| Objective:Hepatocellular carcinoma(HCC)is the sixth most common cancer and the third most common cause of cancer-related death worldwide,according to statistics on cancer incidence,mortality and prevalence.It is well known that HCC is difficult to be detected without symptoms in the early stage.Once found,most patients are already in the advanced stage of HCC and have obvious resistance to conventional chemotherapy drugs and radiotherapy.It is precisely because of these characteristics that liver cancer has a poor prognosis.There are a variety of treatment options available to patients,including liver transplantation,surgical resection and non-drug treatment such as sorafenib and drug treatment,while liver transplantation is only suitable for early stage liver cancer and surgical resection is used to treat patients with advanced liver cancer.Continuous search for a variety of new and advanced diagnostic and therapeutic methods will make new breakthroughs in the treatment of HCC,so as to improve the survival rate and non-recurrence rate of liver cancer patients.Multiple studies have found that mi R-1468-3p plays an important role in the occurrence and development of tumors,but its role in liver cancer remains unclear.By exploring the influence and mechanism of mi R-1468-3p on liver cancer,and further exploring its regulation of downstream FLVCR1 and its influence on the stemness of liver cancer cells,It provides a new way to find a new biomarker for diagnosis and treatment of liver cancer.Methods:Extract the human normal liver epithelial cells LO2,human liver cancer cell SMMC-7721 and Hep G2 RNA,RT-q PCR experiment tests FLVCR1 endogenous expression;The overexpression and knockdown plasmids of FLVCR1 were constructed,and the expression of cancer stem marker genes in tumor cells was detected by Western Blot assay.The effects of FLVCR1 on hepatocellular carcinoma cells were verified by scratch,Transwell,Ed U,colony formation and pellet suspension tests.The effect of FLVCR1 on the growth of transplanted tumor in nude mice was verified in vivo.Wild type and mutant luciferase reporter gene plasmoids of binding sites between FLVCR1 and mi R-1468-3p were constructed,and their binding status was verified by Luciferase reporter activity detection.The endogenous expression of mi R-1468-3p was detected by extracting mi RNA from LO2,SMMC-7721 and Hep G2 cells,and the expression of cancer stem marker gene protein in tumor cells was detected by Western Blot.The effects of mi R-1468-3p on hepatoma cells were verified by scratch,Transwell,Ed U,colony formation and pellet suspension experiments.A nude mouse transplanted tumor model was established to verify the effect of mi R-1468-3p on the growth of transplanted tumor in vivo.Results: RT-q PCR showed that the expression of FLVCR1 was up-regulated in hepatocellular carcinoma cells,and Western Blot showed that over-knockdown of FLVCR1 inhibited the expression of stem-related marker genes in tumor cells,while over-expression showed the opposite result.The results of scratch test,Transwell test and Ed U showed that FLVCR1 knockdown inhibited the migration and proliferation of hepatocellular carcinoma cells,while overexpression of FLVCR1 inhibited the migration and proliferation of hepatocellular carcinoma cells.The results of colony formation and pellet suspension experiments showed that knockdown of FLVCR1 inhibited the stemness of cancer cells,and overexpression of Fl VCR1 enhanced the stemness of cancer cells of hepatoma cells.The model of transplanted tumor in nude mice was established.The experimental results showed that FLVCR1 knockdown inhibited the growth of transplanted tumor in nude mice.The results of Luciferase assay showed that mi R-1468-3p could target binding FLVCR1 3’UTR.RT-q PCR showed that the expression of mi R-1468-3p was down-regulated in liver cancer cells.Western Blot assay results showed that overexpression of mi R-1468-3p inhibited the expression of stem-related marker genes in cancer cells,while knockdown of mi R-1468-3p showed opposite results.Scratch experiment,Transwell experiment and Ed U experiment results also showed that overexpression of mi R-1468-3p could inhibit the migration and proliferation of liver cancer cells,but the results were opposite after knockdown of mi R-1468-3p.Colony formation and pellet suspension experiments showed that overexpression of mi R-1468-3p could inhibit the assay stemness of tumor cells,while knockdown of mi R-1468-3p could enhance the stemness of hepatoma cells.A nude mouse transplanted tumor model was established,and the experimental results showed that overexpression of mi R-1468-3p inhibited the growth of transplanted tumor in nude mice.Western Blot response assay results showed that overexpression of FLVCR1 reversed the inhibitory effect of overexpression of mi R-1468-3p on the expression of stem-related gene protein in hepatocellular carcinoma cells.Scratch,Transwell and Ed U experiments showed that overexpression of FLVCR1 reversed the inhibitory effect of overexpression of mi R-1468-3p on the migration and proliferationof hepatoma cells.Colony formation and pellet suspension experiments showed that overexpression of FLVCR1 reversed the stemness inhibition effect of overexpression of mi R-1468-3p on hepatoma cells.The experimental results showed that overexpression of FLVCR1 reversed the inhibitory effect of overexpression of mi R-1468-3p on the growth of transplanted tumor in nude mice.Conclusions:The results suggest that mi R-1468-3p is down-regulated in liver cancer cells and acts as a tumor suppressor to inhibit the proliferation and migration of liver cancer cells,which has a certain impact on the occurrence and development of liver cancer and is closely related to the prognosis and survival rate of patients.mi R-1468-3p inhibits the expression of FLVCR1 by absorbing the 3’UTR of downstream target gene FLVCR1 by sponge.This study is the first to deeply explore the role of mi R-1468-3p in the progression of liver cancer,providing a new idea and direction for the subsequent prevention and treatment of liver cancer.The results suggest that mi R-1468-3p may be a new target for the diagnosis and treatment of liver cancer. |
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