{"paper_id":"077b22eb-9857-4919-9860-ef935060bcdc","body_text":"Research Article \nELUCIDATION OF THE ROLE OF miRNA 4263 IN DYSREGULATED MITOCHONDRIAL ENERGETICS AND \nCARCINOGENESIS \nASHUTOSH KUMAR MAURYA1, SRUTHI TV.2 , V.B. SAMEER KUMAR3,* \n \n1 Department of Biochemistry & Molecular Biology, Central University of Kerala, Kasaragod, 671320, India  \n2 John Hopkins University, USA \n3 Department of Genomic Science, Central University of Kerala, Kasaragod, 671320, India \n \n \nAbstract: Dysfunctional mitochondria have  been reported to be associated with several pathological conditions and \nin cancer, dysregulated mitochondrial metabolism is considered as an important hallmark of the disease. Cancer \ncells alter their mitochondrial machinery and activate glycolytic pathway as an alternate source of continuous \nenergy, required for their indefinite growth. This modulation of the mitochondria could be due to the dysrupted \nexpression of important mitochondrial genes involved in the normal functioning of the mitochondria. MicroRNAs \nare known to regulate the expression pattern of a variety of genes. With our in-silico analysis, we found that miR 4263 \nhas targets on important mitochondrial genes, involved in mitochondrial energetics. Next, we checked the role of miR 4263 \nin modulating the mitochondrial metabolism and impact of this alteration on carcinogenesis. The results revealed that miR \n4263 contributes to carcinogenesis in hepatic cells by altering the mitochondrial energetics. \n \nKey words:  \nmiRNA, Carcinogenesis,  Exosomes, Oxygraph, HCC \nIntroduction \nMicroRNAs are small non -coding RNA molecule of ∼ 22 \nnucleotides (Shoubin Jhan et.al, 2020). MicroRNAs possess \nthe ability of regulating the expression pattern of a variety \nof genes involved in normal functioning of the cells, thus \nplays a very important role in cells survival (Lyudmilla \net.al, 2016). MicroRNAs are coded by nuclear DNA as well \nas mitochondrial DNA (Isabelle D. et.al, 2021).  \nVarious reports suggests that nuclear coded microRNAs \nare localised in to the mitochondria and regulate the \nexpression of target mitochondrial genes (Chiara Giordani \net. al, 2021). Thus, nuclear coded microRNAs play \nimportant role in the normal functioning of the \nmitochondria, along with their mitochondrial counterpart, \nas they too possess the targets on mitochondrial protein \ncoding genes (Goud et.al, 2015).  \nMicroRNAs, capable of changing the fun ctioning of \nmitochondria, are called MitomiRs (Bandiera S., 2013). \nThese MitomiRs either target internal proteins that are \ndirectly involved in ATP generation or mitochondrial mem- \nbrane proteins that are involved in ATP transfer outside \nthe mitochondria ( Das S. et.al, 2012, Purohit P.K., et.al, \n2021), so they play important role in various diseases \nassociated with mitochondrial malfunctioning including \ncancer (Bienertova-Vasku J. et.al, 2013).  \nDysregulated energetics and altered mitochondrial \nmetabolism are signif- icant cancer hallmarks (Sheng -Fan \nWang et.al, 2023). In case of cancer, the mitochondrial \nmachinery is altered and glycolytic pathway is activated to \nmeet the energy requirement to support the infinite \ncellular growth (Narayan - swami Badrinath e t.al, 2018). \nSeveral studies have linked the altered mitochondrial \nmetabolism with a variety of cancers (Fan S et.al, 2019). \nMitochondrial machinery is affected either by critical gene \nmutations or by silencing of genes involved in nor - mal \nmitochondrial f unctioning (Evanthia Pangou et.al, 2021, \nChaojun Y et.al,2019).  \nMicroRNAs could be a key player in the suppression of \nexpression pattern of the crucial mitochondrial genes \ninvolved in electron transport chain (ETC), thereby altering \nthe rate of ATP generation (Wee Lin Tan et.al, 2023). Several \nmalignancies have been reported to have altered \nmitochondrial machinery together with elevated amounts of \noncogenic microRNAs (Sheng -Fan Wang et.al, 2023). \nMicroRNA 4263 is an important oncomiR reported to be \nfound a t higher levels in exosomes derived from hypoxic \ntumor colony of HCC and plays an important role in \ncarcinogenesis by inducing angiogenesis, an important \nhallmark of cancer (Sruthi TV, 2019).  \nThis study deals with elucidation of the role of miR 4263 in \ncarcinogenesis and modulation of mitochondrial \nfunctioning.  \nMethodology \nCell culture: Hela (cervical cancer cell line), WRL-68 (Human \nhepatic non cancerous cell line) and HepG2 cells (Hepatic \ncarcinoma cell line) were cultured in DMEM supplemented \nwith 10% FBS, antibiotic -antimycotic solution and L -\nGlutamine. The cells were maintained under standard \nculture conditions at 37°C with 5% CO2 and 95% humidity. \nFor experiments, seeding den- sity of 0.4 x 104 cells (96 well), \n0.6×106 cells (30mm dish), 0.8×10 6 cells (60mmdish), \n2.2×106 cells (100 mm dish) were used.  \nmiR 4263 cloning: miRNA 4263 was cloned in pCMV miR \nvector between BamH1 and Xho1 restriction sites and \nsuccessful cloning was confirmed by sequencing.  \nTransformation: The competent cells (DH5 𝛼 ) were \ntransformed with miR 4263 plasmid by heat shock method \nwhere the plasmid was incubated with the competent cells \nfollowed by a quick heat shock at 90°C for 2 minutes and then \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \nimmediately transferring it on ice. The transformed cells \nwere then plated on agar plate containing kanamycin. \n \nPlasmid isolation: A Single colony was picked from the agar \nplate and grown in the LB broth containing Kanamycin. The \nbroth was incubated at 37°C in a shaking incubator. The \nplasmid was isolated b y using Himedia midi k it following \nmanufacturers protocol. \n \nTransfection: HeLa cells were seeded in 6 well plates and \ngrown in a monolayer. After reaching 70% confluency, the \ncells were transfected with miR 4263 plasmid using PEI \nreagent and incubated for 24 hours in a CO2 incubator. \nAfter 6 hours of the incubation, the medium was replaced \nwith fresh media and further incubated for 24  hours. \nFollowing this, the cells transfected with plasmids having \nfluorescent tags were observed under fluorescent \nmicroscope to check the efficiency of transfection \n \nIsolation of mitochondria: The mitochondria were isolated \nfrom the transfected cells using hypotonic buffer, where \nthe cells were allowed to swell in the buffer for 10 minutes \nand then break open the cells to release the mitochondr ia. \nThe cell suspension was then centrifuged at 1300g to \nremove the cell debris, followed by centrifugation at \n12000g to get the mitochondrial pellet. The mitochondrial \npellet was suspended in the mitochondrial resuspension \nbuffer. \n \nSonication: \nThe mitochondrial pellet was mixed with Lysis buffer and \nsonicated for 2 minutes at 70% amplitude with 15 sec ON \nand 30 sec OFF cycle on 4°C. The solution obtained, was \ncentrifuged at 12000g for 10 minutes. The supernatant was \ncollected and protein estimation was don e followed by \nsample preparation for SDS PAGE. \nProtein estimation: \nProtein level of mitochondria was estimated by Bradford \nmethod (Bradford etal,1976). To achieve this, 10μl of \nsample and 90μl of bradford reagent (50 mg Coomasie \nBrilliant Blue -G250 in 25ml  ethanol and 50ml of \nphosphoric acid made upto 100ml with water) was added \nin triplicates in 96 well plate and the absorbance was taken \nat 595nm by multimode plate reader. The concentration of \nprotein was calculated from the standard plot to BSA with \nconcentration range from 10μg-100μg.  \nSDS-PAGE: \nProtein sample was prepared by mixing of 6x SDS loading \ndye and boiling it at 90°C for 10 minutes in water bath. The \nsample was immediately kept on ice and briefly centrifuged \nbefore loading on SDS - PAGE gel. The electrophoresis was \ncarried out by using Bi o-Rad electrophoresis unit. The \nprotein samples were run through the stacking gel at 80V \nfor 15 minutes and through the resolving gel at 100V at \nroom temperature until the dye reached the end of the gel.  \nWestern blot analysis: The purity of the mitochondr ial \npellet was checked by western blot using mitochondria \nspecific antibody (VDAC). Also, the mitochondrial pellet \nwas checked for the nuclear and cytoplasmic contaminants \nusing Histone H3 antibody for Nucleus and Hexokinase \nHK3 antibody for the cytoplasm.  \n \nRNA Isolation: RNA was isolated from the mitochondrial \npellet as well as from the total cell using trizole reagent. \nFollowing this, the concentration of the RNA was checked by \nusing nano drop. \n \nPolyadenylation of RNA: Poly A tail was added to the RNA by \nPoly A Polymerase  enzyme, using manufacturers protocol.  \nThis reaction set up was incubated at 37°C for 30 minutes \nfollowed by heat inactivation for 5 minutes at 65°C.  \ncDNA synthesis: The polyadenylated RNA were used for the \nsynthesis of miRNA 4263 specific cDNA by Kang method. \nApart from this, total RNA was used to synthesize the cDNA \nfor checking the expression of mitochondrial genes. \n \nReal Time PCR( qRT PCR): Quantative real time PCR was \nperformed to check the expression pattern of the microRNA \n4263 and other mitochondrial genes in mitochondria before \nand after over expression of miR 4263. \n \nmRNA stability assay: To elucidate the targeting of \nmitochondrial genes by miR 4263, mRNA stability assay was \nperformed. The cells were transfected with miR 4263 using \nPEI method. 