace metal. Experiments using 32 P-5′-end-labeled DNA fragments revealed that the degraded DMF induced DNA damage in the presence of Cu(II). However, purified DMF did not induce DNA damage even in the presence of Cu(II). Addition of purified DMF enhanced DNA damage induced by H 2 O 2 in the presence of Cu(II). The degraded DMF caused Cu(II)-mediated DNA cleavage frequently at thymine and cytosine residues. The similar pattern of site-specific DNA damage was observed with purified DMF and H 2 O 2. Bathocuproine and catalase inhibited the DNA damage, indicating the involvement of Cu(I) and H 2 O 2. A typical free hydroxy radical scavenger showed no inhibitory effect on the DNA damage. Addition of purified DMF enhanced about 3-4-fold 8-oxo-7,8-dihydro-2′-deoxyguanosine formation induced by H 2 O 2 and Cu(II). ESR spectroscopic study demonstrated that carbon-centered radicals and nitrogen-centered radicals were generated in the reaction mixture of DMF, H 2 O 2 , and Cu(II). Inhibitory effects of scavengers on radical formation and DNA damage suggest that carboncentered radicals and/or nitrogen-centered radicals may contribute to the DNA damage. These results suggest that H 2 O 2 generation during DMF degradation is related to the possible carcinogenic activity of DMF.
The soy isoflavones, genistein (5,7,4'-tr... more The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs.
Biochemical and Biophysical Research Communications, 2005
Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxici... more Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5 0-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2 0-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.
Biochemical and Biophysical Research Communications, 2003
Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethyl... more Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu 2þ , but unirradiated ethylbenzene did not. A Cu þ-specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H 2 O 2 were formed in ethylbenzene exposed to sunlight. These results suggest that Cu þ and alkoxyl radical mainly participate in DNA damage, and H 2 O 2 partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2 0-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H 2 O 2 and acetophenone were produced by the sunlight-irradiation of 1phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H 2 O 2 derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.
Background The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer... more Background The transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer. TfR is highly expressed in many cancers and is expected to be a promising new target for cancer therapy; however, its role in nasopharyngeal carcinoma (NPC) remains unknown. Methods The TfR levels were investigated in NPC tissues and cell lines using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Knockdown of TFRC using two siRNA to investigate the effects on intracellular iron level and biological functions, including proliferation by CKK-8 assay, colony formation, cell apoptosis and cell cycle by flow cytometry, migration and invasion, and tumor growth in vivo by nude mouse xenografts. RNA sequencing was performed to find possible mechanism after TFRC knockdown on NPC cells and further verify by western blotting. Results TfR is overexpressed in NPC cell lines and tissues. Knockdown of TFRC inhibited cell proliferation concomitant with increased...
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