| Hepatocellular carcinoma is currently the most common type of malignant tumors in the world.It has the characteristics of difficult early diagnosis and poor prognosis.In-depth study of the influencing factors and mechanism of liver cancer,exploration of early diagnosis of liver cancer,targets for targeted therapy,and biomarkers related to predicting metastasis and recurrence have become one of the main directions of liver cancer research in recent years.Due to the continuous progress and improvement of high-throughput sequencing technology and bioinformatics,the role and value of lncRNA in the occurrence and development of liver cancer have gradually attracted public attention.It has been found to play an important role in the proliferation,apoptosis,invasion and metastasis of liver cancer,and it is expected to serve as a molecular marker and potential therapeutic target for the diagnosis and treatment of liver cancer.In the early stage,through high-throughput second-generation sequencing technology and bioinformatics,the lncRNA closely related to liver cancer:lnc145801 was screened out.Through the study of clinical liver cancer tissue specimens,it was verified that it was highly expressed in liver cancer tissues,and Related to recurrence and metastasis.ObjectiveFirstly,a stable liver cancer cell line with lnc145801 knockdown was constructed to study the migration and invasion ability,proliferation and apoptosis of lnc145801 knockdown stable cell line,and then to detect the effect of lnc145801 knockdown on markers related to epithelial-mesenchymal transition,and to explore lnc145801 The molecular mechanism that affects the migration and invasion of liver cancer.To provide a theoretical basis for studying the function of lncRNA in liver cancer and its clinical value.Methods1.Using real-time quantitative PCR to detect the expression of lnc145801 in eight hepatocellular carcinoma cell lines(Hep G2,SMMC7721,Huh7,MHCC97 L,Hep3B,MHCC97 H,SK-Hep-1,HCCLM3)The screening was used to construct lnc145801 knock-down stable transfection HCC cell line.2.Constructing the sh RNA-lnc145801 lentiviral vector and the empty vector as the control,and then using lentivirus to infect human liver cancer cell lines.Finally,the stable transfection cell lines of liver cancer with lnc145801 knock-down and its empty vector were constructed by puromycin screening program.3.The migration and invasion ability of cells were detected by Transwell assay.Cell proliferation was detected by plate clone formation experiment and immunohistochemistry(ki-67).Cellapoptosis was detected by Annexin V-PE/7AAD flow cytometry,in order to observe the changes of migration,invasion,proliferation and apoptosis of lnc145801 knockdown stable hepatocellular carcinoma cell line.4.The effects of EMT-related markers E-cadherin,β-catenin,N-cadherin,vimentin and TGF-β1 on the transcription and translation levels of lnc145801knock-down stable transgenic plants were detected by qPCR and Western Blot.5.Immunohistochemistry to detect EMT markers by making cell wax blocks of lnc145801 knockdown and empty vector stable cell lines.The expression differences of EMT-related markers β-catenin,N-cadherin,vimentin and TGF-β1were determined by observing the difference in DAB positive optical density.Results1.qPCR showed that the expression levels of lnc145801 in HCC cell lines from low to high were SMMC7721,Hep G2,Huh7,SK-Hep-1,MHCC97L,Hep3B,MHCC97H,and HCCLM3.Therefore,MHCC97H and HCCLM3 with high lnc145801 expression levels were selected for subsequent experiments.2.sh RNA-lnc145801 lentiviral vector and corresponding empty vector were successfully constructed.After infection with HCCLM3 and MHCC97 H,a total of four lnc145801 knock-down and its empty vector stable cell lines were successfully constructed,named as LM3-sh-lnc145801,LM3-sh-NC and97H-sh-lnc145801,97H-sh-NC.3.Detecting the biological function of lnc145801 knockdown stably transfected cells,compared with NC group,the proliferation,migration and invasion of lnc145801 knockdown stably transfected cells were decreased,and the difference was statistically significant(P<0.05).Annexin V-PE/7AAD flow cytometry showed that knocking down lnc145801 promoted the apoptosis of liver cancer cells,and the difference was statistically significant(P<0.05).4.qPCR and Western Blot were used to detect the EMT-related markers of lnc145801 knockdown stably transfected cells.Compared with NC group,the m RNA and protein expression of epithelial markers E-cadherin and β-catenin increased,the m RNA and protein expression of mesenchymal markers N-cadherin and vimentin decreased,and the m RNA and protein expression of cytokine TGF-β1 decreased.5.EMT markers were detected by immunohistochemistry.Compared with NC group,lnc145801 knockdown resulted in increased expression of epithelial markerβ-catenin and decreased expression of mesenchymal markers N-cadherin,vimentin and TGF-β1.Conclusions1.Successfully construct a lentiviral expression vector that knocks down lnc145801 and stably transfects liver cancer cell lines,laying a foundation for further research on the function and mechanism of lnc145801.2.The proliferation,migration and invasion ability of lnc145801 knockdown stable transgenic strain is reduced,and the apoptotic ability is improved.3.Lnc145801 knockdown inhibits the related proteins of epithelial-mesenchymal transition,suggesting that lnc145801 knockdown inhibits the invasion and migration of liver cancer cells may be related to EMT.The detection of lncRNA can provide new ideas for clinical judgment of HCC metastasis and recurrence. |
PDF Full Text Request