24 hours post transfection the cells were treated \nwith actinomycine D at 0, 1, 3, 6 and 12 hours. The samples \nwere collected at each time point for gene expression study. \n \nOxygraph analysis: To check the phenotypic effects of the \ndown regu lation of the mitochondrial genes by miR 4263, \nthe oxygraph analysis was performed, where the oxygen \nconsumption level was checked in the miR 4263 over \nexpressed samples and compared with the control samples. \nIn brief, the cells were grown in a 6 well plate and transfected \nwith the candidate microRNAs using PEI method. After 48 \nhours of incubation at 37°C, the cells were trypsinized and \nthe cell pellet was resuspended in respiration buffer. Later, \n1ml of the cellular suspension was added to oxymeter and \noxygen intake reading was recorded for 10 minutes. The \nreadings were used to plot the graph to represent the oxygen \nconsumption by the mitochondria.  \nExosome isolation: The exosomes were isolated from the miR \n4263 over expressing cells using PEG method. To achieve \nthis the cells were transfected with miR 4263 and media was \nreplaced with serum free medium. 48 hours post \ntransfection; the spent media was collected and centrifuged \nat 2000g for 1/2 hour, to remove cell  debris. Following this, \nthe cells were mixed with PEG solution in 1:2 ratios and \nincubated at 4 degree C overnight. Finally, the solution was \ncentrifuged at 12000g for 1 hour. The exosomal pellet was \ndissolved in PBS and protein estimation was done.  \n \nCell migration Assay: Cancer cells have the metastatic \nproperties, where they move from its origin to another place \nand form a secondary tumor. To mimic this in -vitro we \nperform the cell migration assay with an objective to check, \nif the exosomes from miR 4263 over expressing cancer cells \ncould induce cellular migration of normal Hepatic cells. A \nscratch was made in the WRL monolayer and treated with \nexosomes isolated from miR 4263 over expressing cancer \ncells. The cells were allowed to fill the gap formed by the \nscratch for 48 hours and the images were tak en 0, 24, 48 \nhours respectively and quantified using ImageJ. \n \nSoft Agar Colony formation assay: To elucidate the role of miR \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \n4263 in inducing carcinogenic properties in normal heaptic \ncells, the colony formation assay was performed where the \nWRL cells were suspended in the low melting agarose and \ntreated with the exosomes. The cells were incubated at \n37°C for 28 days and allowed to form the colonies.  The \ncolonies were stained with Coomasie brilliant blue and \nimages were taken from 20 different locations and \nquantification was done by using ImageJ. \n \nIn-Silico analysis: miR 4263 target prediction analysis and \nscoring for mitochondrial genes was done using five  \nalgorithms i.e. MirBase, miRanda, Target Scan, miRDB  and \nMicroRNA.org. Five highest scored mitochondrial genes \n(ND6, ATP6, Cyto -B, Cox1, ND4L) were selected for target \nvalidation.  \nStatistical analysis: \nAll the data in the study were expressed as the mean with \nthe standard error mean of at least three experiments, each \ndone in triplicates. SPSS 11.0 software was used for \nanalysis of statistical significance of difference by Duncan’s \nOne way Analysis of Variance (ANOVA). A value of P<0.05 \nwas considered significant.  \nResults \nCellular and mitochondrial levels of miR 4263 in cancerous \nand non-cancerous hepatic cell lines  \nAs first part of the study, we checked the levels of \nmicroRNA 4263 in HepG2 and non - cancerous hepatic cell \nline (WRL) by qRT -PCR. The results revealed that, there \nwas a higher level of miR 4263 in HepG2 cells when \ncompared with the control cells. Following this, we checked \nthe levels of miR 4263 in the mitochondria of cancerous & \nnon-cancerous hepatic cells (i.e. HepG2 & WRL) a nd the \nresults suggested that level of miR 4263 was found \nsignificantly lower in the mitochondria of the HepG2 cells \nwhen compared with the control cells (WRL), even though \nthe total cellular levels miR 4263 was higher.  \n (Figure 1) \nOver expression of m iR 4263 resulted in its prefer ential \ntargeting to the mitochondria  \nAs we found  that the microRNA  4263 was found \nsignificantly low in the mitochondria, we over expressed \nmiR 4263 in HepG2 cells and checked its relative levels in \nthe cytoplasm & mito chondria. The results revealed that, \nmiR 4263 gets targeted to the mitochondria in a selective \nmanner when compared with the mock transfected cells, \nwith a 4.5 fold increase in mitochondria and 3 fold increase \nin the cytoplasm, when compared to the levels in the moc k \ntransfected cells.  \n (Figure 2) \n miRNA 4263 target the mitochondrial genes  \nAfter confirmation of the localization of miR 4263 to the \nmitochondria, next we checked the effect of this \nenrichment on the expression level of mitochondrial target \ngenes. To check this, we performed target validation study. \nThe results of this study revealed that level of all the target \ngenes of miR 4263 were significantly lowered when \ncompared with the control cells, confirming our in -silico \ntarget prediction analysis.  \n (Figure 3) \nmRNA stability assay revealed the targeting of ND6 gene by \nmiR 4263  \nTarget validation results revealed that levels of all the \nmitochondrial target genes were significantly lowered by \nmiR 4263. To further chec k the targeting of mitochondrial \ngenes by miR 4263, we performed mRNA stability assay and \nthe results revealed that level of all the target genes went \ndown, upon miR 4263 over expression, most effectively ND6, \nsuggesting that 3’ UTR of these genes harbour putative miR \n4263 binding sites.  \n (Figure 4) \nOxygen consumption by mitochondria of HepG2 cells went \nsignificantly lower in miR 4263 over ex- pressing cells  \nThe mRNA stability assay suggested that miR 4263 targets \nthe ND6 gene which is very crucial for complex 1 of electron \ntransport chain in the mito chondria. Any alteration in the \nexpression level of this gene may dysregulate the normal \nfunctioning of the mitochondria. So, to analyze the \nphenotypic effect of the down regulation of this gene on the \nmitochondrial metabolism, we perform ed the oxygraph \nanalysis, where we checked the levels of the oxygen \nconsumption by the HepG2 cells.  \nThe result of the oxygraph analysis revealed  that the levels \nof oxygen consumption by the HepG2 cells went significantly \ndown upon miR 4263 over expression when compared with \nthe mock transfected controls , suggesting its role in \nmodulating the mitochondrial machinery.  \n (Figure 5) \nMicroRNA 4263 increases t he migratory properties of \ncancerous and non-cancerous hepatic cells:  \nFrom our target validation and mRNA stability assay results, \nwe found that miR 4263 get s targeted to the mitochondria \nand down regulate the expression of ND6 gene important in \ncomplex 1 of ETC and further it reduced the oxygen \nconsumption by the HepG2 cells, probably due to the altered \nmitochondrial metabolism. So, we next checked the impact \nof this altered mitochondrial metabolism in carcinogenesis. \nTo achieve this, we performed the cell migration assay using \nnon cancerous & cancerous hepatic cells (WRL & HepG2) \nand treated it with the exosomes isolated from miR 4263 \nover expressing cancer cells or mock transfected Hela cells. \nThe results of cell migration assay revealed an increase in the \nrate of cellular migration when treated with exosomes \nisolated from the miRNA 4263 over expressing cells, when \ncompared to the cells treated with the exosomes isolated \nfrom the mock transfected cells and the pattern was found to \nbe persistent in both, cancerous as well as non -cancerous \nhepatic cell lines.  \n(Figure 6 & 7) \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \nExosomes from miR 4263 over expressing cells enhances \ncolonization property  \nWe performed colony formation assay to check the \ncolonogenic ability of miR 4263 in normal and cancerous \nhepatic cells. Here, we treated hepatic cell colonies with the \nexosomes isolated from miR 4263 over expressing cells or \nmock transfected control cells. Colony formation assay \nresult showed an increase in the number and size of the \ncolonies when treated with the exosomes enriched with \nmiR4263 as compared with the colonies treated with \nexosomes isolated from the mock transfected cells.  \n(Figure 8) \nDiscussion  \nDysregulated mitochondrial  energetics has been \nestablished as one of the major hallmark of the cancer \n(Shan-Millan I, et.al, 2023). By modifying mitochondrial \nmetabolism, cancer cells change how they produce energy \nin more advanced stages of the disease. (Sheng -Fan Wang \net.al, 2023). In a variety of cancer, the glycolytic pathway \nhas been reported as an alternate mode of energy, \nemployed by the cancer cells for their exponential growth. \n(Ganpathi K et.al, 2013).  \nThe modulation of mitochondrial machinery could either \nresult due to the mutations in important genes involved in \nmitochondrial metabolism or due to the suppression of \nexpression of the gene s engaged in the energy produc tion \n(Amanda Lopes, 2020). The suppression of mitochondrial \nactivity could be brought in by the action of the microRNAs \n(Zhang 2021). The microRNAs are small non-coding RNAs, \ncapable of regulating the expression pattern of a variety of \nthe genes involved in various cellular functions (Kioomars \nS. et. al, 2019). MicroRNAs which could alter the \nmitochondrial metabolism are called mitomiRs (Isabelle D. \net.al, 2021). MitomiRs can either be coded by \nmitochondrial genome or nuclear genome. From the \ncytoplasm, nuclear coded miRNAs are transported to the \nmitochondria where they function by controlling the \nexpression le vels of metabolism related mitochondrial \ngenes, resulting in reprogramming of the mitochondrial \nmachinery and replacement of electron transport chain \nwith glycolytic pathway (Purohit P.K., et.al, 2021).  \nHepatocellular carcinoma (HCC) is 3rd most common ty pe \nof cancer, with more than 1 million estimated yearly cases \nby the 2025 (Josep M et.al, 2021). The key role of various \nmicroRNAs including mitomiRs, have been reported to be \nimportant in the initiation and progression of HCC (Yi Fu \net.al, 2019, Zhang Lisheng et.al, 2013). MicroRNA 4263 has \nbeen found at higher levels in the exosomes isolated from \nhypoxic tumor colonies of HCC, and was found to be \ninvolved in the process of angiogenesis, (Sruthi TV, 2019), \nthereby acting as a key player in carcinogenesis.  \nThe main objective of this study was to check the role of \nmiR 4263 in the modulation of the mitochondrial \nmetabolism and its impact on carcinogenesis. Since miR \n4263 is a nuclear coded miRNA, first we checked its level in \nthe mitochondria of HepG2 by qRT -PCR. The results \nrevealed that miR 4263 was significantly low in the \nmitochondria. As established by the various studies, that \nnuclear coded miRNAs gets localized to the mitochondria, \nnext we over expressed miR 4263 in HepG2 cells and qRT - \nPCR results sugg ested that miR 4263 got enriched in the \nmitochondria by 4.5 folds in a preferential manner.  \nOur bioinformatic results revealed that miR 4263 has targets \non impor tant mitochondrial genes, and also we found that \nmiR 4263 gets targeted to the mitochondria, so next we \nperformed target validation study to check if miR 4263 could \nalter the expression of mitochondrial genes upon reaching \nthe mitochondria. The qRT -PCR res ults r evealed that, miR \n4263 signifi cantly lowered the expression levels of all \nmitochondrial target genes. Next, we performed mRNA \nstability assay for all mitochondrial target genes, to further \ncheck their targeting by miR 4263 and the results revealed \nthat ND6 is most effectively targeted by miR 4263, whereas \nCox1, CytoB and ATP6 gets targeted however to non \nsignificant extent and the result falls in line with the 3’ UTR \nanalysis that revealed that 3’ UTR of ND6 harbour putative \nmiR4263 binding sites. Since, ND6 gene plays important role \nin electron transport chain and is crucial for the \nmitochondrial machinery, its down regulation could directly \nimpact the mitochondrial metabolism.  \nSo, next we checked the phenotypic effects of this down \nregulation by performing the oxygraph analysis, to check the \noxygen consumption by mitochondria of miR  4263 \noverexpressed cells and found that oxygen consumption \nlevel went significantly down, suggesting its role in altering \nthe functioning of mitochondrial machinery by ta rgeting \nND6, which is involved in complex 1 of electron transport \nchain. Several studies have established the role microRNAs \nin improving the migratory properties of hepatic cancer \ncells, e.g. miR 221 has been found to promote HCC cells \nmigration by targeting Plant homeo domain finger 2 (PHF2), \na tumor suppressor, regulating P53 (Yi Fu et.al, 2019) and \nmiR-665 promotes proliferation and migration of HCC cells \nby targeting PTPRB involved in hippo signalling (Yuanchang \nHu et.al, 2018). MicroRNAs have also bee n found in \nescalating the colonogenic properties of HCC, for example \nmicroRNA 657 has been reported to promote tumorigenesis \nin HCC by targeting transducing -like enhancer protein -1 \n(TLE-1) (Zhang Lisheng et.al, 2013), and also, miR-125b has \nbeen suggested to be involved in HCC progression (Dorothy \nFan et.al , 2012).  \nSince, these studies demonstrated that under over expressed \nconditions, the microRNAs promoted cell migration and \ncolony formation, so next we checked if the over expression \nof miR 4263 too, ca n induce carcinogenesis in hepatic cell \nlines. To achieve this, we performed cell migration assay and \ncolony formation assay by treating the hepatic cells with the \nexosomes isolated from miR 4263 over expressing cells. The \nresults suggested that migratory and colony formation \nability of hepatic cells got enhanced when treated with the \nexosomes enriched with miR 4263.  \nThese results therefore suggest that miR 4263 possess \ncarcinogenic ability and that it possibly involve the \nmodulation of mitochondrial metabolism.  \nAcknowledgment \nWe acknowledge Indian Council of Medical Research, \nMinistry of Health, Govt. of India for th e financial assistance \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \nin form SRF and Kerala state council for Science \nTechnology & Environment, Govt. of Kerala for fellowship \nin the form of JRF and SRF to Mr. Ashutosh K. Maurya. We \nalso acknowledge Central University of Kerala for \nproviding all the ne cessary facilities to carry out this \nresearch work. \nAuthor Contributions  \nThe authors  confirm contrib ution to the paper as \nfollows: S tudy conception and design: VBSK, \nBioinformatics and wet lab work: AKM. Cloning of miR \n106b: STV. All authors reviewed the results and \napproved the final version of the manuscript.  \nConflicts of Interest \nThe authors declare that they have no conflicts of \ninterest to report regarding the present study.  \nReference: \n Zhan, S., Wang, Y. & Chen, X. RNA virus -encoded \nmicroRNAs: biogene sis, functions and \nperspectives on application. ExRNA 2, 15 (2020).  \n Gulyaeva, L.F., Kushlinskiy, N.E. Regulatory \nmechanisms of microRNA expression.  J Transl \nMed 14, 143 (2016  \n Giordani C, Silvestrini A, Giuliani A, Olivieri F and \nRippo MR (2021) MicroRNAs as Factors in \nBidirectional Crosstalk Between Mitochondria and \nthe Nucleus During Cellular Senescence.  Front. \nPhysiol. 12:734976.  \n Bandiera, S., Matégot, R., Girard, M., Demongeot, J. \nand Henrion -Caude, A. (2013). MitomiRs \ndelineating the intracellular localization of \nmicroRNAs at mitochondria. Free Radic. Biol. Med. \n64, 12-19.  \n Bienertova-Vasku, J., Sana, J. and Slaby, O. (2013). \nThe role of microRNAs in mitochondria in cancer. \nCancer Lett. 336, 1-7.  \n Goud, M. R. and Hua, Z. A. (2015).  Role of \nmicroRNA in the regulation of mitochondrial \nfunctions. Sci. Lett 3, 83-88. \n Das, S., Ferlito, M., Kent, O. A., Fox-Talbot, K., Wang, \nR., Liu, D., Raghavachari, N., Yang, Y., Wheelan, S. J., \nMurphy, E. et al.  (2012). Nuclear miRNA regulates \nthe mitochondrial genome in the heart. Circ. Res. \n110, 1596-1603.  \n Fan, S., Tian, T., Chen, W., Lv, X., Lei, X., Zhang, H., \nSun, S., Cai, L., Pan, G., He, L. et al.  (2019). \nMitochondrial miRNA determines \nchemoresistance by reprogramming metabolism \nand regulating mitochondrial transcription. \nCancer Res. 79, 1069-1084. \n Narayanasamy Badrinath, So Young Yoo, \nMitochondria in cancer: in the aspects of \ntumorigenesis and targeted therapy, \nCarcinogenesis, Volume 39, Issue 12, Dec ember \n2018, Pages 1419–1430  \n Luo, Y.; Ma, J.; Lu, W. The Significance of \nMitochondrial Dysfunction in Cancer. Int. J. Mol. \nSci. 2020, 21, 5598. \nhttps://doi.org/10.3390/ijms21165598 \n Yan, C.; Duanmu, X.; Z eng, L.; Liu, B.; Song, Z. \nMitochondrial DNA: Distribution, Mutations, and \nElimination. Cells 2019, 8, 379.  \n Pangou E and Sumara I (2021) The Multifaceted \nRegulation of Mitochondrial Dynamics During \nMitosis. Front. Cell Dev. Biol. 9:767221.  \n Tan, W.L., Sub ha, S.T., Mohtarrudin, N. et al. An \ninsight into the associations between microRNA \nexpression and mitochondrial functions in cancer \ncell and cancer stem cell. Mol Biol Rep 50, 5395 –\n5405 (2023).  \n San-Millán I. The Key Role of Mitochondrial \nFunction in Healt h and Disease. Antioxidants \n(Basel). 2023 Mar 23;12(4):782. PMID: 37107158; \nPMCID: PMC10135185 \n Wang, SF., Tseng, LM. & Lee, HC. Role of \nmitochondrial alterations in human cancer \nprogression and cancer immunity.  J Biomed Sci  30, \n61 (2023).  \n Ganapathy-Kanniappan, S., Geschwind, JF.H. Tumor \nglycolysis as a target for cancer therapy: progress \nand prospects. Mol Cancer 12, 152 (2013).  \n F. C. Lopes, A. Mitochondrial metabolism and DNA \nmethylation: a review of the interaction between \ntwo genomes. Clin Epigenet 12, 182 (2020).  \n Zhang G-Q, Wang S -Q, Chen Y, Fu L -Y, Xu Y -N, Li L, \nTao L and Shen X -C (2021) MicroRNAs Regulating \nMitochondrial Function in Cardiac Diseases.  Front. \nPharmacol. 12:663322.  \n Saliminejad K, Khorram Khorshid HR, Soleymani \nFard S, Ghaffari SH. An overview of microRNAs: \nBiology, functions, therapeutics, and analysis \nmethods. J Cell Physiol. 2019 May;234(5):5451 -\n5465. Epub 2018 Nov 23. PMID: 30471116. \n Duroux-Richard I, Apparailly F and Khoury M \n(2021) Mitochondrial MicroRNAs Contribut e to \nMacrophage Immune Functions Including \nDifferentiation, Polarization, and Activation.  Front. \nPhysiol. 12:738140.  \n Purohit, P.K., Saini, N. Mitochondrial microRNA \n(MitomiRs) in cancer and complex mitochondrial \ndiseases: current status and future \nperspectives. Cell. Mol. Life Sci.  78, 1405 –1421 \n(2021).  \n Llovet, J.M., Kelley, R.K., Villanueva, A.  et \nal. Hepatocellular carcinoma. Nat Rev Dis Primers 7, \n6 (2021).  \n Sruthi T.V., Identification and functional analysis of \ntumor derived miRNA relevant in t he process of \nangiogenesis, 2019,  \n Yi Fu, Mingyan Liu, Fengxia Li, Li Qian, Ping \nZhang, Fengwei Lv, Wenting Cheng, Ruixing Hou, \n\"MiR-221 Promotes Hepatocellular Carcinoma \nCells Migration via Targeting PHF2\",  BioMed \nResearch International , vol. 2019, Article ID \n4371405, 11 pages, 2019.  \n Hu, Y., Yang, C., Yang, S.  et al.  RETRACTED \nARTICLE: miR -665 promotes hepatocellular \ncarcinoma cell migration, invasion, and \nproliferation by decreasing Hippo signaling \nthrough targeting PTPRB.  Cell Death D is 9, 954 \n(2018).  \n Zhang, Lisheng1; Yang, Lixin 1; Liu, Xiyong1; Chen, \nWei1; Chang, Lufen1; Chen, Linling1; Loera, Sofia2; \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \nChu, Peiguo 2; Huang, Wei -Chien3,4; Liu, Yun -\nRu5; Yen, Yun 1,5. MicroRNA-657 promotes \ntumorigenesis in hepatocellular carcinoma by \ntargeting transducin -like enhancer protein 1 \nthrough nuclear factor kappa B pathways. \nHepatology 57(5):p 1919-1930, May 2013.  \n Ngo-Yin Fan, D., Ho -Ching Tsang, F., Hoi -Kam \nTam, A., Leung -Kuen Au, S., Chak -Lui Wong, C., \nWei, L., Man-Fong Lee, J., He, X., Oi-Lin Ng, I. and \nWong, C. -M. (2013), Histone lysine \nmethyltransferase, suppressor of variegation 3-\n9 homolog 1, promotes hepatocellular \ncarcinoma progression and is negatively \nregulated by  microRNA-125b. Hepatology, 57: \n637-647.   \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n  \n \n \n \nSUPPLEMENTARY DATA: \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n                \n                                                 A.                                                                  B. \nFigure 1: Differential expression pattern of miR 4263 in cytoplasm and mitochondria. RT PCR \nanalysis was performed to check the levels of miR 4263 in mitochondrial    and cellular fraction of HepG2 \ncells, keeping WRL as control. The results suggested that level of miR 4263 was significantly low in \nthe mitochondria of HepG2 cells. A.) Relative expression levels of miR 4263 in the cells B.) Relative \nlevels of miR 4263 in the mitochondria. Results presented are average of three experiments ± SEM each \ndone at least in triplicate, p<0.05. *Statistically significant when compared to control. \n \n0\n0.5\n1\n1.5\n2\nWRL HepG2\nRelative Expression of \n4263\nCELL\nWRL\nHepG2\n*\n0\n0.2\n0.4\n0.6\n0.8\n1\n1.2\n1.4\nWRL HepG2\nRelative Expression of \n4263\nMITOCHONDRIA\nWRL\nHepG2\n*\n                     \nFigure 2: miR 4263 gets targeted to the mitochondria in a selective manner. miR 4263 was over expressed \nin HepG2 cells followed by purification of mitochondria & isolation of RNA from mitochondrial as well as cytoplasmic \nfractions and, qRT PCR was then performed to check the levels of miR 4263. The results revealed that miR 4263 \ngets targeted to mitochondria in a selective manner. Results presented   are average of three experiments ± SEM \neach done at least in triplicate, p< 0.05.*Statistically significant when compared to control. \n \n \n \n \n \n \n \n \n \n \n \n-1\n0\n1\n2\n3\n4\n5\n6\n7\nNormal miR 4263 Level miR 4263 Over\nexpression\nRelative Expression of miR 4263\nCytoplasm\nMitochondria\n*\n*\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nFigure 3: miR 4263 down regulates all its target genes. miRNA 4263 was over expressed in HepG2 \ncells and the  mitochondria isolated. RT-PCR analysis was performed to check the impact of  increased \nmiR 4263 level on the expression pattern of target mitochondrial genes. The result revealed  that the \nexpression level of all the mitochondrial target genes of miR 4263 went significantly down. Results  \npresented are average of three experiments ± SEM each done at least in triplicate, p<0.05.*Statistically \nsignificant when compared to control. \n \n \n \n \n \n \n  \nFigure 4: 3’ UTR analysis and mRNA stability assay revealed the direct targeting of ND6 by miR 4263. miR \n4263 was overexpressed in HepG2 cells and 24 hours post transfection, the cells were treated with actinomycin D \nand RNA samples were collected at 0, 1, 6 and 12 hours respectively. Following this, RT -PCR analysis was \nperformed to check the expression p attern of target genes. Results revealed that expression of ND6 went \nsignificantly down with time suggesting its direct targeting by miR4263. The expression pattern of Cox1, ATP6 \nand Cyto-B also was altered, but not significant. A.) Relative expression of ND6 B.) 3’ UTR analysis of ND6 C.) \nRelative expression of ATP6 D.) 3’ UTR analysis of ATP6 E.) Relative expression of Cox1 F.) 3’ UTR analysis \nof Cox1 G.) Relative expression of Cyto-B H.) 3’ UTR analysis of Cyto-B. Results presented are average of three \nexperiments ± SEM each done at least in triplicate, P<0.05.