Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes
Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apopt...
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| Veröffentlicht in: | Oncology letters 2019-11, Vol.18 (5), p.4495-4502 |
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| creator | Zhang, Lele Wang, Longlong Lei, Mengyuan Ma, Runsheng Yu, Fangqin Liu, Chenguang Yin, Detao |
| description | Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0x[10.sup.8] TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P |
| doi_str_mv | 10.3892/ol.2019.10839 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_6781759</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A605884828</galeid><sourcerecordid>A605884828</sourcerecordid><originalsourceid>FETCH-LOGICAL-c446t-fbd4d6a4d70806374b7b30f1b1f555a8ec721f0d4983c08866464d96f1e088303</originalsourceid><addsrcrecordid>eNptkkFvFSEQxzdGY5vao3cSE-Nln7AsLFxMmlWrSRMveiYsO7yl4cETdq3108v2vVSfEQ4ww2_-kxmmql4SvKFCNm-j3zSYyA3Bgson1TnpZFMXo3n6eO_as-oy51tcFuNECP68OqOEEyKZOK9-XUOApGcXA9JhRG6EMDvrzMEVLdJonu5TdCMyOhhIyID3yLsA6M7NE8qzHjwg-LlPkPMxqO_f97x7UPSLcbakyIAS7GOai8S2JM0vqmdW-wyXx_Oi-vbxw9f-U33z5fpzf3VTm7blc22HsR25bscOC8xp1w7dQLElA7GMMS3AdA2xeGyloAaX-njL21FyS6BYFNOL6t1Bd78MOxhNKTBpr_bJ7XS6V1E7dfoS3KS28YfinSAdk0XgzVEgxe8L5FntXF67oAPEJauGYtZJJhkr6Kt_0Nu4pFDKKxTBvCFM8D_UVntQLthY8ppVVF1xzIRoRSMKtfkPVfYIO2diAOuK_yTg9V8BE2g_Tzn6Zf3JfArWB9CkmHMC-9gMgtU6WCp6tQ6Wehgs-hsFML06</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2310621586</pqid></control><display><type>article</type><title>Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes</title><source>Spandidos Publications Journals</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><creator>Zhang, Lele ; Wang, Longlong ; Lei, Mengyuan ; Ma, Runsheng ; Yu, Fangqin ; Liu, Chenguang ; Yin, Detao</creator><creatorcontrib>Zhang, Lele ; Wang, Longlong ; Lei, Mengyuan ; Ma, Runsheng ; Yu, Fangqin ; Liu, Chenguang ; Yin, Detao</creatorcontrib><description>Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0x[10.sup.8] TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo. Key words: thyroid cancer, coiled-coil domain containing 67, luciferase reporter gene, puromycin, TPC-1</description><identifier>ISSN: 1792-1074</identifier><identifier>EISSN: 1792-1082</identifier><identifier>DOI: 10.3892/ol.2019.10839</identifier><identifier>PMID: 31611958</identifier><language>eng</language><publisher>Athens: Spandidos Publications</publisher><subject>Antibiotics ; Antigens ; Apoptosis ; Biotechnology industries ; Cancer cells ; Cancer genetics ; Cancer metastasis ; Cancer research ; Deoxyribonucleic acid ; DNA ; Drug resistance ; EDTA ; Electrophoresis ; Enzyme-linked immunosorbent assay ; Experiments ; Fluorescence ; Genes ; Genetic aspects ; Luciferase ; Medical prognosis ; Metastasis ; Oncology ; Pathogenesis ; Proteins ; T cells ; Thyroid cancer ; Thyroid diseases ; Thyroid gland ; Transcription (Genetics) ; Tumors</subject><ispartof>Oncology letters, 2019-11, Vol.18 (5), p.4495-4502</ispartof><rights>COPYRIGHT 2019 Spandidos Publications</rights><rights>Copyright Spandidos Publications UK Ltd. 2019</rights><rights>Copyright: © Zhang et al. 2019</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c446t-fbd4d6a4d70806374b7b30f1b1f555a8ec721f0d4983c08866464d96f1e088303</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781759/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781759/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids></links><search><creatorcontrib>Zhang, Lele</creatorcontrib><creatorcontrib>Wang, Longlong</creatorcontrib><creatorcontrib>Lei, Mengyuan</creatorcontrib><creatorcontrib>Ma, Runsheng</creatorcontrib><creatorcontrib>Yu, Fangqin</creatorcontrib><creatorcontrib>Liu, Chenguang</creatorcontrib><creatorcontrib>Yin, Detao</creatorcontrib><title>Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes</title><title>Oncology letters</title><description>Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0x[10.sup.8] TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo. Key words: thyroid cancer, coiled-coil domain containing 67, luciferase reporter gene, puromycin, TPC-1</description><subject>Antibiotics</subject><subject>Antigens</subject><subject>Apoptosis</subject><subject>Biotechnology industries</subject><subject>Cancer cells</subject><subject>Cancer genetics</subject><subject>Cancer metastasis</subject><subject>Cancer research</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Drug resistance</subject><subject>EDTA</subject><subject>Electrophoresis</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Experiments</subject><subject>Fluorescence</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Luciferase</subject><subject>Medical