Effect Of Metformin On Hepatocellular Carcinoma Hepg2 Cells And Its Mechanism

Background: Hepatocellular carcinoma(HCC)is one of the serious human malignant diseases,ranked as the second most common cause of cancer-related death,and the incidence and mortality of HCC have been increasing in recent years.Biological advances in HCC shows that the molecular mechanisms involved in genetic alterations of various somatic cells,subtypes of HCC,diagnosis,drug resistance and recurrence in the development of HCC are very complex,which is a major medical problem requiring further study.At present,effective anti-HCC drugs are insufficient.Metformin(N,N-metformin)is one of the most widely used drugs for the treatment of type II diabetes mellitus,mainly by reducing hepatic gluconeogenesis and increasing glucose storage in skeletal muscle.Epidemiological studies have found that metformin reduces the incidence of various cancers,including HCC,while treating type II diabetes mellitus,compared with those not treated with metformin.In recent years,with the indepth study of metformin,it has been found that it also has a very significant effect in inhibiting the growth of tumors,and long-term use of metformin can reduce the risk of multiple tumors.The effect of metformin on HCC has been basically clear.Studies have shown that metformin can reduce the incidence of primary HCC.In vitro cell studies have shown that metformin can inhibit the expression of Bcl-2 gene,inhibit the proliferation of Hep G2 cell line and exert anti-tumor effects.However,little is known about the antitumor mechanism of metformin.In recent years,studies have shown that there is also a certain degree of autophagy in HCC cells.When the degree of autophagy increases,it can induce apoptosis of HCC cells and inhibit the proliferation of HCC cells.It is well known that the hypoglycemic effect of metformin is produced by initiating the AMPK signal transduction pathway.The initiation of AMPK can phosphorylate downstream TSC2 protein to produce TSC1/TSC2 heterodimer complex with GTPase promoter protein activity,which promotes Rheb inactivation and thus inhibits mTOR complex 1.Moreover,the inhibition of mTOR by metformin is independent of the initiation of AMPK,which is achieved by regulating insulin receptor or insulin-like growth factor,that is,metformin can lead to the inhibition of mTOR signaling pathway through various channels,suggesting that metformin may have a very important regulatory role on autophagy.Therefore,we propose a scientific hypothesis that metformin inhibits the proliferation,invasion and migration of HCC cells by enhancing autophagy of HCC cells.To confirm this scientific hypothesis,we conducted the following experiments:(1)to clarify the effect of metformin on the proliferation,invasion and migration of hepatoma cells.(2)to clarify the effect of metformin on apoptosis of HCC cells.(3)to clarify whether metformin exerts anti-tumor effects by promoting autophagy.Objective: 1.To explore the effects of metformin on the proliferation,migration,invasion and other malignant biological behaviors of HCC Hep G2 cells.2.To explore the effect of metformin on apoptosis of HCC cells.3.To explore the effect of metformin on autophagy-related proteins such as P-AMPK,Atg14 and LC3-II in Hep G2 cells.4.To analyze the potential mechanism of metformin inhibiting HCC.Methods: Hep G2 cells were randomly divided into four groups: saline group,metformin 1 m M group,metformin 5 m M group and metformin 10 m M group.Hep G2 cells were cultured routinely,and cells in saline group,metformin 1 m M group,metformin 5 m M group and metformin 10 m M group were treated with saline,1 m M metformin,5m M metformin and 10 m M metformin,respectively.The viability of cells in each group was detected and the inhibition of proliferation was calculated,and the best dose was selected.The effect of metformin on the invasion ability of Hep G2 cells was detected by Scratch assay and Transwell assay.The effect of metformin on apoptosis of Hep G2 cells was detected by Annexin V-FITC method and flow cytometry.The effect of metformin on autophagy-related proteins such as P-AMPK,Atg14 and LC3-II in Hep G2 cells was detected by Western blot hybridization.Results: 1.MTT results showed that there were statistically significant differences in cell viability of saline group,metformin 1 m M group,metformin 5 m M group and metformin 10 m M group after 24 h,48h,72 h and 96 h of treatment(P<0.05).Besides,the viability and the proliferation rate of Hep G2 cells treated with saline were high.After treatment with metformin,the viability and the proliferation rate of Hep G2 cells decreased.With the increase of the concentration of metformin,the viability of Hep G2 cells decreased(P<0.05).Plate cloning experiment showed that there was a significant difference in the proliferation rate of Hep G2 cells between saline group and metformin 10 m M group(P<0.05),and the proliferation rate of Hep G2 cells in metformin 10 m M group was significantly lower than that in saline group(P<0.05).2.Scratch test showed that there was a significant difference in the cell migration distance between saline group and metformin 10 m M group after 24 h of treatment.When Hep G2 cells were treated with saline,the migration ability of the cells was relatively stronger.When Hep G2 cells were treated with metformin,the migration distance was significantly shorter than that of saline group(P<0.05).3.Transwell assay showed that there was a significant difference in cell invasion rate between saline group and metformin 