Expressional studies of microRNAs in Hepatitis B Patients of Quetta , Pakistan

Hepatitis B (HB) is a severe and widespread infectious liver disease affecting millions of people throughout the globe and caused by hepatitis B virus (HBV). Persistent HBV infections results severe pathologies which include development of chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma (HCC). In response of the HBV, patient cells show differential expression of microRNAs (miRNAs) to manage and fight the HB. miRNAs are small, endogenous and regulatory RNAs, that play an important role in post-transcriptional gene regulation. The aim of this research is to study the expressional pattern of fifteen miRNAs; hsa mir-20, 21, 30, 99, 122, 141, 190, 200, 223, 320, 574, 1228, 4532, 6127 and 6852 in hepatitis B patients of Quetta, Pakistan, selected through literature survey on the basis of their significant role in hepatitis. Briefly, the total RNA from the hepatitis B patient of Quetta, Pakistan and healthy person extracted and subjected to the stem-loop RT-PCR (reverse transcriptional-PCR) for the selected fifteen miRNAs and GAPDH-as internal control gene. The eight miRNAs; hsamir-20, 21, 30, 99, 141, 574, 4532 and 6852 showed upregulation, whereas, the six miRNAs; hsa-mir-122, 190, 200, 223, 320, and 6127 showed down-regulation in HB-patient. One miRNA; 1228 is observed with equal expression in both patient and healthy person. Hepatitis responding miRNAs would be useful to better understand the disease for early diagnose and cure.


Introduction
MicroRNAs (miRNAs) with other tiny RNAs such as; short interfering RNAs (siRNAs) constitute an important regulatory class of RNA, called as, small RNAs.The miRNAs are non-coding, endogenously expressed by cells with length ranges between 18 to 26 nucleotides (nts) and involve in post-transcriptional gene regulation [1].In human, 30% of the whole gene set regulation by miRNAs, is the evidence of its significance for the maintaining of life [2].The miRNAs have been reported to be produced cell and tissue specific, indicating temporal phases expression and as well as role in several biological processes, such as development, differentiation, proliferation, response to biotic and abiotic stresses and the establishment of pathological states [2-5].Hepatitis B (HB) is an infectious liver damaging disease and its main reason is the hepatitis B virus (HBV).HBV on the basis of the genome is a DNA virus with 3.2 kilobases size and has a relaxed-circular and closed architecture with four gene capacity.These are S, Core, P and X genes and involved in the coding of an envelope, a core with e, reverse transcriptase with DNA polymerase and X-proteins respectively [6].After the invasion in the liver cells, HBV relaxed-circular DNA reshaped itself and transcribed into viral messenger RNAs and can cause both acute and chronic disease [6, 7].HB initially with no indications or symptoms, later show the symptoms as; jaundice, fatigue, blackish urine color, abdominal pain, nausea and illness and if not cured may cause death within few weeks [8].Chronic HB has affected more than 350 million people worldwide and thus cause a serious threat to public health.The majority, 75% of 350 million people are from South East Asia and the Western Pacific region.This is clearly showing that why countries like Pakistan should pay more attention for research and awareness for the HB.The alteration in the expression of specific miRNAs to respond the viral invasion by the host cell is one of the prime areas of today gene regulation research.These studies on the relationship between miRNAs and human hepatitis B virus (HBV) infection; have produced convincing evidence that the interplay between HBV and cellular miRNAs is important to many facets of HBV replication and pathogenesis [9].The present research is aimed to study and understand the expressional response of the significant selected miRNAs in the HB patients of Quetta, Pakistan.

Blood sampling
Total six blood (serum) samples of the HB patients, three male and three female, diagnosed by experienced pathologists for HBV and showing the positive test for Hepatitis B surface antigen (HBsAgpositive) were collected from outdoor patients of Al-Shafi hospital, Quetta, Balochistan-Pakistan. Their ages were ranged from 30-50 years.The control (healthy) blood sampling is done from the healthy, non-HB and HBV negative volunteer students and scholars of the Department of Botany, University of Balochistan Quetta.All the blood samples, in anticoagulant tubes, were stored at -20ºC in the freezer of Molecular Biology and Bioinformatics Lab (MBBL) of the Department of Botany, University of Balochistan Quetta.

RNA extraction and cDNA synthesis
Total RNAs from the blood (serum) samples of Hepatitis B patient and volunteer healthy persons were extracted by the Trizol method.Briefly, 1ml Trizol is added to a 1.5ml tube containing 0.4 ml of the sample, vortex for 20-30 second and incubated on ice for 15 minutes.The tubes were centrifuged at 13000g for 10 minutes at 4ºC.The supernatant was extracted first with an equal volume of Chloroform Isoamyl alcohol (CI) (24:1, v/v) and secondly with an equal volume of Phenol Chloroform Isoamyl (PCI) (25:24:1 v/v/v).The total RNA was precipitated from the upper aqueous layer by twice volume of the 100% ice chilled Ethanol.The RNA pellet was dissolved in 40µl DEPC treated water and stored at -20 0C.Quantitative and qualitative analyses of RNAs were performed by measuring optical density at 260 nm and 280 nm using Genova Nano spectrophotometer.Total RNAs integrity were checked by loading and running on a 1% agarose gel and visualized under UV light for ribosomal RNA bands.The cDNAs from the 1µg HB patient and control total RNAs were synthesized by using the Thermo Scientific™ RevertAid™ H Minus First Strand cDNA Synthesis Kit, according to supplier's protocol.The invasion of the HBV in the host cell triggers an alteration in the expression of the host cell's miRNAs.This alteration of host cell's miRNAs may be either up or down regulation.These differentially expressed miRNAs are the important source to unravel the precise mechanism between miRNA and HBV.And it will also lead to developing new molecular techniques for the novel diagnosis and therapy of HB.

Upregulated miRNAs
The cDNAs synthesized from the total RNAs of HB patients and healthy persons were subjected to the expressional analysis of selected 15 miRNAs and an internal control GAPDH.The eight out of fifteen miRNAs showed upregulated expression in HB patients from Quetta-Pakistan (Figure 1).1).Due to the key functions of its target proteins in different signaling pathways, miR-21 has become an attractive candidate for genetic and pharmacological modulation in various disease conditions.

Figure 1 .
Figure 1.Experimental analysis of the selected human miRNAs in hepatitis B patients from Quetta, Pakistan by RT-PCR.The fifteen human significant miRNAs, demonstrating strong expressional response to HBV, identified through literature survey and a GAPDH-as internal control gene, were subjected to the expression analysis through RT-PCR.The eight miRNAs; hsa-mir-20, 21, 30, 99, 141, 574, 4532 and 6852 showed upregulation, whereas, the six miRNAs; hsa-mir-122, 190, 200, 223, 320, and 6127 showed downregulation in HB-patient.The one miRNA; 1228 is observed with equal expression in both patient and healthy person.

The fifteen human selected miRNAs' primers for RT-PCR expression analysis in hepatitis B patients from Quetta, Pakistan. The fifteen human significant miRNAs demonstrating strong expressional response to HBV, identified through literature survey, were subjected to expression analysis through RT-PCR. The selected miRNAs, their primers, TM (melting temperature) and the product size (in base pair, bp) are shown here
These eight miRNAs are hsa-mir-20, 21, 30, 99, 141, 574, 4532 and 6852.