Part Ⅰ:A Study Of Function Of LKB1 In Colorectal Cancer Part Ⅱ:the Role Of Splicing Isoform CHK1-S In Liver Cancer Audits Clinical Significance

LKB1,also known as serine/threonine kinase 11(STK11),was first identified as an inactivating mutation in Peutz-Jeghers syndrome(PJS).LKB1 is often found in complex with pseudokinase STRAD and scaffold protein M025 to regulate its stability,kinase activity,and subcellular localization.LKB1 directly phosphorylates AMPK and 12 other AMPK-like kinases and is involved in the regulation of cell growth,metabolism,autophagy and polarity.A large number of studies showed that LKB1 was a tumor suppressor gene,and its inactivating mutations were associated with the development of various tumors,such as lung cancer and skin cancer.Although studies showed that a low rate LKB1 mutation in colorectal cancer,its low expression was associated with poor prognosis in colorectal cancer patients.To study the role of LKB1 in colorectal cancer,we first constructed a colorectal cancer cell line that LKB1 was stably knocked out using CRSPR-Cas9 technology.The effect of LKB1 on the proliferation of colorectal cancer cells was studied by CCK-8 assay.It was found that the deletion of LKB1 had no significant effect on the proliferation of colorectal cancer cells.Furthermore,the mice model of colorectal cancer inducted by AOM/DSS was used and the results showed that the number of colorectal tumors was no significantly difference between the mice of conditional knockout of LKB1 gene in intestine and the normal control mice.Through cell migration and invasion experiments,we found that LKB1 deletion promoted migration and invasion of colorectal cancer cells.Using mouse tail vein injection experiments,LKB1 deletion promoted lung metastasis of colorectal cancer cells in vivo.To investigate how LKB1 loss promotes migration and invasion of colorectal cancer cells,the differential expression genes was analyzed in HCT116 and HCT15 cells with LKB1 deletion or not,respectively,by using RNA-seq sequencing.The results showed that there were 44 differentially expressed genes,both in the two groups,including TNIK,a gene associated with colorectal caner.To investigate the relationship between LKB1 and TNIK,we found that LKB1 negatively regulated TNIK expression in colorectal cancer cells.LKB1 kinase inactivating mutations could not effectivly inhibit the expression of TNIK,and the results indicated that LKB1 negatively regulated TNIK expression depending on its kinase activity.To clarify the effect of TNIK on the migration and invasion of colorectal cancer cells,we firstly constructed cells that TNIK was stably knocked down.Migration and invasion experiments showed that TNIK knockdown could inhibit the migration and invasion of colorectal cancer cells.To investigate whether LKB1 affects the migration and invasion of colorectal cancer cells by regulating TNIK expression,we knocked down TNIK expression by siRNA in LKB1-deficient HCT116 cells.Then the results showed that knockdown of TNIK inhibited the metastasis of LKB1-deficient colorectal cancer cells migration,both in vivo and in vitro.To further explore the mechanism by which TNIK affects the migration and invasion of colorectal cancer cells,we analyzed the protein interacting with TNIK by mass spectrometry and found that TNIK interacted with the cytoskeleton-associated protein ARHGAP29.Previous studies showed that ARHGAP29 cloud inhibit the activity of RhoA,thereby inhibiting the phosphorylation of the actin depolymerization factor cofilin,which acts to depolymerize actin and ultimately accelerate the remodeling process of the actin skeleton.When we overexpressed TNIK or deleted LKB1 in colorectal cancer cells and we found that the phosphorylation of cofilin was decreased.In addition,we found that overexpression of TNIK or deletion of LKB1 in colorectal cancer cells significantly increased the pseudopods around the cells by using immumnofluorescence.These results suggested that TNIK may play a role in promoting the migration and invasion of LKB1-deficient colorectal cancer cells.In addition,we also preliminarily explored the effect of LKB1 on macrophages in the tumor microenvironment.Macrophages in the tumor microenvironment are usually divided into two