prognosis</subject><subject>Metastasis</subject><subject>Oncology</subject><subject>Pathogenesis</subject><subject>Proteins</subject><subject>T cells</subject><subject>Thyroid cancer</subject><subject>Thyroid diseases</subject><subject>Thyroid gland</subject><subject>Transcription (Genetics)</subject><subject>Tumors</subject><issn>1792-1074</issn><issn>1792-1082</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNptkkFvFSEQxzdGY5vao3cSE-Nln7AsLFxMmlWrSRMveiYsO7yl4cETdq3108v2vVSfEQ4ww2_-kxmmql4SvKFCNm-j3zSYyA3Bgson1TnpZFMXo3n6eO_as-oy51tcFuNECP68OqOEEyKZOK9-XUOApGcXA9JhRG6EMDvrzMEVLdJonu5TdCMyOhhIyID3yLsA6M7NE8qzHjwg-LlPkPMxqO_f97x7UPSLcbakyIAS7GOai8S2JM0vqmdW-wyXx_Oi-vbxw9f-U33z5fpzf3VTm7blc22HsR25bscOC8xp1w7dQLElA7GMMS3AdA2xeGyloAaX-njL21FyS6BYFNOL6t1Bd78MOxhNKTBpr_bJ7XS6V1E7dfoS3KS28YfinSAdk0XgzVEgxe8L5FntXF67oAPEJauGYtZJJhkr6Kt_0Nu4pFDKKxTBvCFM8D_UVntQLthY8ppVVF1xzIRoRSMKtfkPVfYIO2diAOuK_yTg9V8BE2g_Tzn6Zf3JfArWB9CkmHMC-9gMgtU6WCp6tQ6Wehgs-hsFML06</recordid><startdate>20191101</startdate><enddate>20191101</enddate><creator>Zhang, Lele</creator><creator>Wang, Longlong</creator><creator>Lei, Mengyuan</creator><creator>Ma, Runsheng</creator><creator>Yu, Fangqin</creator><creator>Liu, Chenguang</creator><creator>Yin, Detao</creator><general>Spandidos Publications</general><general>Spandidos Publications UK Ltd</general><general>D.A. Spandidos</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AN0</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>M0S</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20191101</creationdate><title>Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes</title><author>Zhang, Lele ; Wang, Longlong ; Lei, Mengyuan ; Ma, Runsheng ; Yu, Fangqin ; Liu, Chenguang ; Yin, Detao</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c446t-fbd4d6a4d70806374b7b30f1b1f555a8ec721f0d4983c08866464d96f1e088303</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Antibiotics</topic><topic>Antigens</topic><topic>Apoptosis</topic><topic>Biotechnology industries</topic><topic>Cancer cells</topic><topic>Cancer genetics</topic><topic>Cancer metastasis</topic><topic>Cancer research</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Drug resistance</topic><topic>EDTA</topic><topic>Electrophoresis</topic><topic>Enzyme-linked immunosorbent assay</topic><topic>Experiments</topic><topic>Fluorescence</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Luciferase</topic><topic>Medical prognosis</topic><topic>Metastasis</topic><topic>Oncology</topic><topic>Pathogenesis</topic><topic>Proteins</topic><topic>T cells</topic><topic>Thyroid cancer</topic><topic>Thyroid diseases</topic><topic>Thyroid gland</topic><topic>Transcription (Genetics)</topic><topic>Tumors</topic><toplevel>online_resources</toplevel><creatorcontrib>Zhang, Lele</creatorcontrib><creatorcontrib>Wang, Longlong</creatorcontrib><creatorcontrib>Lei, Mengyuan</creatorcontrib><creatorcontrib>Ma, Runsheng</creatorcontrib><creatorcontrib>Yu, Fangqin</creatorcontrib><creatorcontrib>Liu, Chenguang</creatorcontrib><creatorcontrib>Yin, Detao</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>ProQuest_Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central UK/Ireland</collection><collection>British Nursing Database</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Oncology letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zhang, Lele</au><au>Wang, Longlong</au><au>Lei, Mengyuan</au><au>Ma, Runsheng</au><au>Yu, Fangqin</au><au>Liu, Chenguang</au><au>Yin, Detao</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes</atitle><jtitle>Oncology letters</jtitle><date>2019-11-01</date><risdate>2019</risdate><volume>18</volume><issue>5</issue><spage>4495</spage><epage>4502</epage><pages>4495-4502</pages><issn>1792-1074</issn><eissn>1792-1082</eissn><abstract>Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0x[10.sup.8] TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo. Key words: thyroid cancer, coiled-coil domain containing 67, luciferase reporter gene, puromycin, TPC-1</abstract><cop>Athens</cop><pub>Spandidos Publications</pub><pmid>31611958</pmid><doi>10.3892/ol.2019.10839</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Antibiotics Antigens Apoptosis Biotechnology industries Cancer cells Cancer genetics Cancer metastasis Cancer research Deoxyribonucleic acid DNA Drug resistance EDTA Electrophoresis Enzyme-linked immunosorbent assay Experiments Fluorescence Genes Genetic aspects Luciferase Medical prognosis Metastasis Oncology Pathogenesis Proteins T cells Thyroid cancer Thyroid diseases Thyroid gland Transcription (Genetics) Tumors |
| title | Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes |
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