*Statistically significant when compared to control.  \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n  \n \nFigure 5: Levels of oxygen consumption by the cells went significantly down, when  miR 4263 was \nover expressed. miR 4263 was over expressed in HepG2 cell line and 24 hour post  transfection, cells \nwere harvested and oxygraph analysis was performed. The results revealed a decrease in O2 consumption \nby the mitochondria of miR 4263 overexpressing cells when compared with the mock transfected cell. \nResults presented are average of three experiments ± SEM each done at least in triplicate, p<  \n0.05.*Statistically significant when compared to control. \n \n \n \n \n \n \n \n \n325\n330\n335\n340\n345\n350\ncontrol miR4263\nOxygen\nconcentration\nnmol/ml\n \nFigure 6: Exosomes isolated from miR 4263 over expressing cells were enriched \nwith miR 4263. Real time PCR analysis was performed  to check the enrichment of \nmiR 4263 i n the exosomes isolated from miR 4263 over express ing cells and \ncompared with the exosomes isolated from the mock transfected control c ells. The \nresults revealed that exosomes isolated from microRNA 4263 over expressing cell s, \nshown 7 fold higher level of microRNA 4263. Results presented are average of three  \nexperiments ± SEM each done at least in triplicate , p< 0.05.*Statistically significant \nwhen compared to control. \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nFigure 7: miR 4263 escalates the cellular migration when treated with exosomes isolated from microRNA \n4263 over expressing cells. Cells were grown in a monolayer and a scratch was made, followed by exosome \ntreatment. The microphotographs were taken at 0, 24, and 48 hours. The distance/gap covered by the cells with time \nwas estimated by image -J software. The result revealed that the rate of the cellular migration got escalated when \ntreated with exosomes enriched with miR 4263 . A.) Microphotograph of cell migration pat tern (HepG2) with \nrespect to time B.) Microphotograph of cell migration pattern (HepG2) with respect to time C.) Relative percentage \nof migration by HepG2 cells, at 0, 24 & 48 hours D.) Relative percentage of migration by WRL cells at 0, 24 & 48 \nhours E.) Relative percentage of wound healing at 0, 24 & 48 hours (HepG2). F.) Relative percentage of wound \nhealing at 0, 24 & 48 hours (WRL). Results presented are average of three experiments ± SEM each done at least \nin triplicate, p<0.05.*Statistically significant when compared to control.  \n \n \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n        \n                      A.                                                                                                      B.                                                             \n         \n                                      C.                                                                                   D. \n          \n                                    E.                                                                           F. \nFigure 8: miR 4263 enhances the tumor size when treated with exosomes isolated from microRNA \n4263 over expressing cells. HepG2 and WRL colonies were treated with exosomes  isolated from miR \n4263 overexpressing cells and allowed to grow for 28 days. Following this, microphotographs were \ntaken at 20 different regions and 20 colonies from each region was taken  for the size estimation by \nimage-J software. The number of colonies were counted manually from  20 regions to estimate the \nnumber of tumor colonies. The results revealed that the size as well as the   number of colonies got \nenhanced in HepG2 and WRL cells, when treated with exosomes enriched  with miR4263. A.) \nRepresentative microphotograph of HepG2 cell colony B.) Representative microphotograph of WRL \ncell colony C.) Comparative colony size of   HepG2 cells. D.) Comparative colony size of WRL cells \nE.) Number of colonies of HepG2 cells, when compared with control F.) Number of colonies of  WRL \ncells, when compared  with control. Results presented are average of three experiments ± SEM each \ndone at least in triplicate, p<0.05. *Statistically significant when compared to control. \n \n    \n    \n0\n50\n100\n150\n200\n250\nControl Test\nSize of the colony (nm)\nHepG2 Cell\nControl\nTest\n*\n0\n20\n40\n60\n80\n100\n120\nControl Test\nSize of the colony (nm)WRL CELL\nControl\nTest\n*\n0\n50\n100\n150\n200\n250\nControl Test\nNo. of colonies\nHepG2 cell\nControl\nTest\n*\n0\n50\n100\n150\nControl Test\nNo. of colonies\nWRL Cell\nControl\nTest\n*\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted April 27, 2024. ; https://doi.org/10.1101/2024.04.25.591199doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}