10 m M group after 24 h of treatment,and the cell invasion ability of metformin 10 m M group was significantly lower than that of saline group(P<0.05).4.Flow cytometry showed that apoptotic Hep G2 cells were fewer in saline group.When metformin was administered,apoptotic Hep G2 cells was significantly increased,with statistically significant difference(P<0.05).Western blot showed that the expression of cleaved caspase-3 in Hep G2 cells was significantly increased after treatment with metformin(P<0.05).5.Western blot detection of autophagy-related proteins showed that the expression of P-AMPK,Atg14 and LC3-II in metformin 10 m M group was significantly higher than that in saline group(P<0.05).Conclusion: The study confirms that the inhibition of the viability of Hep G2 cells increases with the increase of the concentration of metformin,and the optimal dose is 10 m M.Metformin can significantly inhibit the migration distance of Hep G2 cells,and the invasion ability of cells treated with metformin is significantly lower than that of saline group.Flow cytometry results showe that metformin significantly increases the apoptosis of Hep G2 cells.Western blot detected that the expression of P-AMPK,Atg14 and autophagy marker LC3-II is significantly increased in metformin 10 m M group,suggesting that metformin may increase apoptosis by promoting autophagy of Hep G2 cells,and then inhibit cell viability,migration and invasion of Hep G2 cells.It is a potential drug for clinical treatment of HCC.These findings may be helpful in elucidating the mechanism of action of metformin in inhibiting HCC cells,which deserves further study.Background: Hepatocellular carcinoma(HCC)is a malignant tumor with high morbidity and mortality in the world,and about 50% of the new cases and deaths of HCC occur in China.At present,surgical resection is still the main treatment for liver cancer.Compared with traditional laparotomy,laparoscopic hepatectomy for hepatocellular carcinoma has many advantages,such as less trauma,faster recovery,less postoperative complications,lower infection rate of surgical incision,and greatly shorter hospital stay.Therefore,laparoscopic hepatectomy has been gradually carried out worldwide.Over the past ten years,with the improvement of laparoscopic surgical instruments and the advancement of surgical technology,laparoscopic hepatectomy has been rapidly developed,and many forbidden areas have been breached one by one.Laparoscopic hepatectomy seeks to be based on the physiological anatomy of the liver,ensuring complete resection of the main cancer focus and its intrasegmentalmicrometastases,while maximizing the integrity of the remaining liver structure and function and obtaining a safe margin.At present,laparoscopic hepatectomy is still in the exploratory stage,and the key problem of limited technical popularization is the lack of accurate laparoscopic liver segment marking methods.At present,the commonly used methods of hepatic segment marking are: ischemic line marking,methylene blue marking and liver surface marking.However,the above three markers all have defects.Because the liver is uneven three-dimensional,the anatomical boundary between the liver segment or lobe in the liver parenchyma and the liver surface is not in the same plane,the ischemic line and the liver surface markers are only visible on the liver surface,but not when the liver parenchyma is severed.The severed surface of the liver parenchyma during the operation still needs to be judged by the course of the hepatic vein and the operator's experience,and only depends on the ischemic line and surface of the liver surface.The difficulty in achieving accurate markers for precise anatomical hepatectomy increases the risk of positive margins.Therefore,finding an effective,safe,stable and precise method of laparoscopic liver segmentation marking,and then completely resecting tumor lesions is the future development direction of laparoscopic hepatectomy.Indocyanine green(ICG),also known as indocyanine green,is a near-infrared fluorescent contrast agent,which can display fluorescence or color fluorescence on the imaging equipment after being ingested by a special fluorescence acquisition system,and can be used for the development of tissues and organs.The indocyanine green fluorescent dye has good biosafety.After the protein-bound ICG enters the hepatic sinusoids,it is mainly taken up by hepatocytes,and then discharged into the biliary tract through the capillary bile duct in its original form.It does not participate in lymphatic reflux and intestinal-hepatic circulation.Therefore,it has broad application prospects in the field of liver surgery.The earliest application of ICG imaging in liver surgery was mainly to assess liver function by ICG clearance rate before surgery.At the beginning of this century,studies have shown that ICG fluorescence imaging can identify liver cancer lesions in real time and with high sensitivity,especially small liver cancer lesions,and improve the accuracy of hepatectomy and surgical staging.In recent years,the emergence of indocyanine green fluorescence imaging(ICG-FI)under laparoscopy has synchronously displayed the field of view of the laparoscopic field and ICG fluorescence fusion image on the same screen.By injecting ICG positive and negative staining technology,not only the liver surface but also the liver parenchyma can be accurately and persistently stained.The method solves the defects of traditional laparoscopic hepatectomy in liver segment marking,realizes three-dimensional stereo positioning