PKM2 Modulates Proliferation And Apoptosis Via Regulating ER Stress In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the highest incidence of digestive system malignant tumor in the world.Due to lack of specific clinical manifestations in the early stagy,the majority of patients have been found to be medium or advanced stage of HCC.Because there have no effective treatment,the mortality of HCC ranks to the third cause of cancer-related deaths in the world.According to statistics,China has more than230000 people die from hepatocellular carcinoma every year.HCC has become one of the most important diseases affecting human health.In the past few decades,the development of surgical technology,radiotherapy,chemotherapy and other treatments make that a part of patients had an extended life.However,overall effect is not ideal.Therefore,it is urgently needed to explore the molecular mechanisms of liver cancer,improve the effect of the treatment of liver cancer and prolong patient's life.Altered metabolism is one of the hallmarks that cancer cells differ from normal cells.Unlike normal cells,tumor cells favor high rates of aerobic glycolysis which generally involves an increased uptake of glucose and conversion of pyruvate into lactate in the presence of sufficient oxygen.Pyruvate kinase(PK)catalyzes the final and rate-limiting reaction of glycolysis,converting phosphoenolpyruvate to pyruvate by transferringthe high-energy phosphate group to adenosine diphosphate to produce adenosine triphosphate.There are four isoforms of PK in mammals,and PKM2 is characteristic of all proliferating cells especially in tumor.PKM2 has intimate relationship with tumor and its specific role in hepatocellular carcinoma and potential mechanism has unclear.Above all,exploring the function of PKM2 in liver cancer has a great significance for the treatment of liver cancer.Endoplasmic reticulum is one of the important organelles of eukaryotic cells.It is the sites of protein translation and calcium ion storage.In the condition of hypoxia or calcium balance disorders,unfolded proteins or misfolded proteins accumulate in endoplasmic reticulum.The structure and function of endoplasmic reticulum would be disordered if unfolded proteins and misfolded proteins beyond to the processing capacity of endoplasmic reticulum which called as ERS.In mammalian,there are three kinds of ERS receptors,including IRE1?,ATF6 and PERK.In general,these receptors combined with partner molecules GRP78(glucose regulated protein78)in the inactive state.The expression of IRE1?,ATF6,PERK,GRP78 incresed in ERS.At present,a large number of studies have found that ERS has dual effects on cell survival and apoptosis.Therefore,our studies research the effect of PKM2 and its potential mechanism in HCC progression,elucidate whether PKM2 modulated ERS.Our study mainly divided into the following several parts:1.The expression of PKM2 in human HCC tissues and cell linesTo identify the change of PKM2 expression between HCC tissues and adjacent noncancerous tissues,the degree of liver carcinoma was determined by H&E staining firstly.Histopathological analysis showed that human HCC tissues displayed hepatic steatosis,cellular edema,focal nodular hyperplasia and inflammatory infiltration,while normal lobular architecture with central veins and radiating hepatic cords.Secondly,the immunohistochemical analysis showed that the expression of PKM2 was up-regulated in the HCC group compared with corresponding normal liver tissues.Western Blot also indicated PKM2 expression was up-regulated in the HCC group.What is more,we collected HCC cell lines including Hep G2,BEL-7402 and SMMC-7721 for investigation.Meanwhile,L02 cell was included as the normal control.Results suggested that protein expressions of PKM2 in HCC cell lines were much higher in comparison with normal liver cell line L02 cell.2.The effect of PKM2 drown-regulation on cell proliferation in SMMC-7721 cells.PKM2-si RNA was transfected into SMMC-7721 cells by Lipofectamine TM2000.In order to confirm the effection of PKM2 knockdown,we detected the level of PKM2 in SMMC-7721 after transfection for 24 h.Compared with control-si RNA group,the expression of PKM2 was significantly decreased in PKM2-si RNA group after detecting PKM2 expression by Western blot and q RT-PCR.Taken together,PKM2 could be down-regulated or over-expressed successfully.To explore the effect of PKM2drown-regulation in SMMC-7721 on cell proliferation and apoptosis,cells were transfected with PKM2-si RNA or control-si RNA.As a result,PKM2-si RNA significantly down-regulated the expressions of proliferation-related protein C-myc and Cyclin D1 by Western blot.Furthermore,knocking down PKM2 decreased the ability of SMMC-7721 cells to form colonies.There were increased PKM2-si RNA SMMC-7721 cells arrested in G1 phase(82.2%)compared with control-si RNA cells(73.9%).Moreover,Cell apoptosis analysis indicated that PKM2-si RNA increased the percentage of early apoptosis and advanced apoptosis in SMMC-7721 cells.These results suggested that PKM2 repression inhibited cell proliferation and promoted cell apoptosis of SMMC-7721 cells.3.The effect of PKM2 up-regulation on cell proliferation in SMMC-7721 cells.GV230-PKM2 was transfected into SMMC-7721 cells by Lipofectamine TM2000.In order to confirm the effection of PKM2 up-regulation,we detected the level of PKM2 in SMMC-7721 after transfection for 24 h.Compared with GV230-control group,the expression of PKM2 was significantly increased in GV230-PKM2 group after detecting PKM2 expression by Western blot and q RT-PCR.Taken together,PKM2 could be over-expressed successfully.To explore the effect of PKM2 up-regulation in SMMC-7721 on cell proliferation and apoptosis,cells were transfected with GV230-PKM2 or GV230-control.As a result,GV230-PKM2 significantly up-regulated the expressions of proliferation-related protein C-myc and Cyclin D1 by Western blot.Furthermore,PKM2 over-expression increased the ability of SMMC-7721 cells to form colonies.There were decreased GV230-PKM2 SMMC-7721 cells arrested in G1 phase(63.5%)compared with control-si RNA cells(77.6%).Moreover,Cell apoptosis analysis indicated that GV230-PKM2 decreased the percentage of early apoptosis and advanced apoptosis in SMMC-7721 cells.These results suggested that PKM2 over-expression promoted cell proliferation and inhibited cell apoptosis in SMMC-7721 cells.4.The effect of PKM2 drown-regulation on ERSTo investigate whether PKM2 modulates cell proliferation and apoptosis were mediated through ERS,we measured the protein levels of ER Stress marker protein GRP78,ATF6 and CHOP when PKM2 expression levels were modulated in SMMC-7721 cells.Knocking down PKM2 by si RNA significantly increased the protein levels of GRP78,ATF6 and CHOP compared with that transfected with control-si RNA in SMMC-7721 cells.QRT-PCR experimental results further confirmed that m RNA lever of GRP78 was increased when PKM2-si RNA were transfected into cells.5.The effect of PKM2 up-regulation on ERSER Stress mark protein GRP78,ATF6 and CHOP were obviously down-regulated after transfection with GV230-PKM2 compared with that derived from GV230-PKM2 transfected SMMC-7721 cells.The m RNA lever of GRP78 was decreased when GV230-PKM2 were transfected into cells.These results suggested that PKM2 modulates cell proliferation and apoptosis via regulating ER Stress in SMMC-7721 cells.