polarization forms,the anti-tumor M1 phenotype and the tumor-promoting M2 phenotype.We induced macrophages using conditioned medium of LKB1-deficient colorectal cancer cell,and detected Ml and M2 related molecules expression by Q-PCR and immunofluorescence staining.As a result,it was found that the polarity of macrophages was more toward the M2 phenotype after being induced by the conditioned medium of LKB1 deletion colorectal cancer cells.In summary,our studies showed that LKB1 deletion had no significant effect on the proliferation of colorectal cancer cells,but LKB1 deletion significantly enhanced the metastatic ability of colorectal cancer cells partly depending on the expression of TNIK and the cytoskeleton remodeling affected by TNIK experssion.This study could provide new ideas for the treatment of colorectal cancer by revealing the possible mechanism of LKB1 deletion enhancing the metastatic ability of colorectal cancer cells.In addition,the role of LKB1 in colorectal cancer was enriched by preliminary study of the effect ofLKB1-deficient colorectal cancer cell conditioned medium on macrophage polarity.Alternative splicing of genes is the selective inclusion or deletion of different exons and introns during pre-mRNA processing.This process produces different mature mRNAs,greatly enriching the transcriptome and proteome diversity of the gene.It is estimated that up to 94%of genes in humans undergo alternative splicing.Alternative splicing of genes is involved in normal life activities such as tissue-specific formation and normal developmental processes.Aberrant RNA splicing is associated with a variety of diseases,including tumors.The alternative splicing abnormality of genes is closely related to tumorigenesis and tumor progression,and its splicing isoforms can also be used as tumor molecular markers and therapeutic targets.Alternative splicing abnormalities of genes are involved in the development of tumors,including cell proliferation,apoptosis,tumor cell metabolism,angiogenesis and tumor metastasis.At present,gene splicing abnormalities have been reported and studied in various tumors,but little is known about the significant alternative splicing events in hepatocellular carcinoma.To investigate aberrant splicing in HCC,we used the HTA2.0 microarray to analyze alternative splicing in hepatocellular carcinoma tissue samples of patients who received hepatectomy relapsed(5 patients)and non-relapsed(5 patients)within 2 years,and found differential splicing of CHK1,a cell cycle-associated gene.CHK1 is a serine/threonine kinase,and regulates DNA damage response.In this study,we measured the expression of an alternative CHK1 transcript(CHK1-S,excluded exon 3)in hepatocellular carcinoma(HCC)tissues and found that CHK1-S was significantly upregulated in HCC tissues compared with paired adjacent noncancerous tissues.The high ratio of CHK1-S was associated with relapse free survival(RFS)of postoperative HCC patients.To further explore the role of CHK1-S in HCC,we used CCK-8 assays,EdU incorporation assays and colony formation assays.The results showed that overexpression of CHK1-S significantly accelerated HCC cell proliferation,compared with overexpression of normal CHK1(CHK1-L).In addition,to explore the splicing mechanism of CHK1-S,we analyzed differentially expressed genes,particularly splicing-related genes from the microarray data that mentioned above,including SRPK1,as a splicing factor kinase regulating the activity of splicing factors.Next,our results showed that both mRNA and protein levels of SRPK1 in tumor tissues were significantly higher than the paired non-tumor tissues.Furthermore,the mRNA level of SRPK1 was positively correlated with CHK1-S mRNA levels(P=0.016).Overexpression of SRPK1 could upregulate the expression of CHK1-S mRNA in HepG2 and QSG-7701 cells,suggesting that SRPK1 was involved in the regulation of splicing CHK1-S.In conclusion,our study demonstrated that CHK1-S,acts as an oncogene,which was upregulated and associated with RFS in HCC patients.SRPK1 may mediate its mRNA splicing in HCC.All these data indicated that CHK1-S expression would have potential values in the prognostic assessment of HCC pateints and splicing factors kinase SRPK1 could be used as a potential therapeutic target.