of liver segment,clearly identifies and has a long marking duration,which greatly promotes the clinical application of laparoscopic precise hepatectomy.Objective: 1.To explore the application value of ICG-FI technology in laparoscopic hepatectomy for hepatocellular carcinoma,and to analyze the value of ICG-FI in determining tumor boundary and real-time staining of liver segment.2.To retrospectively analyze and compare the differences between ICG-FI laparoscopy group and common laparoscopy group in preoperative,perioperative,postoperative and other related indicators,in order to further explore the advantages of ICG-FI-guided laparoscopy.3.To analyze the complications,safety and effectiveness of ICG-FI laparoscopic group,and evaluate the clinical application value of ICG-FI technology in laparoscopic hepatectomy.4.To further analyze the value of positive staining and reverse staining in ICG-FIguided laparoscopic hepatectomy.Methods:1.This study retrospectively analyzed the clinical data of 22 patients with liver cancer who underwent ICG-FI laparoscopic hepatectomy and 38 patients with liver cancer who underwent general laparoscopic hepatectomy from April 2017 to January 2020 in the First Affiliated Hospital of Anhui Medical University.2.Normality test was performed for measurement data,and measurement data conforming to normal distribution were described as mean standard deviation(mean±SD),while measurement data of non-normal distribution were described as median(interquartile range);difference between two groups was compared between ICG-FI laparoscopy group and common laparoscopy group,measurement data conforming to normal distribution were compared using independent t test,measurement of non-normal distribution was used Mann Whitney nonparametric test,chi-square test or Fisher's exact test were used to compare the index data,with P < 0.05 as the statistical difference.Results:1.Preoperative clinical data of patients: A total of 60 patients with liver cancer who underwent hepatectomy were included in this study,including 20 male patients(90.9%)and 2 female patients(9.1%)in the ICG-FI laparoscopic group,with an average age of 57.50±10.09 years,33 male patients(86.8%)in the general laparoscopic group and 5 female patients(13.2%)with an average age of 59.42±10.65 years.There were no significant differences between the two groups in the history of upper abdominal surgery,alpha-fetoprotein index,hepatitis B surface antigen,Child-Pugh classification of liver function,cirrhosis,preoperative prothrombin time,total bilirubin,alanine aminotransferase,aspartate aminotransferase,AST and albumin(p > 0.05).2.There was no significant difference between the two groups in the distribution of tumors and the way of operation between the ICG-FI laparoscopic group and the general laparoscopic group(p > 0.05).3.Relevant data of patients during perioperative period: 2 cases were converted to open operation in ICG-FI laparoscopy group and 6 cases were converted to open operation in general laparoscopy group.The conversion rate of two groups during operation(9.1% vs 15.8%,p=0.733),intraoperative blood loss(261.90±148.24 vs 273.78±185.63,p=0.803),perioperative blood transfusion rate(27.3% vs 26.3%,p=0.936),hilar blockage rate(59.1% vs 50.0%,p=0.P=0.496),portal occlusion time [26.00(15.00-34.00)vs 26.00(15.00-33.00),p=0.688] was no significant difference between the two groups(p > 0.05).There was no significant difference between the two groups(p > 0.05).The operation time(225.45±35.71 vs 258.76±28.55,P = 0.047),positive resection margin rate(0 vs 10.5%,P = 0.032),complication rate(18.2% vs 36.8%,P = 0.032)of ICG-FI laparoscopy group were lower than those of common laparoscopy group,with statistical difference.4.The recovery of total bilirubin,ALT and AST in both groups showed a downward trend,while the recovery of Alb showed an upward trend.There was no significant difference between the two groups(p > 0.05).During the postoperative follow-up,3 patients in the ICG-FI laparoscopic group had tumor recurrence,and 14 patients in the general laparoscopic group had tumor recurrence.The postoperative tumor recurrence rate in the ICG-FI laparoscopic group was better than that in the general laparoscopic group(13.6% vs 36.8%,p=0.045),and the difference between the two groups was statistically significant.5.Fluorescent staining method and results of anatomical hepatectomy in ICG-FI laparoscopic group: 9 cases of anatomical hepatectomy in ICG-FI laparoscopic group were selected,including 3 cases of positive staining method,1 case of failure,6 cases of reverse staining method,and 1 case of failure.Conclusion:Indocyanine green fluorescence fusion imaging(ICG-FI)guided laparoscopic hepatectomy has important clinical application value.In this study,22 patients in the ICG-FI laparoscopic group achieved complete tumor resection.Compared with the common laparoscopic group,ICG-FI laparoscopic group helps to determine the tumor boundary,significantly reduces the positive rate of tumor margin,shortens the operation time,reduces the incidence of postoperative complications,and reduces the postoperative tumor recurrence.It shows that the indocyanine green fluorescence fusion imaging(ICG-FI)guided laparoscopic hepatectomy has good safety,has important clinical application value,and is worthy of wide application in hepatobiliary surgery.In addition,positive staining and reverse staining methods are suitable for different types of hepatectomy,and the key to success is to accurately grasp the puncture site of the target liver pedicle.